ISOTHIOCYANATE CONTAINING BRASSICACEAE PRODUCTS AND METHOD OF PREPARATION THEREOF

Information

  • Patent Application
  • 20250197898
  • Publication Number
    20250197898
  • Date Filed
    December 20, 2024
    10 months ago
  • Date Published
    June 19, 2025
    4 months ago
Abstract
The present invention relates to methods for producing isothiocyanate containing products from Brassicacede material and lactic acid bacteria for use in such methods. The present invention also relates to isothiocyanate containing products from Brassicacede material produced by such methods.
Description
FIELD OF THE INVENTION

The present invention relates to methods for producing isothiocyanate containing products from Brassicaceae material and lactic acid bacteria for use in such methods. The present invention also relates to isothiocyanate containing products from Brassicaceae material produced by such methods.


BACKGROUND OF THE INVENTION

Brassicaceae family members are rich in glucosinolates which can be converted by the enzyme myrosinase to isothiocyanates which have been noted to have beneficial effects on some types of cancer (Moktari et al., 2017; Capuano et al., 2017; Kim and Park, 2016). Sulforaphane, for example, has been found to reduce hepatic glucose production and improve glucose control in obese patients with type 2 diabetes (Axelsson et al., 2017). However, many Brassicaceae family members are highly perishable after harvest with the quality and quantity of nutrients declining rapidly if the product is not stored well.


Brassicaceae are often processed to increase the shelf life which can result in the loss of nutrients. The main methods to obtain a longer shelf life include thermal processing, freezing, modified and controlled atmosphere storage and the addition of chemical preservatives which also would bring undesirable changes in chemical composition.


These processes can result in the loss of glucosinolates or reduce the ability of the enzyme myrosinase to convert glucosinolates to isothiocyanates. For example, conventional broccoli processing/preservation involves blanching prior to freezing to inactivate quality degrading enzymes such as lipoxygenase. Peroxidase inactivation is commonly used as an indicator of the adequacy of blanching. The condition for inactivation of peroxidase leads to the inactivation of myrosinase and thus the resulting product is devoid of isothiocyanates (Dosz and Jeffery, 2013).


Accordingly, there remains a need for improved methods for producing Brassicaceae products comprising phytonutrients such as isothiocyanates.


SUMMARY OF THE INVENTION

The present inventors have developed methods for preparing isothiocyanate containing products from Brassicaceae material.


In an aspect, the present invention provides a method of preparing an isothiocyanate containing product from Brassicaceae material comprising:

    • i) pre-treating the Brassicaceae material to improve the access of myrosinase to a glucosinolate;
    • ii) fermenting the material obtained by step i) with lactic acid bacteria to form the isothiocyanate containing product.


In an embodiment, pre-treating comprises one or more of the following:

    • i) heating;
    • ii) macerating;
    • iii) microwaving;
    • iv) exposure to high frequency sound waves (ultrasound); or
    • v) pulse electric field processing wherein the temperature of the Brassicaceae material does not exceed about 75° C. during pre-treating.


In an embodiment, pre-treating reduces epithiospecifier protein (ESP) activity while maintaining endogenous myrosinase activity.


In an embodiment, pre-treating comprises heating and macerating the Brassicaceae material and wherein the temperature of the Brassicaceae material does not exceed about 75° C. during pre-treating. In an embodiment, heating occurs before macerating or wherein heating and macerating occur at the same time. In an embodiment, pre-treating comprises heating the Brassicaceae material to a temperature of about 50° C. to about 70° C. followed by maceration. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is heated in a sealed package.


In an embodiment, the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than macerated Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises at least about 12 times more isothiocyanate than macerated Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises at least about 14 times more isothiocyanate than macerated Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises at least about 16 times more isothiocyanate than macerated Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.


In an embodiment, lactic acid bacteria was isolated from a broccoli and/or the lactic acid bacteria lacks myrosinase activity.


In an aspect, the present invention provides a method of preparing a isothiocyanate containing product from Brassicaceae material comprising:

    • i) pre-treating the Brassicaceae material to improve the access of myrosinase to a glucosinolate; and
    • ii) acidifying the material obtained by step i) forming the isothiocyanate containing product.


In an aspect, the present invention provides a method of preparing an isothiocyanate containing product from broccoli material comprising fermenting the material with lactic acid bacteria Leuconostoc mesenteroides and/or Lactobacillus plantarum to form the isothiocyanate containing product, wherein the method optionally comprises pre-treating the broccoli material to improve the access of myrosinase to a glucosinolate.


In an aspect, the present invention provides a method of preparing an isothiocyanate containing product from a Brassicaceae material comprising fermenting the material with lactic acid bacteria Leuconostoc mesenteroides and/or Lactobacillus plantarum isolated from broccoli to form the isothiocyanate containing product, wherein the method optionally comprises pre-treating the Brassicaceae material to improve the access of myrosinase to a glucosinolate.


In an aspect, the present invention provides an isolated strain of lactic acid bacteria selected from:

    • i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia; and
    • ii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia.


In an aspect, the present invention provides an isolated strain of lactic acid bacteria selected from:

    • i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • ii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iii) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iv) B2 deposited under V17/021732 on 25 Sep. 2017 at the National
    • v) B3 deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • vi) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; and
    • vii) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.


In an aspect, the present invention provides a starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria selected from one or more or all of:

    • i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • ii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iii) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iv) B2 deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • v) B3 deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • vi) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; and
    • vii) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.


In an embodiment, the starter culture comprises lactic acid bacteria at a concentration of at least about 108 cfu/mL.


In an aspect, the present invention provides a probiotic composition comprising lactic acid bacteria selected from one or more or all of:

    • i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • ii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iii) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iv) B2 deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • v) B3 deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • vi) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; and
    • vii) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.


In an aspect, the present invention provides an isothiocyanate containing product obtained by the method as described herein.


In an aspect, the present invention provides an isothiocyanate containing product obtainable by the method as described herein. 10 In an aspect, the present invention provides an isothiocyanate containing Brassicaceae product comprising at least about 10 times more isothiocyanate than the macerated Brassicaceae material.


In an aspect, the present invention provides an isothiocyanate containing Brassicaceae product comprising about 10 times to about 16 times more isothiocyanate than the macerated Brassicaceae material.


In an aspect, the present invention provides an isothiocyanate containing Brassicaceae product comprising at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.


In an aspect, the present invention provides an isothiocyanate containing Brassicaceae product comprising about 2 times to about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.


In an aspect, the present invention provides an isothiocyanate containing Brassicaceae product comprising at least 150 mg/kg dw of isothiocyanate.


In an embodiment, the present invention provides an isothiocyanate containing product comprises at least 150 mg/kg dw, at least 200 mg/kg dw, at least 300 mg/kg dw, at least 400 mg/kg dw, or at least 450 mg/kg dw, or at least 500 mg/kg dw, or at least 550 mg/kg dw, or at least 600 mg/kg dw, or at least 650 mg/kg dw, or at least 700 mg/kg dw, or at least 1000 mg/kg dw, or at least 2000 mg/kg dw, or at least 3000 mg/kg dw, or at least 4000 mg/kg dw, or at least 5000 mg/kg dw, or at least 6000 mg/kg dw, or at least 7000 mg/kg dw sulforaphane.


In an embodiment, the isothiocyanate containing product comprises Leuconostoc mesenteroides and/or Lactobacillus plantarum.


In an embodiment, the isothiocyanate containing product has one or more or all of the following features:

    • i) is stable for at least 4 weeks, or for at least 8 weeks, or for at least 12 weeks when stored at about 4° C. to about 25° C.;
    • ii) is resistant to yeast, mould and/or coliform growth for at least 4 weeks, or for at least 8 weeks, or for at least 12 weeks when stored at about 4° C. to about 25° C.; and
    • iii) comprises at least 107 CFU/g Leuconostoc mesenteroides and/or Lactobacillus plantarum.


Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise. For instance, as the skilled person would understand examples of lactic acid bacteria outlined above for the methods of the invention equally apply to products of the invention.


The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.


Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.


The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.





BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS


FIG. 1. A) Shows the pathways of hydrolysis of glucoraphanin to sulforaphane and sulforaphane nitrile. B) Shows the effects of maceration and fermentation on sulforaphane content (mg/kg, DW) in broccoli puree. C) Shows the effect of fermentation on lactic acid bacteria count (log CFU/gm) of broccoli puree during storage.



FIG. 2. A) Shows the effects of fermentation on the stability of sulforaphane in broccoli puree stored at 4° C. and 25° C. (RT). B) Effects of heat treatment condition on the conversion of glucoraphanin into sulforaphane in broccoli matrix.



FIG. 3. A) Shows the total phenolic content (mg GAE/100 g DW) of raw broccoli and its changes during fermentation and storage at 25° C. and 4° C., respectively. B) Shows the ORAC (oxygen radical absorbance capacity) antioxidant capacity (μmol TE/g DW) of raw broccoli and its changes during fermentation and storage at 25° C. and 4° C., respectively.



FIG. 4. Shows the fermentation time taken to reach a pH of 4.4 or lower for different combinations of lactic acid bacteria strains.



FIG. 5. A) Shows sulforaphane yield (μmol/kg DW) under different heat treatment conditions of broccoli with a sealed bag. B) Shows sulforaphane yield (μmol/kg DW) under different heat treatment conditions of broccoli immersed directly in water.



FIG. 6. Shows the comparative effects of the combined effects of maceration, pre-heating and fermentation with just maceration and preheating and maceration, preheating and chemical acidification on sulforaphane yield (μmol/kg DW) just after processing and during storage at 4° C. and 25° C. Samples were pre-treated at 65° C. for 3 min in sealed packs.



FIG. 7. Shows the effect of fermentation and storage on glucoraphanin content. Glucoraphanin content is reduced in fermented samples stored at 25° C. and 4° C. compared to raw samples.



FIG. 8. PLS-DA score plot showing the difference in polyphenolic metabolite profile of raw and fermented broccoli puree.



FIG. 9. Important features differentiating fermented and non-fermented samples identified by PLS-DA. The boxes on the right indicate the relative concentration of the respective metabolites in each group.



FIG. 10. Shows the effect of lactic acid fermentation on metabolite profile of broccoli puree-based on untargeted LC-MS analysis. It demonstrates that fermentation releases bound phytochemicals such as polyphenolic glycosides and glucosinolates and enhances their bioaccessibility.



FIG. 11. Shows a volcano plot indicating metabolites with significant (p<0.05) fold changes after fermentation based on untargeted LC-MS analysis. The top 50 metabolites with significant fold changes and their individual fold changes are recited in Table 8.



FIG. 12. Shows the effect of lactic acid fermentation on broccoli polyphenols based on targeted LC-MS analysis. A 6.6 fold change is observed in chlorogenic acid (2.4 to 15.8 μg/mg), a 23.8 fold increase is observed in sinapic acid (3.6 to 86.6 μg/mg), a 10.5 increase in kaempferol (12.7 to 134.6 μg/mg) and a 0.48 fold decrease is observed in p-coumaric acid.



FIG. 13. Shows the SmaI and NotI restriction enzyme digestion from the genomic DNA of BF1 and BF2 obtained with pulse filed gel electrophoreses.





DETAILED DESCRIPTION
General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., enzyme, fermentation, inoculation).


The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.


Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.


As used herein, the term “about”, unless stated to the contrary, refers to +/−10%, more preferably +/−5%, even more preferably +/−1%, of the designated value.


An “allele” refers to one specific form of a genetic sequence (such as a gene) within a cell, an individual plant or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene. The sequences at these variant sites that differ between different alleles are termed “variances”, “polymorphisms”, or “mutations”.


Brassicaceae

A person skilled in the art will appreciate that the methods as described herein are suitable for producing an isothiocyanate containing product from any Brassicaceae material comprising glucosinolate/s. As used herein, “Brassicaceae” refers to members of the Family Brassicaceae commonly referred to as mustards, cruicifers or the cabbage family. A person skilled in the art would appreciate that material can be from more than one Brassicaceae.


In an embodiment, the Brassicaceae is selected from the genus Brassica or Cardamine. In an embodiment, the Brassica is selected from Brassica balearica, Brassica carinata, Brassica elongate, Brassica fruticulosa, Brassica hilarionis, Brassica juncea, Brassica napus, Brassica narinosa, Brassica nigra, Brassica oleracea, Brassica perviridis, Brassica rapa, Brassica rupestris, Brassica septiceps, and Brassica tournefortii.


In an embodiment, the Brassica is Brassica oleracea.


In an embodiment, the Brassica is selected from Brassica oleracea variety oleracea (wild cabbage), Brassica oleracea variety capitate (cabbage), Brassica rapa subsp. chinensis (bok choy), Brassica rapa subsp. pekinensis (napa cabbage), Brassica napobrassica (rutabaga), Brassica rapa var. rapa (turnip), Brassica oleracea variety alboglabra (kai-lan), Brassica oleracea variety viridis (collard greens), Brassica oleracea variety longata (jersey cabbage), Brassica oleracea variety acephala (ornamental kale), Brassica oleracea variety sabellica (kale), Brassica oleracea variety palmifolia (lacinato kale), Brassica oleracea variety ramose (perpetual kale), Brassica oleracea variety medullosa (marrow cabbage), Brassica oleracea variety costata (tronchuda kale), Brassica oleracea variety gemmifera (brussels sprout), Brassica oleracea variety gongylodes (kohlrabi), Brassica oleracea variety italica (broccoli), Brassica oleracea variety botrytis (cauliflower, Romanesco broccoli, broccoli di torbole), Brassica oleracea variety botrytis x italica (broccoflower), and Brassica oleracea variety italica×alboglabra (Broccolini).


In an embodiment, the Brassica is Brassica oleracea, variety italica (broccoli).


In an embodiment, the Brassicaceae is selected from Cardamine hirsuta (bittercress), Iberis sempervirens (candytuft), Sinapis arvensis (charlock), Armoracia rusticana (horseradish), Pringlea antiscorbutica (Kerguelen cabbage), Thlaspi arvense (pennycress), Raphanus raphanistrum subsp. sativus (radish), Eruca sativa (rocket), Anastatica hierochuntica (rose of Jericho), Crambe maritima (sea kale), (akile maritima (sea rocket), Capsella bursa-pastoris (shepherd's purse), sweet Alyssum, Arabidopsis thaliana (thale cress), Nasturtium officinale (watercress), Sinapis alba (white mustard), Erophila verna (whitlow grass), Raphanus raphanistrum (wild radish), Isatis tinctoria (woad), and Nasturtium microphyllum (yellow cress).


In an embodiment, the Brassicaceae has a high level of one or more glucosinolate/s. In an embodiment, the Brassicaceae has been selectively bred to have a high level of one or more glucosinolate/s. In an embodiment, “high level” of a glucosinolate can comprise a higher level of a glucosinolate than shown in Table 2 of Verkerk et al. (2009) in the corresponding Brassicaceae. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 3400 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 4000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 5000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 8000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 10,000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 12,000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 15,000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 18,000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 20,000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 25,000 μmol/kg dry weight. In an embodiment, a high level of glucosinolate is a level of glucosinolate higher than 30,000 μmol/kg dry weight. In an embodiment, the Brassicaceae has been genetically modified or subjected to biotic or abiotic stress to have a high level of one or more glucosinolate/s. A person skilled in the art will appreciate that the Brassicaceae can be modified by any method known to a person skilled in the art.


In an embodiment, the glucosinolate is glucoraphanin (4-Methylsulphinylbutyl). In an embodiment, the glucosinolate is glucobrassicin (3-Indolylmethyl).


As used herein “Brassicaceae material” refers to any part of the Brassicaceae which comprises a glucosinolate, including, but not limited to, the leaves, stems, flowers, florets, seeds, and roots or mixtures thereof.


A person skilled in the art will appreciate that the methods as described herein are suitable for use with different volumes of Brassicaceae material, for example, but not limited to, at least 30 kg, or at least 50 kg, or at least 80 kg, or at least 100 kg, or at least 1,000 kg, or at least 2,000 kg, or at least 5,000 kg, or at least 8,000 kg, or at least 10,000 kg, or at least 15,000 kg, or at least 20,000 kg.


In an embodiment, the Brassicaceae material has been washed. As used herein “washing” removes visible soil and contamination. In an embodiment, the Brassicaceae material has been sanitized. As used herein “sanitized” refers to a reduction of pathogens on the Brassicaceae material.


In an embodiment, the Brassicaceae is mixed with other plant material. In an embodiment, the other plant material is vegetable or fruit material. In an embodiment, the vegetable is a carrot or beetroot.


Glucosinolates

As used herein “glucosinolate” refers to a secondary metabolite found at least in the Brassicaceae family that share a chemical structure consisting of a β-D-glucopyranose residue linked via a sulfur atom to a (Z)—N-hydroximinosulfate ester, plus a variable R group derived from an amino acid as described in Halkier et al. (2006). Examples of glucosinolates are provided in Halkier et al. (2006) and Agerbirk et al. (2012). The hydrolysis of glucosinolate can produce isothiocyanates, nitriles, epithionitrile, thiocyanate and oxazolidine-2-thione (FIG. 1A). Many glucosinolates play a role in plant defence mechanisms against pests and disease.


Glucosinolates are stored in Brassicaceae in storage sites. As used herein, a “storage site” is a site within the Brassicaceae where glucosinolates are present and myrosinase is not present.


As used herein “myrosinase” also referred to as “thioglucosidase”, “sinigrase”, or “sinigrinase” refers to a family of enzymes (EC 3.2.1.147) involved in plant defence mechanisms that can cleave thio-linked glucose. Myrosinases catalyze the hydrolysis of glucosinolates resulting in the production of isothiocyanates. Myrosinase is stored sometimes as myrosin grains in the vacuoles of particular idioblasts called myrosin cells, but have also been reported in protein bodies or vacuoles, and as cytosolic enzymes that tend to bind to membranes. Thus, in an embodiment, myrosinase is stored in a myrosin cell in Brassicaceae.


In an embodiment, pre-treating as described herein improves the access of myrosinase to a glucosinolate. As used herein “improves the access” or “access is improved” refers to increasing the availability of glucosinolate to the myrosinase enzyme allowing for the production of an isothiocyanate. In an embodiment, access is improved by the release of a glucosinolate from a glucosinolate storage site. In an embodiment, the glucosinolate storage site is mechanically ruptured (i.e. by maceration) or enzymatically degraded. In an embodiment, glucosinolate is released from a glucosinolate storage site by the activity of one or more polysaccharide degrading enzymes e.g. a cellulase, hemicellulase, pectinase and/or glycosidase. In an embodiment, access is improved by allowing the entry of myrosinase into a glucosinolate storage site. In an embodiment, access is improved by the release of myrosinase from myrosin cells. In an embodiment, about 10% to about 90% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 20% to about 80% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 30% to about 70% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 40% to about 60% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 45% to about 55% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 10% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 20% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 30% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 40% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 50% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 60% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 70% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 80% of a glucosinolate is released from a glucosinolate storage site. In an embodiment, about 90% of a glucosinolate is released from a glucosinolate storage site.


In an embodiment, the Brassicaceae material comprises one or more glucosinolate/s selected from an aliphatic, indole or aromatic glucosinolate.


In an embodiment, the aliphatic glucosinolate is selected from one or more of glucoraphanin (4-Methylsulphinylbutyl or glucorafanin), sinigrin (2-Propenyl), gluconapin (3-Butenyl), glucobrassicanapin (4-Pentenyl), progoitrin (2(R)-2-Hydroxy-3-butenyl, epiprogoitrin (2(S)-2-Hydroxy-3-butenyl), gluconapoleiferin (2-Hydroxy-4-pentenyl), glucoibervirin (3-Methylthiopropyl, glucoerucin (4-Methylthiobutyl), dehydroerucin (4-Methylthio-3-butenyl, glucoiberin (3-Methylsulphinylpropyl), glucoraphenin (4-Methylsulphinyl-3-butenyl), glucoalyssin (5-Methylsulphinylpentenyl), and glucoerysolin (3-Methylsulphonylbutyl, 4-Mercaptobutyl).


In an embodiment, the indole glucosinolate is selected from one or more of glucobrassicin (3-Indolylmethyl), 4-hydroxyglucobrassicin (4-Hydroxy-3-indolylmethyl), 4-methoxyglucobrassicin (4-Methoxy-3-indolylmethyl), and neoglucobrassicin (1-Methoxy-3-indolylmethyl).


In an embodiment, the indole glucosinolate is selected from one or more of Glucotropaeolin (Benzyl) and Gluconasturtiin (2-Phenylethyl).


In an embodiment, the Brassicaceae material comprises one or more glucosinolate/s selected from benzylglucosinolate, allylglucosinolate and 4-methylsulfinylbutyl. In an embodiment, the glucosinolate is glucoraphanin (4-Methylsulphinylbutyl). In an embodiment, the glucosinolate is glucobrassicin (3-Indolylmethyl).


In an embodiment, pre-treating as described herein increases the extractable glucosinolate content compared to the extractable glucosinolate content of the Brassicaceae material before pre-treatment.


As used herein “extractable glucosinolate content” refers to the level of glucosinolate accessible in the Brassicaceae material for conversion to isothiocyanate. Excluding conversion into nitriles and other compounds the expected maximum yield of isothiocyanate from 1 mole of glucosinolate is 1 mole of isothiocyanate (1 mole of glucosinolate can maximally be converted to 1 mole of isothiocyanate, 1 mole of glucose and 1 mole of sulphate ion). Thus, in one example, the extractable glucoraphanin content of a commercial broccoli cultivar is 3400 μmol glucoraphanin/kg dw and the expected maximum yield of sulforaphane from the commercial broccoli cultivar is 3400 μmol sulforaphane/kg dw.


Isothiocyanates

As used herein “isothiocyanate” refers to sulphur containing phytochemicals with the general structure R—N═C═S which are a product of myrosinase activity upon a glucosinolate and bioactive derivatives thereof. In an embodiment, the isothiocyanate is sulforaphane (1-isothiocyanato-4-methylsulfinylbutane). In an embodiment, the isothiocyanate is allyl isothiocyanate (3-isothiocyanato-1-propene). In an embodiment, the isothiocyanate is benzyl isothiocyanate. In an embodiment, the isothiocyanate is phenethyl isothiocyanate. In an embodiment, the isothiocyanate is 3-Butenyl isothiocyanate. In an embodiment, the isothiocyanate is 5-vinyl-1,3-oxazolidine-2-thione. In an embodiment, the isothiocyanate is 3-(methylthio)propyl isothiocyanate. In an embodiment, the isothiocyanate is 3-(methylsulfinyl)-propyl isothiocyanate. In an embodiment, the isothiocyanate is 4-(methylthio)-butyl isothiocyanate. In an embodiment, the isothiocyanate is 1-methoxyindol-3-carbinol isothiocyanate. In an embodiment, the isothiocyanate is 2-phenylethyl isothiocyanate. In an embodiment, the isothiocyanate is iberin.


In an embodiment, the isothiocyanate containing product, further comprises one or more isothiocyanate bioactive derivative/s or oligomers thereof. In an embodiment, the isothiocyanate bioactive derivative is a derivative of any of the isothiocyanates as described herein. In an embodiment, the isothiocyanate bioactive derivative is a derivative of sulforaphane. In an embodiment, the isothiocyanate bioactive derivative is iberin. In an embodiment, the isothiocyanate bioactive derivative is allyl isothiocyanate. In an embodiment, the isothiocyanate bioactive derivative is indole-3-caribinol. In an embodiment, the isothiocyanate bioactive derivative is methoxy-indole-3-carbinol. In an embodiment, the isothiocyanate bioactive derivative is ascorbigen. In an embodiment, the isothiocyanate bioactive derivative is neoascorbigen.


Pre-Treatment

As use herein “pre-treatment” or “pre-treating” releases or aids in the release of a glucosinolate from glucosinolate storage site and/or allows myrosinase to enter a glucosinolate storage site in the Brassicaceae material. In an embodiment, pre-treating increases the exposure of a glucosinolate to myrosinase allowing myrosinase to convert a glucosinolate to an isothiocyanate.


In an embodiment, pre-treating reduces epithiospecifier protein (ESP) while maintaining endogenous myrosinase activity. As used herein “epithiospecifier protein” or “ESP” refers to a protein that directs myrosinase activity towards the production of nitriles and away from isothiocyanate production. Reducing or inhibiting ESP production (mRNA or protein) or activity can increase production of isothiocyanates.


As used herein, “reduces epithiospecifier protein” refers to decreasing the protein production or activity of ESP. In an embodiment, reducing ESP comprises inactivating (e.g. denaturing) ESP at high temperature. In an embodiment, ESP is denatured at temperatures of about 50° C. to about 80° C.


As used herein, “maintaining endogenous myrosinase activity” means not significantly reducing myrosinase activity compared to an untreated control. In an embodiment, endogenous myrosinase activity is not reduced by about 5% or more. In an embodiment, endogenous myrosinase activity is not reduced by about 10% or more. In an embodiment, endogenous myrosinase activity is not reduced by about 15% or more. In an embodiment, endogenous myrosinase activity is not reduced by about 20% or more. In an embodiment, endogenous myrosinase activity is not reduced by about 30% or more. In an embodiment, endogenous myrosinase activity is not reduced by about 40% or more. In an embodiment, endogenous myrosinase activity is not reduced by about 50% or more.


In an embodiment, pre-treating comprises one or more of the following: i) heating; ii) macerating; iii) microwaving; iv) exposure to high frequency sound waves (ultrasound), or v) pulse electric field processing, wherein the temperature of the Brassicaceae material does not exceed about 75° C. during pre-treating.


In an embodiment, the Brassicaceae material is heated in a fuel based heating system, an electricity based heating system (i.e. an oven or ohmic heating), radio frequency heating, high pressure thermal processing or a steam based heating system (indirect or direct application of steam). In an embodiment, the Brassicaceae material is heated in a sealed package (e.g. in a retort pouch). In an embodiment, the Brassicaceae material is heated in an oven, water bath, bioreactor, stove, water blancher, or steam blancher. In an embodiment, the Brassicaceae material is heated via high pressure thermal heating. In an embodiment, the Brassicaceae material is via ohmic heating. In an embodiment, the Brassicaceae material is via radio frequency heating. In an embodiment, the Brassicaceae material is blanched in water. In an embodiment, the Brassicaceae material is heated via high pressure thermal processing. In an embodiment, the Brassicaceae material is placed in a sealed package for high pressure thermal processing.


In an embodiment, pre-treating comprises heating the Brassicaceae material to about 50° C. to about 70° C. In an embodiment, pre-treating comprises heating the Brassicaceae material to about 50° C. to about 65° C. In an embodiment, pre-treating comprises heating the Brassicaceae material to about 50° C. to about 60° C. In an embodiment, heating comprises heating the Brassicaceae material to about 55° C. to about 70° C. In an embodiment, heating comprises heating the Brassicaceae material to about 60° C. to about 70° C. In an embodiment, heating comprises heating the Brassicaceae material to about 65° C. to about 70° C. In an embodiment, the Brassicaceae material is heated for about 30 seconds. In an embodiment, the Brassicaceae material is heated for about 1 minute. In an embodiment, the Brassicaceae material is heated for about 2 minutes. In an embodiment, the Brassicaceae material is heated for about 3 minutes. In an embodiment, the Brassicaceae material is heated for about 4 minutes. In an embodiment, the Brassicaceae material is heated for about 5 minutes.


In an embodiment, the Brassicaceae material is heated in a sealed package for about 1 min at about 60° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 2 mins at about 60° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 3 mins at about 60° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 4 mins at about 65° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 1 min at about 65° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 2 mins at about 65° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 3 mins at about 65° C. In an embodiment, the Brassicaceae material is heated in a sealed package for about 4 mins at about 65° C.


In an embodiment, the Brassicaceae material is heated in water for about 1 min at about 60° C. In an embodiment, the Brassicaceae material is heated in water for about 2 mins at about 60° C.


In an embodiment, heating comprises steaming the Brassicaceae material. In an embodiment, pre-treating comprises steaming the Brassicaceae material. In an embodiment, the Brassicaceae material is steamed to a temperature of about 50° C. to about 70° C. In an embodiment, the Brassicaceae material is steamed to a temperature of about 60° C. to about 70° C. In an embodiment, the Brassicaceae material is steamed for at least about 30 seconds. In an embodiment, the Brassicaceae material is steamed for at least about 1 minute. In an embodiment, the Brassicaceae material is steamed for at least about 2 minutes. In an embodiment, the Brassicaceae material is steamed for at least about 3 minutes. In an embodiment, the Brassicaceae material is steamed for at least about 4 minutes. In an embodiment, the Brassicaceae material is steamed for at least about 5 minutes.


In an embodiment, pre-treating comprises macerating the Brassicaceae material. As used herein “macerating”, “macerated” or “macerate” refers to breaking the Brassicaceae material into smaller pieces. In an embodiment, macerating comprising decompartmentalizing at least about 30% to about 90% of the cells of the Brassicaceae material to allow myrosinase access to its substrate glucosinolates. In an embodiment, macerating comprising decompartmentalizing at least about 40% to about 90% of the cells of the Brassicaceae material. In an embodiment, macerating comprising decompartmentalizing at least about 50% to about 90% of the cells of the Brassicaceae material. In an embodiment, macerating comprising decompartmentalizing at least about 60% to about 90% of the cells of the Brassicaceae material. In an embodiment, macerating comprising decompartmentalizing at least about 70% to about 90% of the cells of the Brassicaceae material. A person skilled in the art will appreciate that decompartimentalizing a cell comprising breaking open the cell wall and disrupting the compartmentalization of organelles within a cell.


In an embodiment, the Brassicaceae material is macerated with a blender, grinder or pulveriser. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 1 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.5 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.25 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.1 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.05 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.025 mm or less. In an embodiment, the Brassicaceae material is macerated so that at least about 80% of the Brassicaceae material is of a size of about 0.01 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 50% to about 90% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 60% to about 80% of the Brassicaceae material is of a size of about 2 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 50% to about 90% of the Brassicaceae material is of a size of about 1 mm or less. In an embodiment, the Brassicaceae material is macerated so that about 60% to about 80% of the Brassicaceae material is of a size of about 1 mm or less. In an embodiment, the Brassicaceae material is heated to a temperature of about 50° C. to about 70° C. during maceration. In an embodiment, the Brassicaceae material is heated to a temperature of about 55° C. to about 70° C. during maceration. In an embodiment, the Brassicaceae material is heated to a temperature of about 60° C. to about 70° C. during maceration. In an embodiment, the Brassicaceae material is heated to a temperature of about 65° C. to about 70° C. during maceration.


In an embodiment, pre-treating comprises heating and macerating the Brassicaceae material. In an embodiment, pre-treating produces a puree. As used herein a “puree” refers to Brassicaceae material blended to the consistency of a creamy paste or liquid.


A person skilled in the art will appreciate that “microwaves” or “microwaving” heats a substance such as Brassicaceae material by passing microwave radiation through the substance. In an embodiment, pre-treating comprises microwaving the Brassicaceae material. In an embodiment, Brassicaceae material is pre-treated in a consumer microwave or industrial microwave. In an embodiment, the industrial microwave is a continuous microwave system, for example, but not limited to the MIP 11 Industrial Microwave Continuous Cooking Over (Ferrite Microwave Technologies). In an embodiment, pre-treating comprises microwaving the Brassicaceae material. In an embodiment, the Brassicaceae material is microwaved at about 0.9 to about 2.45 GHz. In an embodiment, the Brassicaceae material is microwaved for at least about 30 seconds, or at least about 1 minute, or at least about 2 minutes, or at least 3 minutes.


In an embodiment, pre-treating comprises exposing the Brassicaceae material at low to medium frequency ultrasound waves. In an embodiment, pre-treating comprises exposing the Brassicaceae material with thermosonication (low to medium frequency ultrasound waves with heat of about 30° C. to about 60° C.). In an embodiment, the ultrasound waves are generated with an industrial scale ultrasonic processor. In an embodiment, the ultrasonic processor is a continuous or batch ultrasonic processor. In an embodiment, the ultrasonic processor is for example, but not limited to, UIP500hd or UIP4000 (Hielscher, Ultrasound Technology). In an embodiment, the ultrasounds waves are at a frequency of about 20 kHz to about 600 kHz. In an embodiment, the Brassicaceae material is exposed to sound waves for at least about 30 seconds, or at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or about 5 minutes.


In an embodiment, pre-treating comprises exposing the Brassicaceae material to pulse electric field processing. Pulse electric field processing is a non-thermal processing technique comprising the application of short, high voltage pulses. The pulses induce electroporation of the cells of the Brassicaceae material enhancing the access of myrosinase to glucosinolates. In an embodiment, pulse electric field processing heats the Brassicaceae material to a temperature of about 40 to about 70° C. In an embodiment, pulse electric field processing heats the Brassicaceae material to a temperature of about 50° C. to about 70° C. In an embodiment, pulse electric field processing heats the Brassicaceae material to a temperature of about 60° C. to about 70° C. In an embodiment, pulse electric field processing comprises treating the Brassicaceae material with voltage pulses of about 20 to about 80 kV. In an embodiment, pre-treating converts about 10% to about 90% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 20% to about 80% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 30% to about 70% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 40% to about 60% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 10% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 20% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 30% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 40% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 50% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 60% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 70% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 80% of a glucosinolate to an isothiocyanate. In an embodiment, pre-treating converts about 90% of a glucosinolate to an isothiocyanate.


Fermentation

A person skilled in the art will appreciate that the fermentation method as described herein can comprise the use of any lactic acid bacteria. As used herein, “fermentation” refers to the biochemical breakdown of the Brassicaceae material by lactic acid bacteria. In an embodiment, fermentation with lactic acid bacteria is performed using the addition of exogenous lactic acid bacteria. As used herein, “lactic bacteria” or “lactic acid bacteria” are bacteria that produce lactic acid as an end product of carbohydrate fermentation, and can include, but are not limited to including bacteria from the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus and Weissella. In an embodiment, the lactic acid bacteria comprises myrosinase activity. In an embodiment, the lactic acid bacteria is from the genera Leuconostoc. In an embodiment, the lactic acid bacteria is from the genera Lactobacillus.


In an embodiment, the lactic acid bacteria is selected from one or more of Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus brevis, Lactococus lactis, Pediococcus pentosaceus and Pedicoccus acidilacti.


In an embodiment, the lactic acid bacteria was isolated from a Brassicaceae. In an embodiment, the lactic acid bacteria was isolated from a Brassica oleracea. In an embodiment, the lactic acid bacteria was isolated from broccoli. In an embodiment, the lactic acid bacteria was isolated from broccoli leaves. In an embodiment, the lactic acid bacteria was isolated from broccoli stem. In an embodiment, the lactic acid bacteria was isolated from broccoli puree. In an embodiment, the lactic acid bacteria was isolated from Australian broccoli.


In an embodiment, the lactic acid bacteria lacks myrosinase activity.


In an embodiment, the lactic acid bacteria is a Lactobacillus.


In an embodiment, the lactic acid bacteria is selected from: i) a Leuconostoc mesenteroides; ii) a Lactobacillus plantarum; iii) a Lactobacillus pentosus; iv) a Lactobacillus rhamnosus; v) a combination of i) and ii); vi) a combination of i), ii) and iii); and vii) a combination of i), ii) and iv).


In one embodiment, the lactic acid bacteria is Leuconostoc mesenteroides. In an embodiment, the Leuconostoc mesenteroides is ATCC8293. In an embodiment, the Leuconostoc mesenteroides is BF1 and/or BF2. In an embodiment, the Leuconostoc mesenteroides lacks myrosinase activity.


In one embodiment, the lactic acid bacteria is Lactobacillus plantarum. In an embodiment, the Lactobacillus plantarum lacks myrosinase activity.


In one embodiment, about 50% of the lactic acid bacteria is Leuconostoc mesenteroides and about 50% of the lactic acid bacteria is Lactobacillus sp.


In one embodiment, about 50% of the lactic acid bacteria is Leuconostoc mesenteroides and about 50% of the lactic acid bacteria is Lactobacillus plantarum.


In an embodiment, the Lactobacillus plantarum is selected from one or more or all of B1, B2, B3, B4 and B5. In an embodiment, the Lactobacillus plantarum is B1. In an embodiment, the Lactobacillus plantarum is B2. In an embodiment, the Lactobacillus plantarum is B3. In an embodiment, the Lactobacillus plantarum is B4. In an embodiment, the Lactobacillus plantarum is B5.


In an embodiment, fermentation occurs in the presence of at least 2, or at least 3, or at least 4, or at least 5, or at least 6 strains of lactic acid bacteria selected from BF1, BF2, B1, B2, B3, B4 and B5.


In one embodiment, the lactic acid bacteria is a recombinant bacteria modified to produce a high level of myrosinase activity compared to a control bacteria lacking the modification. A person skilled in the art will appreciate that the recombinant lactic acid bacteria is produced by any technique known to a person skilled in the art.


In an embodiment, the lactic acid bacteria is stressed, for example but not limited to, heat stress, cold stress, sub-lethal ultrasonic waves e.g. about 20 to about 2000 MHz, high pressure, dynamic high pressure or pulsed-electric field, to increase myrosinase activity and the activity of polysaccharide degrading enzymes compared to a control lactic acid bacteria that has not been stressed. In an embodiment, heat stress comprises heating the bacteria to greater than about 40° C. to about 75° C. In an embodiment, heat stress comprises heating the bacteria to greater than about 45° C. to about 65° C. In an embodiment, heat stress comprises heating the bacteria to greater than about 45° C. to about 55° C. In an embodiment, cold stress comprises lower the bacteria to temperature of about 0° C. to about 8° C. In an embodiment, cold stress comprises lower the bacteria to temperature of about 2° C. to about 6° C. In an embodiment, cold stress comprises lower the bacteria to temperature of about 4° C.


In an embodiment, the Brassicaceae material is inoculated with at least about 105 CFU/g of a lactic acid bacteria as described herein. In an embodiment, the Brassicaceae material is inoculated with at least 106 about CFU/g of a lactic acid bacteria as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 107 CFU/g of a lactic acid bacteria as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 108 CFU/g of a lactic acid bacteria as described herein. In an embodiment, the Brassicaceae material has been pre-treated.


In an embodiment, fermentation is at about 20° C. to about 34° C. In an embodiment, fermentation is at about 22° C. to about 34° C. In an embodiment, fermentation is at about 24° C. to about 34° C. In an embodiment, fermentation is at about 24° C. to about 30° C. In an embodiment, fermentation is at about 34° C. to about 34° C. In an embodiment, fermentation is at about 25° C. In an embodiment, fermentation is at about 30° C. In an embodiment, fermentation is at about 34° C.


In an embodiment, fermentation is for about 8 hours to about 17 days. In an embodiment, fermentation is for about 8 hours to about 14 days. In an embodiment, fermentation is for about 8 hours to about 7 days. In an embodiment, fermentation is for about 8 hours to about 5 days. In an embodiment, fermentation is for about 8 hours to about 4 days. In an embodiment, fermentation is for about 8 hours to about 3 days. In an embodiment, fermentation is for about 8 hours to about 30 hours. In an embodiment, fermentation is for about 8 to about 24 hours. In an embodiment, fermentation is for about 10 hours to about 24 hours. In an embodiment, fermentation is for about 10 days. In an embodiment, fermentation is for about 9 days. In an embodiment, fermentation is for about 8 days. In an embodiment, fermentation is for about 7 days. In an embodiment, fermentation is for about 4 days. In an embodiment, fermentation is for about 6 days. In an embodiment, fermentation is for about 5 days. In an embodiment, fermentation is for about 72 hours. In an embodiment, fermentation is for about 60 hours. In an embodiment, fermentation is for about 45 hours. In an embodiment, fermentation is for about 30 hours. In an embodiment, fermentation is for about 24 hours. In an embodiment, fermentation is for about 20 hours. In an embodiment, fermentation is for about 18 hours. In an embodiment, fermentation is for about 15 hours. In an embodiment, fermentation is for about 16 hours. In an embodiment, fermentation is for about 14 hours. In an embodiment, fermentation is for about 12 hours. In an embodiment, fermentation is for about 10 hours. In an embodiment, fermentation is for about 8 hours. In an embodiment, the fermentation culture is stirred. In an embodiment, stirring is intermittent. In an embodiment, stirring is continuous. In a particularly preferred embodiment, fermentation is for 15 hours with intermittent stirring. In a particularly preferred embodiment, fermentation is for 24 hours with intermittent stirring.


In an embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 3.8. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 3.6. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.5 to about 4.04. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of about 4.3 to about 4.04. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.5 or less, or 4.4 or less, or 4.3 or less, or 4.04 or less, or 3.8 or less. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.5 or less. In an embodiment, the fermentation reaction is complete when the composition reaches a pH of 4.4 or less.


In an embodiment, if present fermentation reduces the number of one or more or all of: E. coli, Salmonella and Listeria. In an embodiment, if present fermentation reduces the CFU/g of one or more or all of: E. coli, Salmonella and Listeria.


In an embodiment, no salt is added to the fermentation culture.


In an embodiment, fermentation increases the extractable glucosinolate content compared to the extractable glucosinolate content in the pre-treated Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content is increased by about 100% to about 500% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 200% to about 450% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 250% to about 450% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 300% to about 400% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 300% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, fermentation increases the extractable glucosinolate content by about 400% compared to the extractable glucosinolate content in the Brassicaceae material. In an embodiment, the glucosinolate is glucoraphanin.


Acidification

The pre-treated material can by acidified to improve the microbial safety and stability (susceptibility to microbial degradation) of the product and increase the stability of isothiocyanate in the product. Acidification can be achieved by the addition of organic acids, such as, but not limited to lactic, acetic, ascorbic, and citric acid. In embodiment, acidification can be achieved with the addition of glucono-delta-lactone. In an embodiment, acidification comprises lowering the pH to a pH of about 4.4 to about 3.4. In an embodiment, acidification comprises lowering the pH to a pH of 4.5, or 4.4, or 4.2, or 4, or 3.8, or 3.6, or 3.4 or less. In an embodiment, acidification comprises lowering the pH to a pH of 4.4 of less.


Isothiocyanate Containing Product from Brassicaceae


An isothiocyanate containing product from Brassicaceae as described herein can be produced by the methods as described herein. It will be appreciated be a person skilled in the art that an isothiocyanate containing product produced using the methods as described herein contains higher levels of isothiocyanates, for example sulforaphane, than the Brassicaceae material or Brassicaceae material subjected to fermentation alone (without pre-treatment as described herein). For example, macerated broccoli from a commercial broccoli cultivar has a sulforaphane concentration of ˜800 μmol/Kg dw (˜149.8 mg/Kg dw), fermented macerated broccoli has a sulforaphane concentration of ˜1600 μmol/Kg dw (˜278.8 mg/Kg dw) and pre-treated and fermented broccoli produced using the methods as described herein has a sulforaphane concentration of ˜13100 μmol/Kg dw (˜2318.7 mg/Kg dw).


In an embodiment, the isothiocyanate containing product comprises at least about 4 times more isothiocyanate than macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 6 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 8 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 10 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 12 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 14 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 17 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 4 times to about 17 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 4 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 8 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 10 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 12 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 14 times to about 16 times more isothiocyanate than the macerated Brassicaceae material. In an embodiment, the isothiocyanate is sulforaphane.


In an embodiment, the level of isothiocyanate present in the isothiocyanate containing product is higher than what would be expected from the extractable glucosinolate content of the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 1 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises at least about 3 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises at least about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises at least about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 4 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 2 times to about 3.8 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content. In an embodiment, the isothiocyanate containing product comprises about 2 times to about 3 times the expected maximum yield of isothiocyanate based on the extractable glucosinolate content.


In an embodiment, the level of sulforaphane present in the isothiocyanate containing product is higher than what would be expected from the extractable glucoraphanin content of the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 1 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises at least about 2 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises at least about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises at least about 3.8 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises at least about 4 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 4 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 3.8 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 1 times to about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content. In an embodiment, the isothiocyanate containing product comprises about 2 times to about 3 times the expected maximum yield of sulforaphane based on the extractable glucoraphanin content.


In an embodiment, the isothiocyanate containing product comprises about 100 mg/kg dw to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 500 mg/kg dw to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 1000 mg/kg dw to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 1600 mg/kg dw to about 4000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 1600 mg/kg dw to about 3000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 2000 mg/kg dw to about 4000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 2000 mg/kg dw of to about 7000 mg/kg dw of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 3000 mg/kg dw isothiocyanate to about 7000 mg/kg of isothiocyanate. In an embodiment, the isothiocyanate containing product comprises about 2300 mg/kg dw of the isothiocyanate.


In an embodiment, the isothiocyanate containing product comprises at least about 100 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 200 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 250 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 300 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 350 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 400 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 450 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 500 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 550 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 600 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 650 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 700 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 1000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 2000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 3000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 4000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 5000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 6000 mg/kg dw of the isothiocyanate. In an embodiment, the isothiocyanate containing product comprises at least about 7000 mg/kg dw of the isothiocyanate.


In an embodiment, the isothiocyanate containing product comprises at least about 100 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 150 mg/kg of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 200 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 250 mg/kg of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 300 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 350 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 400 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 450 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 500 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 550 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 600 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 650 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 700 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 1000 mg/kg of sulforaphane dw. In an embodiment, the isothiocyanate containing product comprises at least about 2000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 3000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 4000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 5000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 6000 mg/kg dw of sulforaphane. In an embodiment, the isothiocyanate containing product comprises at least about 7000 mg/kg dw of sulforaphane.


In an embodiment, the isothiocyanate containing product comprises at least about 5% more total fibre than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 10% more total fibre than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 15% more total fibre than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 20% more total fibre than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 4% more protein than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 6% more protein than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 8% more protein than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 10% more protein than the Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises at least about 10% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 20% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 30% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 40% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 45% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises at least about 48% less carbohydrate than the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises about 10% to about 48% less carbohydrate than the Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises an increased level of polyphenolic glycosides compared to the Brassicaceae material. In an embodiment, the polyphenolic glycosides are anthocyanin glycosides. In an embodiment, the polyphenolic glycosides are phenolic acid glycosides. In an embodiment, the polyphenolic glycosides are phenolic acids.


In an embodiment, the isothiocyanate containing product comprises an increased level of glucosinolates compared to the Brassicaceae material. In an embodiment, the glucosinolate is glucoraphanin. In an embodiment, glucoraphanin is increased at least about 25 fold. In an embodiment, the glucosinolate is glucobrassicin. In an embodiment, the glucobrassicin is increased by 26 times. In an embodiment, the isothiocyanate containing product comprises indole-3-carbinol. In an embodiment, indol-3carbinol is increased at least about 2 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material. In an embodiment, indol-3-carbinol is increased at least about 3 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises ascorbigen. In an embodiment, ascorbigen is increased at least about 2 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material. In an embodiment, ascorbigen is increased at least about 3 fold in the isothiocyanate containing product compared to the macerated Brassicaceae material.


In an embodiment, the isothiocyanate containing product comprises an increased level of one or more of ferullic acid, syringic acid, phenyllactic acid, chlorogenic acid rutin, sinapic acid, methyl syringate, hesperetin, quercetin and kaempferol compared to the Brassicaceae material. In an embodiment, the isothiocyanate containing product comprises an increased level of chlorogenic acid compared to the Brassicaceae material. In an embodiment, chlorogenic acid is increased about 6.6 fold. In an embodiment, the isothiocyanate containing product comprises an increased level of sinapic acid compared to the Brassicaceae material. In an embodiment, sinapic acid is increased about 23.8 fold. In an embodiment, the isothiocyanate containing product comprises an increased level of kaempferol compared to the Brassicaceae material. In an embodiment, kaempferol is increased about 10.5 fold.


In an embodiment, the isothiocyanate containing product comprises an decreased level of one or more of protocatechuic acid, gallic acid, 4,hydroxybenzoic acid, vanillic acid, 2,3dihydroxybenzoic acid, p-cuomaric acid, cinnamic acid, catechin, rosmarinic acid, caffeic acid compared to the Brassicaceae material.


In an embodiment, about 40% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 50% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 60% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 70% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 80% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 90% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 95% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 97% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 98% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 99% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 100% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 40% to about 100% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing product. In an embodiment, about 40% to about 80% of a glucosinolate present in the Brassicaceae material is converted to an isothiocyanate in the isothiocyanate containing Brassicaceae product.


In an embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least a week, or for at least two weeks, or for at least 3 weeks, or for at least 4 weeks, or for at least 6 weeks, or for at least 8 weeks, or for at least 10 weeks, or for at least 12 weeks, or for at least 14 weeks when stored at about 4° C. to about 25° C. In an embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least 4 weeks when stored at about 4° C. to about 25° C. In an embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least 8 weeks when stored at about 4° C. to about 25° C. In an embodiment, the isothiocyanate in the isothiocyanate containing product is stable for at least 12 weeks when stored at about 4° C. to about 25° C.


As used herein “stable” refers to no decrease or only a minor decrease in isothiocyanate concentration when stored at 4° C. for six weeks. In an embodiment, a minor decrease refers to a decrease in isothiocyanate concentration of about 1% to about 30%. In an embodiment, a minor decrease refers to a decrease in isothiocyanate concentration of about 5% or less. In an embodiment, a minor decrease refers to a decrease in isothiocyanate concentration of about 10% or less. In an embodiment, a minor decrease refers to a decrease in isothiocyanate concentration of about 15% or less. In an embodiment, a minor decrease refers to a decrease in isothiocyanate concentration of about 20% or less. In an embodiment, a minor decrease refers to a decrease in isothiocyanate concentration of about 30% or less. Isothiocyanate analysis can be performed by any method know to a person skilled in the art and for example as shown in Example 1 for sulforaphane.


In an embodiment, the isothiocyanate is sulforaphane.


In an embodiment, the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least a week, or for at least two weeks, or for at least 3 weeks, or for at least 4 weeks, or for at least 6 weeks, or for at least 8 weeks, or for at least 10 weeks, or for at least 12 weeks, or for at least 14 weeks when stored at about 4° C. to about 25° C.


In an embodiment, the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least 4 weeks when stored at about 4° C. to about 25° C. In an embodiment, the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least 8 weeks when stored at about 4° C. to about 25° C. In an embodiment, the isothiocyanate containing product is resistant to yeast, mould and/or coliform growth for at least 12 weeks when stored at about 4° C. to about 25° C.


As used herein “resistant” to yeast, mould and/or coliform growth means that <1 Log CFU/g of yeast, mould and/or coliform is detectable in the sample after the above listed time periods using the methods described in Example 1. In an embodiment, the isothiocyanate containing product comprises about 20 g/100 gdw to about 32 g/100 gdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 20 g/100 gdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 25 g/100 gdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 28 g/100 gdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 29 g/100 gdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 30 g/100 gdw total fibre. In an embodiment, the isothiocyanate containing product comprises about 32 g/100 gdw total fibre.


In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 14000 μmol TE/100 gdw to about 19000 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 14000 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 15000 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 16000 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 17000 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 18000 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 18695 μmol TE/100 gdw. In an embodiment, the isothiocyanate containing product comprises an ORAC antioxidant capacity of about 19000 μmol TE/100 gdw.


In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 1750 mg GAE/100 gdw to about 2600 mg GAE/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 1750 mg GAE/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2000 mg GAE/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2100 mg GAE/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2200 mg GAE/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2300 mg GAE/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total polyphenol content of about 2360 mg GAE/100 gdw.


In an embodiment, the isothiocyanate containing product comprises a total titratable acidity of about 0.9% to about 1.1% lactic acid equivalent. In an embodiment, the isothiocyanate containing product comprises a total titratable acidity of about 1.1% lactic acid equivalent.


In an embodiment, the isothiocyanate containing product comprises a total protein content of about 23 g/100 gdw to about 39 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 23 g/100 gdw to about 30 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 25 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 27 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 28 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 29 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 30 g/100 gdw. In an embodiment, the isothiocyanate containing product comprises a total protein content of about 32 g/100 gdw.


In an embodiment, the isothiocyanate containing product comprises at least about 100 mg/kg dw of an isothiocyanate and one or more or all of the following.

    • i) total fibre at about 29 to about 36 g/100 gdw;
    • ii) an ORAC antioxidant capacity of about 15000 to about 18695 μmol TE/100 gdw;
    • iii) a total polyphenol content of about 2310 to about 2600 mg GAE/100 gdw;
    • iv) a total titratable acidity of about 0.9 to about 1.1% lactic acid equivalent;
    • v) a total protein content of about 27 to about 39 g/100 gdw; and
    • vi) Leuconostoc mesenteroides and/or Lactobacillus plantarum.


In an embodiment, the isothiocyanate containing product is produced from broccoli.


The Brassicaceae products as described herein can comprise live lactic acid bacteria which can aid the conversion of glucosinolate present in the isothiocyanate containing product to an isothiocyanates during digestion of a glucosinolate containing product in a subject (i.e. they act as a probiotic). In an embodiment, the lactic acid bacteria is a Leuconostoc mesenteroide. In an embodiment, the lactic acid bacteria is Lactobacillus sp. In an embodiment, the lactic acid bacteria is Lactobacillus plantarum.


In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 102 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 102 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 105 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 106 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 107 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 108 CFU/g. In an embodiment, the isothiocyanate containing product comprises lactic acid bacteria at a concentration of at least about 109 CFU/g.


In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product for at least 10 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 20 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 30 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 40 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 50 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 60 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 70 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 80 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 85 days when stored at about 4° C. to about 25° C. In an embodiment, live lactic acid bacteria are present in the isothiocyanate containing product at least 90 days when stored at about 4° C. to about 25° C.


In an embodiment, the lactic acid bacteria is a Lactobacillus sp. In an embodiment, the lactic acid bacteria is Lactobacillus plantarum. In an embodiment, the lactic acid bacteria is Leuconostoc mesenteroides. In an embodiment, the bacteria are present at a concentration of at least about 107 CFU/g.


In an embodiment, the isothiocyanate containing product comprises one or more bacteriocin/s produced by lactic acid bacteria. In an embodiment, the bacteriocin is a Class I bacteriocin. In an embodiment, the bacteriocin is a Class II bacteriocin. In an embodiment, the bacteriocin is a Class III bacteriocin. Examples of bacteriocins produced by lactic acid bacteria can be found in Alvarez-Sieiro et al. (2016).


In an embodiment, the isothiocyanate containing product is a food product. In an embodiment, the isothiocyanate containing product is a nutraceutical. In an embodiment, the isothiocyanate containing product is a supplement. In an embodiment, the isothiocyanate containing product is a food ingredient. In an embodiment, the isothiocyanate containing product is a probiotic. In an embodiment, the isothiocyanate containing product is an animal feed. The animal can be an aquatic animal such as fish or livestock. In an embodiment, the isothiocyanate containing product is a pesticide. In an embodiment, the isothiocyanate containing product is a cosmeceutical. In an embodiment, the isothiocyanate containing product is topically formulated.


In an embodiment, the isothiocyanate containing product is a solid, liquid, puree or a powder. In an embodiment, the isothiocyanate containing product is dried to a powder after fermentation. In an embodiment, the isothiocyanate containing product is freeze dried after fermentation. In an embodiment, the isothiocyanate containing product is microencapsulated as described in WO2005030229 after fermentation. In an embodiment, the isothiocyanate containing product is formulated as a pill.


Post-Treatment

In an embodiment, after fermentation or acidification the isothiocyanate containing product can be post-treated to inactivate microbes that for example contribute to degradation of the product or a pathogenic if consumed.


As used herein “post-treatment” or “post-treating” refers to treatment of the isothiocyanate containing product as described herein after fermentation to inactivate microbes. As used herein “microbes” refers to bacterial, viral, fungal or eukaryotic activity that can result in degradation or spoilage of the isothiocyanate containing product. As used herein “inactivate” or “inactivation” of microbes refers to reducing the viable microbes by about 1 to about 7 logs. In an embodiment, the viable microbes are reduced by about 1 to 6 logs. In an embodiment, the viable microbes are reduced by about 2 to 6 logs. In an embodiment, the viable microbes are reduced by about 3 to 6 logs.


A person skilled in the art will appreciate that the post treatment can be any method that inactivates microbes, including for example, heat treatment, UV treatment, ultrasonic processing, pulsed electric field processing or high pressure processing. In an embodiment, the isothiocyanate containing product is post-treated with heat processing. In an embodiment, the isothiocyanate containing product is post-treated with high pressure processing. In an embodiment, the isothiocyanate containing product is in a sealed package during post-treatment. In an embodiment, the isothiocyanate containing product is in a sealed package during high pressure processing. In an embodiment, the isothiocyanate containing product is in a sealed package during heat treatment. In an embodiment, high pressure processing comprises treating the isothiocyanate containing product with isostatic pressure at about 300 to about 600 MPa. In an embodiment, high pressure processing comprises treating the isothiocyanate containing product with isostatic pressure at about 350 to about 550 MPa. In an embodiment, high pressure processing comprises treating the isothiocyanate containing product with isostatic pressure at about 300 to about 400 MPa. In an embodiment, heat treatment comprises heating the sample to a temperature of about 60° C. to about 121° C. In an embodiment, heat treatment comprises heating the sample to a temperature of about 65° C. to about 100° C. In an embodiment, heat treatment comprises heating the sample to a temperature of about 65° C. to about 80° C. In an embodiment, heat treatment comprises heating the sample to a temperature of about 65° C. to about 75° C.


Isolated Strains and Starter Cultures

In an embodiment, the present invention provides isolated strains of lactic acid bacteria suitable for use in the methods and products as described herein.


In an embodiment, the present invention provides an isolated strain of lactic acid bacteria selected from:

    • i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • ii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iii) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iv) B2 deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • v) B3 deposited under V17/21733 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • vi) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; and
    • vii) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.


In an embodiment, the present invention provides an isolated strain of Leuconostoc mesenteroides comprising genomic DNA which when cleaved with SmaI and/or NotI produces a SmaI and/or NotI fingerprint identical to BF1 or BF2. The SmaI and NotI fingerprints for BF1 and BF2 are shown in FIG. 13.


In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising genomic DNA which when cleaved with SmaI and/or NotI produces a SmaI and/or NotI fingerprint identical to B1, B2, B3, B4 or B5.


In an embodiment, the present invention provides an isolated strain of Leuconostoc mesenteroides comprising one or more or all of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 5 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 10 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 15 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 19 or more of the polymorphisms listed in Table 18 or 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 20 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 30 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 50 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 80 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 100 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 150 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 200 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 300 or more of the polymorphisms listed in Table 19 that differs from ATCC8293. In an embodiment, the isolated strain of Leuconostoc mesenteroides comprises 400 or more of the polymorphisms listed in Table 19 that differs from ATCC8293.


In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising one or more or all the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 5 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 10 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 15 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 20 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 25 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 30 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 35 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014. In an embodiment, the present invention provides an isolated strain of Lactobacillus plantarum comprising 40 or more of the polymorphisms listed in Table 13, Table 14, Table 15, Table 16 or Table 17 that differs from ATCC8014.


In an embodiment, the present invention provides a starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria comprising one or more of the isolated strains as described herein. As used herein a “starter culture” is a culture of live microorganisms for fermentation. In an embodiment, the present invention provides a starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria selected from one or more or all of:

    • i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National
    • ii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iii) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • iv) B2 deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • v) B3 deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • vi) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; and
    • vii) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.


In an embodiment, the Brassicaceae material is inoculated with at least about 105 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least 106 about CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 107 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 108 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with at least about 1010 CFU/g of a starter culture as described herein. In an embodiment, the Brassicaceae material is inoculated with about 105 CFU/g to about 1010 CFU/g of a starter culture as described herein.


Probiotics

In an embodiment, the present invention provides for a probiotic comprising one or more of the lactic acid bacteria isolated from a Brassicaceae. As used herein a “probiotic” refers to a live microorganism which when administered in an adequate amount confers a health benefit to the host. In an embodiment, the lactic acid bacteria was isolated from a Brassica oleracea. In an embodiment, the lactic acid bacteria was isolated from broccoli. In an embodiment, the lactic acid bacteria was isolated from Australian broccoli. In an embodiment, the lactic acid bacteria is selected from: i) a Leuconostoc mesenteroides; ii) a Lactobacillus plantarum; iii) a Lactobacillus pentosus; iv) a Lactobacillus rhamnosus; v) a combination of i) and ii); vi) a combination of i), ii) and iii); and vii) a combination of i), ii) and iv). In one embodiment, the lactic acid bacteria is selected from one or more or all of BF1, BF2, B1, B2, B3, B4 and B5. In an embodiment, the lactic acid bacteria is B1. In an embodiment, the lactic acid bacteria is B2. In an embodiment, the lactic acid bacteria is B3. In an embodiment, the lactic acid bacteria is B4. In an embodiment, the lactic acid bacteria is B5. In an embodiment, the probiotic is a capsule, tablet, powder or liquid. In an embodiment, the probiotic is microencapsulated as described in WO 2005030229.


EXAMPLES
Example 1—Methods
Chemicals and Reagents

HPLC grade methanol, sodium dihydrogen phosphate, sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Merck (Damstadt, Germany). Folin-Ciocalteu's reagent, sodium carbonate (Na2CO3), gallic acid, fluorescein sodium salt and dibasic-potassium phosphate were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium dihydrogen phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 2,20-azobis (2-methylpropionamidine) dihydrochloride (AAPH) were purchased from Sapphire Bioscience (Redfern, NSW, Australia).


Lactic Acid Bacteria

Lactic acid bacteria used during fermentation were selected from one or more of:

    • LP: Lactobacillus plantarum ATCC8014;
    • LGG: Lactobacillus rhamnosus ATCC53103;
    • B1: Lactobacillus plantarum isolated from broccoli deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • B2: Lactobacillus plantarum isolated from broccoli deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • B3: Lactobacillus plantarum isolated from broccoli deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • B4: Lactobacillus plantarum isolated from broccoli deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • B5: Lactobacillus plantarum isolated from broccoli deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • BF1: Leuconostoc mesenteroides isolated from broccoli puree deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • BF2: Leuconostoc mesenteroides isolated from broccoli puree BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia;
    • BP: pooled BF1, BF2; and
    • LAB: pooled B1, B2, B3, B4 and B5.


BF1 and BF2 were identified as Leuconostoc mesenteroides via a 16s-RNA sequence (Australian Genome Research Facility; data not shown). B1 to B5 were identified as Lactobacillus plantarum based on 16S-RNA sequence. The identity of all the isolates were confirmed by whole genome sequence analysis.


Isolation of Lactic Acid Bacteria from Broccoli and Broccoli Puree


The above Lactobacillus plantarum B1, B2, B3, B4 and B5 were isolated from broccoli leaves and stem. The leaves and stem were washed with water and homogenised with added peptone saline using a stomacher. The soaking solution was serially diluted and spread plated on De Man, Rogosa and Sharpe (MRS) agar. The plates were incubated under anaerobic condition for 48 to 72 hrs at 37° C. for isolating presumptive mesophilic lactic acid bacteria. Based on different colonial morphology on MRS plates, colonies were isolated, cultivated in MRS broth, screened using staining and biochemical characterisation techniques, and kept frozen with glycerol at −80° C. The isolates were identified at species level using 16s RNA sequencing at AGRF.


For the isolation of Leuconostoc mesenteroides BF1 and BF2, broccoli floret puree was used after serial dilution instead of the suspension described above for the isolation from broccoli leaves.


Preparation of Starter Cultures

The lactic acid bacteria strains, Leuconostoc mesenteroides and Lactobacillus plantarum, were isolated from broccoli and identified by Australian Genome Research Facility Ltd. To obtain the primary culture, lactic acid bacteria cultures which were stored at −80° C. were inoculated into 10 mL of MRS broth (Oxoid, Victoria, Australia) and incubated at 30° C. for 24 h to obtain an initial biomass of 8 log colony-forming units per milliliter (CFU/mL). Two mL of each primary inoculum was inoculated into 200 mL of MRS broth and incubated for 24 hrs at 30° C. The cultures were collected by centrifugation at 2000 g for 15 min at 4° C., washed twice with sterile phosphate buffer saline (PBS), and all the Lactobacillus plantarum cultures were mixed together and all the Leuconostoc mesenteroides cultures were mixed together. The two culture suspensions were diluted to 10 log CFU/ml and were mixed at the same volumetric proportion and stored with glycerol at −80° C. until use as a mixed starter culture for broccoli fermentation.


Fermentation Method

Broccoli (Brassica oleracea L. ssp. Italic; 30 kg) florets were cut approximately 2 cm from the crown, shredded to smaller pieces and, were macerated with Milli-Q water in ratio of 3:2 for 1 min using magic bullet blender. The broccoli slurry, was mixed well and placed into sterile plastic bottles (200 mL) with screw lids. Each bottle of broccoli puree (200 mL) was inoculated with the prepared starter culture at an initial concentration of 8 log CFU/g. The fermentation experiment was carried out in 48 bottles in parallel at 30° C., until a pH value of about 4.0 was reached (Day 4). After the fermentation phase was completed, 3 samples were taken out as the Day 0 storage samples, the other samples were separated to two lots for the storage experiments: one lot was stored in a refrigerator (4° C.) and another stored in room thermostated at 25° C. Samples were periodically taken over 12 weeks for microbiological, physicochemical and phytochemical analyses. The fermented broccoli puree was compared with raw broccoli puree which was stored at −20° C. after homogenization and puree samples incubated for the same period of time as the fermented samples without inoculation by LAB.


Sampling

For time course experiments, sampling was performed at days 10, 20, 30, 40, 50, 60, 70, 80, and 90, and on days 14, 28, 42, 56, 70 and 84 for samples stored at 25° C. and 4° C., respectively. Sampling was performed in triplicate with color measured on the surface and pH measured immediately after opening the fermentation bottles. Thereafter, samples were taken for microbiological analysis and titratable acidity analysis. The remaining material was separated into two parts, the first portion was frozen and freeze dried, ground to fine powder and stored in a desiccator for further analyses, and the second part was frozen and kept at −20° C. until glucoraphanin and sulforaphane analyses.


Microbiological Analysis

For microbial analysis, three different media were used to measure CFU per g broccoli puree of the different microorganisms; the plate counts for total lactic acid bacteria on DeMan-Rogosa-Sharp (MRS) agar, for total enterobacteria on violet red bile glucose agar (VRBGA), and the yeasts and mould on potato dextrose agar (PDA). For each sample, serial dilution of the broccoli suspension in sterilized peptone saline diluent were made and 0.1 mL of the dilutions were plated onto agar plates in duplicates. After aerobic incubation at 25° C. for 72 h (PDA), 37° C. for 24 h (VRBGA), and anaerobic incubation at 30° C. for 72 h (MRS), respectively, the CFU were counted.


Determination of pH and Titratable Acidity

The pH value was determined directly in fermentation bottles containing broccoli puree by a pH meter (PHM240, MeterLab). Titratable acidity (TA) of broccoli samples was measured with an Automatic titrator (Titralab 854 titration manager, Radiometric Analytical, France). In brief, diluted broccoli puree (10 mL) was titrated using 0.1 M NaOH to the end point pH=8.1 and the result obtained was expressed as gram equivalent of lactic acid per liter of sample in accordance with the following equation:







TA



(
g/L
)


=


[

v
×
acid


factor
×
1000

]


sample


volume






where, v is titer volume of NaOH. The acid factor for lactic acid is 0.009.


Total Protein and Color Analyses

The total protein content of broccoli samples was determined as total nitrogen content multiplied by 6.25. Total nitrogen content of broccoli was analyzed using a Dumas combustion method with LECO TruMac apparatus (LECO Corporation, Michigan, USA). The color indexes (L, a, b) of fermented broccoli sample were determined using a Chroma meter CR-200 tristimulus colorimeter (Minolta, Osaka, Japan). The color values obtained were expressed as lightness/darkness (as L*), redness/greenness (a*) and yellow/blueness (b*). The total color difference (ΔE) was calculated according to the following equation:







Δ

E

=


[



(


L
*

-

L
0


)

2

+


(


a
*

-

a
0


)

2

+


(


b
*

-

b
0


)

2


]


1
/
2






where, L0, a0, b0 are color values of fresh unfermented broccoli.


Determination of Total Polyphenol Content

The total phenolic content (TPC) was measured spectrophotometrically using the Folin-Ciocalteu colorimetric method (Singleton and Rossi, 1965) with modifications. Briefly, 50 mg of broccoli powder was suspended in 10 mL of acidified (1% HCl) methanol/water (70:30, v/v) solution and extracted in ultrasonic bath (IDK technology Pty Ltd, VIC, Australia) for 8 min. The extracts were kept for 16 h at 4° C. and filtered with 0.2 μM filter and stored at 4° C. until analysis. 1 mL of 0.2 N Folin-Ciocalteu reagent, 800 μL of sodium carbonate solution (7.5% p/v) and 180 μL Milli-Q grade water were added to the extract (20 μL). After 1 h of incubation in the dark at 37° C., the absorbance was measured at 765 nm in triplicates using a spectrophotometer (UV-1700 Pharma Spec, SHIMADZU). Gallic acid was used as a standard and TPC was expressed as the gallic acid equivalent (GAE) in mg per 100 g of fresh weight (mg GAE/100 g FW) based on a standard curve developed using known concentrations of gallic acid.


Oxygen Radical Absorbance Capacity Assay

Freeze-dried broccoli powder (10 mg) was suspended in 10 mL of methanol/water (80:20, v/v), the extraction solvent. The slurry was extracted at 650 rpm on a Heidolph Multi-Reax (John Morris Scientific, NSW, Australia) at room temperature for an hour. Then it was centrifuged at 25,000 g for 15 min in 4° C., the supernatant was collected, and was ready for analysis after 100× dilution with 75 mM potassium phosphate buffer (pH 7.4). ORAC analysis was conducted according to the procedure reported by Huang et al. (2002) with minor modifications. The assay was carried out in opaque 96-well plates (dark optical bottom, Waltham, MA, USA). The assay reactants included 81.6 nM of fluorescein, 153 mM of AAPH, Trolox standard of different concentration (100, 50, 25, 12.5, and 6.25 μM), and 75 mM phosphate buffer as the blank. The reactants were added in the following order: 25 μL of diluted sample; either 25 μL of 75 mM phosphate buffer, 25 μL Trolox standard and 150 μL fluorescein. After adding the fluorescein, the plate was incubated at 37° C. for 10 min and then the AAPH (25 μL) was added. Immediately after addition of AAPH, the plate was placed in the fluorescence plate reader (BMG Labtech ClarioStar, Germany) and the fluorescence was measured every 3 min until it decreased to less than 5% of original fluorescence. The ORAC values were calculated as the area under the curve (AUC) and expressed as micromoles of trolox equivalent (TE) per gram dry weight of broccoli (μmol TE/g DW). Each sample was assayed triplicate.


Sulforaphane Analysis

The extraction of sulforaphane from broccoli matrix was conducted following the methods of Li et al. (2012) with some modification. In brief, frozen broccoli (2 g) was mixed with 2 mL of Milli-Q water and vortexed for 1 min. Then 20 mL ethyl acetate was added to the slurry followed by sonication for 5 min and shaking for 20 min at 4° C. The slurry was then centrifuged at 15,000 g for 10 min, and the supernatant was collected. Then another 15 mL ethyl acetate was added to the precipitate to carry out the second extraction. Pooled extracts from each sample were evaporated to dryness with a vacuum spin dryer (SC250EXP, Thermo Fisher Scientific, CA, USA) at room temperature, and stored at −20° C. until analysis. The concentration of sulforaphane was determined using an Acquity™ Ultra Performance LC system (Waters Corporation, Milford, MA, USA), which is equipped with a binary solvent delivery manager and a sample manger. Chromatographic separations were performed on a 2.1×50 mm, Acquity BEH C18 chromatography column. The mobile phase A and B were 0.1% formic acid in millique water and 0.1% formic acid in acetonitrile, respectively. The gradient elution system consisted of mobile phase A (0.1% formic acid in millique water) and B (0.1% formic acid in acetonitrile) and separation was achieved using the following gradient: 0-2 min, 10% B; 2-5 min, 20% B; 5-10 min, 10% B. The column temperature was kept constant at 30° C. The flow-rate was 0.350 mL/min and the injection volume was 5 μL.


Prior to analysis, all samples were dissolved in 1 mL 30% acetonitrile, and filtered through a 0.22 μm membrane filter (Merk Millipore, Billerica, MA, USA). The identification of each peak was based on the retention time and the chromatography of authentic standards. The concentrations of each compound were calculated according to a standard curve, and the results were expressed as micromoles per kilogram DW (μmol/kg DW) of broccoli.


Glucoraphanin Analysis

The extraction of glucoraphanin from raw or fermented broccoli was carried out according to the method of Cai and Wang (2016) with some modification. Accordingly, to 2 g of frozen broccoli puree, 10 mL of boiling Milli-Q water was added, and the mixture was incubated for 5 min in a boiling water bath. It was then cooled and centrifuged at 15000×g for 15 min, and the supernatant was collected. The precipitate was extracted once more with 8 mL of boiling water. Pooled extracts from each sample were evaporated to dryness with a vacuum spin dryer (Speedvac SC250EXP, Thermo Fisher Scientific, CA, USA) at 3° C., and stored at −20° C. until analysis. The concentration of glucoraphanin was quantified using an Alliance HPLC instrument (Waters Corporation, Milford, MA, USA) equipped with Photo Diode Array Detector 2998. A HPLC column-Luna® 3 μM Hydrophilic Interaction Liquid Chromatography (HILIC) 200° A (100×4.6 mm; Phenomenex, Torrance, CA, USA) was used for the analysis at a column temperature of 25° C. The mobile phase consisted of an acetonitrile/water (85:15, v/v) with 30 mM Ammonium formate (solution A) and acetonitrile (solution B) with the following isocratic flow program: solution A 70%; solution B 30%. Other chromatographic conditions included a constant flow rate of 2.0 mL/min, an injection volume of 100 μL, a run time of 8 min, and detection wavelength of 235 nm. Prior to analysis, all samples were dissolved in 1 mL solvent A, and filtered through a 0.22 μm membrane filter (Merk Millipore, Billerica, MA, USA). The identification of each peak was based on the retention time and the chromatography of an authentic glucoraphanin standard. The concentrations of glucoraphanin were calculated using a standard curve, and the results were expressed as micromoles glucoraphanin per kilogram DW (μmol/kg DW) of broccoli.


Statistical Analysis

All experiments were conducted in triplicate and the results were expressed as mean values. A one-way analyses of variance (ANOVA) was applied to evaluate the significance of the differences among the mean values at 0.05 significance level (p<0.05). The statistical analysis was conducted using the statistical software, SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA).


Example 2—Microbial Analysis of Lactic Acid Bacteria Fermented Broccoli Florets

The fermentation of broccoli puree was carried out as described in the fermentation section of Example 1. The counts of total lactic acid bacteria were lower for raw broccoli compared to inoculated broccoli as showed in Table 1. After 4 days of fermentation, the pH of the sample reached 4.04 and fermentation was stopped, and the fermented sample before storage experiments was taken as the Day 0 sample. It is clear from Table 1 and FIG. 1C that the counts of total lactic acid bacteria of the Day 0 sample were significantly increased (8 log CFU/g) compared to the raw broccoli. During the first two weeks of storage, the viable number of total lactic acid bacteria increased to the highest values of 9 log CFU/g for samples stored at both 25° C. and 4° C. (Table 1 and Table 2). During storage at 25° C., the total lactic acid bacteria counts increased to 9 log CFU/g at Day 10 and slowly declined during storage to 5 log CFU/g by Day 50, and declined further to almost undetectable level after Day 70. In contrast,









TABLE 1







Microbiological and physicochemical changes of fermented broccoli during the storage at room temperature (25° C.).











Microbial loads (Log CFU/g)

Color


















MRS
PDA
VRBGA
pH
TA (g/L)
TP (mg/g, FW)
L
a
b
ΔE





Raw broccoli
2.4 ± 0.2
2.5 ± 0.1
3.4 ± 0.1
6.33 ± 0.00
 4.8 ± 0.2
26.9 ± 0.0
48.4 ± 0.4
−13.2 ± 0.1 
17.2 ± 0.2



Day 0
8.4 ± 0.2
<1
<1
4.04 ± 0.00
10.7 ± 0.7
29.6 ± 0.8
48.5 ± 0.7
−2.1 ± 0.1
13.6 ± 0.6
11.7


Days 10
9.4 ± 0.1
<1
<1
3.87 ± 0.02
14.4 ± 0.2
27.8 ± 0.8
47.7 ± 0.8
−1.1 ± 0.2
12.2 ± 0.5
13.1


Days 20
6.2 ± 0.3
<1
<1
3.76 ± 0.02
14.7 ± 0.2
30.5 ± 0.8
47.1 ± 0.5
−1.1 ± 0.0
12.5 ± 0.2
13


Days 30
6.2 ± 0.1
<1
<1
3.78 ± 0.00
15.1 ± 0.3
29.7 ± 1.2
47.2 ± 0.2
−1.0 ± 0.1
10.9 ± 0.5
13.8


Days 40
6.1 ± 0.4
<1
<1
3.79 ± 0.02
15.1 ± 0.4
28.8 ± 1.1
46.3 ± 0.5
−0.8 ± 0.1
11.0 ± 0.9
14


Days 50
5.1 ± 0.6
<1
<1
3.75 ± 0.00
15.2 ± 0.5
28.5 ± 0.1
45.8 ± 0.5
−0.9 ± 0.1
11.0 ± 0.2
14


Days 60
2.4 ± 0.1
<1
<1
3.76 ± 0.01
15.4 ± 0.3
27.3 ± 0.6
45.4 ± 0.1
−0.9 ± 0.1
10.5 ± 0.1
14.3


Days 70
1.5 ± 0.1
<1
<1
3.76 ± 0.01
15.7 ± 0.1
27.7 ± 0.2
45.3 ± 0.5
−0.9 ± 0.1
 9.9 ± 0.4
14.7


Days 80
<1
<1
<1
3.76 ± 0.01
15.7 ± 0.7
28.3 ± 0.2
45.9 ± 0.1
−0.9 ± 0.1
 9.7 ± 0.1
14.6


Days 90
<1
<1
<1
3.71 ± 0.01
15.7 ± 0.3
28.7 ± 0.4
45.0 ± 0.0
−0.8 ± 0.2
 9.3 ± 0.2
15.1





Each value was expressed as mean ± standard deviation (n = 3).


“—”not available.


MRS, de Man-Rogosa-Sharpe agar for LAB; PDA, potato dextrose agar for total yeasts and moulds; VRBGA, violet red bile glucose agar for Enterobacteriaceae; TA, titratable acidity; TP: total protein; ΔE: total color difference.













TABLE 2







Microbiological and physicochemical changes of fermented broccoli during the storage at 4° C.











Microbial loads (Log CFU/g)

Color


















MRS
PDA
VRBGA
pH
TA (g/L)
TP (mg/g, FW)
L
a
b
ΔE





Raw broccoli
2.4 ± 0.2
2.5 ± 0.1
3.4 ± 0.1
6.33 ± 0.00
 4.8 ± 0.2
26.9 ± 0.0
48.4 ± 0.4
−13.2 ± 0.1 
17.2 ± 0.2



Day 0
8.4 ± 0.2
<1
<1
4.04 ± 0.00
10.7 ± 0.7
29.6 ± 0.8
48.5 ± 0.7
−2.1 ± 0.1
13.6 ± 0.6
11.7


Days 14
9.0 ± 0.1
<1
<1
4.04 ± 0.03
12.6 ± 0.8
32.5 ± 1.2
47.2 ± 1.1
−1.9 ± 0.5
12.4 ± 1.5
12.3


Days 28
8.0 ± 0.1
<1
<1
3.95 ± 0.02
13.5 ± 0.8
32.0 ± 0.7
45.9 ± 0.7
−2.2 ± 0.3
13.8 ± 2.5
11.8


Days 42
7.6 ± 0.1
<1
<1
3.89 ± 0.03
13.8 ± 0.2
32.0 ± 0.8
46.7 ± 0.2
−1.5 ± 0.1
12.6 ± 0.5
12.7


Days 56
6.5 ± 0.4
<1
<1
3.89 ± 0.02
13.8 ± 0.5
29.9 ± 0.3
46.6 ± 0.4
−1.7 ± 0.1
13.1 ± 0.5
12.4


Days 70
6.3 ± 0.4
<1
<1
3.86 ± 0.01
13.7 ± 0.1
31.6 ± 0.2
46.7 ± 0.8
−1.6 ± 0.2
12.2 ± 0.4
12.7


Days 84
6.0 ± 0.8
<1
<1
3.85 ± 0.01
13.8 ± 0.1
32.0 ± 0.5
47.6 ± 0.9
−1.9 ± 0.2
14.0 ± 0.6
11.8





Each value was expressed as mean ± standard deviation (n = 3).


“—”not available.


MRS, de Man-Rogosa-Sharpe agar for LAB; PDA, potato dextrose agar for total yeasts and moulds; VRBGA, violet red bile glucose agar for Enterobacteriaceae; TA, titratable acidity; TP: total protein; ΔE: total color difference.







the LAB count in the samples stored at 4° C. remained high (6 log CFU/g) even after storage for 84 days.


The total counts of yeast and moulds in the raw broccoli sample was 2 log CFU/g. The Enterobacteriaceae count in the raw broccoli with 3 log CFU/g. No fungi, moulds and enterobacteria were detected after fermentation or on the fermented samples after storage at both temperature conditions. No pathogenic and spoilage organisms were detected following fermentation and during storage. The results indicate that the fermentation process resulted in a safe and stable product with undetectable level of potentially pathogenic eneterobacteriaceae and spoilage yeast and mould, which maintained high levels of total lactic acid bacteria when stored at 4° C. There are ˜106 CFU/g lactic acid bacteria after ˜3 months at 4° C.


Example 3—Assessment of pH and Titratable Acidity after Storage of Lactic Acid Bacteria Fermented Broccoli Florets

The pH and titratable acidity (TA) of raw broccoli, fermented broccoli and fermented broccoli after storage at 25° C. and 4° C. was analyzed as described in Example 1. The determination of TA was used to estimate the amount of lactic acid and acetic acid, the main acids produced by lactic acid bacteria, during fermentation. During fermentation, the acids produced by the lactic acid bacteria decrease the pH of the sample. As shown in Table 1, the TA was increased to 10.7 g/L in Day 0 samples. When stored in 25° C., the pH was decreased to 3.87 during storage after 10 days, along with the significantly increased values of TA which reached 14.4 g/L (p<0.05; see Table 1). The results indicate that there were still substrates present for lactic acid bacteria to consume and further produce acid during the early days of storage. Neither the pH nor TA value were significantly changed during the remaining storage period (Table 1).


Decreasing the temperature to 4° C. reduced the rate of decrease of pH and TA in the stored samples due to the decreased activity of the lactic acid bacteria at the lower temperature (see Table 2). After nearly 3 months storage at 4° C., the pH was 3.85 and the TA value was 13.7 g/L.


Example 4—Assessment of Broccoli Maceration and Fermentation on the Conversion of Glucoraphanin into Sulforaphane

Broccoli florets were cut into small pieces, mixed with water at 3:2 broccoli: water ratio and the mixture was macerated into a puree using a blender. Puree samples (200 gm) were aliquoted into sterile plastic bottles. The samples were inoculated at 108 CFU/gm with pooled culture of lactic acid bacteria (Leuconostoc mesenteroides and Lactobacillus plantarum) isolated from Australian broccoli. Samples were incubated in a water bath maintained at 30° C. until the pH dropped to ˜4.0, which was attained after four days of fermentation. Control non-inoculated samples were immediately frozen after maceration. A second set of non-inoculated control samples, to which sodium benzoate was added to inhibit microbial growth, were incubated with the inoculated samples at 30° C. for four days until the fermentation of the inoculated samples was completed. Experiments were conducted in triplicate. All samples were kept frozen until sulforaphane and glucoraphanin analysis. As shown in FIG. 1B and Table 3 maceration followed by fermentation increased the sulforaphane yield compared to just maceration and incubation alone.









TABLE 3







Effects of maceration and fermentation on


sulforaphane content in broccoli puree.










25° C.
SF(mg/kg, DW)
4° C.
SF (mg/Kg, DW)





Raw material
149.8 ± 12.4
Raw material
149.8 ± 12.4


Control
86.8 ± 0.6
Control
86.8 ± 0.6


incubated

incubated



 0 days
278.4 ± 1.8 
 0 days
278.4 ± 1.8 


10 days
 189 ± 8.8
14 days
288.6 ± 3.1 


20 days
136.6 ± 6.2 
28 days
218.8 ± 4.3 


30 days
122.2 ± 12.2
42 days
199.4 ± 14.7


40 days
116.3 ± 5.0 
56 days
 190 ± 7.1


50 days
112.3 ± 4.0 
70 days
190.8 ± 10.7


60 days
111.9 ± 11.0
84 days
179.6 ± 10.2


70 days
108.8 ± 15.8




80 days
102.6 ± 14.7




90 days
87.6 ± 3.7









Example 5—Assessment of Total Protein Content and Color after Storage of Lactic Acid Bacteria Fermented Broccoli Florets

The total protein content and color of lactic acid fermented broccoli florets after fermentation was assessed as described above in the methods section. Compared to raw broccoli (26.9±0.03), the total protein content of fermented broccoli was significantly increased (29.6±0.8 mg/g; p<0.05). This could be due to the high number of lactic acid bacteria inoculated into the sample and the growth during fermentation and protein synthesis by the lactic acid bacteria. The total protein content stayed stable during storage both at 25° C. and 4° C. (Table 1 and Table 2), with no significant difference between samples.


The color values (L, a, b) and the total color difference (ΔE) of broccoli samples are summarized in Table 1 and Table 2. As presented in Table 1 and Table 2, significant differences in the color parameters and the total color difference value (ΔE) were recorded between raw and fermented samples. The L* value (lightness) did not change significantly, whereas a* (greenness) and b* (yellowness) values decreased after the fermentation of broccoli puree. The decrease in a* and b* values may be attributed to the degradation in the color pigmented compounds, such as chlorophyll which would convert to pheophytins under the low pH. The high ΔE value (12.5) of Day 0 sample indicate that the color of broccoli puree was significantly changed after fermentation, which was visually noticeable. During storage (Table 1 and Table 2) there was no significant change in the ΔE value in neither 25° C. nor 4° C. samples.


Broccoli after fermentation with LAB+BP (Lactobacillus plantarums B1, B2, B3, B4, B5 and Leuconostoc mesenteroides BF1, BF2 isolated from broccoli) had a brighter, more intense green color more similar in color to raw macerated broccoli compared to broccoli fermented with LAB only (the Lactobacillus plantarums isolated from broccoli (B1, B2, B3, B4, B5)).


Example 6—Changes of Total Phenolic Content and Antioxidant Activity of Lactic Acid Bacteria in Fermented Broccoli Florets

The total phenolic content (TPC) and antioxidant activity of lactic acid fermented broccoli florets after fermentation was assessed as described above in the methods section. The TPC of raw broccoli was 127.6±12.4 mg GAE/100 g (FIG. 3A) of fresh weight. The values of TPC on Day 0 significantly increased to 236.9±23.4 mg GAE/100 g (p<0.05) compared to raw broccoli. There was no significant difference between samples stored at 25° C. and 4° C. in the TPC after storage (FIG. 3A). When stored at 25° C., the value of TPC in fermented broccoli was 246.2±19.3 mg GAE/100 g on Days 10, and 248.1±25.0 mg GAE/100 g on Days 90. When stored at 4° C., the values of TPC was 274.1±20.2 and 267.2±3.3 mg GAE/100 g for Days 14 and Days 84, respectively.


The antioxidant activities of sample expressed as ORAC values are shown in FIG. 3B. The ORAC value of the raw sample was 110.1±0.05 μmol TE/g. Fermentation significantly increased the ORAC value by ˜70% to 186.9±3.3 μmol TE/g when compared to raw broccoli. This result suggested that antioxidant compounds may have increased during fermentation and was consistent with the change in TPC after fermentation.


During storage, the antioxidant activity of fermented broccoli did not change significantly. As shown in FIG. 3B, when stored at 25° C., the values of ORAC at Days 10 and Days 90 were 173.0±14.4 and 150±5.5 μmol TE/g, respectively. Similar results were obtained for samples stored at 4° C. The ORAC value was 172.0=15.5 μmol TE/g at the beginning of storage, which increased to a maximum value of (188.7±12.9 μmol TE/g) after storage.


Example 7—Assessment of Fermentation Time for Different Combinations of Lactic Acid Bacteria

Macerated broccoli was prepared as described above in the methods section with a broccoli to water ratio of 3:2 and a maceration time of 1 min. The broccoli material was inoculated with either 107 CFU/g or 108 CFU/g with one of: LGG, LAB (Lactobacillus plantarum (B1, B2, B3, B4, B5) isolated from Australian broccoli, LAB+LP (Lactobacillus plantarum isolated from broccoli and Lactobacillus sp. ATCC 8014), BP (Leuconostoc mesenteroides isolated from broccoli), LAB+BP (a mixture of the two groups as described in the methods sections) and fermented at either 25° C., 30° C. or 34° C. to reach a target pH of 4.4. As shown in FIG. 4 the addition of lactic acid bacteria isolated from broccoli and/or broccoli puree significantly reduced the time taken for the fermentation with the combination of LAB+BP reaching a pH of 4.4 after fermenting for about 4 days. An example composition of fermented broccoli product is shown in Table 4.









TABLE 4







Composition of the fermented broccoli product.










Quality attributes
Value






Total fibre
    ~29.5 g/100 gdw



ORAC antioxidant capacity
18695 μmol TE/100 gdw 



Total polyphenol content
2369 mg GAE/100 gdw



Total titratable acidity
1.1% lactic acid equiv.



Lactic acid bacteria count
~108 CFU/gm



Total protein
    30 g/100 gdw



Broccoli to water ratio in puree
3 to 2



by mass









Example 8: Effect of Storage on Sulforaphane Content of Fermented Broccoli


FIG. 2A shows the effects of storage at 4 and 25° C. on sulforaphane content of fermented broccoli puree. As can be seen in the FIG. 2A, the sulforaphane content of samples stored at 25° C. dramatically decreased to 770.7±34.9 μmol/kg (a 52% loss) after 20 days storage, followed by a slower decline during the rest of the storage period, reaching a total loss of 69.5%. Interestingly, no statistically significant change in sulforaphane content was observed during the first 2 weeks of storage of fermented broccoli samples at 4° C. A significant decrease of ˜23.7% occurred during the subsequent two weeks followed by a slow degradation during the rest of the storage period. At the end of the storage (Day 84), the sulforaphane content was 1012.9±57.6 μmol/kg in samples stored at 4° C., making the total loss of sulforaphane ˜37.4% compared to the Day 0 samples. The sulforaphane content during the first two weeks of storage was maintained perhaps due to simultaneous production and degradation of sulforaphane since some decrease in glucoraphanin content was observed in the 4° C. stored samples over the same period.


Example 9: Effect of Fermentation and Storage on Glucoraphanin Content


FIG. 7 shows the effect of maceration and fermentation on glucoraphanin content and its stability during storage at 4° C. and 25° C. The glucoraphanin content of raw broccoli was 3423.7±39.7 μmol/kg (FIG. 7), After fermentation, the glucoraphanin content sharply decreased to 712.4±64.2 μmol/kg (Day 0 sample). Glucoraphanin is relatively stable in intact tissue and the degradation in this case can be attributed to myrosinase catalyzed hydrolysis due to increased enzyme-substrate interaction in the macerated tissue during fermentation. The period of sharp decrease in glucoraphanin coincided with the fermentation period.


No significant change in glucoraphanin content was observed in fermented samples during storage at 25° C. and 4° C. However, slightly higher glucoraphanin content was observed in samples stored at 25° C. This could be related to the faster decline in pH of the samples stored at 25° C. (pH 3.87 at the second time point) compared to samples stored at 4° C. (pH 4.04 at the second time point). The optimal pH for myrosinase catalyzed hydrolysis of glucoraphanin ranges from 5 to 6 decreasing to the lowest value at pH 3.0 (Dosz & Jeffery, 2013). The relatively higher pH of the samples stored a 4° C. may have contributed to the slightly higher degradation of glucoraphanin during storage at 4° C. compared to 25° C.


Example 10—Assessment of Heat Treatment Conditions to Maximise Conversion of Glucoraphanin into Sulforaphane in Broccoli Matrix

Broccoli florets packed in retort pouches were subjected to thermal processing at temperatures ranging from 60° C. to 80° C. and treatment times of 0 to 5 minutes. The treatment involved pre-heating to the experimental temperature in a water bath maintained at 5° C. higher than the experimental temperature followed by incubation in a second water bath maintained at the experimental temperature. Following thermal treatment, samples were cooled in ice-water and were macerated with water added at 2:3 water to broccoli ratio as described above. The macerated samples were incubated for 1 hr at 30° C. and kept frozen until sulforaphane analysis. Results are shown in FIG. 2B and Table 5. As shown in Table 5 pre-heating the sample at 60° C., 65° C. or 80° C. followed by maceration increased the sulforaphane yield relative to raw broccoli floret which was macerated without pre-heating.









TABLE 5







Effects of heat treatment on sulforaphane production in broccoli matrix.












Heat treatment
Sulforaphane
Sulforaphane
Sulforaphane


Temperature
time (minute)
(μmol/kg, DW)
(mg/kg, DW)
(mg/g, DW)





Raw broccoli floret

817.5 ± 9.29
 145 ± 1.6
0.145 ± 0.002


60° C.
0
2343.5 ± 124.1
415.5 ± 22.0
0.415 ± 0.022



1
2661.5 ± 10.9 
471.9 ± 1.9 
0.472 ± 0.002



3
2780.9 ± 270.7
493.0 ± 48.0
0.493 ± 0.048



5
3147.6 ± 148
558.1 ± 26.2
0.558 ± 0.026


65° C.
0
3585.9 ± 119.2
635.8 ± 21.1
0.636 ± 0.021



1

3673 ± 144.8

651.2 ± 25.7
0.651 ± 0.026



3
3983.4 ± 30.5 
706.3 ± 5.4 
0.706 ± 0.005



5
3620.1 ± 240.7
641.8 ± 42.7
0.642 ± 0.043


80° C.
0
1451.5 ± 43.5 
257.3 ± 7.7 
0.257 ± 0.008



1
1446.8 ± 17.5 
256.5 ± 3.1 
0.257 ± 0.003



2
1043.1 ± 94.2 
184.9 ± 16.7
0.185 ± 0.017



3
981.2 ± 35.1
 174 ± 6.2
0.174 ± 0.006









Example 11—Assessment of Preheating Prior to Lactic Acid Bacterial Fermentation on the Sulforaphane Content of Broccoli

This study evaluated the impact of mild preheating treatment of broccoli florets to inactivate the Epithiospecifier protein (ESP) combined with lactic acid bacteria on sulforaphane content of broccoli puree.


Materials

Broccoli (cv. ‘Viper’) was purchased from a local supermarket (Coles, Werribee South, VIC, Australia). DeMan-Rogosa-Sharp (MRS) broth (1823477, CM0359, Oxoid) was purchased from Thermo Fisher Scientific (Australia). DL-Sulforaphane was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All the other chemical and biochemical reagents were analytical grade or higher and were purchased from local chemical vendors.


Experiments to Optimize the Mild Pre-Heating Conditions to Maximize Sulforaphane Yield

Broccoli florets were cut at approximately 2 cm below the head, and each 30 g of randomly mixed broccoli florets were used in the pre-heating experiments. Two types of pre-heating experiments were conducted; in-pack processing and direct water blanching. In the case of the in-pack experiments, broccoli florets were packed in retort pouches (Caspak Australia, Melbourne), sealed and pre-heated for various time points in a thermostated water batch maintained at 60° C., 65° C. and 80° C. The temperature of the broccoli samples at the slowest heating point was measured by using a thermometer. Time 0 was defined as the time for the core temperature to reach the designated experimental temperature. The treatment time were 0, 1, 3, and 5 min for 60° C. and 65° C. and 0, 1, 2, 3 min for 80° C. With the direct water-blanching experiments, the broccoli florets were immersed in Milli-Q water in a glass beaker that was heated in a thermosated water-bath. The direct water blanching experiments were conducted at 60° C. and 65° C. The temperature of the broccoli samples was continuously measured using a thermometer and timing started once the temperature at the slowest heating point attained the designated experimental temperature as described above. All thermal treatment experiments were carried out in triplicate. Unheated broccoli florets were used as controls. Immediately following the heat treatment, the samples were cooled in ice water and were homogenized with Milli-Q water in ratio of 3 parts broccoli to 2 parts of water for 1 min using a kitchen scale magic bullet blender (Nutribullet pro 900 series, LLC, USA). The homogenized samples were incubated in the dark for 4 h at 25° C. to allow the enzymatic hydrolysis of glucoraphanin. After incubation, all the samples were frozen in −20° C. until sulforaphane analysis.


Preparation of Starter Cultures

Pooled cultures of Leuconostoc mesenteroides (BF1, BF2) and Lactobacillus plantarum (B1, B2, B3, B4, B5) isolated from broccoli as described in the methods in Example 1. were used in the fermentation experiments. The lactic acid bacteria stock cultures, which were stored at −80° C., were activated by inoculation into 10 mL MRS broth (Oxoid, Victoria, Australia) and incubation at 30° C. for 24 hours to get the primary inoculum. 2 mL of the primary cultures were inoculated into 200 mL of MRS broth to obtain the secondary cultures. After 24 h incubation, the 6 secondary cultures were centrifuged, washed twice with sterile phosphate buffer saline (PBS) and each of the culture was resuspended in Milli-Q water at a concentration of 10 log colony-forming units per millilitre (CFU/mL) to obtain an initial biomass of 8 log CFU/mL in 100 gm broccoli puree samples. The L. plantarum cultures were mixed with the L. mesenteroides cultures at 1:1 proportion prior to inoculation into the broccoli puree samples.


Sample Preparation

Broccoli florets were cut at approximately 2 cm below the crown and were separated into two lots; heat treated and non-treated. After heat treatment at the optimal condition selected based on the results of the experiments as described above, the samples were cooled in ice-water, shredded and homogenized with Milli-Q water in ratio of 3:2 for 1 min using a kitchen scale magic bullet blender (Nutribullet pro 900 series, LLC, USA). The non-treated broccoli were also homogenized in a similar way. The broccoli puree, after mixing well, was aliquoted into sterile plastic containers (100 mL) with screw lids (Technoplast Australia) for further experiments.


Fermentation

Broccoli puree samples (pre-heated and untreated) were inoculated with the LAB culture prepared as described above in this example. Preheating of broccoli florets was conducted in-pack at 65° C. for 3 min based on the result of the experiment to optimise the pre-heating condition. In order to evaluate the impact of acidification without fermentation on conversion of glucoraphanin into sulforaphane, acidification experiments were conducted on pre-heated and untreated broccoli puree using glucono-delta-lactone (GDL) to attain the pH of the fermented broccoli puree. Preheated broccoli puree and untreated broccoli puree without further treatment were used as controls.


For the fermentation experiment, each broccoli puree sample was inoculated with the prepared starter culture at an initial level of 8 log CFU/g. The fermentation experiment was carried out at 30° C. until the pH reached ˜4.0 after 15 hrs of incubation. Once the fermentation was completed, 3 samples (day 0 samples) of each fermented group were taken and stored at −20° C. until analysis. The rest of the ferments were randomly separated into two lots for the storage trials: one lot was stored under refrigerated condition (4° C.) and the second lot was stored at 25° C. for the assessment of the sulforaphane stability of the samples after 14 days storage. Similarly, the untreated broccoli puree, preheated broccoli puree and the preheated-GDL treated broccoli puree were also sampled at time zero and stored at 25 and 4° C. for the 14 days storage trials. After 14 days storage, all the samples were frozen and kept at −20° C. until sulforaphane analyses.


Sulforaphane Analysis and Statistical Analysis

Was performed as described in Example 1.


Optimization of Heat Treatment Conditions for Improving Sulforaphane Yield

The influence of heat treatment on the formation of sulforaphane of the heated-in-pack broccoli florets at three different temperatures (60, 65 and 80° C.) for various processing times (0, 1, 3 and 5 min for 60 or 65° C.; 0, 1, 2 and 3 min for 80° C.) are shown in FIG. 5A. The results showed that compared to the raw broccoli the sulforaphane yield increased in all of the heat treated samples. Time 0 designate samples that were heated until their core reached the experimental temperature.


As shown in FIG. 5A, an increase in sulforaphane yield occurred when the packed broccoli samples were heated at 60° C. for 0, 1, 3, and 5 min. The concentration of sulforaphane in these samples were 2343.5±124.1, 2661.5±10.9, 2780.9±270.8, and 3147.7±148.0 μmol/kg DW, respectively. On the other hand, when broccoli was processed at 65° C., the sulforaphane yield initially increased with processing time from 3585.9±119.2 (0 min) to the highest value of 3983.4±30.5 μmol/kg DW (3 min). Further increase in treatment time resulted in lower yield with the lowest value of 3620.1±240.7 μmol/kg observed after 5 min treatment time. In contrast to treatments at 60 and 65° C., for samples that were processed at 80° C., a steady decrease in sulforaphane yield was observed with longer treatment times; with sulforaphane content of 1451.5±43.5, 1446.8±17.5, 1043.1±94.2, and 981.2±35.1 μmol/kg DW after 0 min, 1 min, 2 min and 3 min treatment respectively. Overall, the highest yield of sulforaphane (3983.4±30.5 μmol/kg) for in-pack treatment of broccoli was obtained for samples pre-heated at 65° C. for 3 min, which is ˜5 fold higher than raw broccoli (817.5±9.3 μmol/kg DW). In contrast, heating broccoli directly in water, generally resulted in a lower yield of sulforaphane compared to in-pack processing as shown in FIG. 5B. For direct water blanching at 60° C., the sulforaphane yield increased with treatment time from 1698.00±121.9 μmol/kg DW (0 min), to 2833.3±118.6 μmol/kg DW (1 min) and then steadily decreased to the lowest value of 2345.8±57.7 μmol/kg DW for 5 min treatment at 60° C. A sharp drop in sulforaphane yield compared to 60° C. was observed when samples were blanched at 65° C. The sulforaphane yield was 503.7±23.8 μmol/kg DW of broccoli after 5 min thermal treatment at 65° C., which was even lower than the value obtained for raw broccoli. The reason could be the leaching of glucoraphanin into the blanching water resulting in low yield of sulforaphane. For direct water blanching, the optimum treatment temperature for maximizing sulforaphane yield was 60° C. compared to 65° C. for the in-pack processing.


In this study, the highest yield of sulforaphane was obtained for broccoli florets processed in-pack for 3 min at 65° C., indicating that the condition favors the inactivation of ESP to a larger extent while maintaining sufficient myrosinase activity resulting in optimal conversion into sulforaphane. Under this condition, it seems that most of the extractable glucoraphanin is converted to sulforaphane assuming 1 to 1 conversion, since the glucoraphanin content of the broccoli samples were determined to be 3423.7±39.7 μmol/kg DW.


The observation that the exposure of the heat-treated broccoli to fermentation resulted in higher levels of sulforaphane than would be predicted from the level of extractable glucoraphanin from raw broccoli suggests heat-treatment may have increased the accessibility of glucoraphanin to myrosinase, resulting in higher sulforaphane yield than would be expected based on the quantifiable amount of glucoraphanin present in the untreated broccoli.


Less sulforaphane yield was obtained for broccoli florets directly blanched in water, most probably due to leaching into the blanching water, since glucoraphanin is soluble in water. It is also interesting to note that when broccoli florets were heated directly in water, the maximum amount of sulforaphane was obtained by heating at 60° C. for 1 min compared to 65° C. for 3 min when heat treatment of broccoli florets was done in-pack. This may be due to the higher leaching rate into the blanching water at 65° C. which counteracted the effects of higher level of inactivation of ESP at 65° C.


The Effect of LAB Fermentation and Chemical Acidification on Sulforaphane Yield

Broccoli florets were pre-heated in-pack at the best treatment condition selected above (65° C., 3 min). Samples were then either fermentation by lactic acid bacteria or acidified using the acidulant (GDL). Consistent with the pre-treatment experiments, the sulforaphane value of broccoli significantly increased (p<0.05) after the heat treatment; with 806.2±7.0 μmol/kg DW and 3536.0±136.9 μmol/kg DW of sulforaphane yield for raw and pre-heated broccoli, respectively. The value of 3536 μmol/kg DW obtained with this separate batch of broccoli preheated prior to fermentation is of the same order obtained when a different batch of broccoli was used, where 3983 μmol/kg DW was obtained indicating slight batch to batch variation.


As shown in Table 6, after the fermentation, the sulforaphane content of broccoli samples varied depending on the treatment of the broccoli prior to fermentation. The sulforaphane content of raw broccoli puree after fermentation (1617.4±10.2 μmol/kg DW) was approximately twice the sulforaphane content of raw broccoli puree. Pre-heating of broccoli prior to pureeing resulted in much higher increase in sulforaphane content after fermentation. The sulforaphane content of preheated-fermented broccoli (13121.3±440.8 μmol/kg DW) was about 8 times of the raw-fermented broccoli puree. The observed sulforaphane yield after the combined preheating-fermentation treatment is much higher than what would be expected based on the quantifiable amount of glucoraphanin (3423.7±39.7 μmol/kg) in the raw broccoli sample. It seems that the combined preheating and fermentation process enhances the release and accessibility of glucoraphanin for conversion over and above the inactivation of ESP by the pre-heating process. The pre-heating process coupled with microbial cell wall degrading enzymes may have enhanced the disruption of the cell compartment and release of bound glucosinolates in the matrix, that were not extractable or accessible in the raw broccoli. Some lactic acid strains produce polysaccharide degrading enzymes such as cellulases and pectinases capable of degrading the cell wall structure and enhance the release of wall bound components.


In contrast, chemical acidification of preheated broccoli puree by GDL resulted in a significantly lower (p<0.05) content of sulforaphane compared to pre-heated and preheat-fermented samples (Table 6). The sulforaphane content of the GDL acidified samples were 2169.4±176.0 μmol/kg DW, which is 40% lower than the preheated broccoli sample (3536.0±136.9 μmol/kg DW) (P<0.05). It appears that the fast reduction to pH 4.04 during acidification may have reduced the conversion of glucoraphanin into sulforaphane in the GDL samples. It is well known that the conversion of glucosinolates is highly dependent on pH and acidic pH favours conversion into nitriles (Latte et al., 2011).


In the case of the pre-heated fermented samples, the acidification occurs gradually over a period of >15 hr enabling the conversion of glucoraphanin mainly to sulforaphane since the activity of ESP is expected to be significantly reduced after preheating at 65° C. for 3 min.


Changes of Sulforaphane Content During Storage

The concentration of sulforaphane of all the samples declined after 14 days storage at 25° C. (see Table 6 and FIG. 6). Interestingly, an increase in sulforaphane content was observed in all samples except the fermented samples during 14 days storage at 4° C. The sulforaphane content of the raw puree almost doubled during storage at 4° C. Similarly, the sulforaphane content of the pre-heated samples increased by ˜2.6 times whereas the sulforaphane content of the preheated GDL samples increased by ˜2.3 times, which suggests continuous release of glucoraphanin from the matrix during storage allowing further conversion to sulforaphane and increase in concentration counteracting the consequence of sulforaphane degradation during storage. With respect to the preheated-fermented samples, reduction in sulforaphane content was observed during storage at both temperatures. All the accessible glucoraphanin may have been converted to sulforaphane during fermentation so much so that no further conversion occurred during storage but rather degradation albeit to a different extend depending on the temperature. As such, only a slight decline (˜6%) was observed during storage at 4° C. whereas the decline during storage at 25° C. was ˜70%.


This study showed that pre-heating coupled with lactic acid bacteria fermentation substantially enhances the sulforaphane content of broccoli based products. In-pack pre-heating treatment of broccoli florets at 65° C. for 3 min followed by maceration and fermentation resulted in as much as ˜16 times higher yield of sulforaphane compared to raw broccoli puree. Preheating under this condition increased the sulforaphane yield in broccoli puree from 806 μmol/KgDW (dry weight) in the untreated broccoli to 3536 μmol/KgDW, indicating that the treatment substantially inhibits ESP while maintaining sufficient myrosinase activity for the conversion of glucoraphanin into sulforaphane. The best preheating condition during direct water blanching was 1 min at 60° C. and resulted in sulforaphane yield of 2833 μmol/KgDW. The lower yield during direct blanching can be attributed to leaching of the water-soluble glucoraphanin into the blanching media. Preheating of broccoli florets in-pack (65° C./3 min) combined with lactic acid bacteria fermentation further enhanced the sulforaphane content to 13121 μmol/KgDW, which is ˜16 times increase compared to raw broccoli. Chemical acidification of in-pack preheated (65° C., 3 min) combined with acidification of the broccoli puree by glucono-delta-lactone resulted in sulforaphane yield of 2169 μmol/KgDW, which is lower than pre-heating alone. The sulforaphane content of the preheated-fermented puree remained stable (˜94% retention) during two weeks storage at 4° C.









TABLE 6







Sulforaphane yield (μmol/Kg DW) of broccoli before and after processing.









Sulforaphane (μmol/kg, DW)














Raw-
Preheatnot

Preheat-



Raw
Fermented
GDL
Preheat GDL
Fermented





Day 0
806.2 ± 7.0
1617.4 ± 10.2
 3536.0 ± 136.9
2169.4 ± 176.0
13121.3 ± 440.8


Days 14_4° C.
1409.8 ± 82.7
1627.7 ± 17.5
9149.4 ± 63.6
4994.8 ± 291.2
12301.3 ± 443.5


Days 14_25° C.
1268.2 ± 0.1 
1065.8 ± 49.8
3338.2 ± 93.9
2593.1 ± 97.7 
3974.2 ± 71.2





DW: dry weight, GDL: acidified using glucono-delta-lactone. Preheating was conducted at 65° C. in pack for 3 minutes.






Example 12—Effect of Lactic Acid Bacteria Fermentation on Polyphenolic Profile of Broccoli

In order to determine the effects of fermentation on the polyphenolic metabolites of broccoli samples, targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis of the raw and fermented broccoli puree samples was conducted. The resulting multivariate data was analysed using Metaboanalyst software (Metaboanalyst 3.0, Xia and Wishart, 2016). Fermentation resulted in a significant change in the metabolite profile of the broccoli samples. The partial least square discriminant analysis (PLS-DA) of the data shows a clear distinction between the polyphenolic profile of the fermented and the non-fermented samples (FIG. 8).


The top 15 metabolites that were identified to be responsible for the differences between the two groups are shown in FIG. 9. They are phenolic acids and phenolic aglycones, with higher bioactivity and bioavailability compared to their phenolic acid ester and phenolic glycoside precursors. The concentrations of most of these metabolites showed substantial increase following fermentation indicating the beneficial effect of fermentation on the polyphenol profile of broccoli puree. The fold changes for some of the metabolites are shown in Table 7.


A substantial increase in sinapic acid and kaempferol, 24 fold and 16 fold respectively was observed following fermentation. Similarly, fermentation induced an 8 fold increase in chlorogenic acid and phenyllactic acid. The concentrations of hesperetin, quercetin, methyl syringate and syringic acid also increased substantially after fermentation. The increase in the concentration of aglycones such as kaempferol, hesperetin and quercetin can be attributed to conversion of their glycoside precursors by the activity of microbial glycosidases. The increase in the concentration of phenolic acids such as sinapic acid could be due to the conversion of phenolic acid esters in broccoli by the activity of microbial esterases. Some decrease in caffeic acid and gallic was observed following fermentation. The activity of microbial decarboxylases convert caffeic acid into the corresponding vinyl catechol and gallic acid into pyrgallol, which may be responsible for the decrease in their concentration (Filanino et al., 2015; Guzman-Lopez et al., 2009).









TABLE 7







Fold changes in the top 13 polyphenols responsible for differences


between fermented and non-fermented broccoli puree.












Fold change




Compounds
(FC)
Log2(FC)













1
Sinapic acid
24.1
4.6


2
Kaempferol
16.1
4.0


3
Chlorogenic acid
8.3
3.1


4
Phenyllactic acid
7.9
3


5
Hespertin
3.7
1.9


6
Methyl syringate
3.3
1.7


7
Syringic acid
3.3
1.7


8
Caffeic acid
0.32
−1.6


9
Ferullic acid
2.7
1.4


10
4, hydroxybenzoic acid
0.4
−1.4


11
Quercetin
2.6
1.3


12
Rutin
2.5
1.3


13
Gallic acid
0.5
−1.1









Example 13—Identification of Metabolites Produced by Lactic Acid Bacteria Fermentation of Broccoli by Targeted and Untargeted LC MS Analyses of Samples

The fermented and non-fermented broccoli puree samples were frozen and freeze dried. The samples (100 mg freeze dried powder each) were extracted using 1 ml of ice-cold methanol and Milli-Q water (50:50, v: v), which comprised 100 mg/ml of caffeine as an internal standard. The samples were then vortexed for 2 minutes prior to being sonicated (40 Hz) for 30 minutes. Samples were then centrifuged at 20,000 rpm at 4° C. for 30 minutes, and the supernatant transferred to clean silanised LC-MS vials. Samples were analyzed by injecting 1.4 μl into an Agilent 6410 LC-QQQ HPLC (Agilent Technologies, Santa Clara, California, USA). The analyses were performed using a reversed-phase Agilent Zorbax Eclipse Plus C18, Rapid Resolution HD, 2.1×50 mm, 1.8 um (Agilent Technologies, Santa Clara, California, USA), with a column temperature of 30° C. and a flow rate of 0.3 ml/min. The mobile phase was operated isocratically for 1 min 95:5 (A: B) then switched to 1:99 (A: B) for a further 12 min before returning back to 95:5 (A: B) for an additional 2 min; providing a total run time of 15 min. Mobile phase ‘A’ consisted of 100% H2O and 0.1% formic acid, and mobile phase ‘B’ contained 75% acetonitrile, 25% isopropanol and 0.1% formic acid. The MS was collecting data in the mass range 50-1000 m/z. Qualitative identification of the compounds was performed according to the Metabolomics Standard Initiative (MSI) Chemical Analysis Workgroup using several online LC-MS metabolite databases, including Massbank and METLIN. Overall, the instrumental conditions were similar for both positive electrospray (+ESI) and negative electrospray (−ESI) modes. Scan time was 500, the source temperature was maintained at 350° C., the gas flow was 12 L/min and the nebuliser pressure was 35 psi.


For the identification of compounds in the untargeted analysis, the criteria was set at >90% match rate. Where the match rate dropped to between 70-89%, the compounds are identified with brackets (for example, if a compound was between 70-89% they are annotated as “<name>”). Any matches below 70% were removed. In total, there was ca. 1000-1500 fatures to identify; many were poorly matched (and removed) or were less than 10×S/N ratio from the baseline. As such, the compounds/peaks used were actual peaks and the IDs are fairly strong (i.e. >70%).


Untargeted LC-MS metabolomics study showed a 2 to 360 fold increase in certain polyphenolic glycosides including anthocyanin glycosides, phenolic acid glycosides, phenolic acids, a 5 to 60 fold increase in some glucosinolates with glucoraphanin increasing 27 fold and about a 3 to 4 fold increase in indol-3carbinol and ascorbigen. Results are summarised in Table 8 and are shown in FIG. 10 and in a volcano plot in FIG. 11. The top 50 metabolites that increased after fermentation include several polyphenol glycosides and glucosinolates indicating that the process enhances their extractability and bioaccessibility.









TABLE 8







Fold changes in different metabolites between fermented and non-


fermented broccoli puree based on untargeted LC-MS analysis.











Metabolite
FC
log2(FC)
raw. pval
(−LOG10(p))














Benzoic acid
4670.1
12.189
5.50E−08
7.2593


Cyanidin 3-O-rutinoside
361.03
8.496
0.011951
1.9226


Cyanidin 3-O-6″-p-coumaroyl-glucoside
271.87
8.0868
0.011465
1.9406


molybdopterin
149.51
7.2241
0.00915
2.0386


5-methylthiopentylglucosinolate
59.335
5.8908
0.005835
2.234


5-methylthioribulose 1-phosphate
46.001
5.5236
0.000334
3.4757


Ellagic acid arabinoside
42.956
5.4248
0.002845
2.546


thiamine phosphate
42.436
5.4072
0.005123
2.2905


2-carboxy-D-arabinitol 1-phosphate
41.06
5.3597
0.013093
1.883


N-acetyl-D-glucosamine 1,6-bisphosphate
40.636
5.3447
0.001824
2.739


S-norreticuline
32.883
5.0393
0.000362
3.4412


5-formamido-1-5-phospho-D-ribosyl-
30.585
4.9348
8.28E−06
5.0817


imidazole-4-carboxamide






4-methylumbelliferone 6′-O-
30.436
4.9277
0.001329
2.8765


malonylglucoside






Hydroxytyrosol 4-O-glucoside
28.971
4.8565
0.001319
2.8798


glucoraphanin
27.475
4.7801
0.014685
1.8331


glucobrassicin
26.746
4.7413
0.00441
2.3556


5-hydroxy-CMP
25.864
4.6929
0.004277
2.3689


4alpha-formyl,4beta,14alpha-dimethyl-
18.8
4.2326
0.003497
2.4563


9beta,19-cyclo-5alpha-ergost-24241-en-






3beta-ol






indole-3-acetyl-phenylalanine
17.44
4.1243
2.37E−06
5.6245


N-hydroxypentahomomethionine
16.92
4.0807
0.000559
3.2529


Cyanidin 3-O-arabinoside
16.098
4.0088
0.000413
3.3837


tetrahydrobiopterin
15.412
3.946
0.015746
1.8028


orotidine 5′-phosphate
14.737
3.8813
0.001699
2.7699


2-2′-methylthiopentylmaleate
14.621
3.87
0.005417
2.2662


S-adenosyl 3-methylthiopropylamine
14.564
3.8644
0.00177
2.752


4-methylthiobutyl glucosinolate
14.183
3.8261
0.011178
1.9516


salicylate
13.59
3.7644
0.000221
3.6556


N-hydroxyhomomethionine
12.902
3.6896
0.004311
2.3654


4′-phosphopantetheine
11.775
3.5576
0.003073
2.5124


5-phospho-beta-D-ribosylamine
10.643
3.4119
0.003185
2.497


D-erythro-imidazole-glycerol-phosphate
10.288
3.3629
0.019147
1.7179


a reduced flavodoxin
10.108
3.3374
0.005373
2.2698


Cyanidin 3-O-6″-dioxalyl-glucoside
9.9207
3.3104
0.000299
3.5242


8-oxo-GMP
9.8883
3.3057
0.008524
2.0694


3-dehydroteasterone
8.985
3.1675
8.33E−09
8.0793


indolylmethylisothiocyanate
7.7651
2.957
0.018337
1.7367


choline
7.7212
2.9488
0.023412
1.6306


carbamoyl phosphate
7.7098
2.9467
0.009139
2.0391


homogentisate
7.6608
2.9375
0.00153
2.8153


S-adenosyl-L-methionine
7.3817
2.8839
2.85E−05
4.5445


oxaloacetate
7.3494
2.8776
0.000538
3.2694


urate
7.2329
2.8546
0.000803
3.0951


coniferaldehyde glucoside
7.1826
2.8445
0.016973
1.7702


pyridoxal 5′-phosphate
7.0734
2.8224
0.021829
1.661


dTMP
6.9501
2.797
0.018743
1.7272


2-oxoglutarate
6.8749
2.7813
0.00019
3.7216


coniferaldehyde
6.6643
2.7365
1.46E−05
4.8345


Petunidin 3-O-rhamnoside
6.0484
2.5965
0.002487
2.6043


6-phospho D-glucono-1,5-lactone
5.8171
2.5403
0.019384
1.7126


dTDP
5.6526
2.4989
0.000837
3.0774


propane-1,3-diamine
5.5793
2.4801
0.001873
2.7275


benzoate
5.4402
2.4437
0.005218
2.2825


xi-progoitrin
5.091
2.3479
0.000107
3.9715


2-phospho-D-glycerate
5.0613
2.3395
0.001146
2.941


R-4′-phosphopantothenoyl-L-cysteine
4.8855
2.2885
0.01357
1.8674


L-arogenate
4.782
2.2576
0.018843
1.7248


L-phenylalanine
4.5585
2.1886
0.000213
3.671


Phenol
4.4651
2.1587
0.002537
2.5956


Gardenin B
4.3888
2.1338
0.012372
1.9076


glucomalcommin
4.1855
2.0654
0.014526
1.8378


Sulfachloropyridazine
4.1627
2.0575
0.013676
1.864


4-methyl-2-oxopentanoate
3.906
1.9657
0.004372
2.3593


ascorbigen
3.7819
1.9191
0.017398
1.7595


2-naphthol
3.6366
1.8626
0.01404
1.8526


Medioresinol
3.6131
1.8532
0.007717
2.1125


E-2-pentenol
3.5473
1.8267
0.012466
1.9043


N-feruloyltyramine
3.3648
1.7505
0.004573
2.3399


2-methyl-6-phytyl-1,4-benzoquinol
3.3442
1.7417
0.000245
3.6101


pyridoxal
3.0278
1.5983
0.00016
3.7954


1D-myo-inositol 1-monophosphate
2.784
1.4771
0.005472
2.2618


N-monomethylethanolamine
2.7546
1.4618
1.55E−05
4.8092


3,4-Dicaffeoylquinic acid
2.7368
1.4525
0.012553
1.9013


Cirsilineol
2.6151
1.3868
0.001515
2.8197


S-methylmalonate-semialdehyde
2.5477
1.3492
0.012237
1.9123


benzaldehyde
2.5268
1.3373
0.01558
1.8074


Unidentified metabolite No. 1
2.3799
1.2509
7.84E−05
4.1056


Isorhamnetin
2.2605
1.1766
0.001828
2.738


AMP
2.1939
1.1335
0.002464
2.6083


2-Hydroxybenzoic acid
2.1338
1.0935
0.006072
2.2167


butan-1-al
2.0853
1.0602
3.16E−07
6.5005


7-Hydroxymatairesinol
2.0626
1.0445
0.008034
2.095


Dimethylmatairesinol
0.43475
−1.2018
0.000284
3.5464


trans-zeatin
0.39207
−1.3508
0.008484
2.0714


Unidentified metabolite No. 2
0.38059
−1.3937
0.000721
3.1421


coniferyl alcohol
0.37824
−1.4026
0.011806
1.9279


papaverine
0.36651
−1.4481
0.012288
1.9105


2,5-diamino-6-5-phospho-D-
0.3594
−1.4763
0.020453
1.6893


ribosylaminopyrimidin-43H-one






S-4-hydroxymandelonitrile
0.32867
−1.6053
0.00375
2.426


22alpha-hydroxy-campest-4-en-3-one
0.32674
−1.6138
0.004969
2.3037


3-cyano-L-alanine
0.32471
−1.6228
0.013212
1.879


Ellagic acid glucoside
0.32466
−1.623
0.022951
1.6392


2-naphthol 6′-O-malonylglucoside
0.30641
−1.7064
0.000709
3.1492


pelargonidin
0.30629
−1.707
0.010379
1.9838


2S-naringenin
0.30353
−1.7201
0.019827
1.7027


8-methylthiooctyl-thiohydroximate
0.28257
−1.8233
0.002811
2.5512


Stigmastanol ferulate
0.28168
−1.8279
0.017703
1.752


Pinosylvin
0.26912
−1.8937
0.01535
1.8139


germacra-110,4,1113-trien-12-ol
0.23506
−2.0889
0.022511
1.6476


indole-3-acetyl-glutamine
0.20278
−2.302
0.006425
2.1921


2-7′-methylthioheptylmalate
0.19682
−2.3451
0.001077
2.968


p-coumaroyltriacetic acid lactone
0.18436
−2.4394
0.0122
1.9136


6″-O-Acetyldaidzin
0.15801
−2.6619
0.008935
2.0489


indole-3-acetyl-glutamate
0.15472
−2.6922
0.003623
2.441


Isorhamnetin 3-O-glucoside 7-O-
0.15357
−2.703
0.002647
2.5773


rhamnoside






olivetol
0.13094
−2.933
0.005902
2.229


N-hydroxy-L-phenylalanine
0.1141
−3.1316
0.000812
3.0905


R-pantothenate
0.10725
−3.221
1.36E−05
4.8679


glucoiberverin
0.087316
−3.5176
0.00014
3.8538


6-O-methylnorlaudanosoline
0.055734
−4.1653
6.96E−05
4.1575


carlactone
0.052932
−4.2397
2.93E−05
4.5332


E,E-geranyllinalool
0.018254
−5.7757
0.004044
2.3932


UDP-alpha-D-xylose
13.367
3.7407
0.0235
1.6289


Z-1-glutathione-S-yl-2-phenyl-
19.906
4.3151
0.026163
1.5823


acetohydroximate






Apigenin 7-O-6″-malonyl-apiosyl-
0.38092
−1.3925
0.02641
1.5782


glucoside






4alpha-formyl-stigmasta-7,24241-dien-
58.691
5.8751
0.026582
1.5754


3beta-ol






soyasapogenol B
0.35836
−1.4805
0.027448
1.5615


dihydroconiferyl alcohol glucoside
5.6248
2.4918
0.027644
1.5584


3-deoxy-alpha-D-manno-octulosonate
6.6012
2.7227
0.027652
1.5583


Anhydro-secoisolariciresinol
2.3975
1.2616
0.027928
1.554


3-isopropyl-7-methylthio-2-oxoheptanoate
0.30287
−1.7232
0.028072
1.5517


Kaempferide
0.15749
−2.6666
0.0281
1.5513


2-aminoprop-2-enoate
2.0003
1.0002
0.029166
1.5351


isoliquiritigenin
2.8505
1.5112
0.029212
1.5344


m-Coumaric acid
2.187
1.129
0.029331
1.5327


indole-5,6-quinone
2.6937
1.4296
0.02956
1.5293


2-4′-methylthiobutylmalate
0.43617
−1.197
0.030711
1.5127


7-methylthioheptyl glucosinolate
0.42422
−1.2371
0.030739
1.5123


camalexin
0.27584
−1.8581
0.030778
1.5118


3-Methoxynobiletin
8.9717
3.1654
0.031528
1.5013


8-methylsulfinyloctyl glucosinolate
0.1694
−2.5615
0.031733
1.4985


ent-cassa-12,15-diene
0.33285
−1.587
0.032806
1.484


Catechol
4.0005
2.0002
0.033382
1.4765


L-aspartate-semialdehyde
2.9298
1.5508
0.033499
1.475


10-methylthio-2-oxodecanoate
4.5655
2.1908
0.033543
1.4744


indole-3-carbinonium ion
2.7807
1.4754
0.033654
1.473


laurate
0.33955
−1.5583
0.034205
1.4659


malonate
9.0975
3.1855
0.035699
1.4473


1-aci-nitro-8-methylsulfanyloctane
8.8356
3.1433
0.035865
1.4453


2-hydroxy-5-methylthio-3-oxopent-1-enyl
13.56
3.7612
0.036727
1.435


1-phosphate






glyoxylate
16.835
4.0734
0.037951
1.4208


Feruloyl tartaric acid
5.5489
2.4722
0.038578
1.4137


3beta-hydroxyparthenolide
8.1691
3.0302
0.038749
1.4117


22R,23R-22,23-dihydroxycampesterol
2.0564
1.0401
0.039305
1.4056


Gallic acid 4-O-glucoside
2.515
1.3306
0.039605
1.4023


E-phenylacetaldoxime
2.1608
1.1116
0.040641
1.391


18-hydroxystearate
0.14519
−2.784
0.042027
1.3765


5′-phosphoribosyl-4-N-
0.4281
−1.224
0.042243
1.3742


succinocarboxamide-5-aminoimidazole






3-Feruloylquinic acid
3.3496
1.744
0.042655
1.37


2-carboxy-L-threo-pentonate
2.0447
1.0319
0.043
1.3665


trans-zeatin riboside
0.40453
−1.3057
0.044527
1.3514


4-fumaryl-acetoacetate
5.0298
2.3305
0.044744
1.3493


2-cis-abscisate
76.81
6.2632
0.044918
1.3476


4-Hydroxycoumarin
0.48212
−1.0525
0.045785
1.3393


Biochanin A
2.1017
1.0716
0.046533
1.3322


S-2,3,4,5-tetrahydrodipicolinate
4.1401
2.0497
0.046976
1.3281


26,27-dehydrozymosterol
14.846
3.892
0.047042
1.3275


N-methylethanolamine phosphate
10.038
3.3273
0.047416
1.3241


Kaempferol 3-O-2″-rhamnosyl-galactoside
2.7008
1.4334
0.048201
1.3169


7-O-rhamnoside






pheophorbide a
6.3398
2.6644
0.049365
1.3066


Chrysoeriol 7-O-6″-malonyl-glucoside
4.8949
2.2913
0.049727
1.3034


allantoate
10.972
3.4557
0.050008
1.301


Ligstroside-aglycone
12.072
3.5936
0.052404
1.2806


cycloeucalenone
3.4926
1.8043
0.052645
1.2786


Unidentified metabolite No. 3
3.5807
1.8403
0.053727
1.2698


laricitrin
0.42811
−1.224
0.05399
1.2677


Sulfadimethoxine
11.488
3.5221
0.05455
1.2632


3,4-Diferuloylquinic acid
5.2839
2.4016
0.054583
1.2629


glucotropeolin
0.47952
−1.0603
0.054637
1.2625


5,6-dihydroxyindole-2-carboxylate
5.2663
2.3968
0.055218
1.2579


S-laudanine
2.8697
1.5209
0.055638
1.2546


L-nicotianamine
0.39854
−1.3272
0.057257
1.2422


5-methylthiopentyl-thiohydroximate
0.30202
−1.7273
0.057551
1.2399


aldehydo-D-galacturonate
2.6643
1.4138
0.05785
1.2377


R-mevalonate 5-phosphate
0.34888
−1.5192
0.058188
1.2352


6-Hydroxyluteolin 7-O-rhamnoside
2.142
1.099
0.05845
1.2332


L-aspartate
3.5705
1.8361
0.061441
1.2115


--Epicatechin 3-O-gallate
2.4481
1.2916
0.063269
1.1988


glycine
0.23586
−2.084
0.065585
1.1832


Episesaminol
2.4077
1.2677
0.065876
1.1813


6alpha-hydroxy-castasterone
3.7782
1.9177
0.068376
1.1651


alpha-D-galacturonate 1-phosphate
11.846
3.5664
0.070966
1.149


R-2,3-dihydroxy-3-methylpentanoate
2.995
1.5825
0.071057
1.1484


cyanidin-3-O-beta-D-glucoside
2.0686
1.0487
0.07128
1.147


D-erythrose 4-phosphate
3.7463
1.9054
0.07247
1.1398


CDP-choline
617.84
9.2711
0.073728
1.1324


adenine
2.0623
1.0442
0.074004
1.1307


raphanusamate
5.5593
2.4749
0.074387
1.1285


3-Methoxysinensetin
2.4046
1.2658
0.075102
1.1243


betaine aldehyde
3.5234
1.817
0.075291
1.1233


E-7-methylthioheptanaldoxime
2.2972
1.1999
0.076906
1.114


6-methylthiohexyl-thiohydroximate
5.5473
2.4718
0.077579
1.1103


6″-O-Malonylglycitin
0.16741
−2.5786
0.080677
1.0933


monodehydroascorbate radical
2.0677
1.048
0.081844
1.087


anthranilate
3.0289
1.5988
0.082088
1.0857


Hydroxycaffeic acid
0.43234
−1.2098
0.082209
1.0851


Myricetin 3-O-arabinoside
2.3978
1.2617
0.086518
1.0629


cis-aconitate
0.18331
−2.4477
0.088998
1.0506


5-phospho-alpha-D-ribose 1-diphosphate
0.47829
−1.064
0.089065
1.0503


Malvidin 3-O-glucoside
0.48171
−1.0538
0.089472
1.0483


N6-delta2-isopentenyl-adenosine 5′-
44.241
5.4673
0.092566
1.0335


monophosphate






Quercetin 3-O-6″-acetyl-galactoside 7-O-
2.9914
1.5808
0.093824
1.0277


rhamnoside






cholesterol
2.816
1.4936
0.095163
1.0215


9-methylthiononyl-thiohydroximate
15.416
3.9464
0.098598
1.0061









In order to determine the effects of fermentation on the polyphenolic metabolites of broccoli samples, targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis of the raw and fermented broccoli puree samples was conducted. Statistical analysis was performed without preprocessing. Fermentation resulted in a significant change in the metabolite profile of the broccoli samples.


In the targeted LC-MS analysis, polyphenol standards were used for the identification and quantification of the metabolites. Increases in chlorogenic acid, ferullic acid, syringic acid, phenyllactic acid, rutin, sinapic acid, methyl syringate, hesperetin, quercetin and kaempferol were confirmed in fermented broccoli (FIG. 12). Decreases in protocatechuic acid, gallic acid, 4,hydroxybenzoic acid, vanillic acid, 2,3dihydroxybenzoic acid, p-cuomaric acid, cinnamic acid, catechin, rosmarinic acid, caffeic acid were confirmed in fermented broccoli (FIG. 12). Of note is that a 6.6 fold change in chlorogenic acid (2.4 to 15.8 μg/mg), a 23.8 fold increase is in sinapic acid (3.6 to 86.6 μg/mg), a 10.5 increase in kaempferol (12.7 to 134.6 μg/mg) and a 0.48 fold decrease in p-Coumaric acid occurred in fermented samples (FIG. 12).


Example 14—Assessment of the Broccoli Fermentation Culture to Inhibit the Growth of Intentionally Introduced Microorganisms

A challenge study was conducted to assess the ability of the broccoli fermentation culture to inhibit the growth of intentionally introduced microorganisms which are often observed and of concern in food preparation.


Lab Culture Starter Culture

10 ml of 1010 cfu/mL of an inoculum comprising B1, B2, B3, B4, B5, BF1 and BF2 to achieve 108 CFU/gm of sample in the ferment.


Pathogen Cultures


E. coli isolates FSAW 1310, FSAW 1311, FSAW 1312, FSAW 1313 and FSAW 1314 were grown separately to 1-4×108 cfu/mL in NB (nutrient broth) overnight at 37° C., static. The cultures were combined (1 mL of each) and the combined culture diluted to 104 with MRD (maximum recovery diluent) for first two dilutions and water for last two dilutions.



Salmonella strains S. infantis 1023, S. Singapore 1234, S. typhimurium 1657 (PT135), S. typhimurium 1013 (PT9) and S. Virchow 1563 were grown separately to 1-4×108 cfu/mL in NB overnight at 37° C., static. The cultures were combined (1 mL of each) and combined culture diluted to 104 with MRD for first two dilutions and water for last two dilutions.



Listeria isolates Lm2987 (7497), Lm2965 (7475), Lm2939 (7449), Lm2994 (7537) and Lm2619 (7514) were grown separately in 10 mL BHI (brain heart infusion broth) overnight at 37° C. under agitation. All cultures were then combined (1 mL of each) and this cocktail was diluted using MRD for first two (1/10) dilutions and sterile deionised water for last two dilutions.



B. cerus spore crops were prepared from isolates B3078, B2603, 2601, 7571 and 7626.


Method

Broccoli puree was prepared prior to preparing the inoculums, Broccoli: Sterile Tap Water 3:2 (900 g broccoli: 600 g water). Broccoli heads were rinsed in tap water, the stalks were cut off the broccoli with a sterile knife on a cutting board sanitised with 80% ethanol. Broccoli florets (900 g) were cut into small pieces. 450 g of broccoli pieces were placed into Thermomix bowl with all 600 g of the water. The translucent Thermomix cup/lid was sanitised with 80% ethanol and placed over the lid hole. The broccoli was chopped at speed 4 for 1 min. The second 450 g of broccoli pieces were added to the Thermomix bowl and chopped at speed 4 for 1 min. The contents were chopped for a further 5 min at speed 10 (max). After making sure the puree was indeed smooth enough, the Thermomix bowl was placed in the cool room to cool down the contents for 30 min. Following this, the bowl was put in the incubator and equilibrated to 30° C. Meanwhile the starter culture and pathogen culture (E. coli, B. cereus, Salmonella, Listeria monocytogenes) were prepared. 10 mL of LAB culture and 7.5 mL of the 10-4-diluted challenge microorganism cocktail (104 cfu/mL culture in water) were added into the broccoli puree (105 of B. cereus). Foil was held down over the large hole in the Thermomix lid prior to mixing culture. The cultures were mixed into the puree for 1 min on maximum speed. The heat setting for the Thermomix was switched off and the Thermomix was placed inside the 30° C. incubator and the fermentation started at 10:45 am. pH and temperature measurements were taken every hour up until 7 h (end of work time) after mixing the puree for 1 min speed 4.5. The pH meter was calibrated and sanitised using 80% ethanol. The temperature probe was also sanitised prior to measurements with 80% ethanol.


The growth of the challenge microorganisms was assessed by counts on growth on the selective media MRS, DRBX and NA+S of raw broccoli, before fermentation (TO) and after fermentation commenced at 4 hours (T4) and 22 hours (T22).


Results

The yeast and mould were significantly reduced by 4 hours, and were not detected at the end of fermentation (T22). E. coli and Salmonella were never detected at the end of fermentation (T22). Listeria was detected in low numbers at the end of fermentation, with a starting inoculum just over 103 cfu/mL. B. cereus spores were generally not affected by the fermentation, but did not germinate. The result of the challenge study indicates that the lactic acid bacteria strains that we isolated from broccoli are able to completely inactivate Salmonella and E. coli and inhibit the growth of the most acid resistant strains of Listeria. They are also able to inhibit the sporulation of B. cerus spores.









TABLE 9







Example of microbial challenge study with E. coli. E. coli


(mix of 5 E. coli strains EC1605, EC1606, EC1607, EC1608 inoculated


(2.2 × 102 CFU/gm) into the macerated broccoli (3:2 broccoli-


water ratio) ferment to evaluate if the fermentation starter (a consortia


of B1, B2, B3, B4, B5, BF1, BF2) inhibits the growth of E.coli.


Experiments were repeated three times. Fermentation was conducted


at 30° C. for 22 hrs to pH below 4.0.












Time
Lactic acid
Yeast and mould

E. coli




(hrs)
bacteria (CFU/gm)
(CFU/gm)
(CFU/gm)















0
1.6 × 108
2.4 × 103
1.6 × 102



4
1.5 × 108

3 × 10

1.2 × 102



22
3.6 × 109
<10
<1
















TABLE 10







Example of microbial challenge study with Salmonella. Salmonella


(A mix of 5 strains S. Infantis 1023, S. Singapore 1234, S. Typhimurium


1657 (PT135), S. Typhimurium 1013 (PT9), S. Virchow 1623) inoculated


(1.1 × 103) into macerated broccoli (3:2 broccoli-water ratio)


ferment to evaluate if the fermentation starter (a consortia of B1,


B2, B3, B4, B5, BF1, BF2) inhibits the growth of Salmonella.


Experiments were repeated three times. Fermentation was conducted


at 30° C. for 22 hrs to pH below 4.0.










Time
Lactic acid
Yeast and mould

Salmonella



(hrs)
bacteria (CFU/gm)
(CFU/gm)
(CFU/gm)













0
3.5 × 108
1.4 × 103
6.4 × 102


4
4.2 × 108

2 × 10

3.3 × 102


22
1.4 × 109
<10
<10
















TABLE 11







Example of microbial challenge study with Listeria monocytogenes.



Listeria monocytogenes (A mix of 5 strains Lm2987 (7497),



Lm2965 (7475), Lm2939 (7449), Lm2994 (7537), Lm2919 (7514))


inoculated (1.9 × 103) into macerated broccoli (3:2 broccoli-


water ratio) ferment to evaluate if the fermentation starter (a consortia


of B1, B2, B3, B4, B5, BF1, BF2) inhibits the growth of acid resistant



Listeria. Experiments were repeated three times and the final




Listeria count at the end of fermentation ranged from <10



(undetected) to 1.1 × 102 CFU/gm. Fermentation was conducted


at 30° C. for 22 hrs to pH below 4.0.












Time
Lactic acid
Yeast and mould

Listeria




(hrs)
bacteria (CFU/gm)
(CFU/gm)
(CFU/gm)















0
5.6 × 108
5.2 × 104
2.1 × 103



4
4.1 × 108
3.6 × 103
2.8 × 103



22
5.1 × 109
<10

2 × 10

















TABLE 12







Example of microbial challenge study with Bacillus cereus.



Bacillus cereus (A mix of 5 strains B3078, B2603, B2601, B7571,



B7626) inoculated (1.9 × 103) into macerated broccoli (3:2


broccoli-water ratio) ferment to evaluate if the fermentation starter


(a consortia of B1, B2, B3, B4, B5, BF1, BF2) inhibits the growth of


acid resistant Listeria. Experiments were repeated three times.


Fermentation was conducted at 30° C. for 22 hrs to pH below 4.0.












Time
Lactic acid
Yeast and mould

Listeria




(hrs)
bacteria (CFU/gm)
(CFU/gm)
(CFU/gm)















0
2.4 × 108
1.2 × 103
3.1 × 103



4
3.3 × 108
9.5 × 10
2.3 × 103



22
1.9 × 109
<10
1.7 × 103









Example 15—Pulse Filed Gel Electrophoreses of Leuconostoc mesenteroides Isolates


Leuconostoc mesenteroides from vegetables was assessed with SmaI and NotI restriction enzyme digestion with pulse filed gel electrophoreses as described in Chat and Dalmasso (2015) with modification.


Methods:
Day 1

Assessed isolates were inoculated into 10 mL MRS broth and incubated overnight at 30° C. in incubator (16 h).


Day 2

Isolates were centrifuge at 3500 g for 10 min and the supernatant discarded. The pellet was mixed and washed with 5 mL deionised water and centrifuged at 3500 g for 10 min and the supernatant discarded. The pellet was mixed with 5 mL TES (1 mM EDTA, 10 mM Tris-HCl, 0.5 M saccharose) and vortexed. Next the samples were centrifuged at 3500 g for 15 min and the supernatant discarded. 700 μL of Lysis solution (TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0, sterilise as normal) with lysozyme at 10 mg/mL) was added to the pellet and mixed and incubated at 56° C. for 2 h to lyse bacteria. Next, 700 μL of agarose (1% SeaChem Gold agarose with 50 μL EDTA/100 mL) was added to the cell mixture, mix and dispensed into plug moulds and 2 mL of deproteinisation (660 μL of proteinase K buffer, 11 μL proteinase K) solution added all plugs for one sample placed in the tube and incubated at 55° C. overnight.


Day 3

Next the plugs were heated in 100 mL of sterile deionised water at 55° C., the deproteinisation solution was removed and the plugs transferred to 15 mL centrifuge tubes, washed with 4 mL of sterile deionised water and heated to 55° C. for 10 min at room temperature followed by washing four times with 4 mL TE buffer for 10 min at room temperature.


Restriction Digests

2 mm slice off plug was placed in an eppendorf tube with 100 μL 1× restriction buffer, incubated for 20 min at room temperature, restriction buffer was removed and replaced with 40-100 μL of SmaI (20 U) or NotI in restriction buffer and incubated for 4 h at the optimum temperature (25° C.).


Day 4
Separation of Restriction Fragments

1 mL 0.5×TBE buffer to each tube and allowed to sit for at least 15 min to stop reaction and the bacteriophage A DNA ladder (New England Biolab) was incubated in TBE buffer. The buffer was removed and the slices loaded onto comb, with the ladder in every five lanes. 1.0% ultra-pure DNA grade agarose (pulsed field certified agarose) was prepared in 0.5×TBE running buffer.


Electrophoresis Conditions

Buffer maintained at 14° C. (model 1000 Mini-chiller, BioRad).BioRad “Chef Mapper™”, select Two State Program (not Auto Algorithm). Pulse time ramped linearly (press enter when “a” appears) from 2 to 25 s. Gradient 6 V/cm (voltage), Included angle 120°, Running time of 24 h.


Day 5

Gels stained ˜30 min in GelRed, destained, visualised


Results

The restriction fingerprint for BF1 was district but similar to Leuconostoc mesenteroides isolated from carrot (FIG. 13). The restriction fingerprint for BF2 was district from all Leuconostoc mesenteroides strains assessed (FIG. 13).


Example 16—Variant Analysis of Leuconostoc mesenteroides and Lactobacillus plantarum Isolates

For the SNP analysis of the Lactobacillus plantarum isolates (B1 to B5), B1 Prokka gbk was used as reference for Snippy SNP analysis-standard method. Single comparisons were performed using read data for each strain. B1 reads were ran as a control.


Example command was:

    • snippy --cpus 24 --outdir B5 --ref B1_S1mod.gbk --pe1
    • B5_S17_L001_R1_001.fastq.gz --pe2 B5_S17_L001_R2_001.fastq.gz


Calculated individual comparisons and core using B1 gbk as reference snippy-core --prefix core B1 B2 B3 B4 B5


Comparisons were also performed between B1 and the reference strain read data downloaded from the SRA for Lactobacillus plantarum ATCC 8014 (SRR1552613). Downloading was performed using standard method with prefetch and conversion to fastq using—sratoolkit.2.9.2-win64. Similar approaches were used for comparison of the Leuconostoc mesenteroides isolates BF1 and BF2 with Leuconostoc mesenteroides ATCC 8293 as reference.


Results

Variants (41) were observed between B1 and ATCC 8014 (Table 13). Variants (1 to 4) were observed between B1 and the other B isolates B2, B3, B4 and B5 (Table 14 to 17). BF1 and BF2 are very different from one another. Variants (19) were observed between BF1 and ATCC 8293 (Table 18). Variants (˜7000) were observed between BF2 and ATCC 8293. 459 complex variants were identified between BF2 and ATCC8293 which are summarized in Table 19.









TABLE 13







Polymorphisms identified by variant analysis B1 compared to ATCC8014.


















POS
TYPE
REF
ALT
EVIDENCE
FTYPE
STRAND
NT_POS
AA_POS
EFFECT
LOCUS_TAG
GENE





















292863
complex
GTCG
ATCT
ATCT: 96
CDS
+
292/
98/
missense_variant
JBMIHLAL_00290
ohrR_1






GTCG: 0


477
158
c.292_295delGTCGinsATCT













p.ValAla98IleSer







21413
snp
C
T
T: 204













C: 1












49138
snp
T
G
G: 226
CDS
+
771/
257/
missense_variant
JBMIHLAL_00337
lacR_1






T: 2


1011
336
c.771T>G p.Asn257Lys







68529
del
TATTAATGGCT
TA
TA: 97











CGCGTCATTAA

TATTAATGGCTCG













CGTCATTAA: 0












70435
snp
G
A
A: 199
CDS

95/
32/
missense_variant
JBMIHLAL_00352
lacS_2






G: 1


1959
652
c.95C>T p.Thr32Ile







70584
snp
T
C
C: 154













T: 1












71677
snp
T
C
C: 201
CDS

209/
70/
missense_variant
JBMIHLAL_00353







T: 0


1029
342
c.209A>G p.Tyr70Cys







72030
del
CGCTCAACCAG
CG
CG: 91
CDS

978/
320/
inframe_deletion
JBMIHLAL_00354
lacR_3




ATTAGTACCCA

CGCTCAACCAGAT


996
331
c.958_978delCTGGGTACTAATCTGGTT






G

TAGTACCCAG: 0




GAG













p.Leu320_Glu326del







136221
snp
C
A
A: 178
CDS

559/
187/
missense_variant
JBMIHLAL_00407
gatC_1






C: 1


1272
423
c.5596>T p.Ala187Ser







15092
snp
C
A
A: 102













C: 1












153210
snp
G
T
T: 117
CDS

385/
129/
missense_variant
JBMIHLAL_00681
gabR






G: 1


1365
454
c.385C>A p.Gln129Lys







38124
snp
C
T
T: 264













C: 1












128067
snp
G
A
A: 261
CDS

208/
70/
missense_variant
JBMIHLAL_01118
yjjP_1






G: 1


1344
447
c.208C>T p.Arg70Cys







188850
snp
A
C
C: 241
CDS

491/
164/
missense_variant
JBMIHLAL_01179
oppA_2






A: 0


1617
538
c.491T>G p.Ile164Ser







2322
snp
A
G
G: 107
CDS

397/
133/
missense_variant
JBMIHLAL_01186
adcR






A: 1


474
157
c.397T>C p.Phe133Leu







111662
ins
CAA
CAAA
CAAA: 133
CDS
+
10/
4/
frameshift_variant
JBMIHLAL_01302
mntB






CAA: 11


876
291
c.9dupA p.Ser4fs







11376
snp
G
A
A: 115
CDS

1831/
611/
synonymous_variant
JBMIHLAL_01356







G: 0


1947
648
c.1831C>T p.Leu611Leu







115510
snp
G
A
A: 199
CDS

95/
32/
missense_variant
JBMIHLAL_01453







G: 1


411
136
c.95C>T p.Thr32Ile







143457
snp
G
C
C: 264
CDS
+
1122/
374/
synonymous_variant
JBMIHLAL_01479
pepD






G: 0


1416
471
c.1122G>C p.Val374Val







111973
snp
G
A
A: 118
CDS

731/
244/
missense_variant
JBMIHLAL_01603
murA1






G: 1


1317
438
c.731C>T p.Ala244Val







27553
snp
C
T
T: 104
CDS

472/
158/
missense_variant
JBMIHLAL_01677
wbnH






C: 1


1092
363
c.472G>A p.Gly158Ser







80888
snp
T
C
C: 84
CDS
+
256/
86/
stop_lost&splice_region_
JBMIHLAL_01727
ytlR_1






T: 0


258
85
variant













c.256T>C p.Ter86Glnext*?







133147
snp
A
C
C: 76
CDS

443/
148/
missense_variant
JBMIHLAL_01777
yjbM






A: 0


663
220
c.443T>G p.Phe148Cys







74711
snp
C
T
T: 212
CDS
+
874/
292/
missense_variant
JBM1HLAL_01855
murF_2






C: 1


1389
462
c.874C>T p.Leu292Phe







19793
snp
T
C
C: 114
CDS

925/
309/
missense_variant
JBMIHLAL_01907
sigA






T: 1


1107
368
c.925A>G p.Asn309Asp







60643
snp
C
T
T: 89
CDS

242/
81/
missense_variant
JBMIHLAL_01945
dnaK






C: 1


1869
622
c.242G>A p.Ser81Asn







10806
ins
GTTTTTTTTG
GTTTTTTTTTG
GTTTTTTTTTG: 













49













GTTTTTTTTG: 1












50276
complex
CG
CACCACCAGGC
CACCACCAGGCCG
CDS

341/
114/
missense_variant&inframe_
JBMIHLAL_02031
ribU





CGATTGTGGCG
ATTGTGGCGA: 


555
184
insertion







A
39




c.341delCinsTCGCCACAATCGGCCTGG








CG: 0 




TGGT













p.Ala114delinsValAlaThrIleGly













LeuValVal







50325
snp
A
C
C: 99
CDS

293/
98/
stop_gained
JBMIHLAL_02031
ribU






A: 1


555
184
c.293T>G p.Leu98*







64233
snp
A
G
G: 77
CDS

2516/
839/
missense_variant
JBMIHLAL_02043
clpB






A: 1


2604
867
c.2516T>C p.Val839Ala







79046
snp
G
C
C: 140
CDS
+
394/
132/
missense_variant
JBMIHLAL_02139
ygaZ_2






G: 1


765
254
c.394G>C p.Ala132Pro







14904
snp
G
A
A: 82
CDS

113/
38/
missense_variant
JBMIHLAL_02340







G: 0


876
291
c.113C>T p.Pro38Leu







45542
snp
T
G
G: 158
CDS

1312/
438/
missense_variant
JBMIHLAL_02365
pgcA






T: 0


1728
575
c.1312A>C p.Lys438Gln







21706
ins
TAT
TAAT
TAAT: 122
CDS
+
872/
291/
frameshift_variant
JBMIHLAL_02489
mprF






TAT: 1


2604
867
c.871dupA p.Ile291fs







29454
del
TGA
TA
TA: 73
CDS
+
94/
32/
frameshift_variant
JBMIHLAL_02559







TGA: 0


132
43
c.94delG p.Asp32fs







27619
snp
A
G
G: 134
CDS

78/
26/
synonymous_variant
JBMIHLAL_02812







A: 1


588
195
c.78T>C p.Gly26Gly







4360
snp
C
T
T: 96













C: 1












8851
del
CGG
CG
CG: 117
CDS

82/
28/
frameshift_variant
JBMIHLAL_02963
tcaR






CGG: 0


513
170
c.82delC p.Pro28fs







19068
del
CTTGCCGAAAT
CT
CT: 51
CDS
+
154/
52/
frameshift_variant
JBMIHLAL_02974





TCGACAAACAA

CTTGCCGAAATTC


564
187
c.154_185delGAAATTCGACAAACAACC






CCCTCGGATTG

GACAAACAACCCT




CTCGGATTGTTGCC






T

CGGATTGT: 0




p.Glu52fs







17533
ins
ATTTTTTG
ATTTTTTTG
ATTTTTTTG: 













220













ATTTTTTG: 2
















TABLE 14







Polymorphism identified by variant analysis B2 compared to B1.


















POS
TYPE
REF
ALT
EVIDENCE
FTYPE
STRAND
NT_POS
AA_POS
EFFECT
LOCUS_TAG
GENE




















8417
snp
C
T
T:105 C:0
CDS
+
105/264
35/87
synonymous_variant
JBMIHLAL_02984











c.105C > T p.Asp35Asp
















TABLE 15







Polymorphisms identified by variant analysis B3 compared to B1

























NT_
AA_





POS
TYPE
REF
ALT
EVIDENCE
FTYPE
STRAND
POS
POS
EFFECT
LOCUS_TAG
GENE





4326
del
TATAAAAAAAGC
TA
TA: 31











GACCCCCGTTCA

TATAAAAAAAGC











TTAACGGTGCCG

GACCCCCGTTCA











CTCACAGATCAT

TTAACGGTGCCG











TATTAGTGAAAA

CTCACAGATCAT











TCACCCGGCA

TATTAGTGAAAA













TCACCCGGCA: 













0












8417
snp
C
T
T: 135 C: 0
CDS
+
105/
35/
synonymous_
JBMIHLAL_










264
87
variant
02984












c.105C>T













p.Asp35Asp
















TABLE 16







Polymorphism identified by variant analysis B4 compared to B1.


















POS
TYPE
REF
ALT
EVIDENCE
FTYPE
STRAND
NT_POS
AA_POS
EFFECT
LOCUS_TAG
GENE




















8417
snp
C
T
T:93 C:0
CDS
+
105/264
35/87
synonymous_variant
JBMIHLAL_02984











c.105C > T p.Asp35Asp
















TABLE 17







Polymorphisms identified by variant analysis B5 compared to B1.


















POS
TYPE
REF
ALT
EVIDENCE
FTYPE
STRAND
NT_POS
AA_POS
EFFECT
LOCUS_TAG
GENE





















199035
snp
T
C
C:124
CDS
+
 368/1206
123/401
missense_variant
JBMIHLAL_00946







T:0




c.368T > C













p.Val123Ala




143457
snp
G
C
C:158
CDS
+
1122/1416
374/471
synonymous_variant
JBMIHLAL_01479
pepD






G:0




c.1122G > C













p.Val374Val




23797
snp
A
C
C:146
CDS
+
 71/666
 24/221
missense_variant
JBMIHLAL_02490
immR_1






A:0




c.71A > C













p.Gln24Pro




8417
snp
C
T
T:131
CDS
+
105/264
35/87
synonymous_variant
JBMIHLAL_02984







C:0




c.105C > T













p.Asp35Asp
















TABLE 18







Polymorphisms identified by variant analysis BF1 compared to ATCC8293.


















POS
TYPE
REF
ALT
EVIDENCE
FTYPE
STRAND
NT_POS
AA_POS
EFFECT
LOCUS_TAG
GENE





















197592
del
TGT
TT
TT:178













TGT:0









269841
del
TGG
TG
TG:305
CDS
+
 33/306
 11/101
frameshift_variant
LEUM_0316







TGG:0




c.33delG













p.Asn12fs




338699
snp
G
T
T:239
CDS
+
 764/1719
255/572
missense_variant
LEUM_0385







G:0




c.764G > T













p.Trp255Leu




410044
snp
C
A
A:210
CDS
+
2229/2457
743/818
synonymous_variant
LEUM_0448
pheT






C:0




c.2229C > A













p.Thr743Thr




558511
ins
CAT
CAAT
CAAT:140
CDS
+
204/261
68/86
frameshift_variant
LEUM_0587







CAT:0




c.203dupA













p.His68fs




559188
snp
A
G
G:169
CDS
+
601/981
201/326
missense_variant
LEUM_0588







A:0




c.601A > G













p.Ile201Val




615572
del
TCC
TC
TC:245













TCC:5









755527
snp
A
T
T:196
CDS
+
351/993
117/330
missense_variant
LEUM_0777







A:0




c.351A > T













p.Leu117Phe




796683
del
GCC
GC
GC:207
CDS
+
2986/3009
 996/1002
frameshift_variant
LEUM_0814







GCC:0




c.2986delC













p.Glu997fs




953160
snp
G
T
T:178
CDS
+
805/843
269/280
missense_variant
LEUM_0952







G:0




c.805G > T













p.Ala269Ser




1009293
snp
C
A
A:1652
CDS
+


no annotation
LEUM_1009







C:171









1094250
snp
T
A
A:188
CDS
+


no annotation
LEUM_1090







T:0









1236979
snp
G
T
T:194













G:1









1237016
del
CAA
CA
CA:183













CAA:6









1291050
del
CGT
CT
CT:177













CGT:0









1600218
del
AGG
AG
AG:168













AGG:2









1624087
ins
GA
GTA
GTA:205













GA:0









1693283
snp
T
A
A:247
CDS



no annotation
LEUM_1724







T:0









1993032
snp
G
A
A:209
CDS



no annotation
LEUM_2026







G:0
















TABLE 19







Polymorphisms identified by variant analysis BF2 compared to ATCC8293.

















POS
REF
ALT
EVIDENCE
FTYPE
STRAND
NT_POS
AA_POS
EFFECT
LOCUS_TAG
GENE




















1737
TTCA
ATCC
ATCC: 151
CDS
+
63/
21/
synonymous_variant 
LEUM_0002






TTCA: 0


1137
378
c.63_66delTTCAinsATCC












p.IleSer21IleSer







11810
CATG
TATA
TATA: 216
CDS
+
144/
48/
missense_variant 
LEUM_0010






CATG: 0


1626
541
c.144_147delCATGinsTATA












p.AsnMet48AsnIle







12635
ACGT
GCGC
GCGC: 255 
CDS
+
969/
323/
synonymous_variant 
LEUM_0010






ACGT: 0


1626
541
c.969_972delACGTinsGCGC












p.GlnArg323GlnArg







20351
TCT
GCG
GCG: 230 
CDS
+
172/
58/
missense_variant 
LEUM_0017






TCT: 0


795
264
c.172_174delTCTinsGCG 












p.Ser58Ala







22033
AGCTA
GGCTG
GGCTG: 214
CDS
+
1047/
349/
missense_variant
LEUM_0018






AGCTA: 0


1185
394
c.1047_1051delAGCTAinsGGCTG












p.GluAlaAsn349GluAlaAsp







36499
TATT
CATC
CATC: 289 
CDS
+
564/
188/
synonymous_variant 
LEUM_0044






TATT: 0


1062
353
c.564_567delTATTinsCATC












p.ArgIle188ArgIle







45902
GTAATGTGA
CCACATTAC
CCACATTAC: 251












GTAATGTGA: 0












47145
TAT
TTCAG
TTCAG: 241 












TAT: 0












64340
CTGT
TTGC
TTGC: 335 
CDS

205/
68/
missense_variant 
LEUM_0076






CTGT: 0


915
304
c.202_205delACAGinsGCAA












p.ThrAsp68AlaAsn







70144
GGTATGGGATGGGA
CGTATGGGA
CGTATGGGA: 233












GGTATGGGATGGGA: 0












75797
AGAG
GGAT
GGAT: 179 
CDS
+
51/
17/56
missense_variant 
LEUM_0091






AGAG: 0


171

c.51_54delAGAGinsGGAT












p.LeuGlu17LeuAsp







97951
TAAT
CAAG
CAAG: 197 
misc_
+


no annotation







TAAT: 0
bind-












ing











138065
GGCG
TGCA
TGCA: 279 
CDS

1002/
333/
synonymous_variant
LEUM_0153






GGCG: 0


1431
476
c.999_1002delCGCCinsTGCA 












p.ValAla333ValAla







138074
ATTG
GTTC
GTTC: 276 
CDS

993/
330/
synonymous_variant 
LEUM_0153






ATTG: 0


1431
476
c.990_993delCAATinsGAAC












p.ValAsn330ValAsn







138092
AACT
GACC
GACC: 278 
CDS

975/
324/
synonymous_variant 
LEUM_0153






AACT: 0


1431
476
c.972_975delAGTTinsGGTC












p.ProVal324ProVal







140746
GGGT
AGGC
AGGC: 196 
CDS
+
366/
122/
synonymous_variant
LEUM_0156






GGGT: 0


540
179
c.366_369delGGGTinsAGGC 












p.GluGly122GluGly







140797
CGCC
TGCT
TGCT: 208 
CDS
+
417/
139/
synonymous_variant 
LEUM_0156






CGCC: 0


540
179
c.417_420delCGCCinsTGCT












p.AspAla139AspAla







142611
GTT
CTG
CTG: 135 
CDS
+
271/
91/
missense_variant 
LEUM_0158






GTT: 0


375
124
c.271_273delGTTinsCTG 












p.Val91Leu







142687
CAAAAAG
CAAAAAAA
CAAAAAAA: 178
CDS
+
353/
118/
frameshift_variant&
LEUM_0158






CAAAAAG: 0


375
124
missense_variant












c.353delGinsAA p.Ser118fs







145324
CAG
AAA
AAA: 292 
CDS
+
505/
169/
missense_variant 
LEUM_0161
gltX





CAG: 0


1497
498
c.505_507delCAGinsAAA 












p.Gln169Lys







162834
TGAT
GGAC
GGAC: 260 
CDS
+
2400/
800/
missense_variant 
LEUM_0185






TGAT: 0


2481
826
c.2400_2403delTGATinsGGAC












p.AspAsp800GluAsp







192260
ATAAA
GTAAC
GTAAC: 301
CDS
+
433/
145/
missense_variant 
LEUM_0228
truA





ATAAA: 0


768
255
c.433_437delATAAAinsGTAAC












p.IleAsn145ValThr







196751
CTAT
ATAC
ATAC: 138 
CDS

55/
18/67
missense_variant 
LEUM_0234






CTAT: 0


204

c.52_55delATAGinsGTAT












p.IleAla18ValSer







196918
AATA
GATG
GATG: 246 












AATA: 0












216494
CACG
TACC
TACC: 230 
CDS
+
108/
36/
synonymous_variant 
LEUM_0256
nrdF





CACG: 0


978
325
c.108_111delCACGinsTACC












p.AspThr36AspThr







231792
ATCTC
GTCTT
GTCTT: 235 
CDS
+
553/
185/
missense_variant 
LEUM_0276






ATCTC: 0


1728
575
c.553_557delATCTCinsGTCTT












p.IleSer185ValLeu







231812
GCTC
ACTT
ACTT: 229 
CDS
+
573/
191/
synonymous_variant 
LEUM_0276






GCTC: 0


1728
575
c.573_576delGCTCinsACTT












p.AlaLeu191AlaLeu







234250
ACTT
CCTG
CCTG: 217 
CDS
+
336/
112/
synonymous_variant 
LEUM_0279
tmk





ACTT: 0


642
213
c.336_339delACTTinsCCTG












p.GlyLeu112GlyLeu







242029
CTAT
TTAC
TTAC: 265 
CDS

664/
221/
missense_variant 
LEUM_0287






CTAT: 0


966
321
c.661_664delATAGinsGTAA












p.IleAla221ValThr







244287
GACT
AACC
AACC: 251 
CDS
+
1436/
479/
missense_variant 
LEUM_0288






GACT: 0


1962
653
c.1436_1439delGACTinsAACC












p.ArgLeu479LysPro







250392
GGCG
AGCT
AGCT: 182 
CDS
+
345/
115/
synonymous_variant
LEUM_0295
proA





GGCG: 0


1242
413
c.345_348delGGCGinsAGCT 












p.ValAla115ValAla







271910
TTA
CTG
CTG: 297 
CDS
+
358/
120/
synonymous_variant 
LEUM_0318






TTA: 0


843
280
c.358_360delTTAinsCTG 












p.Leu120Leu







288308
ATA
AC
AC: 232 












ATA: 0












318676
GATTAG
AATCAA
AATCAA: 121
CDS
+
14/
5/101
missense_variant 
LEUM_0366






GATTAG: 0


306

c.14_19delGATTAGinsAATCAA












p.GlyLeuVal5GluSerIle







341498
GTTTTTTTTTA
GTTTTTTTTC
GTTTTTTTTC: 114












GTTTTTTTTTA: 0












359500
GCAAG
ACAAC
ACAAC: 238
CDS
+
3034/
1012/
missense_variant
LEUM_0399






GCAAG: 0


3540
1179
c.3034_3038delGCAAGinsACAAC












p.AlaSer1012ThrThr







366821
ACATC
GCATT
GCATT: 250 
CDS
+
957/
319/
synonymous_variant
LEUM_0406
lysS





ACATC: 0


1488
495
c.957_961delACATCinsGCATT












p.LysHisLeu319LysHisLeu







366884
AGAAGCA
GGATGCG
GGATGCG: 217
CDS
+
1020/
340/
missense_variant
LEUM_0406
lysS





AGAAGCA: 0


1488
495
c.1020_1026delAGAAGCAinsGGA












TGCG












p.GluGluAla340GluAspAla







366896
GTTGGCC
ATTAGCA
ATTAGCA: 225
CDS
+
1032/
344/
synonymous_variant
LEUM_0406
lysS





GTTGGCC: 0


1488
495
c.1032_1038delGTTGGCCinsATT












AGCA












p.LysLeuAla344LysLeuAla







366971
ATTTGTA
GTTCGTT
GTTCGTT: 225
CDS
+
1107/
369/
synonymous_variant
LEUM_0406
lysS





ATTTGTA: 0


1488
495
c.1107_1113delATTTGTAinsGTT












CGTT












p.GluPheVal369GluPheVal







371223
CTTC
ATTT
ATTT: 226 
CDS
+
273/
91/
synonymous_variant 
LEUM_0414






CTTC: 0


1449
482
c.273_276delCTTCinsATTT












p.GlyPhe91GlyPhe







395520
CTCT
ATCC
ATCC: 206 
CDS

525/
174/
missense_variant 
LEUM_0436






CTCT: 0


942
313
c.522_525delAGAGinsGGAT












p.IleGlu174MetAsp







395821
ACCA
GCCG
GCCG: 177 
CDS

224/
74/
missense_variant 
LEUM_0436






ACCA: 0


942
313
c.221_224delTGGTinsCGGC












p.MetVal74ThrAla







410847
CGGT
TGGC
TGGC: 232 
CDS
+
495/
165/
synonymous_variant 
LEUM_0449






CGGT: 0


1287
428
c.495_498delCGGTinsTGGC












p.ValGly165ValGly







420486
CGCAC
AGCAT
AGCAT: 187
CDS
+
200/
67/
missense_variant 
LEUM_0457






CGCAC: 0


609
202
c.200_204delCGCACinsAGCAT












p.AlaHis67GluHis







455735
GTG
CTT
CTT: 112 
CDS

1922/
640/
missense_variant 
LEUM_0497






GTG: 0


2088
695
c.1920_1922delCACinsAAG












p.AsnThr640LysSer







457087
GCCAT
ACCAC
ACCAC: 262
CDS

570/
189/
missense_variant 
LEUM_0497






GCCAT: 0


2088
695
c.566_570delATGGCinsGTGGT












p.AspGly189GlyGly







490235
GCG
ACA
ACA: 136 
CDS
+
142/
48/
missense_variant 
LEUM_0524






GCG: 0


738
245
c.142_144delGCGinsACA 












p.Ala48Thr







493487
TGGT
CGGC
CGGC: 189 
CDS
+
168/
56/
synonymous_variant 
LEUM_0527






TGGT: 0


834
277
c.168_171delTGGTinsCGGC












p.ArgGly56ArgGly







500830
GCT
ACC
ACC: 176 
CDS
+
352/
118/
missense_variant 
LEUM_0536






GCT: 0


2031
676
c.352_354delGCTinsACC 












p.Ala118Thr







502254
CGAA
TGAG
TGAG: 214 
CDS
+
1776/
592/
synonymous_variant
LEUM_0536






CGAA: 0


2031
676
c.1776_1779delCGAAinsTGAG












p.ValGlu592ValGlu







502272
CATTC
TCTCT
TCTCT: 187 
CDS
+
1794/
598/
missense_variant
LEUM_0536






CATTC: 0


2031
676
c.1794_1798delCATTCinsTCTCT












p.PheIleLeu598PheLeuLeu







502291
TTG
CTA
CTA: 215 
CDS
+
1813/
605/
synonymous_variant 
LEUM_0536






TTG: 0


2031
676
c.1813_1815delTTGinsCTA 












p.Leu605Leu







505441
AGG
GGA
GGA: 156 
CDS
+
826/
276/
missense_variant 
LEUM_0540






AGG: 4


834
277
c.826_828delAGGinsGGA 












p.Arg276Gly







507015
ACCAC
GCCAA
GCCAA: 199
CDS

507/
168/
missense_variant 
LEUM_0543






ACCAC: 0


1098
365
c.503_507delGTGGTinsTTGGC












p.SerGly168IleGly







508582
TGCT
CGCG
CGCG: 163 
CDS
+
861/
287/
synonymous_variant 
LEUM_0544






TGCT: 0


1008
335
c.861_864delTGCTinsCGCG












p.ProAla287ProAla







509588
TTG
CTA
CTA: 171 
CDS
+
751/
251/
synonymous_variant 
LEUM_0545






TTG: 0


1866
621
c.751_753delTTGinsCTA 












p.Leu251Leu







510386
GTCATA
ATCTTG
ATCTTG: 158
CDS
+
1549/
517/
missense_variant
LEUM_0545






GTCATA: 0


1866
621
c.1549_1554delGTCATAinsATCT












TG












p.ValIle517IleLeu







511743
CAGC
AAGT
AAGT: 187 
CDS
+
927/
309/
synonymous_variant 
LEUM_0546






CAGC: 0


1347
448
c.927_930delCAGCinsAAGT












p.LeuSer309LeuSer







519040
TCGT
CCGC
CCGC: 165 
CDS
+
210/
70/
synonymous_variant 
LEUM_0553






TCGT: 0


1371
456
c.210_213delTCGTinsCCGC












p.GlyArg70GlyArg







530354
TTGG
GTGA
GTGA: 118 
CDS
+
193/
65/
missense_variant 
LEUM_0562






TTGG: 0


1728
575
c.193_196delTTGGinsGTGA












p.LeuVal65ValMet







536863
AAGA
GAGG
GAGG: 178 
CDS
+
1959/
653/
synonymous_variant
LEUM_0566






AAGA: 0


2301
766
c.1959_1962delAAGAinsGAGG












p.SerArg653SerArg







560132
AAC
TAT
TAT: 202 
CDS
+
423/
141/
missense_variant 
LEUM_0589






AAC: 0


882
293
c.423_425delAACinsTAT












p.ValThr141ValMet







603339
AAT
GAC
GAC: 238 
CDS
+
673/
225/
missense_variant 
LEUM_0636






AAT: 0


1944
647
c.673_675delAATinsGAC 












p.Asn225Asp







607531
GAGC
AAGT
AAGT: 217 
CDS
+
438/
146/
missense_variant 
LEUM_0640






GAGC: 0


894
297
c.438_441delGAGCinsAAGT












p.MetSer146IleSer







610263
TAACA
CAACG
CAACG: 174
CDS
+
773/
258/
missense_variant 
LEUM_0643






TAACA: 0


1464
487
c.773_777delTAACAinsCAACG












p.LeuThr258SerThr







610344
TAGCTGCAAGTGCTGC
CAGCTGCAAGTG
CAGCTGCAAGTG: 127
CDS
+
854/
285/
missense_variant&inframe_
LEUM_0643




AAGTG

TAGCTGCAAGTGCTGCAA


1464
487
deletion







GTG: 0




c.854_864delTAGCTGCAAGTins












CA












p.Ile285_Ser288delinsThr







613023
CGGC
AGGT
AGGT: 209 
CDS
+
801/
267/
synonymous_variant
LEUM_0645






CGGC: 0


1143
380
c.801_804delCGGCinsAGGT 












p.ProGly267ProGly







613326
GACG
AACA
AACA: 160 
CDS
+
1104/
368/
synonymous_variant
LEUM_0645






GACG: 0


1143
380
c.1104_1107delGACGinsAACA












p.AlaThr368AlaThr







615534
GTTG
ATTA
ATTA: 217 












GTTG: 0












615580
GCCC
CCCT
CCCT: 199 












GCCC: 0












641900
TCCG
CCCA
CCCA: 199 
CDS
+
417/
139/
synonymous_variant 
LEUM_0673






TCCG: 0


570
189
c.417_420delTCCGinsCCCA












p.TyrPro139TyrPro







642442
CAGTA
TAGCG
TAGCG: 148
CDS
+
282/
94/
missense_variant 
LEUM_0674






CAGTA: 0


684
227
c.282_286delCAGTAinsTAGCG












p.GlySerThr94GlySerAla







654478
CTTC
TTTT
TTTT: 217 
CDS
+
597/
199/
synonymous_variant 
LEUM_0686






CTTC: 0


795
264
c.597_600delCTTCinsTTTT












p.AsnPhe199AsnPhe







658429
TCG
GCA
GCA: 147 
CDS
+
622/
208/
missense_variant 
LEUM_0689






TCG: 0


4314
1437
c.622_624delTCGinsGCA












p.Ser208Ala







671357
CAGTTAT
AAGCTAC
AAGCTAC: 180
CDS
+
432/
144/
synonymous_variant
LEUM_0698






CAGTTAT: 0


891
296
c.432_438delCAGTTATinsAAGCT












AC












p.LeuSerTyr144LeuSerTyr







697054
AAT
CAG
CAG: 204 
CDS
+
2160/
720/
missense_variant 
LEUM_0723






AAT: 0


2217
738
c.2160_2162delAATinsCAG












p.LeuIle720PheSer







700692
ACCC
CCCT
CCCT: 206 
CDS
+
378/
126/
synonymous_variant 
LEUM_0727
purH





ACCC: 0


1527
508
c.378_381delACCCinsCCCT












p.GlyPro126GlyPro







700713
AGCT
TGCC
TGCC: 209 
CDS
+
399/
133/
synonymous_variant 
LEUM_0727
purH





AGCT: 0


1527
508
c.399_402delAGCTinsTGCC












p.AlaAla133AlaAla







701025
CGGCAAA
TGGTAAG
TGGTAAG: 121
CDS
+
711/
237/
synonymous_variant
LEUM_0727
purH





CGGCAAA: 0


1527
508
c.711_717delCGGCAAAinsTGGTA












AG












p.HisGlyLys237HisGlyLys







723536
CACTG
TACTC
TACTC: 162 
CDS
+
326/
109/
missense_variant 
LEUM_0746






CACTG: 0


534
177
c.326_330delCACTGinsTACTC












p.ThrLeu109IleLeu







726007
ATAAA
TTTAT
TTTAT: 130 












ATAAA: 0












745561
ATAAT
GTAAC
GTAAC: 87 












ATAAT: 0












751089
ACTG
GCTA
GCTA: 157 
CDS
+
2232/
744/
synonymous_variant
LEUM_0774






ACTG: 0


3339
1112
c.2232_2235delACTGinsGCTA












p.GluLeu744GluLeu







769650
GCCA
ACCG
ACCG: 139 
CDS

27/
8/277
synonymous_variant 
LEUM_0791






GCCA: 0


834

c.24_27delTGGCinsCGGT












p.AspGly8AspGly







784937
CCCG
TCCA
TCCA: 96 
CDS

1608/
535/
synonymous_variant
LEUM_0807






CCCG: 0


1674
557
c.1605_1608delCGGGinsTGGA 












p.IleGly535IleGly







787928
AAACG
GAACC
GAACC: 132
CDS
+
1190/
397/
missense_variant
LEUM_0808






AAACG: 0


1701
566
c.1190_1194delAAACGinsGAACC












p.GlnThr397ArgThr







788232
TATCATC
CATCTTG
CATCTTG: 120
CDS
+
1494/
498/
missense_variant
LEUM_0808






TATCATC: 0


1701
566
c.1494_1500delTATCATCinsCAT












CTTG












p.ThrIleIle498ThrIleLeu







796989
ATTAGGC
GCTGGGT
GCTGGGT: 149












ATTAGGC: 0












797082
GGGA
TGGG
TGGG: 154 












GGGA: 0












797274
TAAAA
GAAAC
GAAAC: 136












TAAAA: 0












800184
ACAAT
GCAAG
GCAAG: 171
CDS
+
900/
300/
missense_variant 
LEUM_0818






ACAAT: 0


4521
1506
c.900_904delACAATinsGCAAG












p.ProGlnSer300ProGlnAla







829273
CATTAT
AAGTAC
AAGTAC: 116
CDS
+
211/
71/
missense_variant
LEUM_0842






CATTAT: 0


909
302
c.211_216delCATTATinsAAGTAC












p.HisTyr71LysTyr







831087
TAGC
CAAT
CAAT: 103 
CDS

408/
135/
synonymous_variant 
LEUM_0844






TAGC: 0


897
298
c.405_408delGCTAinsATTG












p.ValLeu135ValLeu







831917
GAACAGGT
AAACCGGC
AAACCGGC: 130
CDS
+
300/
100/
synonymous_variant
LEUM_0845






GAACAGGT: 0


2025
674
c.300_307delGAACAGGTinsAAAC












CGGC












p.GlyAsnArgLeu100GlyAsnArg












Leu







832789
GAGC
CAGT
CAGT: 158 
CDS
+
1172/
391/
missense_variant 
LEUM_0845






GAGC: 0


2025
674
c.1172_1175delGAGCinsCAGT












p.GlyAla391AlaVal







833573
TATGG
CATGA
CATGA: 172
CDS
+
1956/
652/
missense_variant
LEUM_0845






TATGG: 0


2025
674
c.1956_1960delTATGGinsCATGA












p.HisMetAla652HisMetThr







835366
GCAT
ACAA
ACAA: 139 
CDS
+
459/
153/
missense_variant 
LEUM_0847






GCAT: 0


1149
382
c.459_462delGCATinsACAA












p.GlyHis153GlyGln







838604
AAGT
GAGC
GAGC: 132 
CDS
+
687/
229/
synonymous_variant
LEUM_0849






AAGT: 0


729
242
c.687_690delAAGTinsGAGC 












p.GlySer229GlySer







838832
GGTAC
AGCAT
AGCAT: 131
CDS
+
185/
62/
missense_variant 
LEUM_0850






GGTAC: 0


330
109
c.185_189delGGTACinsAGCAT












p.GlyTyr62GluHis







843675
CAGATTAACG
AAAATCAAAA
AAAATCAAAA: 133
CDS
+
256/
86/
missense_variant
LEUM_0854






CAGATTAACG: 0


1620
539
c.256_265delCAGATTAACGinsAA












AATCAAAA












p.GlnIleAsnAla86LysIleLys












Thr







843731
GAAT
AAAC
AAAC: 158 
CDS
+
312/
104/
synonymous_variant 
LEUM_0854






GAAT: 0


1620
539
c.312_315delGAATinsAAAC












p.LysAsn104LysAsn







847585
AACA
GACG
GACG: 149 
CDS
+
660/
220/
synonymous_variant
LEUM_0857






AACA: 0


8466
2821
c.660_663delAACAinsGACG 












p.ThrThr220ThrThr







853659
ATA
GTG
GTG: 201 
CDS
+
6734/
2245/
missense_variant 
LEUM_0857






ATA: 0


8466
2821
c.6734_6736delATAinsGTG












p.AsnAsn2245SerAsp







863407
GTAA
TTGC
TTGC: 77 












GTAA: 0












870920
TC
TAT
TAT: 106 












TC: 0












876892
ATAGCTCA
CTAGATCG
CTAGATCG: 171
CDS
+
367/
123/
missense_variant
LEUM_0882






ATAGCTCA: 0


2223
740
c.367_374delATAGCTCAinsCTAG












ATCG












p.IleAlaHis123LeuAspArg







877704
CGCC
TGCT
TGCT: 185 
CDS
+
1179/
393/
synonymous_variant
LEUM_0882






CGCC: 0


2223
740
c.1179_1182delCGCCinsTGCT












p.TyrAla393TyrAla







880042
ACTAT
TCTAC
TCTAC: 151 
CDS
+
77/
26/
missense_variant 
LEUM_0884






ACTAT: 0


1506
501
c.77_81delACTATinsTCTAC












p.AsnTyr26IleTyr







883034
ACCACTT
GCCGCTC
GCCGCTC: 136
CDS
+
1422/
474/
missense_variant
LEUM_0885






ACCACTT: 0


2253
750
c.1422_1428delACCACTTinsGCC












GCTC












p.IleProLeu474MetProLeu







883123
GAGA
AAGG
AAGG: 126 
CDS
+
1511/
504/
missense_variant 
LEUM_0885






GAGA: 0


2253
750
c.1511_1514delGAGAinsAAGG












p.ArgGlu504LysGly







893725
TAA
CAG
CAG: 132 
CDS
+
1167/
389/
missense_variant 
LEUM_0894






TAA: 0


2259
752
c.1167_1169delTAAinsCAG












p.AlaLys389AlaArg







894794
AAA
GAG
GAG: 173 
CDS
+
2236/
746/
missense_variant 
LEUM_0894






AAA: 0


2259
752
c.2236_2238delAAAinsGAG 












p.Lys746Glu







895508
CAAG
TAAA
TAAA: 112 
CDS
+
675/
225/
synonymous_variant 
LEUM_0895






CAAG: 0


687
228
c.675_678delCAAGinsTAAA












p.IleLys225IleLys







895583
ATTAAGCG
GTCAAGTT
GTCAAGTT: 92
CDS

996/
330/
missense_variant
LEUM_0896






ATTAAGCG: 0


1008
335
c.989_996delCGCTTAATinsAACT












TGAC












p.ThrLeuAsn330LysLeuAsp







895607
CGGT
TGGG
TGGG: 101 
CDS

972/
323/
synonymous_variant 
LEUM_0896






CGGT: 0


1008
335
c.969_972delACCGinsCCCA












p.ValPro323ValPro







903892
CTTTGCCTT
TTTTACCTC
TTTTACCTC: 158
CDS
+
1215/
405/
missense_variant
LEUM_0901






CMGCCTT: 0


1839
612
c.1215_1223delCTTTGCCTTinsT












TTTACCTC












p.AlaPheAlaLeu405AlaPheThr












Ser







907285
GCTAC
ACTAT
ACTAT: 127 












GCTAC: 0












911930
CAGC
TAGT
TAGT: 94 
CDS
+
39/
13/
synonymous_variant 
LEUM_0909






CAGC: 0


822
273
c.39_42delCAGCinsTAGT












p.SerSer13SerSer







933210
CAGGGC
GAGCGT
GAGCGT: 156
CDS
+
1909/
637/
missense_variant
LEUM_0929






CAGGGC: 0


1992
663
c.1909_1914delCAGGGCinsGAGC












GT












p.GlnGly637GluArg







945839
TAG
TAAA
TAAA: 60 












TAG: 0












945853
GAT
AAC
AAC: 61 












GAT: 0












972869
CATT
TATC
TATC: 142 
CDS
+
168/
56/
synonymous_variant 
LEUM_0972






CATT: 0


480
159
c.168_171delCATTinsTATC












p.HisIle56HisIle







980203
TTAGTA
CTGGTG
CTGGTG: 85
CDS
+
220/
74/
synonymous_variant
LEUM_0980






TTAGTA: 0


513
170
c.220_225delTTAGTAinsCTGGTG












p.LeuVal74LeuVal







980531
TCATTA
CAATTG
CAATTG: 125












TCATTA: 0












982914
AGCT
GGCA
GGCA: 58 
CDS
+


no annotation
LEUM_0984






AGCT: 0












986252
GGTCC
TGTCT
TGTCT: 31 
CDS
+


no annotation
LEUM_0987






GGTCC: 0












986279
CGAAACGCTCATTC
TGAGACACTAATTA
TGAGACACTAATTA: 30
CDS
+


no annotation
LEUM_0987






CGAAACGCTCATTC: 0












986308
GGTC
AGAT
AGAT: 30 












GGTC: 0












986319
ATT
GTC
GTC: 31 
CDS
+


no annotation
LEUM_0988






ATT: 0












986356
CGTT
TGTG
TGTG: 30 
CDS
+


no annotation
LEUM_0988






CGTT: 0












986375
GTTTCAGAAAAA
ATGTCGGAAGAG
ATGTCGGAAGAG: 25
CDS
+


no annotation
LEUM_0988






GTTTCAGAAAAA: 0












1008480
CAAG
TAAA
TAAA: 14 
CDS
+


no annotation
LEUM_1008






CAAG: 0












1008786
CCTG
TCTA
TCTA: 1619 
CDS
+


no annotation
LEUM_1009






CCTG: 0












1008954
ACCC
GCCA
GCCA: 1877 
CDS
+


no annotation
LEUM_1009






ACCC: 0












1022214
TTTG
ATTA
ATTA: 76 












TTTG: 0












1135118
TGG
CGA
CGA: 83 












TGG: 0












1135159
TCGT
CCGC
CCGC: 83 












TCGT: 0












1135269
TTAC
CTAT
CTAT: 123 
CDS
+


no annotation
LEUM_1138






TTAC: 0












1138281
GTTT
ATTC
ATTC: 201 
CDS



no annotation
LEUM_1142






GTTT: 0












1139585
CAACC
TAACT
TAACT: 197 
CDS



no annotation
LEUM_1143






CAACC: 0












1155368
AGCG
GGCA
GGCA: 141 
CDS



no annotation
LEUM_1157






AGCG: 0












1157871
ATTT
GTTG
GTTG: 155 
CDS



no annotation
LEUM_1161






ATTT: 0












1169465
GTCG
TTCT
TTCT: 178 
CDS



no annotation
LEUM_1172






GTCG: 0












1170652
GCG
TCA
TCA: 135 
CDS



no annotation
LEUM_1173






GCG: 0












1170669
TATC
CATT
CATT: 124 
CDS



no annotation
LEUM_1173






TATC: 0












1170980
TTTA
CTCG
CTCG: 123 
CDS



no annotation
LEUM_1174






TTTA: 0












1174201
GAC
AAT
AAT: 87 












GAC: 0












1174261
CGTG
AGTA
AGTA: 130 
CDS



no annotation
LEUM_1177






CGTG: 0












1183816
GGTA
AGTG
AGTG: 139 
CDS



no annotation
LEUM_1187






GGTA: 0












1194019
GCAAT
ACAAC
ACAAC: 139 
CDS



no annotation
LEUM_1195






GCAAT: 0












1238393
GGCAGG
AGTAGA
AGTAGA: 81 












GGCAGG: 0












1238441
TAAT
GATA
GATA: 47 












TAAT: 0












1258437
CTT
TTG
TTG: 43 












CTT: 0












1263043
TGGG
CGGA
CGGA: 194 
CDS
+


no annotation
LEUM_1275






TGGG: 0












1267583
TGGGCAG
GGGTCAA
GGGTCAA: 131
CDS
+


no annotation
LEUM_1279






TGGGCAG: 0












1289296
TCTC
CCU
CCTT: 197 
CDS



no annotation
LEUM_1302






TCTC: 0












1294486
ACAA
GCA
GCA: 189 












ACAA: 0












1296449
CAGCTGTA
TATCCGTG
TATCCGTG: 188
CDS



no annotation
LEUM_1309
aspS





CAGCTGTA: 0












1302442
TCCG
ACCA
ACCA: 161 
CDS



no annotation
LEUM_1314






TCCG: 0












1303222
AGTA
GGTG
GGTG: 220 
CDS



no annotation
LEUM_1314






AGTA: 0












1306063
TACC
GACA
GACA: 193 
CDS



no annotation
LEUM_1316
lacZ





TACC: 0












1319219
TACAGCAA
CACATCAC
CACATCAC: 135












TACAGCAA: 0












1319558
ATTTAAGTTCAGTCAC
CTACAATATCACTTCC
CTACAATATCACTTCCC:










A
C
109












ATTTAAGTTCAGTCACA:












0












1319611
ACGTCT
CCGTTC
CCGTTC: 146












ACGTCT: 0












1319951
ACGC
GCGT
GCGT: 150 
CDS
+


no annotation
LEUM_1334






ACGC: 0












1345228
ACTTG
GCTTA
GCTTA: 204 
CDS



no annotation
LEUM_1363






ACTTG: 0












1346846
TGGG
CGGA
CGGA: 191 
CDS



no annotation
LEUM_1363






TGGG: 0












1392214
TAAA
AAGC
AAGC: 157 
CDS



no annotation
LEUM_1404






TAAA: 0












1396399
CGC
TGT
TGT: 177 
CDS



no annotation
LEUM_1408






CGC: 0












1407216
TGA
AGC
AGC: 120 
CDS



no annotation
LEUM_1412






TGA: 0












1407234
TGTTAGT
AGCTAAC
AGCTAAC: 94
CDS



no annotation
LEUM_1412






TGTTAGT: 0












1407252
AATG
GATA
GATA: 112 
CDS



no annotation
LEUM_1412






AATG: 0












1410440
GCTT
ACTC
ACTC: 158 
CDS



no annotation
LEUM_1415






GCTT: 0












1410471
CTT
ATC
ATC: 162 
CDS



no annotation
LEUM_1415






CTT: 0












1415069
TTTC
CTTA
CTTA: 140 
CDS



no annotation
LEUM_1420






TTTC: 0












1415084
CACT
AACA
AACA: 142 
CDS



no annotation
LEUM_1420






CACT: 0












1415294
AAGT
TAGC
TAGC: 163 
CDS



no annotation
LEUM_1420






AAGT: 0












1415654
GTAC
ATAA
ATAA: 203 
CDS



no annotation
LEUM_1420






GTAC: 0












1415711
AGCT
CGCC
CGCC: 184 
CDS



no annotation
LEUM_1420






AGCT: 0












1415881
AAC
GAA
GAA: 192 












AAC: 0












1416065
GCCT
TCCA
TCCA: 207 
CDS



no annotation
LEUM_1421






GCCT: 0












1416263
GTTT
ATTA
ATTA: 191 
CDS



no annotation
LEUM_1421






GTTT: 0












1416317
GATG
AATA
AATA: 199 
CDS



no annotation
LEUM_1421






GATG: 0












1416380
CAAA
TAAG
TAAG: 211 
CDS



no annotation
LEUM_1421






CAAA: 0












1416695
TGTT
GGTC
GGTC: 168 
CDS



no annotation
LEUM_1421






TGTT: 0












1417341
AUG
GTTA
GTTA: 195 
CDS



no annotation
LEUM_1422






ATTG: 0












1417434
ATTA
GTTG
GTTG: 217 
CDS



no annotation
LEUM_1422






ATTA: 0












1417596
CAG
TAA
TAA: 222 
CDS



no annotation
LEUM_1423






CAG: 0












1417722
AAGGAGA
GAGAAGT
GAGAAGT: 134
CDS



no annotation
LEUM_1423






AAGGAGA: 0












1417734
CAACGTT
GTGTGTC
GTGTGTC: 128
CDS



no annotation
LEUM_1423






CAACGTT: 0












1417782
GTCT
ATCC
ATCC: 185 
CDS



no annotation
LEUM_1423






GTCT: 0












1417965
CTTGTCA
TTTATCG
TTTATCG: 206
CDS



no annotation
LEUM_1423






CTTGTCA: 0












1418013
GCCA
ACCG
ACCG: 208 
CDS



no annotation
LEUM_1423






GCCA: 0












1418025
GGCG
AGCA
AGCA: 180 
CDS



no annotation
LEUM_1423






GGCG: 0












1418040
TAAAGCCTCTTG
CAGAGCAGCTTC
CAGAGCAGCTTC: 88
CDS



no annotation
LEUM_1423






TAAAGCCTCTTG: 0












1418061
TTG
CTC
CTC: 91 
CDS



no annotation
LEUM_1423






TTG: 0












1418069
GACCGGCA
ACCCTGCG
ACCCTGCG: 89
CDS



no annotation
LEUM_1423






GACCGGCA: 0












1418094
TCCC
ACCT
ACCT: 100 
CDS



no annotation
LEUM_1423






TCCC: 0












1418103
TAAG
CAGA
CAGA: 87 
CDS



no annotation
LEUM_1423






TAAG: 0












1418148
CGCG
TGCA
TGCA: 197 
CDS



no annotation
LEUM_1423






CGCG: 0












1418160
GCCA
ACCG
ACCG: 194 
CDS



no annotation
LEUM_1423






GCCA: 0












1418193
GTGCAA
ATTTAG
ATTTAG: 162
CDS



no annotation
LEUM_1423






GTGCAA: 0












1418208
ATGG
CTGA
CTGA: 175 
CDS



no annotation
LEUM_1423






ATGG: 0












1418271
TTTT
ATCC
ATCC: 170 
CDS



no annotation
LEUM_1423






TTTT: 0












1418322
TTTA
CTTG
CTTG: 167 
CDS



no annotation
LEUM_1423






TTTA: 0












1418385
AGAG
GGAA
GGAA: 118 
CDS



no annotation
LEUM_1423






AGAG: 0












1418582
ACC
GCT
GCT: 210 
CDS



no annotation
LEUM_1424






ACC: 0












1418878
TGCCTCG
AGTCTCA
AGTCTCA: 149
CDS



no annotation
LEUM_1424






TGCCTCG: 0












1418950
ACTC
GCTT
GCTT: 163 
CDS



no annotation
LEUM_1424






ACTC: 0












1419097
CCTA
TCTG
TCTG: 175 
CDS



no annotation
LEUM_1424






CCTA: 0












1419197
GTGCT
TTGCC
TTGCC: 208 
CDS



no annotation
LEUM_1424






GTGCT: 0












1419226
GTTA
ATTG
ATTG: 221 
CDS



no annotation
LEUM_1424






GTTA: 0












1419311
TCG
GCC
GCC: 230 
CDS



no annotation
LEUM_1424






TCG: 0












1419388
GCTT
ACTG
ACTG: 223 
CDS



no annotation
LEUM_1424






GCTT: 0












1419438
TTTTAG
GTTG
GTTG: 162 
CDS



no annotation
LEUM_1424






TTTTAG: 0












1429917
TGGCTCCTCTATTTGT
AGGCACCTTTAGTCGT
AGGCACCTTTAGTCGTTT
CDS



no annotation
LEUM_1434




CTTT
TTTA
TA: 173












TGGCTCCTCTATTTGTCT












TT: 0












1429993
TGTG
CGTA
CGTA: 204 
CDS



no annotation
LEUM_1434






TGTG: 0












1430085
AGAGT
GGAGC
GGAGC: 169
CDS



no annotation
LEUM_1434






AGAGT: 0












1430128
GTTG
ATTA
ATTA: 172 
CDS



no annotation
LEUM_1434






GTTG: 0












1430143
AGACGTG
GGCTGTA
GGCTGTA: 153
CDS



no annotation
LEUM_1434






AGACGTG: 0












1430176
CTCT
TTCA
TTCA: 177 
CDS



no annotation
LEUM_1434






CTCT: 0












1430203
CCCG
TCCA
TCCA: 186 
CDS



no annotation
LEUM_1434






CCCG: 0












1430314
AGCTGTGACC
GGCAGTCACT
GGCAGTCACT: 192
CDS



no annotation
LEUM_1434






AGCTGTGACC: 0












1430344
CAAC
TAAG
TAAG: 206 
CDS



no annotation
LEUM_1434






CAAC: 0












1430374
TTCG
CTCA
CTCA: 216 
CDS



no annotation
LEUM_1434






TTCG: 0












1430413
TAAA
CAAG
CAAG: 214 
CDS



no annotation
LEUM_1434






TAAA: 0












1430623
CTCT
TTCA
TTCA: 192 
CDS



no annotation
LEUM_1435






CTCT: 0












1430785
AACCAATCCT
TACAAAACCA
TACAAAACCA: 159
CDS



no annotation
LEUM_1435






AACCAATCCT: 0












1430806
CAA
TAG
TAG: 183 
CDS



no annotation
LEUM_1435






CAA: 0












1430942
TTAGAATC
GTAGGATT
GTAGGATT: 180
CDS



no annotation
LEUM_1435






TTAGAATC: 0












1431011
CTTTTT
TCTTTC
TCTTTC: 161
CDS



no annotation
LEUM_1435






CTTTTT: 0












1431073
CTTA
TTTT
TTTT: 160 
CDS



no annotation
LEUM_1435






CTTA: 0












1431088
CAGA
TAGG
TAGG: 142 
CDS



no annotation
LEUM_1435






CAGA: 0












1431356
AAC
TAT
TAT: 129 
CDS



no annotation
LEUM_1435






AAC: 0












1431525
TTT
CTC
CTC: 143 
CDS



no annotation
LEUM_1436






TTT: 0












1431755
CACC
TACT
TACT: 154 
CDS



no annotation
LEUM_1436






CACC: 0












1431803
CGTA
TGTG
TGTG: 139 
CDS



no annotation
LEUM_1436






CGTA: 0












1432287
GCAAA
ACAAT
ACAAT: 162












GCAAA: 0












1432326
AAAC
TACT
TACT: 140 












AAAC: 0












1432336
TAAAA
GAAAG
GAAAG: 143












TAAAA: 0












1432349
TATG
CATA
CATA: 141 
CDS



no annotation
LEUM_1437






TATG: 0












1432378
CTGA
TTGG
TTGG: 207 
CDS



no annotation
LEUM_1437






CTGA: 0












1432717
AAT
CAC
CAC: 213 
CDS



no annotation
LEUM_1437






AAT: 0












1433379
CCA
GCG
GCG: 209 
CDS



no annotation
LEUM_1438






CCA: 0












1433417
GGACTTA
AGATTTG
AGATTTG: 205
CDS



no annotation
LEUM_1438






GGACTTA: 0












1433441
CACA
TACG
TACG: 222 
CDS



no annotation
LEUM_1438






CACA: 0












1433984
CGTG
TGTA
TGTA: 206 
CDS



no annotation
LEUM_1438






CGTG: 0












1436006
AAAG
GAAA
GAAA: 254 
CDS



no annotation
LEUM_1440






AAAG: 0












1436796
CAA
TAC
TAC: 92 












CAA: 0












1437736
CAAA
TAAG
TAAG: 245 
CDS



no annotation
LEUM_1443






CAAA: 0












1437751
CTTA
TTTG
TTTG: 249 
CDS



no annotation
LEUM_1443






CTTA: 0












1441725
CGCTT
TGCTTT
TGCTTT: 165












CGCTT: 0












1444575
CAAAAAAAAAAAAAC
CAAAAAAAACAAAC
CAAAAAAAACAAAC: 












127












CAAAAAAAAAAAAAC: 0












1447932
AAAC
GAAT
GAAT: 203 
CDS



no annotation
LEUM_1454






AAAC: 0












1474016
TTAAC
CTAAT
CTAAT: 171 
CDS



no annotation
LEUM_1480






TTAAC: 0












1475011
TAGT
CAGC
CAGC: 175 
CDS



no annotation
LEUM_1481






TAGT: 0












1475048
TGTG
CGTT
CGTT: 194 
CDS



no annotation
LEUM_1481






TGTG: 0












1475219
TTGT
CTGC
CTGC: 188 
CDS



no annotation
LEUM_1481






TTGT: 0












1477474
TTAAC
CTAAA
CTAAA: 148 
CDS



no annotation
LEUM_1481






TTAAC: 0












1501570
AGATC
GCATG
GCATG: 145
CDS



no annotation
LEUM_1502






AGATC: 0












1501590
ACA
GCG
GCG: 140 
CDS



no annotation
LEUM_1502






ACA: 0












1510576
TAAT
CAAA
CAAA: 199 
CDS



no annotation
LEUM_1513






TAAT: 0












1518189
AGGC
GGGT
GGGT: 152 
CDS



no annotation
LEUM_1520
engB





AGGC: 0












1519140
AGCA
GGCT
GGCT: 222 
CDS



no annotation
LEUM_1521
clpX





AGCA: 0












1519209
GGAG
AGAT
AGAT: 236 
CDS



no annotation
LEUM_1521
clpX





GGAG: 0












1527336
GTCC
ATCT
ATCT: 171 
CDS



no annotation
LEUM_1529






GTCC: 0












1539200
GAAA
AAAG
AAAG: 234 
CDS



no annotation
LEUM_1539






GAAA: 0












1548015
CAAACT
AGAACA
AGAACA: 112
CDS
+


no annotation
LEUM_1546






CAAACT: 0












1553910
AATT
GATA
GATA: 154 
CDS



no annotation
LEUM_1554






AATT: 0












1563023
ATAG
TTAA
TTAA: 147 












ATAG: 0












1563156
CCCC
TCCT
TCCT: 161 
CDS



no annotation
LEUM_1564






CCCC: 0












1563399
ACCG
GCCC
GCCC: 202 
CDS



no annotation
LEUM_1564






ACCG: 0












1570912
GGGA
AGGG
AGGG: 201 
CDS



no annotation
LEUM_1569






GGGA: 0












1575438
GCAAA
ACAAG
ACAAG: 118












GCAAA: 0












1576436
TTCT
CTCC
CTCC: 188 
CDS



no annotation
LEUM_1575






TTCT: 0












1576450
GTATA
ATATC
ATATC: 188 
CDS



no annotation
LEUM_1575






GTATA: 0












1576582
CCTC
ACTT
ACTT: 201 
CDS



no annotation
LEUM_1575






CCTC: 0












1582261
CACA
GACG
GACG: 210 
CDS



no annotation
LEUM_1578






CACA: 0












1582441
TACTGCA
CACCGCG
CACCGCG: 178
CDS



no annotation
LEUM_1578






TACTGCA: 0












1589522
ACTGC
GCCGT
GCCGT: 119
CDS



no annotation
LEUM_1586






ACTGC: 0












1622472
TTATAT
ACGTAC
ACGTAC: 247
CDS



no annotation
LEUM_1624






TTATAT: 0












1624045
AGCCTAC
GCCCGAT
GCCCGAT: 111
CDS



no annotation
LEUM_1627






AGCCTAC: 0












1624058
CAAG
GAGA
GAGA: 110 
CDS



no annotation
LEUM_1627






CAAG: 0












1624079
TATT
AATCA
AATCA: 164 












TATT: 0












1624096
ATTA
GTTG
GTTG: 184 












ATTA: 0












1624117
TAG
CAA
CAA: 203 












TAG: 0












1624234
GCCGCCA
ACCACCG
ACCACCG: 231
CDS



no annotation
LEUM_1628






GCCGCCA: 0












1624336
TTGA
CTGG
CTGG: 149 
CDS



no annotation
LEUM_1628






TTGA: 0












1624351
ATTACCA
GTTCCCG
GTTCCCG: 149
CDS



no annotation
LEUM_1628






ATTACCA: 0












1624431
TGTTG
AGTTA
AGTTA: 98 
CDS



no annotation
LEUM_1628






TGTTG: 0












1624459
CTTA
TTGT
TTGT: 84 
CDS



no annotation
LEUM_1628






CTTA: 0












1624574
TTG
GTA
GTA: 149 
CDS



no annotation
LEUM_1628






TTG: 0












1624609
GCCG
TCCA
TCCA: 180 
CDS



no annotation
LEUM_1628






GCCG: 0












1624618
TCCG
GCCA
GCCA: 193 
CDS



no annotation
LEUM_1628






TCCG: 0












1624654
GTTGGAA
ATTTGAG
ATTTGAG: 220
CDS



no annotation
LEUM_1628






GTTGGAA: 0












1624720
TAA
CAT
CAT: 230 
CDS



no annotation
LEUM_1628






TAA: 0












1624729
AGCG
GGCA
GGCA: 229 
CDS



no annotation
LEUM_1628






AGCG: 0












1624843
TAG
CAA
CAA: 250 
CDS



no annotation
LEUM_1628






TAG: 0












1624858
ATTA
GTTG
GTTG: 243 
CDS



no annotation
LEUM_1628






ATTA: 0












1624900
TGCG
AGCA
AGCA: 250 
CDS



no annotation
LEUM_1628






TGCG: 0












1624918
GGCTAGC
AGCCAGT
AGCCAGT: 239
CDS



no annotation
LEUM_1628






GGCTAGC: 0












1624978
CACCGAG
GACTGAA
GACTGAA: 222
CDS



no annotation
LEUM_1628






CACCGAG: 0












1625140
AAACGAA
GAATGAG
GAATGAG: 202
CDS



no annotation
LEUM_1628






AAACGAA: 0












1625152
ATAATTTGC
GTAGCTTGT
GTAGCTTGT: 206
CDS



no annotation
LEUM_1628






ATAATTTGC: 0












1625209
CACG
TACA
TACA: 233 
CDS



no annotation
LEUM_1628






CACG: 0












1629235
GATG
TATA
TATA: 176 
CDS



no annotation
LEUM_1635






GATG: 0












1629250
ATTA
GTTG
GTTG: 180 
CDS



no annotation
LEUM_1635






ATTA: 0












1629328
TGTGTTCAAAGAT
CATATTTAGAGAC
CATATTTAGAGAC: 159
CDS



no annotation
LEUM_1635






TGTGTTCAAAGAT: 0












1629619
TAATGCG
CAGTGCA
CAGTGCA: 203
CDS



no annotation
LEUM_1635






TAATGCG: 0












1629658
TATC
GATT
GATT: 223 
CDS



no annotation
LEUM_1635






TATC: 0












1629722
ACACCTG
TCTGCTAA
TCTGCTAA: 130
CDS



no annotation
LEUM_1635






ACACCTG: 0












1629759
ATGA
GTGC
GTGC: 191 
CDS



no annotation
LEUM_1635






ATGA: 0












1650708
TAAC
AAAT
AAAT: 59 
CDS



no annotation
LEUM_1656






TAAC: 0












1650750
AGGAATCGTTCA
ATAGATTGGCTCG
ATAGATTGGCTCG: 35












AGGAATCGTTCA: 0












1650948
ACGCATT
GCGCCTC
GCGCCTC: 199
CDS



no annotation
LEUM_1657






ACGCATT: 0












1651008
ATTG
GTTA
GTTA: 221 
CDS



no annotation
LEUM_1657






ATTG: 0












1651041
TAT
CAC
CAC: 223 
CDS



no annotation
LEUM_1657






TAT: 0












1651098
ATA
GTC
GTC: 188 












ATA: 0












1651117
GTGCA
GATA
GATA: 133 












GTGCA: 0












1651140
GCCA
ACCG
ACCG: 210 












GCCA: 0












1651201
TTCC
CTCT
CTCT: 224 
CDS



no annotation
LEUM_1658






TTCC: 0












1656232
GCCT
ACCC
ACCC: 197 
CDS



no annotation
LEUM_1671






GCCT: 0












1661069
CACT
AACC
AACC: 262 
CDS



no annotation
LEUM_1680






CACT: 0












1665094
TTTTAAACCGTCA
CTTCAAATCATCG
CTTCAAATCATCG: 164
CDS
+


no annotation
LEUM_1690






TTTTAAACCGTCA: 0












1665117
CTTCC
ATTCA
ATTCA: 176 
CDS
+


no annotation
LEUM_1690






CTTCC: 0












1665274
GTACGGC
ATATGGG
ATATGGG: 200
CDS
+


no annotation
LEUM_1690






GTACGGC: 0












1665286
CCAC
TCAT
TCAT: 208 
CDS
+


no annotation
LEUM_1690






CCAC: 0












1665328
CGGA
TGGC
TGGC: 200 
CDS
+


no annotation
LEUM_1690






CGGA: 0












1665337
GAAAGACGCT
AAAGGATGCC
AAAGGATGCC: 196
CDS
+


no annotation
LEUM_1690






GAAAGACGCT: 0












1665424
GAAA
AAAG
AAAG: 171 
CDS
+


no annotation
LEUM_1690






GAAA: 0












1665436
GTATG
ATACA
ATACA: 144
CDS
+


no annotation
LEUM_1690






GTATG: 0












1665448
CAAGCGC
TAAACGT
TAAACGT: 139
CDS
+


no annotation
LEUM_1690






CAAGCGC: 0












1665484
ACCTACC
GCCAACT
GCCAACT: 153
CDS
+


no annotation
LEUM_1690






ACCTACC: 0












1665529
TTTA
ATTG
ATTG: 168 
CDS
+


no annotation
LEUM_1690






TTTA: 0












1665572
AGAAC
GGAAT
GGAAT: 198
CDS
+


no annotation
LEUM_1690






AGAAC: 0












1665664
GGG
AGA
AGA: 206 
CDS
+


no annotation
LEUM_1690






GGG: 0












1665752
TTACAA
CTGCAG
CTGCAG: 201
CDS
+


no annotation
LEUM_1690






TTACAA: 0












1665790
GATTACT
AATAACA
AATAACA: 195
CDS
+


no annotation
LEUM_1690






GATTACT: 0












1665814
TAGT
CAGC
CAGC: 202 
CDS
+


no annotation
LEUM_1690






TAGT: 0












1666025
TTAT
ATAC
ATAC: 134 












TTAT: 0












1667151
TAAAAAAT
TAAAAAAAG
TAAAAAAAG: 78












TAAAAAAT: 0












1669413
AAACA
GAACG
GAACG: 158
CDS
+


no annotation
LEUM_1695






AAACA: 0












1670484
ACCT
TCCC
TCCC: 177 
CDS



no annotation
LEUM_1696






ACCT: 0












1672983
ACTGG
GCTGT
GCTGT: 189
CDS
+


no annotation
LEUM_1698






ACTGG: 0












1684163
GTCTC
ATCTT
ATCTT: 153 












GTCTC: 0












1695377
ACCG
GCCA
GCCA: 273 
CDS



no annotation
LEUM_1726






ACCG: 0












1696196
GGCCGCTAGCATG
TGCAGCCAACATA
TGCAGCCAACATA: 189
CDS



no annotation
LEUM_1726






GGCCGCTAGCATG: 0












1696244
TCGCAA
CCGTAG
CCGTAG: 215
CDS



no annotation
LEUM_1726






TCGCAA: 0












1716146
TAATT
CAATC
CAATC: 45 












TAATT: 0












1717930
ATCA
GTCT
GTCT: 47 
CDS



no annotation
LEUM_1748






ATCA: 0












1717975
ATCGATG
GTCTATA
GTCTATA: 22
CDS



no annotation
LEUM_1748






ATCGATG: 0












1718317
ATCG
GTCT
GTCT: 10 
CDS



no annotation
LEUM_1748






ATCG: 0












1718353
ATTT
GTTC
GTTC: 22 
CDS



no annotation
LEUM_1748






ATTT: 0












1719685
GGA
AGG
AGG: 289 
CDS



no annotation
LEUM_1748






GGA: 2












1725927
TAGCC
CAGCT
CAGCT: 186
CDS



no annotation
LEUM_1752






TAGCC: 1












1726130
GCTA
TCTG
TCTG: 43 
CDS



no annotation
LEUM_1752






GCTA: 0












1726179
TATCC
CAGCT
CAGCT: 65 
CDS



no annotation
LEUM_1752






TATCC: 0












1726202
GCTA
TCTG
TCTG: 90 
CDS



no annotation
LEUM_1752






GCTA: 0












1726215
TAGCC
CAGCT
CAGCT: 95 
CDS



no annotation
LEUM_1752






TAGCC: 0












1726251
CAGCT
TAGCC
TAGCC: 143
CDS



no annotation
LEUM_1752






CAGCT: 2












1756654
TCTAC
GCTAT
GCTAT: 128 












TCTAC: 0












1756824
ATC
GTA
GTA: 145 
CDS



no annotation
LEUM_1786






ATC: 0












1757247
GAAA
AAAG
AAAG: 196 
CDS



no annotation
LEUM_1786






GAAA: 0












1759552
TACT
CACC
CACC: 256 
CDS
+


no annotation
LEUM_1788






TACT: 0












1759606
GGCG
AGCA
AGCA: 266 
CDS
+


no annotation
LEUM_1788






GGCG: 0












1760925
ACCCGATGGGTTGTAT
GCCACTAGGCTGCAT
GCCACTAGGCTGCAT: 










T

37












ACCCGATGGGTTGTATT:












0












1760955
CAAATGA
TAAGTGG
TAAGTGG: 35
CDS



no annotation
LEUM_1791






CAAATGA: 0












1760994
GGCTGCAAACGCTGCA
AGCAGCGAAAGCAGCG
AGCAGCGAAAGCAGCGCG
CDS



no annotation
LEUM_1791




CGCAGGCGCAGC
CGTAAACGAAGT
TAAACGAAGT: 37












GGCTGCAAACGCTGCACG












CAGGCGCAGC: 0












1761057
CTTGGGG
TTTTGGT
TTTTGGT: 167
CDS



no annotation
LEUM_1791






CTTGGGG: 0












1761069
CTGGGGTATCAAAACG
TTGTGGAATTAATACT
TTGTGGAATTAATACTGT
CDS



no annotation
LEUM_1791




GTTACA
GTCACT
CACT: 168












CTGGGGTATCAAAACGGT












TACA: 0












1761096
GTTA
ATTG
ATTG: 166 
CDS



no annotation
LEUM_1791






GTTA: 0












1761107
CTGCCTGC
TTGCTTGT
TTGCTTGT: 173
CDS



no annotation
LEUM_1791






CTGCCTGC: 0












1764663
TTC
CTG
CTG: 125 
CDS
+


no annotation
LEUM_1793






TTC: 0












1766295
TAA
CAG
CAG: 302 
CDS



no annotation
LEUM_1794






TAA: 0












1776537
CGA
AGC
AGC: 191 
CDS



no annotation
LEUM_1803






CGA: 0












1790033
CTGT
TTGC
TTGC: 198 
CDS



no annotation
LEUM_1817






CTGT: 0












1824412
CAA
AAG
AAG: 178 
CDS



no annotation
LEUM_1850






CAA: 0












1830003
GAGA
AAGG
AAGG: 208 












GAGA: 0












1842065
ACCA
GCCC
GCCC: 231 
CDS



no annotation
LEUM_1868
atpC





ACCA: 0












1857246
ATTACCTTTGATAAC
GTTATCAAAGGTAAT
GTTATCAAAGGTAAT: 












71












ATTACCTTTGATAAC: 0












1860337
AGA
GGG
GGG: 145 
CDS



no annotation
LEUM_1886






AGA: 0












1861225
CTTTGCA
TTTTACG
TTTTACG: 221
CDS



no annotation
LEUM_1888






CTTTGCA: 0












1875169
ATT
GTC
GTC: 252 
CDS



no annotation
LEUM_1900






ATT: 0












1878574
ACG
AA
AA: 157 












ACG: 1












1878900
GCAAGT
ATAAGC
ATAAGC: 121
CDS
+


no annotation
LEUM_1905






GCAAGT: 0












1878918
GTG
TTT
TTT: 121 
CDS
+


no annotation
LEUM_1905






GTG: 0












1878926
CTTT
TTTC
TTTC: 114 
CDS
+


no annotation
LEUM_1905






CTTT: 0












1878938
ATAGA
GTAA
GTAA: 113 
CDS
+


no annotation
LEUM_1905






ATAGA: 0












1878945
TCCC
GACG
GACG: 112 












TCCC: 0












1878959
GTAT
TTAA
TTAA: 139 












GTAT: 0












1879309
CCTAGCCA
TCTGGCCT
TCTGGCCT: 176












CCTAGCCA: 0












1882947
AGTAGT
GGTTGC
GGTTGC: 244












AGTAGT: 0












1882969
TACAT
GACAC
GACAC: 243












TACAT: 0












1886783
CCAATCA
TCGATCG
TCGATCG: 207
CDS
+


no annotation
LEUM_1917






CCAATCA: 0












1887546
TAGG
CAAA
CAAA: 137 
CDS



no annotation
LEUM_1919






TAGG: 0












1887555
ACGTGTT
TCGCGTA
TCGCGTA: 147
CDS



no annotation
LEUM_1919






ACGTGTT: 0












1887567
CAATGAACCG
TAGAGAGCCA
TAGAGAGCCA: 147
CDS



no annotation
LEUM_1919






CAATGAACCG: 0












1887582
TTCA
CTCG
CTCG: 153 
CDS



no annotation
LEUM_1919






TTCA: 0












1887645
GGCT
AGCC
AGCC: 249 
CDS



no annotation
LEUM_1919






GGCT: 0












1887654
CTTG
TTTA
TTTA: 252 
CDS



no annotation
LEUM_1919






CTTG: 0












1887666
ACGAAGC
GCGCAAT
GCGCAAT: 172
CDS



no annotation
LEUM_1919






ACGAAGC: 0












1887684
CTGG
TTGT
TTGT: 196 
CDS



no annotation
LEUM_1919






CTGG: 0












1887711
TGTCACTTGA
AGTTACCTGG
AGTTACCTGG: 239
CDS



no annotation
LEUM_1919






TGTCACTTGA: 0












1887732
GCCG
ACCA
ACCA: 275 
CDS



no annotation
LEUM_1919






GCCG: 0












1887771
CTTC
TTTT
TTTT: 299 
CDS



no annotation
LEUM_1919






CTTC: 0












1887795
CGCTCCA
TGCACCG
TGCACCG: 316
CDS



no annotation
LEUM_1919






CGCTCCA: 0












1887821
ATTTA
GCTTG
GCTTG: 277 
CDS



no annotation
LEUM_1919






ATTTA: 0












1887831
GTTTCCA
ATTACCG
ATTACCG: 281
CDS



no annotation
LEUM_1919






GTTTCCA: 0












1887852
GTGA
ATGT
ATGT: 312 
CDS



no annotation
LEUM_1919






GTGA: 0












1887867
ACTG
GCTA
GCTA: 324 
CDS



no annotation
LEUM_1919






ACTG: 0












1887897
TAG
CAA
CAA: 307 
CDS



no annotation
LEUM_1919






TAG: 0












1887906
AGCA
GGCG
GGCG: 305 
CDS



no annotation
LEUM_1919






AGCA: 0












1896684
TCAGC
CCAGA
CCAGA: 220
CDS



no annotation
LEUM_1927






TCAGC: 0












1897538
GCGC
ACGT
ACGT: 286 
CDS



no annotation
LEUM_1928






GCGC: 0












1915818
AGTT
GGTC
GGTC: 305 
CDS



no annotation
LEUM_1944






AGTT: 0












1917475
TTA
CTC
CTC: 134 
CDS



no annotation
LEUM_1945






TTA: 0












1933246
TCA
CCG
CCG: 225 
CDS
+


no annotation
LEUM_1960






TCA: 0












1933618
CATT
TATA
TATA: 200 
CDS
+


no annotation
LEUM_1960






CATT: 0












1933723
GCCCA
TCCCG
TCCCG: 175
CDS
+


no annotation
LEUM_1960






GCCCA: 0












1933941
GTCT
ATT
ATT: 134 












GTCT: 0












1934018
ATATTAC
TTGTTAT
TTGTTAT: 133












ATATTAC: 0












1934029
ACAA
GTAT
GTAT: 135 












ACAA: 0












1934072
GTAA
ATA
ATA: 142 












GTAA: 0












1934080
ATGTGGC
GTGTTGT
GTGTTGT: 142












ATGTGGC: 0












1952692
GAATA
TAATG
TAATG: 97 












GAATA: 0












1952721
GAAG
AAAT
AAAT: 82 












GAAG: 0












1952732
GTGTT
TCGTC
TCGTC: 78 












GTGTT: 0












1953810
CGGTG
TTGTA
TTGTA: 462












CGGTG: 0












1960043
CAATT
TAATC
TAATC: 36 












CAATT: 0












1960073
TTTGGG
AAGGGA
AAGGGA: 39












TTTGGG: 0












1960134
TGTGTTAAATAC
AGTGCTATATTT
AGTGCTATATTT: 34
CDS



no annotation
LEUM_1991






TGTGTTAAATAC: 0












1960163
GTCA
ATCT
ATCT: 36 
CDS



no annotation
LEUM_1991






GTCA: 0












1960179
ATTGC
CTTAA
CTTAA: 39 
CDS



no annotation
LEUM_1991






ATTGC: 0












1960376
TGCT
AGCA
AGCA: 107 
CDS



no annotation
LEUM_1991






TGCT: 0












1960390
GTCTT
ACCTC
ACCTC: 106 
CDS



no annotation
LEUM_1991






GTCTT: 0












1960567
AAA
CAC
CAC: 136 












AAA: 0












1960585
CTGCA
TTGCG
TTGCG: 122












CTGCA: 0












1960664
TGTC
CGTT
CGTT: 161 












TGTC: 0












1969902
GTC
ATT
ATT: 182 
CDS
+


no annotation
LEUM_2001






GTC: 0












1969941
GTTTA
ATTTT
ATTTT: 173 
CDS
+


no annotation
LEUM_2001






GTTTA: 0












1970013
TTAT
CTGC
CTGC: 152 
CDS
+


no annotation
LEUM_2001






TTAT: 0












1978224
AGTAT
GGTAC
GGTAC: 277
CDS



no annotation
LEUM_2010






AGTAT: 0












1980589
CTTGT
TTTGC
TTTGC: 192 












CTTGT: 0












1994040
TAATT
GAATC
GAATC: 291 
CDS



no annotation
LEUM_2027






TAATT: 0












1996966
GTGG
ATGA
ATGA: 363 
CDS



no annotation
LEUM_2030






GTGG: 0












1996984
GATT
AATC
AATC: 258 
CDS



no annotation
LEUM_2030






GATT: 0












1996993
GGCAGGC
AGCTGGT
AGCTGGT: 241
CDS



no annotation
LEUM_2030






GGCAGGC: 0












1997007
GACCCCGTTCAGGC
ATCCTCGCTCCGGT
ATCCTCGCTCCGGT: 
CDS



no annotation
LEUM_2030






235












GACCCCGTTCAGGC: 0












1997032
CACA
AACG
AACG: 318 
CDS



no annotation
LEUM_2030






CACA: 0












2025691
GCTA
ACTG
ACTG: 240 
CDS



no annotation
LEUM_2060






GCTA: 0












2025829
AACA
GACG
GACG: 213 
CDS



no annotation
LEUM_2060






AACA: 0












2026633
GCAG
ACAA
ACAA: 327 
CDS



no annotation
LEUM_2061






GCAG: 0












2036598
GCCT
ACCC
ACCC: 291 
CDS



no annotation
LEUM_2072






GCCT: 0












2037136
TCGA
CCGT
CCGT: 198 












TCGA: 0












2037152
TAACA
GAACG
GAACG: 210












TAACA: 0












2037383
TCCA
CCCT
CCCT: 285 
CDS



no annotation
LEUM_2073






TCCA: 0












2037417
CGT
TGC
TGC: 259 
CDS



no annotation
LEUM_2073






CGT: 0












2037438
GTATC
TTATT
TTATT: 286 
CDS



no annotation
LEUM_2073






GTATC: 0

















It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.


This application claims priority from Australian Provisional Application No. 2017903944 entitled “Isothiocyanate containing Brassicaceae products and method of preparation thereof” filed on 28 Sep. 2017, the entire contents of which are hereby incorporated by reference.


All publications discussed and/or referenced herein are incorporated herein in their entirety.


Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.


REFERENCES



  • Agerbirk et al. (2012) Phytochemisty 77:16-45.

  • Alvarez-Sieiro et al. (2016) Applied Microbiology and Biotechnology 7:2939-2951.

  • Axelsson et al. (2017) Sci Transl Med 9 (394).

  • Cai and Wang (2016) Food Chem 1; 210:451-6.

  • Capuano et al. (2017) Curr Pharm Des 19:2697-2721.

  • Chuat and Dalmasso (2015) p. 241-251. In Jordan and Dalmasso (ed.), Pulse Field Gel Electrophoresis: Methods and Protocols, vol. 1301. Springer, New York, NY.

  • Dosz and Jeffery (2013) Journal of Functional Foods 5:987-990.

  • Filannino et al. (2015). Food microbiology 46:272-279.

  • Guzman-Lopez et al. (2009). J Ind Microbiol Biotechnol 36:11-20.

  • Halkier et al. (2006) Annual Reviews in Plant Biology 57:303-33.

  • Huang et al. (2002) Journal of agricultural and food chemistry 50 (16), 4437-4444.

  • Jeffery and Araya (2009) Phytochemistry Reviews 8:283-298.

  • Kim and Park (2016) Excli J 15:571-577.

  • Latte et al. (2011) Food & Chemical Toxicology, 49 (12), 3287-3309.

  • Li et al. (2012) Journal of Medicinal Plants Research 6:4796-4803.

  • Moktari et al. (2017) J Cell Commun Signal July 23.

  • Singleton and Rossi (1965) American Journal of Enology and Viticulture 16:144-158.

  • Verkerk et al. (2009) Molecular Nutrition and Food Research 53: S219-S265.

  • Xia and Wishart (2016) Current Protocols in Bioinformatics 55:14.10.1-14.10.91.


Claims
  • 1.-41. (canceled)
  • 42. An isolated strain of lactic acid bacteria selected from: i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia; andii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia.
  • 43. (canceled)
  • 44. A starter culture for producing an isothiocyanate containing product or a probiotic comprising lactic acid bacteria selected from one or more or all of: i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia; andii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia.
  • 45. (canceled)
  • 46. A probiotic composition comprising lactic acid bacteria selected from one or more or all of: i) BF1 deposited under V17/021729 on 25 Sep. 2017 at the National Measurement Institute Australia; andii) BF2 deposited under V17/021730 on 25 Sep. 2017 at the National Measurement Institute Australia.
  • 47.-54. (canceled)
  • 55. The starter culture of claim 44, further comprising a lactic acid bacteria selected from one or more of: i) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;ii) B2 deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;iii) B3 deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;iv) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; andv) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.
  • 56. The probiotic composition of claim 46, further comprising a lactic acid bacteria selected from one or more of: i) B1 deposited under V17/021731 on 25 Sep. 2017 at the National Measurement Institute Australia;ii) B2 deposited under V17/021732 on 25 Sep. 2017 at the National Measurement Institute Australia;iii) B3 deposited under V17/021733 on 25 Sep. 2017 at the National Measurement Institute Australia;iv) B4 deposited under V17/021734 on 25 Sep. 2017 at the National Measurement Institute Australia; andv) B5 deposited under V17/021735 on 25 Sep. 2017 at the National Measurement Institute Australia.
Priority Claims (1)
Number Date Country Kind
2017903944 Sep 2017 AU national
Continuations (1)
Number Date Country
Parent 16651974 Mar 2020 US
Child 18990423 US