Patent Grant
11272716
The present application is the U.S. National Stage of International Application PCT/EP2017/071352, filed Aug. 24, 2017, and claims priority to International Application No. PCT/DK2016/000031, filed Sep. 1, 2016.
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 18, 2019, is named 030427-0292_SL.txt and is 1,748 bytes in size.
The present invention relates to a bacterial cell with texturizing property, starter cultures comprising the cell, and dairy products manufactured using the bacterial cell.
The food industry uses numerous bacteria, in particular lactic acid bacteria (LAB), in order to improve the taste and the texture of foods but also in order to extend the shelf life of these foods. In the case of the dairy industry, LAB are used extensively in order to bring about the acidification of milk (by fermentation) but also in order to texturize the product into which they are incorporated.
Among the LAB used in the food industry, there can be mentioned the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Bifidobacterium.
The LAB are used extensively alone or in combination with other bacteria for the production of food products, in particular fermented products. They are used in particular in the formulation of the starter cultures used for the production of fermented milks, for example yogurts.
Certain of them play a dominant role in the development of the texture of the fermented product. This characteristic is closely linked to the production of polysaccharides. Among the strains of Streptococcus thermophilus and Lactobacillus bulgaricus it is possible to distinguish texturizing and non-texturizing strains.
In order to meet the requirements of the industry, it has become necessary to propose novel texturizing strains of LAB, in particular of Streptococcus thermophilus and Lactobacillus species. One specific focus could be to reduce for example the amount of proteins or pectin (both improving texture) in dairy products, as these are both costly.
Thus the problem that the invention proposes to resolve is to provide a strain of lactic acid bacterium having good properties for texturizing food products, especially products where the texturizing strain of the lactic acid bacterium (such as Streptococcus thermophilus) is used together with a strain of a Lactobacillus species.
The present inventors have surprisingly found that the concentration of metal ions in lactic acid bacteria cells, or in starter cultures containing lactic acid bacteria cells, influence the formation of texture when such bacteria cells, or starter cultures, are used for the production of fermented milk.
In accordance with this surprising finding, the present invention relates to novel LAB strains with improved texturizing properties, and a method for producing such strains, and to fermented milk products made using such strains.
Thus, an aspect of the present invention relates to a composition of lactic acid bacteria that are able to texturize a milk substrate, said composition comprises lactic acid bacteria having a lowered intracellular concentration of metal ions, and/or said composition comprises a lowered amount of metal ions.
It is contemplated that such cells can be obtained in different ways, such as:
Relating to alternative A), the present inventors describe the finding that mutants of texturizing LAB which are perturbed (such as decreased, suppressed) in their ion metabolism will give superior texture in a fermented milk product.
In a second aspect, the present invention relates to a method for increasing the viscosity of a dairy product fermented with LAB, said method comprises fermenting a milk substrate with reduced iron (esp. Fe2+) and/or manganese (esp. Mn2+) content, or fermenting a milk substrate where at least one of said metal ions are made partly or fully inaccessible for the LAB.
The present invention is based on the surprisingly finding that milk fermented with S. thermophilus strain CHCC15712—isolated as a mutant of the exopolysaccharide (EPS) producing strain S. thermophilus CHCC9844—showed increased shear stress as well as increased gel stiffness, two important rheology parameters which are believed to be related with texturing properties in yoghurt and yoghurt type products. Viscosity, measured by monitoring the efflux time from a volumetric pipette, was increased as well (see example 1).
The isolation and characterisation of EPS from CHCC9844 and CHCC15712 showed an increase of EPS production for CHCC15712 by 9% which correlates well with the viscosity increase (example 2).
Analyzing the mutant CHCC15712, the inventors surprisingly revealed that the cells contained an abnormal low concentration of Fe2+ and other metal ions compared to CHCC9844.
The inventors investigated the possible mechanism underlying the surprising finding, and in the genome sequence of CHCC9844 they identified in the upstream region of the EpsA gene, a part of the CHCC9844 eps operon, a sequence which is highly homologous to the Fur (ferric uptake regulator) box, indicating that Fur is involved in the regulation of EPS expression of CHCC9844 (
Without wishing to be bound to a specific theory or hypothesis, it is believed that EpsA expression is under control of Fur and de-repression of EpsA leads to increased EPS production and increased texture. Any mutant with impaired divalent metal uptake will therefore give increased texture, compared to a mother strain which has normal uptake.
By DNA array (expression microarray) analysis of CHCC15712 in comparison with CHCC9844 it was shown that a specific iron uptake gene cassette (Fat operon) was down regulated. The fatABC genes were about 4-fold lower expressed in CHCC15712 and fatD was 2-fold lower expressed compared to CHCC9844.
Investigations Relating to Alternative A) Above:
The inventors speculated that this down-regulation of the iron transporter will result in a reduced intracellular Fe2+ or Fe3+ concentration which again will lead to less binding of the Fur regulator to Fur boxes. Since Fur works as a repressor, genes under control of Fur are therefore de-repressed by reduced Fe2+/3+ concentrations.
A down regulation of all other genes involved in uptake of iron, like e.g. the divalent metal ion ABC uptake system (NRAMP), would also lead to a lower intracellular iron concentration and, via Fur regulation, to increased texture.
The hypothesis was tested by making a genetically engineered mutant of CHCC9844 where the fur gene was inactivated. The effect on texture was measured using the pipette efflux test. The wt strain CHCC9844 had a pipette value of 24±1 s whereas the fur mutant KA509 measured 38±0 s corresponding to a 58% increase (example 3).
The effect of the fur inactivation on gene expression was also estimated using DNA microarrays. The genes fatABDC involved in iron uptake are known to be regulated by Fur. In KA509 these genes were down regulated 4-fold compared to the CHCC9844 wt strain. When KA509 was grown in presence of 10 mM Fe2+ there was no substantial changes in expression of the fat gene cluster. Contrarily, when the mother strain CHCC9844 was grown in presence of iron, the fatC and fatD genes were induced 4-fold.
Investigations Relating to Alternative B) Above:
The hypothesis was tested further by performing milk acidification experiments in the presence of EDTA, a metal chelator. Addition of EDTA should reduce the amount of available iron and therefore result in de-repressing of EpsA resulting in increased texture. Indeed it was observed that the addition of 1 mM EDTA to milk increased the texture of milk fermented with CHCC15712 by 25% (example 5).
Investigations Relating to Alternative C) Above:
Based on the finding that mutants from CHCC9844 showed reduced expression of genes related to iron uptake, tellurite resistant mutants were isolated from CHCC9844.
The efflux time using the pipette test, and by this the viscosity, was increased for the tellurite resistant mutants. The highest increase was measured for mutant 9844-K2 with 47% (example 4).
With this experiment it was demonstrated that it is possible to increase the texturing properties of eps positive S. thermophilus strains further by the isolation of tellurite resistant mutants. The present invention also includes this embodiment.
Based on the above surprising findings, the present invention in a first aspect relates to a bacterium cell, such as lactic acid bacterium (LAB), which intracellularly contains less than 4200 ppm (parts per million, mg/Kg dry weight) in total of all divalent metal ions, such as less than 4100, less than 4000, 3000 or less than 2000 ppm. Interesting embodiments of such a bacterial cell are:
Further interesting embodiments are:
Mn2+, Te2+, Ca2+, Zn2+, Co2+, Ni2+ and Cu2+.
Interesting embodiments of the second and third aspects are:
Other interesting aspects (claim aspects) of the invention are listed below:
and/or which optionally is in the form of a stirred type product, a set type product, or a drinkable product.
As used herein, the term “lactic acid bacterium” (short LAB) designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order “Lactobacillales” which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp., are generally included in the group of lactic acid bacteria. These are frequently used as culture in food fermentations alone or in combination with other lactic acid bacteria. Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus, are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called “Direct Vat Set” (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product. Such cultures are in general referred to as “starter cultures” or “starters”. Some dairies also employ “bulk starters” where the culture is propagated onsite before inoculation into for example milk.
In the present context, the term “milk substrate” may be any raw and/or processed milk material that can be subjected to fermentation according to the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions/suspensions of any milk or milk like products comprising protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk substrate may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder. Preferably, at least part of the protein in the milk substrate is proteins naturally occurring in milk, such as casein or whey protein. However, part of the protein may be proteins which are not naturally occurring in milk. Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.
The term “milk” is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also comprises milk made of plant materials, such as soy milk. Optionally the milk is acidified, e.g. by addition of an acid (such as citric, acetic or lactic acid), or mixed, e.g. with water. The milk may be raw or processed, e.g. by filtering, sterilizing, pasteurizing, homogenizing etc., or it may be reconstituted dried milk. An important example of “bovine milk” according to the present invention is pasteurized cow's milk. It is understood that the milk may be acidified, mixed or processed before, during and/or after the inoculation with bacteria.
Cow milk contains iron in a concentration about 0.03 mg per 100 g=0.3 μg/g=0.3 ppm Cow milk contains manganese in a concentration about 0.03 μg/g=0.03 ppm
In the present context, the term “mutant” should be understood as a strain derived from a strain of the invention by means of e.g. genetic engineering, radiation and/or chemical treatment, and/or selection, adaptation, screening, etc. The term also includes mutants with improved or altered phage resistance, e.g. phage hardened mutants. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding yield, viscosity, gel stiffness, mouth coating, flavor, post acidification, acidification speed, and/or phage robustness) as the mother strain. In the present context a mutant of the invention is preferable a mutant with same or improved properties with respect to EPS production, and/or for generating viscosity when a milk substrate is fermented. Such a mutant is a part of the present invention. Especially, the term “mutant” refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. The term mutant also includes a tellurite resistant mutant. By the term tellurite resistant mutant is understood a mutant which is able to grow (form a colony) on M17 agar plates containing 0.1 mM K2TeO3.
A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenisation step followed by a screening/selection step), but it is presently preferred that no more than 1000, no more than 100, no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion or a combination thereof) compared to the mother strain.
In the present context, the term “variant” should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties e.g. regarding viscosity, gel stiffness, mouth coating, flavor, post acidification, acidification speed, and/or phage robustness). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.
By inactivation of a gene is meant that the gene (including promotor and other regulatory regions) is amended in such a way that the gene or its product loses its activity (partly or fully). Inactivation can, for instance, be deletion (partly or fully) of the gene, mutation of the gene (e.g., by genetic engineering or mutagenisis), provision of knock-out mutants, introduction of frame shift, insertion of e.g. stop codons, etc.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having”, “including” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
S. thermophilus CHCC15712 was isolated as a mutant of the exopolysaccharide producing strain S. thermophilus CHCC9844. Milk fermented with CHCC15712 showed increased shear stress as well as increased gel stiffness, two important rheology parameters which are believed to be related with texturing properties in yoghurt type products. Viscosity, measured by monitoring the efflux time from a volumetric pipette was increased as well.
Viscosity is measured by monitoring the efflux time from a 25 ml volumetric pipette, which has been filled with 25 ml of a fermented milk prepared by 16 hours of growth of the bacterium in 9.5% reconstituted skim milk at 37 degrees C., when the milk is inoculated with 10E8 CFU per ml.
The rheological properties of the sample were assessed on a rheometer (StressTech, Reologica Instruments, Sweden) equipped with a C25 coaxial measuring system. The viscometry test was made with shear rates varying from 0.27 to 300 1/s in 21 steps. Shear rates were increased and then decreased and the upward and downward curves of shear stress and apparent viscosity were recorded. Delay and integration times were 5 s and 10 s, respectively. For further analysis, shear stress at 300 s-1 was chosen.
The rheometer results showed that CHCC15712 had shear stress and gel stiffness increased by 20% compared to CHCC9844.
Viscosity was also measured by calculating the efflux time from a 25 ml volumetric pipette. For CHCC15712 an increase of viscosity by 9% was measured compared to the wild type strain CHCC9844 (see
The two strains CHCC9844 and CHCC15712 were grown at 37° C. in M17 broth with 1% lactose for 16 hours. The cells were removed by centrifugation and EPS was extracted from the supernatant by precipitating proteins with 96% trichloroacetic acid solution (TCA), and afterwards precipitating EPS with 96% ethanol. The EPS samples were then dialyzed against Milli Q-water at 4° C. for at least 24 hours (Slide-A-lyzer dialysis cassette, 0.5-3 ml, MWCO 10000, Thermo scientific). EPS was then hydrolysed with 4M TFA (trifluoroacetic acid) and analysed for composition and amount of monosaccharides by HPLC.
The HPLC analysis revealed the presence of rhamnose, galactosamine, galactose, glucose, and mannose in the EPS of CHCC9844 and CHCC15712 (see
Strain KA509 is a mutant of CHCC9844 where the fur gene was genetically inactivated. This was done by single crossover of a plasmid vector harbouring an internal fragment containing bases 2-385 of the CHCC9844 fur gene. The resulting mutant has the vector inserted after the first nucleotide of the 485 bp long fur CDS (codon sequence). This results in disruption of the gene which is hence rendered inactive. Correct insertion was checked by PCR using one primer placed in the vector and one placed in the fur gene downstream of the internal fragment.
The effect on texture was checked using the pipette efflux test. The wt strain CHCC9844 had a pipette value of 24±1 s whereas the fur mutant measured 38±0 s corresponding to 58% increase (
The effect of the fur inactivation on gene expression was also estimated using DNA microarrays. In KA509 the genes fatABDC were down regulated 4-fold compared to the CHCC9844 wt strain. When KA509 was grown in presence of 10 mM Fe2+ there was no substantial changes in expression of the fat gene cluster. Contrarily, when the mother strain CHCC9844 was grown in the presence of iron, the fatC and fatD genes were induced 4-fold.
Based on our finding that mutants from CHCC9844 showed reduced expression of genes related to iron uptake, tellurite resistant mutants were isolated from CHCC9844.
The mutants were directly screened on M17 agar plates containing 0.1 mM K2TeO3. Four mutants were purified demonstrating a stable tellurite resistant phenotype. The four CHCC9844 mutants were inoculated from an M17 overnight culture containing 0.1 mM K2TeO3 1% in milk, and incubated at 37° C. for 24 hours. The viscosity of the fermented milk was then determined by measuring efflux time from a 25 ml pipette. The longer the efflux time the higher is the viscosity of the test medium: for each strain the efflux time is indicated as an average of three measurements (see
The efflux time, and by this the viscosity, was increased for the four tellurite resistant mutants. The highest increase was measured for mutant 9844-K2 with 47%.
With this experiment it was demonstrated that it is possible to increase the texturing properties of eps positive S. thermophilus strains further by the isolation of tellurite resistant mutants.
The hypothesis that a reduced iron concentration modulates viscosity was tested by performing milk acidification in the presence of EDTA, a metal chelator. Addition of EDTA should reduce the amount of available iron and therefore result in a de-repressing of EpsA resulting in increased texture. Indeed it was observed that the addition of 1 mM EDTA to milk increased the texture of CHCC15712 by 25% (
Strains of Streptococcus thermophilus without a perturbed divalent ion metabolism have been analysed for content of intracellular divalent ion concentrations.
For determination of intracellular ion concentration cells were grown over night in M17-2% lactose.
After centrifugation of 50 ml for 10 min at 5000 rpm, the cells were washed with 50 ml M17-2% lactose medium. All tubes were centrifuged at 5000 rpm for 10 minutes, supernatant discarded, and cells were then washed with 5 ml PBS buffer (treated o/n with Chelex, 10 g in 1 Litre buffer). All tubes were centrifuged for 10 minutes at 5000 rpm, and then washed with 5 ml PBS buffer, centrifuged for 10 minutes at 5000 rpm and washed/resuspended in 1 ml PBS buffer and transferred to a 2 ml Eppendorf tube. The tubes were then centrifuged for 15 minutes at 15.000×g and the supernatants were discarded as much as possible.
The tubes were dried in a speedvac o/n at RT (room temperature). All pellets were weighed.
After weighing, the pellets were solubilized by the addition of 200 μL concentrated (65%) Nitric acid with 0.1% Triton X-100, shaking the tubes at 95° C. for 10 minutes and afterwards vortexing each tube for 20 seconds.
After vortexing, the tubes were centrifuged at 15.000×g for 5 minutes and the supernatant was taken off in a new tube.
Metal ion concentration of the samples was determined by ICP-MS (Inductively Coupled Plasma Mass Spectrometry).
The results (see
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The strain Streptococcus thermophilus CHCC15712 was deposited at DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM25955, on Apr. 27, 2012.
CHCC4895 was deposited with DSMZ under the accession no. DSM19242, on 29 Mar. 2007
CHCC8833 was deposited with DSMZ under the accession no. DSM17876, on 11 Jan. 2006.
The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
The Applicant requests that a sample of the deposited microorganism should be made available only to an expert approved by the Applicant.
All references cited in this patent document are hereby incorporated herein in their entirety by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/DK2016/000031 | Sep 2016 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/071352 | 8/24/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/041717 | 3/8/2018 | WO | A |
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|---|---|---|---|
| 6087559 | Nichols | Jul 2000 | A |
| 7582743 | Horvath et al. | Sep 2009 | B2 |
| 9453231 | Kibenich et al. | Sep 2016 | B2 |
| 20040146983 | Caufield et al. | Jul 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| 102965318 | Mar 2013 | CN |
| WO-03102204 | Dec 2003 | WO |
| WO-2004085607 | Oct 2004 | WO |
| WO-2011026863 | Mar 2011 | WO |
| WO-2012052557 | Apr 2012 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20190320673 A1 | Oct 2019 | US |
