Patent Application
20150272144
The present invention relates to a composition comprising at least three different strains of Lactobacillus sakei (L. sakei) bacteria, and its use for preserving edible products, preferably meat products, in particular beef meat and even more particularly beef carpaccio.
Meat products are food which, without preservation treatment, is quickly spoiled, at a rate which depends on various factors: product acidity, ambient moisture content, modified atmosphere, type and load of bacteria, temperature . . . .
Numerous treatments for preserving food, and in particular meat products, are known (fermentation, smoking, salting, drying, addition of preservatives, vacuum packaging . . . ). However, the use of preservatives is regulated. Moreover, most of these treatments cause a transformation of the fresh product (taste, color, texture). It is also known to use protective cultures for bio-preservation purposes, the intended effect being then to insure the microbiological quality without organoleptic change of the product, so as to preserve its fresh character. This implies therefore the use of bacteria, harmless to human health, which will prevent other undesirable bacterial species from being developed, without modifying the food base. Research for engineering good protective cultures has been developing for several years, and the major difficulty lays in the fact that the competition between different bacterial species on complex food substrates results from a set of mechanisms still poorly understood (Devlieghere et al., 2004). Some examples exist (in France: lactic ferment LLO for prawn preservation; in the USA: BovamineMeat™ for meat preservation; in Europe Holdbac™ and SafePro Ranger™ marketed by Danisco and Ch Hansen for meat products preservation). However, existing products only target a single bacterial species (Listeria monocytogenes) and are not concerned with beef carpaccio.
More particularly, as regards meat products, the meat intended to be consumed raw or undercooked (steaks tartare or beef burger, carpaccio for example) or ready-cooked food, are those that can have some significant contamination problems with a repercussion on the consumer health. Indeed, in the case of products consumed raw or undercooked, pathogenic bacteria are not destroyed during cooking, and in the case of ready-cooked food, made sterile by prior cooking, a subsequent contamination can be hazardous because in the absence of competing species, newly contaminating bacteria can develop up to a high degree. This is thus substantially on these types of products that research on protective cultures of meat products has been developed (Bredholt et al., 2001; Vermeiren et al., 2004; Vold et al., 2000). It has been observed that the endogenous flora of meat, and in particular L. sakei, contributes to preventing pathogenic bacteria such as Escherichia coli O157:H7 from being developed in chopped meat. Tests made using anti-Listeria bacteriocin producing strains have shown that they limited the development of Listerias on cooked meat products. However, the effectiveness of bactericins in food bases remains poorly controlled and the range of targets is restricted to a few bacterial species.
There is a very wide diversity of strains of L. sakei the size of the genome of which ranges from 1.8 Mb to 2.3 Mb. This wide diversity is divided into around ten genetic classes within the species. Thus, the inventors have identified different genetic markers enabling the strains of L. sakei to be characterized (international applications WO 2009/106491 and WO 2011/023675).
In spite of the knowledge of this diversity, it is currently impossible to determine whether there is an “ideal” assembly of strains which could fight most efficiently against the alteration of one or more altering species in a meat product, in particular of the beef carpaccio type.
Surprisingly, the inventors have engineered a specific composition comprising 3 classes of L. sakei, well identified and traceable in the product, which allows to fight against several food-altering bacterial species, in particular for meat products, more particularly raw or undercooked meat products and most particularly beef carpaccio and beef burger. These different classes of L. sakei are identifiable by specific markers.
Thus, the present invention relates to a composition comprising at least three different strains of L. sakei bacteria, wherein:
Preferably, said first strain of L. sakei bacteria comprises in its genome the following sequences:
preferably said first strain of L. sakei bacteria comprises in its genome the following sequences:
particularly preferably, said strain is the strain deposited with the CNCM (Collection Nationale de Cultures de Microorganismes; Institut Pasteur, 28 rue du Docteur Roux, F-75724 Paris CEDEX 15) on Nov. 19, 2010 as CNCM I-4396.
Preferably, said second strain of L. sakei bacteria comprises in its genome the following sequences:
preferably said second strain of L. sakei bacteria comprises in its genome the following sequences:
preferably said second strain of L. sakei bacteria comprises in its genome the following sequences:
preferably said strain further comprises one, preferably, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and even preferably 7, 8, 9, 10, 11, 12 of the sequences selected from the group consisting of the sequences: SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, and SEQ ID NO: 41, and/or a sequence exhibiting at least 95% identity thereto respectively,
and particularly preferably, said strain is the strain deposited with the CNCM on Nov. 19, 2010 as CNCM I-4393.
Preferably, said third strain of L. sakei bacteria comprises in its genome the following sequences:
preferably said third strain of L. sakei bacteria comprises in its genome at least the following sequences:
preferably said third strain of L. sakei bacteria comprises in its genome at least the following sequences:
and preferably said strain further comprises one, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 and even preferably 5, 6, 7, 8, 9 of the sequences selected from the group consisting of the sequences: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 41 or a sequence exhibiting at least 95% identity thereto respectively,
and particularly preferably, said strain is the strain deposited with the CNCM on Nov. 19, 2010, as CNCM I-4394.
Thus, preferably, the composition according to the present invention comprises:
Particularly preferably, the composition according to the present invention comprises:
Particularly preferably, the composition according to the present invention comprises:
preferably, said strain further comprising one, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and even preferably 7, 8, 9, 10, 11, 12 of the sequences selected from the group consisting of the sequences: SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, and/or a sequence exhibiting at least 95% identity thereto respectively, and
preferably said third strain of L. sakei bacteria comprising in its genome at least the following sequences:
and preferably said strain further comprises one, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 and even preferably 5, 6, 7, 8, 9 of the sequences selected from the group consisting of the sequences: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 41 or a sequence exhibiting at least 95% identity thereto respectively.
Preferably, the present invention relates to a composition as defined above comprising at least one fourth strain of L. sakei bacteria, different from the other strains defined above.
Preferably, said fourth strain of L. sakei bacteria comprises in its genome at least the sequence SEQ ID NO: 39 or a sequence exhibiting at least 95% identity thereto.
In a preferred embodiment, the fourth strain comprises in its genome at least the following sequences:
and preferably said strain further comprises one, preferably, 1, 2, 3, 4, 5, and even preferably 1 or 2 of the sequences selected from the group consisting of the sequences: SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 32 and SEQ ID NO: 38 or a sequence exhibiting at least 95% identity thereto respectively, and particularly preferably, said strain is the strain deposited with the CNCM on Nov. 19, 2010 as CNCM I-4395.
The genome of the 4 strains of L. sakei deposited with the CNCM as the following numbers: CNCM I-4393, CNCM I-4394, CNCM I-4395 and CNCM I-4396 has been sequenced by the inventors.
Preferably, said composition is a food composition. For the purposes of the present invention, by “food composition”, it is intended a composition likely to be integrated in a food product, that is a composition suitable for consumption. By “suitable for consumption”, it is meant a product assimilable by an individual, preferably a human, safely, that is not likely to generate a pathology in the short term.
Preferably, said composition is formulated so as to guarantee the viability of said at least three strains, preferably of said at least four strains.
Preferably, for the purposes of the present invention, the nucleotide sequences used as markers of the strains of L. sakei according to the present invention correspond to a nucleotide sequence exhibiting at least 95%, 96%, 97%, 98%, 99% identity with the sequences defined herein above.
By nucleotide sequence exhibiting at least 95% identity, preferably at least 96%, 97%, 98%, 99% identity to a sequence, it is intended the polynucleotides exhibiting, with respect to the reference polynucleotide, some modifications such as in particular a deletion, truncation, elongation, chimeric fusion and/or substitution, in particular a point one. These are preferably sequences coding the same amino acid sequences as the reference sequence, this related to the degeneracy of the genetic code, or complementary sequences which are likely to specifically hybridize to the reference nucleotide sequences, preferably under high stringency conditions.
A hybridization under high stringency conditions means that the temperature and ionic strength conditions are selected such that they enable the hybridization to be held between two fragments with complementary DNAs.
The percent identities referred to within the scope of the disclosure of the present invention are determined based on an overall alignment of sequences to be compared, that is an alignment of the sequences taken in their entirety, using for example the NEEDLEMAN and WUNSCH algorithm (J. Mol. Biol. 48, 443-453, 1970). This sequence comparison can be performed for example with the needle software using the parameter “Gap open” equal to 10.0, the parameter “Gap Extend” equal to 0.5, and a matrix “Blosum 62”. Needle software is for example available on the web site www.ebi.ac.uk, under the name “Align”.
Particularly preferably, the composition as previously defined comprises at least one, preferably two, preferably three, and even preferably four strains of L. sakei selected from the group consisting of: the strains deposited with the CNCM on Nov. 19, 2010 as the following numbers: CNCM I-4393, CNCM I-4394, CNCM I-4395 and CNCM I-4396.
Thus, preferably, the composition as previously defined comprises at least the 4 strains of L. sakei deposited with the CNCM as the following numbers: CNCM I-4393, CNCM I-4394, CNCM I-4395 and CNCM I-4396.
In a particular embodiment, the composition as previously defined does not comprise strains of L. sakei other than the four strains as defined above.
Preferably, the composition according to the present invention can comprise other preservative agents selected from the group comprising: pickles, such as red wine or soya based pickles, essential oils and bacteriocins.
Preferably, the composition according to the present invention comprises the at least four strains at equivalent concentrations.
Preferably, said strains are present at concentrations enabling the target flora to be desirably reduced in the edible product. Preferably, each of said strains is present in the composition at a concentration between about 103 and 108 cfu/mL, preferably at a concentration between about 104 and 107 cfu/mL, particularly preferably at a concentration between 105 and 106 cfu/mL.
Another aspect of the present invention relates to a method for preparing the composition according to the present invention comprising a step of culturing at least three strains, preferably at least four strains of L. sakei such as defined herein before in a food composition.
Preferably, said at least three strains, preferably at least four strains of L. sakei are introduced at equivalent concentrations.
Preferably, the at least three strains, preferably at least four strains of L. sakei are introduced at a concentration between 103 and 108 cfu/mL of food composition, preferably at a concentration between 104 and 107 cfu/mL of food composition, particularly preferably at a concentration between 105 and 106 cfu/mL of food composition.
Another aspect of the present invention relates to the use of a composition as defined herein before for preserving an edible product, preferably a meat product, in particular beef meat and even more particularly beef carpaccio or chopped beef and particularly preferably beef carpaccio.
Preferably, the meat product is raw or undercooked.
The chopped beef can for example be intended to be consumed as a beef tartare.
The composition according to the present invention enables the edible product to be preserved by virtue of a growth inhibition of some microorganisms likely to generate an alteration of said edible product, in particular during product storage.
The measurement of the alteration can be evaluated by the difference in the concentration of said microorganisms between the storage initial and final times, and can then be expressed as Δ Log10 CFU/g of edible product between the final time and initial time.
The alteration of said edible product can in some cases generate a pathological response in an individual.
By way of example of microorganisms likely to generate an alteration of the edible product, there can be mentioned for example Enterobacteria from the group Serratia (Serratia, Hafnia, Obesumbacterium, Rahnella), Leuconostocs from the group mesenteroides (mesenteroides-carnosum-gasicomitatum-gelidum), Pseudomonas selected from the species P. fragi, P. putida and P. fluorescens, and some species, more specifically as Hafnia alvei, Brochothrix thermosphacta and Weisseila viridescens . . . .
Alteration is a phenomenon related to the growth of altering species and the amount of population reached by these species during storage.
For the purposes of the present invention, by “carpaccio”, it is intended thin slices, preferably with a thickness between 0.1 mm and 1 mm, of a raw edible product.
Another aspect of the present invention relates to a method for preserving an edible product as previously defined comprising a step of contacting said edible product with a composition as previously defined.
Preferably, said contacting step is performed before the step of packaging said edible product.
Preferably, said contacting step is performed by dipping said edible product into a composition as previously defined.
Preferably, said edible product is dipped into the composition as previously defined for 1 to 30 seconds, preferably 2 to 10 seconds, even preferably 4 to 6 seconds and particularly preferably for 5 seconds.
Alternatively, the contacting step is performed by spraying the composition according to the present invention onto said edible product.
For the purposes of the present invention, by “spraying”, it is meant atomizing said composition onto said edible product.
Alternatively, the contacting step is performed by blending the composition according to the present invention, preferably in a lyophilized form, with said edible product.
Preferably, the contacting step performed by blending the composition according to the present invention, preferably in lyophilized form, with said edible product is implemented when said edible product is beef burger.
Preferably, the contacting step is performed after the step of slicing or chopping the edible product, preferably meat.
Particularly preferably, the contacting step is performed for less than 1 hour, preferably less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes and preferably less than 1 minute after the step of slicing or chopping the edible product, preferably meat.
Preferably, the contacting step is followed by a step of vacuumizing or placing under protective atmosphere (such as an atmosphere comprising nitrogen, carbon dioxide and oxygen) of said edible product.
Particularly preferably, the vacuumizing step is performed for less than 1 hour, preferably less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes and preferably less than 1 minute after the contacting step.
Finally, the present invention relates to an edible product comprising a composition as previously defined.
Preferably, said edible product is selected from meat products, in particular a raw or undercooked meat product.
Preferably, said meat product, in particular said raw or undercooked meat product is beef meat, more particularly beef carpaccio or chopped beef and particularly preferably beef carpaccio.
The chopped beef can for example be intended to be consumed as a beef tartare.
Preferably, each of said strains is present in said edible product at a concentration between 102 and 107 cfu/g of edible product, preferably at a concentration between 103 and 106 cfu/g of edible product, particularly preferably at a concentration between 104 and 105 cfu/g of edible product.
Preferably, said edible product comprises a total concentration of strains of the composition between 103 and 109 cfu/g of edible product, preferably between 103 and 104 cfu/g and 108 cfu/g of edible product after contacting with the composition according to the present invention.
Preferably, said edible product comprises a total concentration of strains of the composition between 104 cfu/g and 108 cfu/g of edible product, preferably between 105 cfu/g and 107 cfu/g of edible product at the recommended last consumption date.
Each circle corresponds to an experimental datum, the data giving a straight line. The accuracy of 0.1 Log 10 and the error margin of 0.3 Log 10 of data obtained by q-PCR are wholly smaller than those known for bacterial counts made on culture media.
This CGE value is schematized as circles of different colors depending on the type of comparison performed (the scale is expressed in ΣΔ Log 10 CFU/g of meat and is measured at T14 days for the different samples (see storage conditions description in the caption of
The values of control No 2 (6 other batches without adding a composition) can be compared with those obtained with the same batches treated with other 4-strain compositions used during the screening process, that is 3 series (noted PPB1 to PPB3) of 7 compositions.
The experiments have been made on carpaccio purchased in the market, thus naturally contaminated.
a. Quantization of Altering Bacteria Naturally Present in Carpaccio
Some genera or species of altering bacteria were systematically present and readily quantifiable. They have thus been quantified without requiring a further addition to carpaccio (Enterobacteria of the Serratia group (including Hafnia alvei) (SER), Leuconostocs (mesenteroides-carnosum-gasicomitatum-gelidum) and Pseudomonas (fragi-putida-fluorescens) (PSD)).
b. Quantization of Altering Bacteria after Intentional Seeding of the Carpaccio
On the contrary, other genera or species of altering bacteria could be present but more hardly quantifiable, or poorly developing, or at very variable rates. These have been quantified both as natural and after an intentional seeding (Hafnia alvei, strain CIP57.31 (HAL) Brochothrix thermosphacta, strain INRA 160*7 (BTH), Weisseila viridescens, strain CIP102810 (WVI)) contaminants.
Therefore, an intentional inoculation protocol has been engineered where a bacterial suspension of a known load (in CFU/mL of suspension) was used to seed the carpaccio by dipping/dripping and then the load of carpaccio was measured (in CFU/g of carpaccio). Then, a chart has been used where the initial concentration of the bacterial suspension used to dip the carpaccios was sufficient to deduce the load of intentionally inoculated carpaccio.
An alteration protocol has been engineered which allowed a measurable development of different species: after inoculation by the compositions to be tested, the samples have been vacuum packaged, and then placed 7 days at 8° C., 24 hours at 22° C. and finally 6 days at 8° C.
The quantitative measurements of different bacterial genera/groups/species are generally performed by spreading on more or less selective culture media. A protocol to set these measurements by quantitative real time PCR (q-PCR) has been developed. It required engineering (including the specificity and effectiveness test) primers which enable bacterial species/genera/groups to be measured from the bacterial DNA extracted from the carpaccios. Once the effectiveness and specificity of the primers have been tested, standard ranges have been made to have the correspondence Ct (q-PCR datum on the DNA extracted from the carpaccios)/CFU (experimental datum corresponding to the number of bacteria inoculated on the standard carpaccios) (for example, quantization of Hafnia alvei CIP57.31 by qPCR, see
The following primers have been used:
The effect of a composition can be directly measured by measuring Δ Log10 CFU/g between T14 days and T0 for altering species.
It appears that the quality of the assembly of the strains is important to fight against alteration and that it is not sufficient to use a single strain of Lactobacillus sakei or to assemble randomly several strains to achieve a barrier effect.
The notion of the most or least effective composition is defined by the minimum or maximum value obtained by cumulating all the values of Δ Log10 CFU/g for the altering bacteria. This arbitrary measurement enables the effect of a composition on all the altering species to be assessed. Thus, an active composition against Brochothrix thermosphacta can turn out to be poorly active against Leuconostocs.
Table 1 hereinafter shows the overall effectiveness of the composition according to the invention by comparison with the effectiveness of the 21 other compositions of L. sakei and controls without adding composition. The results are expressed in Δ Log10 CFU/g for the target species.
Leuconostocs
Brochothrix
Hafnia)
fluorescens)
carnosum)
Hafnia alvei
thermosphacta
aControl = 12 untreated carpaccio samples having a natural population of L. sakei
bComposition of the invention = 6 carpaccio samples treated with the composition of the present invention
cOther compositions = 21 carpaccio samples treated with 21 different cocktails of 4 strains of L. sakei
Table 1 shows, if the median values are considered, that:
The composition according to the invention has a single molecular imprinting, enabling molecular markers specific to each of the strains of the composition to be selected. An accurate quantization of the strains of the composition according to the present invention within a meat product as carpaccio is thus possible.
Marker katA (SEQ ID NO: 1), specific to the L. sakei species (present in all the strains, that is those of the composition according to the present invention but also those of the flora of L. sakei naturally present on meat) has been used as a positive control.
The markers of the sequences SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 21, and SEQ ID NO: 26 are each specific of a single of the 4 strains making up the composition according to the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1257785 | Aug 2012 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/066886 | 8/13/2013 | WO | 00 |
