EP3047015A1

EP3047015A1 - Use of lactobacillus sakei for the biopreservation of products from the sea

Info

Publication number: EP3047015A1
Application number: EP14722297.0A
Authority: EP
Inventors: Gaëtan PODEUR; Françoise LEROI; Marie-France PILET; Hervé PREVOST
Original assignee: Institut Francais de Recherche pour lExploitation de la Mer (IFREMER); Oniris SA
Current assignee: Institut Francais de Recherche pour lExploitation de la Mer (IFREMER); Oniris SA
Priority date: 2013-04-19
Filing date: 2014-04-18
Publication date: 2016-07-27
Also published as: FR3004725A1; US20160143302A1; CA2909790A1; FR3004725B1; WO2014170621A1

Abstract

The invention relates to a strain of Lactobacillus sake called Lactobacillus sakei LHIS2885 strain, and to a strain derived from L. sakei LHIS2885. The invention also relates to the use of a strain of L. sakei, preferably the strain LHIS2885 or a strain derived from LHIS2885, for the preservation of food products, preferably products from the sea.

Description

USE OF LACTOBACILLUS SAKEI FOR BIOPRESERVATION

PRODUCTS OF THE SEA

FIELD OF THE INVENTION The present invention relates to the biopreservation of food products, preferably seafood. More specifically, the present invention relates to the bacterial strain Lactobacillus sakei LHIS2885 and its use for limiting histamine formation in food products. , preferably seafood.

STATE OF THE ART

Seafood is responsible for 10 to 20% of human food poisoning. Of these, histamine is the leading cause of fish-related toxi-infections. Thus, 30 to 40% of the epidemics related to fish consumption are due to histamine. This toxi-infection results in cutaneous (redness, urticaria), neurological (headache), gastrointestinal (diarrhea, vomiting), palpitations and edema symptoms that can sometimes lead to hospitalization and death of weakened patients.

Histamine poisoning is related to the consumption of fish rich in histidine (such as, for example, tuna, mackerel, sardines or anchovies) and contaminated with histamine concentrations greater than 500 mg / kg.

Histamine is formed in fish rich in histidine by a bacterial enzyme, histidine decarboxylase. Since histamine is resistant to cooking, canning, smoking and freezing, the fight against the histamine-induced infections involves the inhibition of the production of this biogenic amine, for example via the inhibition of the development of histamine producing bacteria on the products, or via the inhibition of histidine decarboxylase. Histamine can be formed by mesophilic bacterial species at temperatures above 7-10 ° C. The latter, however, are unable to form histamine at temperatures between 0-5 ° C, that is to say the temperature required for the conservation of seafood according to US or European standards. However, recent studies have demonstrated the existence of psychrotolerant, highly histamine-producing bacteria at 0-5 ° C (Emborg et al., 2005, Kanki et al., 2004). Examples of these bacteria are Morganella morganii and Morganella psychrotolerans which was recently isolated by the team of Paw Dalgaard (Emborg et al., 2006), as well as the bacteria Photobacterium phosphoreum and Photobacterium damselae.

The formation of histamine in the event of a cold chain break, and even at the recommended temperatures for the storage of seafood is therefore a major problem for the fishing industry.

One of the strategies for combating food contamination is biopreservation. Biopreservation is the addition to the product of microorganisms selected for their ability to inhibit the growth of undesirable microorganisms that are responsible for the alteration of the product or pathogenicity inducers. The addition of these microorganisms can thus increase the shelf life of the product, and preserve its organoleptic properties. Thus, for example, US Pat. No. 5,576,035 describes the use of bacteria for the preservation of food products, in particular meat. On the other hand, the European patent EP 1 456 350 describes the use of a strain of lactic acid bacterium, Lactococcus lactis, for the preservation of seafood products.

However, to the knowledge of the Applicant, no biopreservation process has been described to date, to prevent or control the production of histamine in seafood products.

In its search for a ferment that can be used in such a biopreservation process, the Applicant has identified and isolated the bacterium Lactobacillus sakei LHIS2885. The addition of this bacterium to a food product, preferably a seafood, such as, for example, cooked fish, can surprisingly reduce the production of histamine therein. Moreover, this effect is observed not only at the usual preservation temperature, that is to say 4 ° C, but also at a temperature of 15 ° C, which allows the product to be protected from also in the event of a break in the cold chain.

ABSTRACT

The present invention therefore relates to the use of a strain of Lactobacillus sakei, one of its fragments or a composition comprising it for the preservation of food products, preferably seafood. According to one embodiment of the invention, the production of histamine in said food products is inhibited. According to another embodiment of the invention, the growth of histaminogenic bacteria in said food products is inhibited.

According to one embodiment of the invention, the food products are products of the sea, preferably fish, more preferably tuna, mackerel, sardine or herring. According to one embodiment of the invention, said food products are cooked products, fresh products, frozen products, vacuum packaged products, products packaged in a modified atmosphere, smoked products, products packaged in a can and / or marinated products.

According to one embodiment of the invention, a quantity of bacteria ranging from about 10 2 to about 10 12 cfu of L. sakei I g of food product is applied to the food product.

The present invention furthermore relates to a strain of Lactobacillus sakei called strain Lactobacillus sakei LHIS2885 and deposited at the CNCM under the number CNCM I-4704. The present invention also relates to a strain derived from Lactobacillus sakei obtained by mutation, variation or recombination of the strain. as described above. For example, said derived strain may comprise a genome having greater than 70% identity with the genome of the strain Lactobacillus sakei LHIS2885. The The present invention also relates to a composition comprising the bacterial strain or the derived strain as described above or a fragment thereof.

The present invention therefore also relates to the use of strain LHIS2885, a strain derived from LHIS2885, or a composition comprising them for the preservation of food products, preferably seafood products.

According to one embodiment, the production of histamine in said food products is inhibited, preferably the growth of histaminogenic bacteria in said food products is inhibited.

According to one embodiment, the food products are products of the sea, preferably fish, more preferably tuna, mackerel, sardine or herring.

In another embodiment, said food products are cooked products, fresh products, frozen products, vacuum packed products, modified atmosphere packaged products, smoked products, canned goods, and / or or marinated products.

According to one embodiment, a quantity of bacteria ranging from about 10 2 to about 101 ¹ 2 ufc of L. sakei LHIS2885 or from a derived strain / g of food product is applied to the food product.

The present invention also relates to a process for the preparation of food products, preferably seafood products, comprising a step of applying to said products a L. sakei strain or one of its fragments, such that, for example, L. sakei strain LHIS2885, a strain derived from L. sakei LHIS2885 or one of their fragments.

The invention also relates to a food product, preferably seafood, comprising a strain of L. sakei, such as, for example, the strain of L. sakei LHIS2885, a strain derived from L. sakei LHIS2885 or one of their fragments. DEFINITIONS

In the present invention, the terms below are defined as follows:

- "About" placed in front of a number, means plus or minus 10% of the nominal value of that number.

DETAILED DESCRIPTION

The present invention relates to a new strain of lactic bacteria, namely the bacterial strain Lactobacillus sakei LHIS2885. According to one embodiment of the invention, the strain Lactobacillus sakei LHIS2885 is isolated. This bacterial strain belongs to the genus Lactobacillus and the sakei species. It is a Gram bacillus + catalase-oxidase-, homo heterofermentaire optional. If the taxonomy were to be modified, those skilled in the art could adapt the taxonomy modifications to deduce the bacterium usable in the present invention.

L. sakei strain LHIS2885 was isolated from fresh sardine meat packaged under modified atmosphere (50% N ₂ + 50% C0 ₂ ).

A sample of the culture of the microorganism according to the present invention, called strain Lactobacillus sakei LHIS2885 was deposited on December 12, 2012 under the Treaty of Budapest in the national collection of French microorganism culture (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15. The sample received the number N ° CNCM 1-4704.

The inventors have demonstrated that the strain Lactobacillus sakei LHIS2885 was able to inhibit the growth of histaminogenic bacteria, and to inhibit the production of histamine on food products contaminated with histaminogenic bacteria. Examples of histaminogenic bacteria include, but are not limited to, Morganella psychrotolerans, Morganella morganii, Photobacterium phosphoreum, Photobacterium damselae and Hafiiia alvei. According to a first embodiment of the invention, the capacity for inhibiting the growth of histaminogenic bacteria is evaluated by a petri dish inhibition test, by measuring the halos of inhibition.

Bacterial growth inhibition tests on a petri dish are well known to those skilled in the art. An example of a bacterial growth inhibition test on a petri dish is described below.

According to one embodiment, this test can be performed by the double layer method. Advantageously, the L. sakei strain LHIS2885 is cultured in broth, preferably in MRS broth (from Man, Rogosa and Sharpe), preferably for 24 to 48 hours, then spots of the bacterial suspension are deposited on an agar medium preferably agar medium based on tuna juice comprising 1.5% agar.

Advantageously, the volume of the bacterial suspension spots varies from 1 to 50 μl, preferably from 5 to 20 μl, more preferably is approximately 10 μl. After seeding, the petri dishes are incubated under anaerobic conditions, preferably at a temperature of varying from about 10 to 20 ° C, more preferably at a temperature of about 15 ° C, for a period of about 5 to 20 days, preferably about 10 days.

At the end of the incubation period, carpets of histaminogenic bacteria are poured onto the spots. Those skilled in the art know how to evaluate the adequate dilution of the histaminogenic strains to obtain a homogeneous bacterial carpet on the boxes.

The agar plates are then incubated, preferably aerobically, at a temperature ranging from about 10 to 20 ° C., preferably at a temperature of about 15 ° C., for a period of 24 to 192 hours, preferably from 36 to 192 hours. 144 hours, even more preferably about 96 hours. At the end of the incubation period, the inhibition halos are measured.

According to one embodiment of the invention, the strain LHIS2885 of the invention has a halo of inhibition of the histaminogenic strain tested. According to a second embodiment of the invention, the capacity for inhibiting the growth of histaminogenic bacteria is evaluated by an inhibition test on a food sample, for example cooked tuna. An example of an inhibition test on a food sample is shown below. In a first step, bacterial suspensions are prepared. Advantageously, the histaminogenic strain is cultured in broth, preferably salted BHI (Brain Heart Infusion) broth (2% NaCl), preferably at a temperature of approximately 15 to 25 ° C., more preferably at a temperature of approximately 20 ° C. C, to a concentration ranging from about 1.10 8 to about 1.1010 CFU / ml, preferably about 2.10 ⁹ CFU / ml. In parallel, the L. sakei strain LHIS2885 is cultivated, preferably in broth, more preferably in MRS broth, preferably at a temperature ranging from 10 to 37 ° C., preferably from 20 to 30 ° C., more preferably from 20 to 30 ° C. at about 26 ° C, until a concentration of from about 10 8 to about 10 10 CFU / ml is reached, preferably about 2.10 ⁹ CFU / ml.

In a second step, the histaminogenic strain, as well as the strain LHIS2885 are preferably diluted in sterile water, and then seeded on the food product. Advantageously, the amount of inoculated histaminogenic bacteria ranges from about 1 to 100 CFU / g, preferably from about 5 to about 50 CFU / g, more preferably about 10 CFU / g of food product. Preferably, the amount of bacteria L. sakei LHIS2885 inoculated from 10 3 ^J to 108 ° CFU / g, more preferably from about 10 to about 10 ⁷ CFU / g, even more preferably is about 10 ^{5 to} 10 ⁶ CFU / g of food product.

According to one embodiment, the seeded food products are then incubated, preferably at a temperature ranging from about 10 to 20 ° C., more preferably about 15 ° C., for a period ranging from 24 to 120 hours, preferably from 48 to 96 hours, more preferably for 72 hours. Advantageously, the inoculated food products are incubated under vacuum. At the end of the incubation period, the amount of histaminogenic bacteria in the product is measured, for example by the method of enumeration on agar medium, such as, for example, on VRBG medium (Violet Red Bile Glucose) incubated at approximately 20 ° C for 48 hours. According to one embodiment of the invention, the LHIS2885 strain of the invention makes it possible to reduce the growth of the histaminogenic bacterium in the food product relative to a control, said control having been inoculated only with the histaminogenic bacterium.

Another subject of the invention is a bacterial strain obtained by mutation, variation or recombination of the L. sakei strain LHIS2885 as described above. According to the invention, such a strain is called "strain derived from LHIS2885". According to one embodiment of the invention, these strains derived from L. sakei LHIS2885 have a capacity for inhibiting the growth of histaminogenic bacteria and / or an ability to inhibit the production of histamine equivalent, or at least equal to that of L. sakei strain LHIS2885.

According to a first embodiment of the invention, the capacity for inhibiting the growth of histaminogenic bacteria is evaluated by a petri dish inhibition test, as described above. Advantageously, the strain derived from LHIS2885 of the invention has an inhibition halo of the histaminogenic strain tested. According to one embodiment of the invention, the strain derived from LHIS2885 has an inhibition halo equal to or greater than that measured for the L. sakei strain LHIS2885 of the invention.

According to a second embodiment of the invention, the ability to inhibit the growth of histaminogenic bacteria is evaluated by an inhibition test on a food sample as described above.

According to one embodiment of the invention, the strain derived from LHIS2885 of the invention makes it possible to reduce the growth of the histaminogenic bacterium in the food product with respect to a control, said control having been seeded only with the histaminogenic bacterium. Preferably, this growth inhibition is approximately equal to or greater than that measured for L. sakei strain LHIS2885.

Examples of methods for obtaining strains derived from L. sakei LHIS2885 include, but are not limited to, random mutagenesis or site-directed mutagenesis. According to one embodiment of the invention, the strains derived from L. sakei LHIS2885 according to the invention comprise a genome having more than 70% identity with the genome of L. sakei LHIS2885, preferably more than 80%, 90 %, 95%, 96%, 97%, 98% or 99% identity.

For the purpose of the present invention, the term "identity", when used in a relationship between the sequences of two or more nucleotide sequences, refers to the degree of relatedness between these nucleotide sequences, as determined by the number of matches between strings of two or more bases.

According to the invention, "identity" corresponds to a percentage of identity between two or more sequences. This percentage is defined as the number of positions for which the bases are identical when the sequences are optimally aligned, divided by the total number of bases of the smaller of the two sequences. The differences between the sequences can be divided randomly and over all their lengths.

Two sequences are said to be optimally aligned when the percentage of identity is maximum. Moreover, as will become clear to those skilled in the art, it may be necessary to use additions of gaps (gaps) so as to obtain an optimal alignment between the two sequences. The percentage identity between two nucleic acid sequences can therefore be determined by comparing these two optimally aligned sequences in which the nucleic acid sequence to be compared can comprise additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is then calculated by determining the number of identical positions for which the nucleotide is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100 to obtain the percentage identity between these two sequences.

Preferably, the methods of determining the identity are designed to give the greatest possible concordance between the compared sequences. The percentage of identity can be determined by a particular mathematical model or by a computer program (generally referred to as an "algorithm"). Methods for calculating identity between nucleotide sequences are well known to those skilled in the art. Non-limiting examples of such methods include those described in the following documents: Computational Molecular Biology, Lesk, A.M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., eds., Academy Press, New York, 1993; Computer Analysis of Sequence Data Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G, Academy Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math., 48, 1073 (1988).

Methods of determining identity have been described in publicly available computer programs. Preferred examples of methods using computer programs include, but are not limited to, the GCG software package, including GAP (Devereux et al., Nucl. Acid Res., 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol Biol 215, 403-410 (1990)). The BLASTX program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al NCB / NLM / NIH Bethesda, Md 20894, Altschul et al., Supra). The Smith-Waterman algorithm, which is well known to those skilled in the art, can also be used to determine the percent identity between two sequences.

The present invention relates to a composition comprising a strain of L. sakei, preferably L. sakei LHIS2885 bacterium, a strain derived therefrom or a fragment thereof. For the purposes of the present invention, the term "fragment" is intended to mean cellular components, metabolites, secreted molecules, compounds resulting from the metabolism of L. sakei, preferably L. sakei LHIS2885 or a derived strain, L. sakei enzymes, preferably L. sakei LHIS2885 or a derived strain, etc. According to one embodiment of the invention, the fragments of L. sakei, preferably L. sakei LHIS2885 or a derived strain can be obtained by recovering the culture supernatant of L. sakei, preferably L. sakei LHIS2885 or a derived strain, or by extracting a culture of L. sakei, preferably L. sakei. LHIS2885 or a strain derived from components or cell fractions, metabolites or secreted compounds. According to another embodiment of the invention, a fragment may also designate a degradation product of L. sakei, preferably L. sakei LHIS2885 or a derived strain. According to one embodiment, the term fragment may designate a compound in an isolated form, or a mixture of several compounds derived from L. sakei, preferably L. sakei LHIS2885 or a derived strain.

According to one embodiment of the invention, the composition comprises living cells of L. sakei, preferably L. sakei LHIS2885, or a strain derived from LHIS2885, preferably cells in the growth phase, more preferably in stationary phase or exponential phase of growth, and even more preferably in the exponential phase of growth.

According to one embodiment of the invention, the composition comprises viable L. sakei cells, preferably L. sakei LHIS2885, or a strain derived from LHIS2885. According to another embodiment of the invention, the composition comprises non-viable L. sakei cells, preferably L. sakei LHIS2885, or a strain derived from LHIS2885.

According to a first embodiment of the invention, the composition is in liquid form. Examples of liquid vehicles which may be used in the composition of the invention include, but are not limited to, water, phosphate buffer, or any liquid medium suitable for culturing L. sakei bacterial strain, preferably adapted to the cultivation of L. sakei LHIS2885 or a strain derived from LHIS2885, such as for example, MRS medium (from Man, Rogosa and Sharpe) or BHI medium containing 2% NaCl (brain heart infusion). According to one embodiment of the invention, the composition is in the form of a frozen liquid. Preferably, when the composition of the invention is in the form of a frozen liquid, this liquid comprises from 1 to 20% of glycerol approximately, preferably from 5 to approximately 15%, even more preferentially about 10% volume / volume. glycerol.

According to a second embodiment of the invention, the composition is in solid form. Examples of solid forms suitable for conditioning the composition according to the invention include, but are not limited to, a powder, such as, for example, a powder comprising L. sakei cells, preferably L. sakei LHIS2885, or a strain derived from LHIS2885 thereof lyophilized; a paste; one tablet; a solid formulation to be extemporaneously dissolved in a liquid vehicle, for example water; a fragment of solid culture medium inoculated with the L. sakei strain, preferably with L. sakei LHIS2885, or a strain derived from LHIS2885, such as, for example, MRS medium.

According to one embodiment of the invention, the composition comprising a strain of L. sakei, preferably the L. sakei strain LHIS2885, or a strain derived from LHIS2885 is packaged in the form of a unit dosage. Examples of unit dosages include, but are not limited to, a bacterial culture tube, a powder sachet comprising L. sakei cells, preferably L. sakei LHIS2885, or a lyophilized strain of LHIS2885 ...

Usually, the concentration of a bacterial composition is expressed in cfu per unit volume or mass. The term ufc is well known to those skilled in the art and refers to a number of "colony forming units", i.e., a number of living cells, capable of forming a colony on a solid culture medium.

According to one embodiment of the invention, the composition comprises a concentration of cells of a strain of L. sakei, preferably L. sakei strain LHIS2885, or a strain derived from LHIS2885 varying from about 10 to about 10 ¹² cfu / ml, preferably about 10 ⁵ to about ¹⁰ cfu / ml, more preferably from about 10 to about 10 ⁹ cfu / ml, and still more preferably

is about 10 cfu / ml.

According to another embodiment of the invention, the composition comprises a concentration of cells of a strain of L. sakei, preferably L. sakei strain LHIS2885, or a strain derived from LHIS2885 varying from about 10 to about 10 ¹² cfu / g, preferably about 10 ⁵ to about 10 ¹⁰ cfu / g, more preferably from about 10 to about 10 ⁹ cFU / g, and even more preferably the concentration is

boy Wut

about 10 cfu / g.

The inventors have surprisingly demonstrated that the application of the strain Lactobacillus sakei LHIS2885 to food products, such as, for example, seafood products, makes it possible to control the production of histamine in these products, thus making it possible to prevent any toxi-infection.

The subject of the present invention is therefore also the use of a L. sakei bacterial strain, preferably of the L. sakei bacterial strain LHIS2885, or of a strain derived from L. sakei LHIS2885, from one of their fragments. or a composition comprising them, for the preservation, preferably biopreservation of food products, preferably seafood. More specifically, the present invention relates to the use of L. sakei, preferably L. sakei LHIS2885 , or a strain derived from L. sakei LHIS2885, a fragment thereof or a composition comprising them for controlling the production of histamine in a food product, preferably a product of the sea.

For the purposes of the present invention, preservation is a process for treating a food product whose purpose is to preserve its organoleptic characteristics, its taste and nutritional properties, its texture and color characteristics, and to avoid possible food poisoning. According to one embodiment of the invention, the preservation comprises the prevention or the inhibition of the development of microorganisms, in particular undesirable microorganisms, responsible for the deterioration of the product or inducing pathogenicity, such as bacteria or fungi. According to another embodiment of the invention, the conservation includes preventing or inhibiting the production of molecules responsible for the alteration of said product or inducing pathogenicity.

According to one embodiment of the invention, the preservation of a food product within the meaning of the present invention does not correspond to the fermentation of the food product. Preferably, the application of a L. sakei bacterial strain, preferably L. sakei bacterial strain LHIS2885, or a strain derived from L. sakei LHIS2885, a fragment thereof or a composition comprising them on the food product, does not modify the organoleptic properties of said food product.

For the purpose of the present invention, the biopreservation of a product corresponds to the addition to said product of one or more microorganisms in order to preserve said product.

Examples of undesirable microorganisms include, but are not limited to, histaminogenic bacteria such as, for example, Morganella psychrotolerans, Hafnia alvei, Photobacterium phosphoreum, Photobacterium damselae and Morganella morganii. Examples of molecules responsible for the alteration of said product or inducing pathogenicity include, but are not limited to, histamine.

The present invention therefore also relates to a method for preserving, preferably biopreserving, a food product, preferably a product of the sea, comprising adding to said product a strain of L. sakei, preferably of the L. sakei bacterial strain LHIS2885, or a derivative of L. sakei LHIS2885, a fragment thereof or a composition comprising them. More specifically, the method comprises controlling the production of histamine in said product.

According to one embodiment of the invention, the application of a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them inhibits the growth and development of bacterial strains producing histamine. Examples of histamine-producing bacterial strains that can be inhibited include, without to be limited, Morganella psychrotolerans, Hafiiia alvei, Photobacterium phosphoreum, Photobacterium damselae and Morganella morganii.

The present invention therefore relates to a method for preserving, preferably biopreserving, a food product, preferably a product of the sea, comprising adding to said product a strain of L. sakei, preferably bacterial strain L. sakei LHIS2885, or a derivative of L. sakei LHIS2885, a fragment thereof or a composition comprising them, wherein the L. sakei strain, preferably the L. sakei bacterial strain. LHIS2885, or the derivative of L. sakei LHIS2885, or one of their fragments inhibits the growth and development of histamine-producing bacterial strains, preferably selected from Morganella psychrotolerans, Hafiiia alvei, Photobacterium phosphoreum, Photobacterium damselae and / or Morganella morganii, thereby controlling the production of histamine in said product.

According to one embodiment of the invention, the method of preserving a food product does not include, or does not consist in, the fermentation of the food product.

Preferably, the method of preserving a food product does not modify the organoleptic properties of said food product.

According to one embodiment of the invention, the application of a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them makes it possible to maintain the concentration of histamine-producing bacteria in the food product, preferably the product of the sea at a level of less than about 10 ⁵ cfu / g of food product, preferably less than about 10 ⁴ cfu / g, more preferably less than about 10 cfu / g. According to one embodiment, these levels of inhibition are observed after incubation of the food product up to 4 days at about 15 ° C., preferably for at least 5, 6, 7 or 8 days of incubation at about 15 ° C. .

According to one embodiment of the invention, the application of a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, one of of their fragments or a composition comprising them inhibits the production of histamine and / or allows the elimination of the histamine produced in the food product, preferably in the product of the sea.

The presence of histamine in fish rich in histidine is regulated in Europe (EC Regulation No. 1441/2007, 2007) with a limit of 100 mg / kg.

According to one embodiment of the invention, the application of a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them makes it possible to maintain the concentration of histamine in the food product at a level of less than 100 mg / kg, preferably less than 75 mg / kg, more preferably less than 50 mg / kg. According to one embodiment, these histamine levels are measured after incubation of the food product up to 4 days at 15 ° C, preferably for at least 5, 6, 7 or 8 days of incubation at about 15 ° C.

According to one embodiment of the invention, the L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885 or one of their fragments inhibits the development of histamine-producing strains, and / or or inhibits the production of histamine and / or allows the removal of histamine produced in a food product at a temperature ranging from about 0 ° C to about 37 ° C, preferably from about 4 ° C to about 30 ° C. ° C, more preferably from about 4 ° C to about 15 ° C. According to one embodiment, the biopreservation effect of L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition according to the invention are observed at 15 ° C. at least 1 day, preferably at least 2 days, more preferably for at least 4 days, and still more preferably for at least 5, 6, 7 or 8 days. According to one embodiment of the invention, the L. sakei strain, preferably the L. sakei strain LHIS2885, a strain derived from LHIS2885, one of their fragments or a composition comprising them is applied in an effective amount to the food product. For the purpose of the present invention, the term "effective amount" refers to the amount of the L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them to be applied to the product for observing the desired effect of inhibiting histamine producing strains and / or inhibiting the production of histamine and / or the removal of already formed histamine. According to one embodiment of the invention, the effective amount varies from about 10 to about 10 12 cfu of the L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885 / g of food product. , preferably about 10 ⁴ to about 10 ⁹ cFU / g, more preferably from about 10 to about 10 ⁷ cFU / g and even more preferably from about 10 to about 5.10 ⁶ cfu / g. For the purposes of the present invention, the term "food product" denotes a product intended for human or animal consumption (for example for the feeding of pet animals (cat, dog, horse, hamster, guinea pig, ferret , etc.), farm animals (cattle, goats, sheep, pigs, equines, camelids, etc.) or zoo animals). Preferably, the term "food product" refers to a product intended for human consumption.

According to one embodiment of the invention, the food product is a product of the sea, preferably a fish, more preferably a fish rich in histidine. Examples of histidine-rich products include, but are not limited to, tuna and other tunas (such as, for example, skipjack, marlin and swordfish), mackerel, sardines and anchovies.

According to one embodiment of the invention, the food product is a fresh product, a cooked product, a frozen product, a vacuum packaged product, a modified atmosphere packaged product (such as, for example, an atmosphere comprising 50% of N ₂ and 50% of C0 ₂ ), a smoked product, a product packaged in a can, a marinated product or a fermented product ...

According to one embodiment, the food product is not a fermented product.

According to one embodiment, the food product is cooked tuna under vacuum. The subject of the present invention is also a process for the preparation of a food product, preferably a product of the sea for consumption, preferably for human consumption, the said preparation method comprising a step of application to the food product. a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them.

According to one embodiment, the L. sakei strain, preferably the L. sakei LHIS2885 strain, the strain derived from LHIS2885, one of their fragments or a composition comprising them is applied to the food product before packaging, preferably just before packing.

According to one embodiment, the L. sakei strain, preferably L. sakei strain LHIS2885, the strain derived from LHIS2885, one of their fragments or a composition comprising them is added to the fresh food product, with or without packaging.

According to another embodiment of the invention, the L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, one of their fragments or a composition comprising them is applied to the food product. after cooking said product.

According to one embodiment of the invention, the method of the invention comprises the following steps:

(a) cooking the food product;

(b) applying to said cooked food product a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them ; and

(c) packaging of said food product.

According to another embodiment of the invention, the L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, one of their fragments or a composition comprising them is applied to the food product before salting and / or smoking said product.

(a) applying to the food product a L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition comprising them;

(b) salting and / or smoking said food product; and

(c) packaging of said food product. According to another embodiment of the invention, the L. sakei strain, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, one of their fragments or a composition comprising them is applied to the food product. after salting and / or smoking said product.

(a) salting and / or smoking of the food product;

(b) applying to said salt or smoked food product a strain of L. sakei, preferably L. sakei strain LHIS2885, a strain derived from LHIS2885, a fragment thereof or a composition including them; and

(c) packaging of said food product.

(a) applying to said salt or smoked food product a strain of L. sakei, preferably L. sakei strain LHIS2885, of a strain derived from

LHIS2885, one of their fragments or a composition comprising them;

(b) placing a protective atmosphere of said food product; and (c) hermetic packaging of said food product, preferably by capping.

In one embodiment, said food product is tuna, and the modified atmosphere comprises 30 to 70% CO ₂ , preferably 40 to 60% CO ₂ ; from 10 to 70% of O ₂ , preferably from 15 to 60% of O ₂ and from 0 to 30% of N ₂ , preferably from 0 to 25% of N ₂ . According to a first embodiment, said food product is tuna, and the modified atmosphere comprises about 40% of CO ₂ and about 60% of O ₂ . According to a second embodiment, said food product is tuna, and the modified atmosphere comprises about 60% of CO ₂ , about 15% of O ₂ and about 25% of N ₂ . In one embodiment, said food product is herring or sardine, and the modified atmosphere comprises 30 to 70% CO ₂ , preferably about 60% CO ₂ ; and 30 to 70% N ₂ , preferably about 40% N ₂ .

The present invention also relates to a food product, preferably a product of the sea, comprising a strain of L. sakei, preferably the L. sakei strain LHIS2885 of the invention, a strain derived from LHIS2885, or one of their fragments.

The L. sakei strain LHIS2885 according to the invention has the following advantages:

the L. sakei strain LHIS2885 develops in food products, such as, for example, seafood, in particular cooked, smoked or conditioned under controlled atmosphere or under vacuum, at a temperature ranging from about 0 to about 37 ° C;

the application of L. sakei strain LHIS2885 to a food product, such as, for example, a product of the sea, contaminated with a histaminogenic bacterium, inhibits the development of said histaminogenic strain;

the application of L. sakei strain LHIS2885 to a food product, such as, for example, a product of the sea, contaminated with a histaminogenic bacterium, inhibits the production of histamine;

the application of L. sakei strain LHIS2885 to a food product, such as, for example, a product of the sea, does not substantially modify the organoleptic properties of this product. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a histogram showing the growth of M. pyschrotolerans alone (MP) or co-cultured with Lactococcus piscium EU2258 (MP + EU2258) or Lactobacillus sakei LHIS2885 (MP + LHIS2885) (MP) on cooked tuna loins kept at 15 ° C under vacuum.

Figure 2 is a histogram showing histamine production by Morganella psychrotolerans alone (MP) or in coculture with Lactococcus piscium EU2258 (MP + EU2258) or Lactobacillus sakei LHIS2885 (MP + LHIS2885) in cooked tuna stored at 15 ° C under -empty. Figure 3 is a combination of two graphs showing (A) the effect of the strain LHIS2885 of the invention on the growth of M. morganii (Mm) on cooked tuna stored at 15 ° C under vacuum, and (B) the effect of the strain LHIS2885 of the invention on the production of histamine.

Figure 4 is a histogram showing growth of Morganella psychrotolerans in absence (Mp control) or presence (Mp test) of Lactobacillus sakei LHIS2885 at 4 ° C for 18 days.

Figure 5 is a histogram showing the growth of Lactobacillus sakei in the absence (control LHIS2885) or presence (test LHIS2885) of Morganella psychrotolerans at 4 ° C for 18 days. Figure 6 is a histogram showing the production of histamine in tuna cooked at 4 ° C by M. psychrotolerans in absence (control - Mp) or presence of L. sakei (Test - Mp / LHIS2885).

Figure 7 is a combination of two graphs showing the effect of the strain LHIS2885 (Ls) of the invention on the bacterium Photobacterium phosphoreum (Pp) on cooked tuna stored at 15 ° C under vacuum. (A) Effect of the strain LHIS2885 of the invention on the growth of Photobacterium phosphoreum. (B) Effect of the strain LHIS2885 of the invention on the production of histamine. Figure 8 is a graph showing the effect of the strain LHIS2885 of the invention on the growth of the bacterium Photobacterium damselae on cooked tuna stored at 15 ° C under vacuum.

EXAMPLES

The present invention will be better understood on reading the following examples which illustrate the invention in a nonlimiting manner.

This example describes the protocol that allowed the isolation and identification of L. sakei LHIS2885 bacteria as being useful for the biopreservation of food products such as, for example, seafood, and more particularly for preventing or controlling histamine production in these products.

Isolation of lactic acid bacteria on fish rich in histidine Materials and methods

At first, isolation of lactic acid bacteria from fish flesh rich in histidine was achieved. Fresh or smoked seafood (smoked tuna, fresh tuna loin, sardine fillet, mackerel fillet, whole mackerel or smoked herring) were packaged and incubated at 8 ° C for one (fresh) or two ( smoked products) weeks, then lactic acid bacteria were isolated from these incubated products.

Results

132 strains of Gram + bacteria were isolated. Inhibition test on petri dishes

The capacity of the identified 132 strains to inhibit in vitro the histaminogenic strain Morganella psychrotolerans was then tested on a Petri dish by the double layer method. Materials and methods

The inhibition test is carried out according to the method of Matamores et al. (2009). The lactic acid bacteria are cultured in broth in BHI medium (2% NaCl) at 20 ° C. for 24 hours or 48 hours. After this pre-culture time, spots of 10 μl of bacterial suspension are deposited on the medium Tuna Juice (JT) (1.5% Agar). After inoculation, the Petri dishes are incubated at 15 ° C. under anaerobic conditions for 10 days. At the end of the incubation time of the lactic acid bacteria, the bacterial mats are poured on the spots. The proper dilution of the bacterial mat is l / 100 ^th for the strain psychrotolérante Mr. psychrotolerans. The bacterial mats are made in the medium JT. The dishes are aerobically placed for 96 hours at 15 ° C. They are observed at 36, 72 and 96h and the halos of inhibitions are measured.

Results

Of the 132 bacterial strains tested, 39 showed an inhibition of the psychrotolerant strain M. psychrotolerans. Inhibition of Histaminogenic Bacteria Morganella psychrotolerans, Morganella morganii, Photobacterium phosphoreum and Photobacterium damselae by LHIS2885 bacteria on cooked tuna stored under vacuum at 15 ° C

Six strains identified during the petri dish inhibition test, as well as six other previously isolated strains of seafood products, were then tested for their ability to inhibit M. psychrotolerans bacteria on cooked tuna loins.

Materials and methods

Bacteria: The strain of M. psychrotolerans comes from the Institut Pasteur collection, its accession number is CIP109403. It is cultured in salted BHI broth (2% NaCl) at 20 ° C for 24 h for the experiments (approximately 2.10 ⁹ CFU / ml). The twelve strains of lactic acid bacteria are grown on MRS or Elliker medium at

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26 ° C for 24 h for the experiments (final concentration varying from 2.10 to 6.10 ⁹ CFU / ml according to the strains).

Food Matrix: Frozen frozen frozen fresh tuna loins were thawed overnight at 4 ° C in an oven. The loins are cut into cubes of 2 cm ³ and distributed in a tray previously cleaned with alcohol. The cubes are placed in bags at a height of 200 g then sealed and packaged under vacuum. The tuna is cooked for 5 minutes in boiling water. The final core temperature of the product is 81.2 ° C. The cubes of tuna are cooled in a fast cooling cell. The products are stored at 4 ° C if used the next day or at -20 ° C if their use is postponed.

Method: a) Preparation of M. psychrotolerans: The preculture M. psychrotolerans was diluted 6 times in succession to l / ^10th. The concentration obtained is 2.10 ³ CFU / ml. b) Preparation of lactic acid bacteria: Precultures of lactic acid bacteria are

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diluted (or not) to a final concentration of 2.10 CFU / ml. c) Preparation of M. psychrotolerans - lactic bacteria mixtures: 10 ml of the preculture of M. psychrotolerans are mixed with 10 ml of the preculture of lactic acid bacterium. Each lactic acid bacterium is tested separately so that 12 different mixtures are prepared. d) Seeding on tuna: The previously prepared tuna is kneaded in the 200 g bag manually to make a homogeneous paste. With the help of a sterile spoon, the flesh is divided into bags of 40 g of tuna. The starting inoculum is fixed at 5% by volume relative to the total mass of tuna meat (v / m). The M. psychrotolerans - lactic bacteria mixture is prepared extemporaneously and vortexed for 15 seconds at maximum speed. 2 ml of the mixture are added to 40 g of tuna using a sterile 2ml pipette. The positive control is inoculated only with M. psychrotolerans, the 10 ml of lactic bacteria having been replaced by 10 ml of sterile physiological saline. The sterility control consists of uninoculated tuna. The Bacterial concentrations on the matrix are approximately 50 CFU / g of M. psychrotolerans and at least 10 ⁶ CFU / g of lactic acid bacteria. e) Incubation: The inoculated products are evacuated and frozen at -20 ° C. for 48 hours. They are then thawed and stored at 15 ° C for 8 days. Analysis: The products are analyzed on the same day (D0) then at 24 (D1), 48 (D2) hours, 4 days (D4) and 8 days (D8). M. psychrotolerans is specifically counted on the VRBG medium at 20 ° C. (48 h of incubation). Lactic bacteria are enumerated either on MRS or Elliker anaerobically at 20 ° C for 48 h. Histamine production is analyzed after box counting using the Veratox kit, according to the manufacturer's instructions or by HPLC. The primary dilution of the count (1/5) is used to perform this test, a volume of 30 ml is collected on each sample and frozen at -20 ° C.

All experiments are done in duplicate. Results Two strains of lactic acid bacteria exhibited inhibitions of histamine production or M. psychrotolerans growth: EU2258 (Lactococcus piscium, previously isolated from pomegranate under modified atmosphere) and LHIS2885 (Lactobacillus sakei, isolated from this study. sardine samples).

Both strains grow efficiently on cooked tuna, reaching about 9 log CFU / g in 8 days.

At 24 h and 48 h of culture, a statistically significant inhibition of approximately 1 to 2 log CFU / g of M. psychrotolerans is visible for both Lactic Bacteria / M. psychrotolerans cocktails. On the other hand, at 4 days of incubation at 15 ° C., the bacteria Lactobacillus sakei LHIS2885 limit the growth of M. psychrotolerans to 3 log CFU / g whereas the control is at 8 log CFU / g (statistically significant difference). At 8 days of incubation at 15 ° C., the bacterium Lactobacillus sakei LHIS2885 limits the growth of M. psychrotolerans to 6 log CFU / g while the control is at 9 log CFU / g (statistically significant difference). EU2258 does not seem to have any effect statistically significant on the growth of M. psychrotolerans from 4 days (Figure 1).

The effect of lactic acid bacteria on histamine production was then measured before thawing, on the day of thawing (D0), and then 1, 2, 4 and 8 days after thawing (D1, D2, D4 and D8, respectively). . The results are expressed in mg / kg of histamine and are presented in FIG.

As shown in Figure 2, before freezing, the products all contain less than 50 mg / kg of histamine. The histamine level remains constant after thawing, and at 24h and 48h where it is always less than 50 mg / kg. At 4 days of incubation, the amount of histamine increases to 3600 mg / kg of histamine in the control. Lactobacillus sakei LHIS2885 reduces histamine production to 38 mg / kg (statistically significant difference). The lactic acid bacterium EU2258 also reduces histamine production but more slowly with 1279 mg / kg (statistically significant difference).

At 8 days of incubation, this inhibition of production is still well marked for L. sakei LHIS2885 with a histamine quantity of 245 mg / kg whereas the control has reached a concentration of 8900 mg / kg (statistically significant difference ). For strain EU2258, the amount of biogenic amine is equivalent to the control and is 7425 mg / kg.

The L. sakei lactic acid bacterium LHIS2885 is therefore able to inhibit the growth of M. psychrotolerans and the production of histamine by the latter, up to 8 days after thawing the product at 15 ° C.

In a second step, the same type of experiment was carried out with the histaminogenic strain M. morganii to check the effectiveness of the bio-protective strain Lactobacillus sakei, LHIS2885. The results presented in FIG. 3 show the effects of strain LHIS2885 on the growth of M. morganii on samples of cooked tuna (FIG. 3A), as well as the production of histamine in these same samples (FIG. 3B). As shown in Figure 3, LHIS2885 strain of the invention also allows statistically significant inhibition of growth of M. morganii on cooked tuna, as well as statistically significant inhibition of histamine production of tuna samples. cooked contaminated with the histaminogenic bacterium M. morganii. These experiments were then carried out with the histaminogenic strains Photobacterium phosphoreum and Photobacterium damselae. The results shown in Figures 7 and 8 show that strain LHIS2885 inhibits the growth of Photobacterium phosphoreum and Photobacterium damselae (respectively) on cooked tuna samples. In addition, as shown in Figure 7, the LHIS2885 strain of the invention also allows the inhibition of histamine production of cooked tuna samples contaminated with the histaminogenic bacterium Photobacterium phosphoreum.

Inhibition of the histaminogenic bacterium Morsanella psychrotolerans by bacteria LHIS2885 on canned cooked tuna preserved at 4 ° C

Materials and methods Bacteria - culture and isolation media: The pre-cultures of the histaminogenic bacterium Morganella psychrotolerans CIP 109403 were carried out in a 2% Brain Heart Broth medium (2% salted BHI) at 20 ° C for 24 hours ( concentration of 2.10 ⁹ CFU / ml). Pre-cultures of L. sakei bacteria LHIS2885 were made in Man, Rogosa and Sharpe broth (MRS) at 26 ° C for 24 hours (concentration of 1.29.10 ⁹ CFU / ml).

Food Matrix: To carry out these experiments, canned yellowfin tuna meat was selected to test the bioprotective capacity of L. sakei LHIS2885 against M. psychrotolerans CIP109403. 25g of tuna are used to prepare each point of analysis. 6 analysis times and 3 batches of tuna were analyzed:

· Lot T: Positive control (tuna inoculated with M. psychrotolerans CIP 109403 T),

• Lot L: lactic indicator (tuna inoculated with LHIS2588),

• Lot E: Test (LHIS2885 + CIP109403 ^T ). The experiments were carried out in triplicate. Method: a) Preparation of the bags: The canned tuna were opened in a laminar flow hood and the tuna was placed in an alcohol sterilized tank and then mixed. The tuna was then divided into 25g sachets, with a spatula sterilized with alcohol. These bags of tuna were then inoculated and kept under vacuum. b) Preparation of the control M. psychrotolerans pure: The preculture of M. psychrotolerans CIP109403 is diluted in a solution of tryptone-salt at 0.85% to obtain a solution at 10 CFU / ml. The bags of tuna are then seeded at 5% (v / m), or 1.25 ml in 25g of tuna. The bags are then mixed by hand to homogenize the distribution in the flesh (final concentration in the flesh 48 CFU / g). c) Preparation of the Lactic Lot (LHIS2885) and Assay (Mp + LHIS2885): The preculture of L. sakei LHIS2885 is diluted in a 0.85% tryptone-salt solution (TS) to prepare the seeding of the tuna meat. For the lactic lot, a solution of L. sakei is prepared at 6.5.10 CFU / ml. The tuna bags are sown at 5% (v / m), or 1.25 ml in 25g of tuna. The bags are then mixed by hand to homogenize the distribution in the flesh (final concentration in the flesh 3.25 × 10 ⁶ CFU / ml). For the Test batch, a solution with 10 ³ CFU / ml of CIP109403 ^T and 6.5.10 'CFU / ml of LHIS2885 is prepared. The bags of tuna are then seeded at 5% (v / m). They are mixed by hand to homogenize the distribution in the flesh and to obtain a final bacterial concentration of 3.25 × 10 ⁶ CFU / g (LHIS2885) and 48 CFU / g (CIP109403 ^T ). d) Packaging and incubation of the sachets: Once inoculated, the sachets are packaged under vacuum. They are put in the freezer at -20 ° C for 48h (except the bags to be analyzed before freezing). After this time, the samples are thawed at 4 ° C and incubated at 4 ° C for 18 days. e) Microbiological analyzes: Samples are taken on day ₀ after thawing, on day ₄ , day ₇ , day ₁₄ and day ₁₄ . At each analysis time, 3 sachets of each batch were analyzed. 20g of tuna were collected sterilely and are then diluted ^1/5 by adding 80 ml of TS medium. The contents of the bag are crushed with Stomacher at normal speed for 120 seconds.

Samples of the mother solution obtained are then taken: 15 ml of ground tuna solution deposited in an empty Falcon tube for analyzes of biogenic amines by the HPLC method and 10 ml for the microbiological counts. From the 10 ml of ground tuna solution, cascade dilutions are carried out according to the assumed concentrations of bacteria. M. psychrotolerans CIP109403 is enumerated in depth on VRBG medium while L. sakei LHIS2885 is counted on MRS medium on the surface. The media were then incubated for 48 h at 20 ° C for VRBG medium and 20 ° C in anaerobic jar for MRS medium.

Biogenic Amines (HPLC) and pH: The pH was measured from the Stomacher bag of ground tuna solution. A pre-assay with the Neogen Veratox kit was carried out on 3 samples to verify the histamine concentration in the products on day ₄ , ₇ , Jn. The samples on day ₇ , day ₁₄ and day ₁₄ were analyzed by HPLC by dylylation. biogenic amines.

Results

The growths of M. psychrotolerans and L. sakei are observable in Figures 4 and 5. The initial concentration of M. psychrotolerans is less than 1 log CFU / g (Figure 4), after 11 days of incubation, it approaches 7 log CFU / g to reach 18 days at a concentration of 8.3 log CFU / g. In the presence of L. sakei (Figure 4), the growth of M. psychrotolerans is weakly inhibited, the initial concentration is less than 1 log CFU / g, and growth follows that of the control with a maximum inhibition of 0.5 log CFU. / g to J ₇ , J _n , Ji ₄ .

L. sakei develops well in tuna at 4 ° C (Figure 5), the starting concentration is 6.5 log CFU / g and increases to 9 log CFU / g after 11 days of incubation. At 14 and 18 days a decrease in concentration is observed at 8.6 and 7.5 log CFU / g respectively. There is no significant difference in the initial rate and growth of L. sakei between the control and the test. The evolution of the histamine concentration in the products is described in Figure 6. The production of histamine in the presence of M. psychrotolerans is initiated from 7 days with concentrations lower than 50 mg / kg. After 11 days of incubation the average between 3 independent samples is 93 mg / kg, and after 18 days the concentration is above the regulatory threshold and reaches an average of 582 mg / kg. In the presence of L. sakei, this production of histamine is limited after 7 days of incubation (6 mg / kg of difference compared to the control). After 11 and 14 days of incubation, the average histamine concentration is 23 and 249 mg / kg respectively, ie 75% and 57% of inhibition relative to the control. The bacterium L. sakei allows to stay at a lower histamine level than the American and European standard after 11 days of incubation and limits the production in very high concentration to 14 days.

These results demonstrate that L. sakei LHIS2885 exhibits valuable bio-protective capabilities at 4 ° C, with a reduction in histamine production of 75% and 57% after 11 and 14 days of incubation respectively. Sensory analysis of cooked sous-vide tuna in the presence of M. psychrotolerans and Lactobacillus sakei LHIS2885 stored at 15 ° C

We then analyzed the impact of the seeding of a seafood product by L. sakei bacteria LHIS2885 on the sensory qualities of this product.

Materials and Methods Raw material: Albacore tuna loins come from the company Cobreco in Douarnenez. They were recovered from the plant's cutting line after their thawing / trimming process on frozen whole tuna. This raw material has been prepared for testing according to the following steps:

- Thawing of tuna 1 night at 4 ° C,

- Cutting tuna and distribution in 200g sachets,

- Vacuum packaging of sachets,

- Cooking the bags (70 ° C in the heart for 5 minutes) in the water, - Cooling of the bags and storage at 4 ° C.

Sample preparation: 10 batches of samples were prepared to test the sensory changes made to the product by pure strains and cocktail strains.

- Lot 1: Cooked tuna

- Lot 2: Witness + Morganella psychrotolerans (Mp)

- Lot 3: Control + Lactococcus piscium: Lp (EU2258)

- Lot 4: Control + Lactobacillus sakei: Ls (LHIS2885)

- Lot 5: Mp + Lp test (EU2258)

- Lot 6: Mp + Ls test (LHIS2885)

The sachets were inoculated and then stored according to the protocol described above, under the following conditions:

- Weighing of 40g bags of cooked tuna + sowing of bags,

- Vacuum packaging of sachets,

- Freezing of the bags at -20 ° C for 48h,

- Defrosting of the sachets at 8 ° C (except 4 sachets of Control, 2 of which will be thawed on D4 and 2 more on D8, to serve as a reference during sensory tests),

Incubation of the sachets at 15 ° C. Analysis protocol: The products are analyzed after 4 days and 8 days of incubation. At each point, 2 bags of each sample are taken for sensory testing.

The tests were carried out by the internal sensory analysis panel of Ifremer de Nantes. This panel has a long experience of seafood; he is asked once or twice a week to test products or for training sessions. A group of 9 people well trained on the odors of altered seafood participated in this study. The sensory evaluation sessions take place in an air-conditioned room, composed of 10 individual tasting booths, illuminated by a standard white light (T = 6500 ° K) meeting the specifications of the AFNOR V-09-105 standard concerning the recommendations relating to the establishment of premises for sensory analysis. A computer system allows the automatic acquisition of data (Fizz software, Biosystems, Dijon) and their statistical processing.

The contents of the 2 bags of each sample are divided into 5 polystyrene odorless jars with a lid, ie about 15 g of crumbs per jar half of each sachet. The pots are stored in an oven at 18 ° C during the course of the session. Each of the 5 sets of 10 samples is felt by 2 different judges. The reference control, thawed on the morning of the test, is proposed at the same time. The samples are presented to the judges according to a balanced plan to avoid a bias due to the effect of the 1st tested product.

The session consists of 2 events:

a scoring test of the level of deterioration of the odor on an unstructured scale from 0 to 10. It is specified to the panel that a note close to 6 is representative of a relatively strong alteration. This value has been determined in previous tests,

- a characterization test by attributes. A list of 22 scents is offered to judges who must select at least one smell: sardine, mackerel, tuna, pâté, oily fish, marine / iodine, milky, potato, vegetable, mop, brine, fruity, metallic, butter / caramel, pyrrolidine / sperm, rancid / linseed oil, amine / urine, acid / pungent, sour / fermented, foot / cheese, cabbage / gas, H ₂ S / egg. Results

For the attribute characterization test, the "amine / urine" criterion was deleted because it was very rarely cited. Citation frequency groupings were performed for the first 5 descriptors (fatty fish / tuna), for rancid and vegetal, for sour / fermented and foot / cheese (sour / cheese) and for cabbage / gas and H ₂ S (sulfur). Judges cited from 1 to 5 odor descriptors per product, the number of citations is not always the same for each sample. Also, the quoting frequency for a product and a given odor criterion was calculated by taking into account the total number of citations for the product.

Notes obtained from unseeded products (control) or products inoculated with pure strains are presented in Table 1 below:

Table 1

As shown in Table 1, the control changes little between D4 and D8. Indeed, the product having been cooked and frozen, it is very little contaminated when the storage starts at 15 ° C. The product inoculated with the strain M. psychrotolerans (Mp) is strongly altered on day 4 and does not evolve thereafter; the odor is of the sulfur type (mixture of dimethylsulfide and H ₂ S). In tuna inoculated with the EU2258 strain alone, the "tuna" odor persists but the "pyrrolidine" grade was clearly detected on D4; a little less J8 because probably masked by other odors more present. Pyrrolidine is a cyclic amine whose odor in pure form resembles that of ammonia, diluted rather it evokes the smell of sperm or bleach. Finally, the tuna inoculated with the LHIS2885 strain alone does not show any alteration over time. The notes obtained by the products seeded with the strain M. psychrotolerans in mixture with a lactic bacterium (EU2258 or LHIS2885) are presented in Table 2 below:

Table 2

As shown in Table 2, the product inoculated with the M. psychrotolerans (MP) and EU2258 strains has a sulfur content; it can come from M. psychrotolerans or from EU2258. Tuna is heavily altered on D8. On the other hand, the tuna inoculated with M. psychrotolerans and LHIS2885 shows no alteration over time.

These results show that the strain Lactobacillus sakei LHIS2885 inhibits the growth of the histaminogenic bacteria M. psychrotolerans and M. morganii, from 4 to 5 log CFU / g. The most important inhibition is observed after 4 days of incubation of cooked tuna under vacuum at 15 ° C. The production of histamine is less than 50 ppm (the threshold retained by the European legislation is 100 ppm, that retained by the American legislation is 50 ppm) whereas in products which could be naturally contaminated the quantity of histamine would be 4000 ppm. At the sensory level, the strain Lactobacillus sakei LHIS2885 is not altered by itself. The strain can even reduce the alteration of the product at 4 and 8 days of incubation. The strain Lactobacillus sakei LHIS2885 thus provides interesting elements to have a better control of the production of histamine in tuna and to limit the weathering of the product.

Claims

1. Lactobacillus sakei strain called Lactobacillus sakei strain LHIS2885 and deposited at the CNCM under number CNCM 1-4704.

2. A strain derived from Lactobacillus sakei comprising a genome having more than 70% identity with the genome of the strain according to claim 1.

3. Composition comprising the bacterial strain according to claim 1 or 2 or a fragment thereof.

4. Use of a strain according to claim 1 or 2, or a composition according to claim 3 for the preservation of food products, preferably of seafood products.

Use according to claim 4, wherein the production of histamine in said food products is inhibited, preferably wherein the growth of histaminogenic bacteria in said food products is inhibited.

6. Use according to claim 4 or claim 5, wherein the food products are seafood, preferably fish, more preferably tuna, mackerel, sardine or herring.

The use according to any one of claims 4 to 6, wherein said food products are cooked products, fresh products, frozen products, vacuum packed products, modified atmosphere packaged products, smoked products, canned goods, and / or pickled products.

8. Use according to any one of claims 4 to 7, wherein is applied to the food product a quantity of bacteria ranging from about 10 to about 10 1 ¹ 2 "cfu of L. sakei LHIS2885 or a derived strain / g of food product.

9. Use of a strain of Lactobacillus sakei, a fragment thereof or a composition comprising it for preserving unfermented foodstuffs, preferably seafood products.

The use according to claim 9, wherein the production of histamine in said food products is inhibited, preferably wherein the growth of histaminogenic bacteria in said food products is inhibited.

11. Use according to claim 9 or claim 10, wherein the food products are seafood, preferably fish, more preferably tuna, mackerel, sardine or herring.

Use according to any of claims 9 to 11, wherein said food products are cooked products, fresh products, frozen products, vacuum packed products, modified atmosphere packaged products, smoked products, canned goods, and / or pickled products.

13. Use according to any one of claims 9 to 12, wherein is applied to the food product an amount of bacteria ranging from about 10 to about 10 12 cfu of L. sakei I g of food product.

A process for preparing food products, preferably seafood products, comprising a step of applying to said products a strain of L. sakei or a fragment thereof, such as, for example, a strain of

L. sakei LHIS2885 according to claim 1, a strain derived from L. sakei LHIS2885 according to claim 2 or a fragment thereof.

A food product, preferably seafood, comprising a strain of L. sakei or a fragment thereof, such as, for example, a strain of L. sakei LHIS2885 according to claim 1, a strain derived from L sakei LHIS2885 according to claim 2 or a fragment thereof.