Novel strain of carnobacterium maltaromaticum and uses thereof

Abstract

The present application relates to a strain of Carnobacterium maltaromaticum, a bacterial preparation comprising at least said strain of Carnobacterium maltaromaticum, and their use in the preparation of a food product.

Description

[0001] Description

[0002] Title of the invention: New strain of Carnobacterium ma / taromaticum and its uses

[0003] The subject of the invention is a specific strain of Carnobacterium ma / taromaticum, a bacterial preparation comprising at least said strain and their use in the preparation of a food product.

[0004] The presence of pathogenic bacteria in food is a major public health issue. In the cheese industry, the potential presence of Listeria monocytogenes is a constant concern. The presence of this pathogen in processed dairy products is regularly the subject of incidents that jeopardize consumer health and the economic stability of industry stakeholders. For example, the highly publicized withdrawal from sale of several batches of cheese contaminated with L. monocytogenes in December 2015 is worth mentioning. As a result, industry stakeholders are actively seeking solutions to better control the Listeria threat.

[0005] Some companies specializing in the production and marketing of microorganisms offer biopreservative / bioprotective microbial cultures with properties For example, some strains of Lactobacillus piantarum are known to have anti-Listeria activity. One strain of L. piantarum is marketed under the brand name HOLDBAC® as a biopreservation / bioprotection ferment.

[0006] However, despite the use of these biopreservative cultures, the presence of L. monocytogenes persists, probably because the biopreservative cultures proposed are classic lactic acid bacteria which do not provide optimal and lasting protection throughout the life of the food product.

[0007] The a-Listeria activity of lactic acid bacteria strains of the species Carnobacterium maitaromaticum has also been described in the prior art. For example, the inventors have previously identified the C. maitaromaticum strains CNCM I-5242 and CNCM I-5243 having remarkable inhibition properties against L. monocytogenes in cheese matrix (FR 3102489 B1). However, even if these strains effectively inhibit the growth of L. monocytogenes, they do not induce its decrease.

[0008] There is therefore a real need for means of reducing the presence, and in particular of inducing the decrease, of L monocytogenes in food products.

[0009] In this context, the inventors identified a new strain of C. mattaromaticum with particularly useful biopreservation properties. Unexpectedly, the presence of this strain leads to a rapid decrease in the quantity of the pathogen, indicating that this strain can inhibit the population of L. monocytogenes. Moreover, unlike the strains of C. mattaromaticum already identified, this new strain inhibits L. monocytogenes without forming an inhibition halo on agar medium in a competition test, suggesting an original mechanism of action.

[0010] Also, one aspect of the invention relates to the strain C. mattaromaticum CNCM I- 5804.

[0011] This isolated strain was deposited with the National Collection of Cultures of Microorganisms (CNCM) (Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS, France) on January 6, 2022 under number CNCM 1-5804, in accordance with the Budapest Treaty.

[0012] In co-culture with the L. monocytogenes strain \\Avn \esc&n\.e EGDelux, the CNCM 1-5804 strain induced luminescence quenching, demonstrating its ability to inhibit L. monocytogenes.

[0013] In a classical competition test on agar medium with L. monocytogenes, the CNCM 1-5804 strain does not generate an inhibition halo, which indicates a mechanism of inhibition of L monocytogenes different and complementary to that of the C. maitaromaticum strains already identified.

[0014] Furthermore, the combination of CNCM 1-5804 with at least one other C. maitaromaticum strain induces a much more effective inhibition of L monocytogenes than with C. maitaromaticum strains used alone. This synergistic effect of CNCM 1-5804 with other C. maltaromaticum strains reveals a promising potential for reducing the amount of L monocytogenes in a cheese-type food.

[0015] Another aspect of the invention relates to a bacterial preparation comprising strain CNCM 1-5804.

[0016] "Bacterial preparation" means a composition conditioned for the growth of Carnobacterium maltaromaticum, said composition comprising a viable strain of C. maltaromaticum and components essential for the growth of the bacteria, such as a carbon and energy source, a potassium and phosphorus source, a calcium source, a magnesium source, lipids, proteins or amino acids.

[0017] Said bacterial preparation may comprise a culture medium or a culture medium extract. Said medium may be TSB-YE medium (Tryptone Soy Broth-Yeast Extract).

[0018] Said culture medium may be a synthetic medium. According to the present invention, synthetic medium is understood to mean a medium into which individual components characterized and subjected to rigorous quantitative and qualitative control are introduced.

[0019] Said culture medium may also be a natural medium. According to the present invention, the term “natural medium” means a medium which contains one or more natural compounds in its composition. Said natural medium may be milk (whole, semi-skimmed or skimmed), in particular raw, thermized, pasteurized milk or UHT milk (sterilized at Ultra High Temperature).

[0020] Said bacterial preparation can be in liquid or solid form.

[0021] In one embodiment of the invention, the concentration of C. maltaromaticum, in particular of the strain CNCM 1-5804, in said bacterial preparation of the invention may be 10 2 UFC.mL- 1 at 1011 UFC.mL 1 , including 10 5 UFC.mL 1 at 10 7 UFC.mL 1 , more particularly at least 10 5 UFC.mL 1 , or at least 10 6 UFC.mL 1 , or at least 10 7 UFC.mL- 1 , relative to the total volume of the bacterial preparation. In one embodiment of the invention, the concentration of C. maltaromaticum, in particular of the strain CNCM 1-5804, in said bacterial preparation of the invention may be 10 2 UFC.mL- 1 , 10 3 UFC.mL- 1 , 10 4 UFC.mL- 1 , 10 5 UFC.mL- 1 , 106 CFU.mL- 1 , 10 7 UFC.mL- 1 , 10 8 UFC.mL 1 , 10 9 UFC.mL 1 , 10 10 UFC.mL 1 or 10 11 UFC.mL- 1 , relative to the total volume of the bacterial preparation.

[0022] In another embodiment of the invention, said bacterial preparation contains an amount of C. maltaromaticum, in particular of the strain CNCM I-5804, of 10 2 UFC.g 1 at 10 11 UFC.g 1 , including 10 5 UFC.g 1 at 10 7 UFC.g 1 , of at least 10 5 UFC.g- 1 , or at least 10 6 UFC.g 1 , or at least 10 7 UFC.g 1 , relative to the total weight of the bacterial preparation.

[0023] In one embodiment of the invention, the concentration of C. maltaromaticum, in particular of the strain CNCM I-5804, in said bacterial preparation of the invention may be 10 2 UFC.g 1 , 10 3 UFC.g 1 , 10 4 UFC.g 1 , 10 5 UFC.g 1 , 10 6 UFC.g- 1 , 10 7 UFC.g 1 , 10 8 UFC.g 1 , 10 9 UFC.g1 , 10 10 UFC.g 1 or 10 11 UFC.g 1 , relative to the total weight of the bacterial preparation.

[0024] In a particular embodiment, said bacterial preparation is a liquid concentrate, a frozen concentrate or a lyophilisate.

[0025] "Concentrate" means a liquid containing a quantity of bacteria greater than 10 5 UFC.mL 1 , especially greater than 10 10 UFC.mL 1 , especially greater than 10 11 UFC.mL 1 , relative to the total volume of the concentrate.

[0026] The concentrate can be obtained by any conventional method, for example by centrifugation or microfiltration of a culture of C. maltaromaticum after suitable incubation.

[0027] "Lyophilisate" means the product obtained after lyophilization of a culture of C. maltaromaticum after the appropriate incubation.

[0028] "Adapted incubation" means incubation using a conventional method to obtain an initial population of C. maltaromaticum close to 10 7 UFC.mL 1 , relative to the total incubation volume.

[0029] For example, the incubation of C. maltaromaticum can be carried out at 30°C for 48 hours in pasteurized whole milk. In a particular embodiment, said bacterial preparation is a concentrate, optionally frozen, comprising pasteurized whole milk and at least 10 7 UFC.mL- 1 of C. maltaromaticum CNCM I-5804, relative to the total volume of the bacterial preparation.

[0030] In a particular embodiment, said bacterial preparation is a concentrate, optionally frozen, comprising UHT whole milk and at least 10 7 UFC.mL- 1 of C. maltaromaticum CNCM I-5804, relative to the total volume of the bacterial preparation.

[0031] In one embodiment of the invention, said bacterial preparation may further comprise at least one lactic ferment and / or at least one ripening ferment.

[0032] According to one embodiment of the invention, said bacterial preparation is a protective culture of C. maltaromaticum.

[0033] The term "protective culture of Carnobacterium maitaromaticurri" means a composition containing C. maltaromaticum bacteria which can be added to food products to inhibit the growth of microorganisms which are pathogenic or toxic to humans or animals and which are present in said food products, in order to reduce the risk of contamination of the food product by these pathogenic or toxic microorganisms.

[0034] For the purposes of the present invention, said pathogenic or toxic microorganism is a bacterium of the genus Listeria, in particular Listeria monocytogenes.

[0035] Said protective culture can be in the form of liquid concentrate or in the form of lyophilisate.

[0036] According to one embodiment of the invention, the bacterial preparation further comprises at least one strain of C. maltaromaticum different from CNCM I-5804.

[0037] According to one embodiment of the invention, the bacterial preparation further comprises at least one strain of C. maltaromaticum exhibiting an inhibition halo in a competition test on agar medium with L. monocytogenes.

[0038] The inhibition test in agar medium can be easily carried out by a person skilled in the art according to the protocol described in the examples. According to one embodiment of the invention, the bacterial preparation further comprises at least one strain of C. maltaromaticum chosen from the strains CNCM 1-5242 and CNCM 1-5243.

[0039] These two isolated strains were deposited with the National Collection of Microorganism Cultures (CNCM) (Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS, France) on September 26, 2017 under the numbers CNCM I-5242 and CNCM I-5243 respectively, in accordance with the Budapest Treaty.

[0040] According to one embodiment of the invention, the bacterial preparation further comprises the strain CNCM I-5242.

[0041] According to one embodiment of the invention, the bacterial preparation further comprises the strain CNCM I-5243.

[0042] According to one embodiment of the invention, the bacterial preparation further comprises strain CNCM I-5242 and strain CNCM I-5243.

[0043] Another aspect of the invention relates to the use of the strain C. maltaromaticum CNCM I-5804, a bacterial preparation described above or a protective culture described above, in the preparation of a food product.

[0044] More particularly, the CNCM I-5804 strain, the bacterial preparation described above or the protective culture described above is used to inhibit the growth of bacteria of the genus Listeria, in particular L. monocytogenes, in a food product.

[0045] According to the invention, the expression "Listen'd inhibition" (or "Listen'd growth inhibition") means the reduction of growth, the blocking of growth or the decrease of bacteria of the genus Listeria.

[0046] Said protective culture of the invention can be used in the production of fermented or unfermented dairy products, in particular in the production of cheeses or fermented milks.

[0047] In a particular embodiment, said food product is a fermented dairy product, in particular cheeses, such as soft cheeses, cooked or uncooked pressed cheeses, fresh cheeses, goat cheeses, sheep cheeses, or fermented milks. Said protective culture of the invention can also be used in the production of other dairy products, such as cream, ice cream, butter, or secondary products derived from milk, such as whey, casein, or other prepared food products containing milk or milk constituents as an ingredient.

[0048] Said protective culture of the invention can also be used in the production of beverages, for example fruit juices, beverages obtained with mixtures of milk and fruit juices, soy milk, oat milk, almond milk, rice milk or fermented vegetable products.

[0049] Said protective culture of the invention can also be used in the industrial production of cured meats and delicatessen products, or butchery products, such as sausages or minced steaks.

[0050] Said protective culture of the invention can also be used in the production of fish-based food products, such as smoked trout or smoked salmon.

[0051] Said protective culture of the invention can also be used in the production of processed plant food products (plant-based sausages for example).

[0052] The use of the protective culture of the invention during the manufacture of food products allows the inhibition of bacteria of the genus Listeria which remain in a food product in small quantities and to extend the shelf life of said food product.

[0053] The protective culture of C. ma / taromaticum according to the present invention is added into food products depending on the type of product.

[0054] For fermented dairy products, such as cheeses or fermented milks, the protective culture is added before or at the same time as the lactic ferments or after their addition.

[0055] For other food products, the protective culture can be added at the start of the production line, during the production line or at the end of the production line, particularly before the food product is packaged.

[0056] For example, the protective culture of C. ma / taromaticum according to the present invention is added to a liquid food product to reach a level of 102 CFU.mL-i to 10" CFU.mL-i, in particular of 10$ CFU.mL-i to 10 CFU.mL-1, more particularly of at least 10 6 UFC.mL- 1 , especially between 10 6 UFC.mL-1 and 10 7 UFC.mL- 1 , relative to the total volume of said liquid food product; or added to a solid food product to reach a level of 10 2 UFC.g 1 at 10 11 UFC.g 1 , including 10 5 UFC.g 1 at 10 7 UFC.g 1 , of at least 10 5 UFC.g 1 , or at least 10 6 UFC.g 1 , or at least 10 7 UFC.g 1 , relative to the total weight of said solid food product, during the storage period of said food product.

[0057] Another aspect of the invention relates to a method for preparing a cheese, in particular a soft cheese or an uncooked pressed cheese, using the CNCM I-5804 strain or a bacterial preparation containing said strain.

[0058] Said method comprises:

[0059] - inoculation with the CNCM I-5804 strain or with a bacterial preparation described above comprising at least the CNCM I-5804 strain in milk or during milk processing.

[0060] According to the method of the invention, the inoculation with the CNCM I-5804 strain can be carried out in milk, in whey, in brine, or directly on the cheese paste. According to the method of the invention, when the inoculation is carried out on the cheese paste, it can be carried out by spraying or by spraying a liquid preparation comprising the CNCM I-5804 strain onto the cheese paste.

[0061] "Inoculation during milk processing" in cheese making means inoculation during storage, pre-ripening, coagulation, draining, molding, pressing, salting, ripening, curing or washing.

[0062] According to the method of the invention, when the seeding is done in the milk, the seeding can be carried out with or without cold maturation of the milk.

[0063] Furthermore, inoculation of milk with the CNCM I-5804 strain can be carried out before or simultaneously with the inoculation of lactic ferments.

[0064] In another embodiment, the inoculation of the CNCM I-5804 strain is carried out 0 to 8 hours before that of the lactic ferments.

[0065] The milk used in the said process may be milk, whole or not, raw or pasteurized or having undergone heat treatment. "Pasteurized whole milk" means whole milk which has undergone heat treatment, for example at 71.5°C for 15 seconds, then quickly cooled to 4°C.

[0066] In one embodiment of the method, the milk is inoculated with the CNCM I-5804 strain at a rate of 10 2 UFC.mL- 1 at 10 11UFC.mL- 1 , including 10 6 UFC.mL- 1 at 10 7 UFC.mL- 1 , relative to the volume of milk.

[0067] In one embodiment, the seeding with the CNCM I-5804 strain is carried out in conjunction with a seeding of at least one bacterium different from CNCM I-5804, preferably a lactic acid bacterium.

[0068] In one embodiment, the seeding is carried out with a bacterial preparation comprising the strain CNCM I-5804 and at least one bacterium different from CNCM I-5804, preferably a lactic acid bacterium.

[0069] In one embodiment, the bacterium different from CNCM I-5804 is a proteobacterium.

[0070] In one embodiment, the lactic acid bacterium other than CNCM I-5804 is selected from the group consisting of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus and Staphylococcus.

[0071] In a preferred embodiment, the seeding with the CNCM I-5804 strain is carried out in conjunction with seeding with at least one strain of C. maltaromaticum other than CNCM I-5804, in particular the CNCM I-5242 strain and / or the CNCM I-5243 strain.

[0072] In a preferred embodiment, the seeding is carried out with a bacterial preparation comprising the strain CNCM I-5804 and at least one strain of C. maltaromaticum different from CNCM I-5804, in particular the strain CNCM I-5242 and / or the strain CNCM I-5243.

[0073] In one embodiment, the inoculated milk is incubated at a temperature of at least 2°C, preferably at least 30°C.

[0074] In one embodiment, the seeded milk is incubated at a temperature of 2°C to 65°C. In one embodiment, the seeded milk is incubated at a temperature of at least 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C.

[0075] In one aspect, the invention therefore also relates to food products, such as those described above, comprising the CNCM I-5804 strain.

[0076] Another aspect of the invention relates to a method for reducing microbial contamination during the manufacturing process of a fermented milk product comprising the addition of an inhibitory amount of strain CNCM I-5804 or a bacterial preparation containing strain CNCM I-5804.

[0077] The present invention also provides a method for reducing contamination by bacteria of the genus Listeria during the manufacturing process of a fermented milk product characterized in that it comprises the addition of an amount of 10 2 UFC.mL- 1 at 10 11 UFC.mL- 1 , including 10 6 at 10 7 UFC.mL 1 , relative to the volume of the dairy product, of the CNCM I-5804 strain or of a bacterial preparation comprising the CNCM I-5804 strain

[0078] The attached figures illustrate the invention:

[0079] [Fig.1] Demonstration of different types of inhibition mechanisms of L. monocytogenes by strains of C. mattaromaticum: (A), C. mattaromaticum CNCM I-5804; (B), C. mattaromaticum CNCM I-5242

[0080] [Fig.2] Growth of Listeria monocytogenes in cheese matrices previously inoculated with Carnobacterium maitaromaticum strains CNCM I-5804 and CNCM I-5242 alone or in a mixture: (A), at 12°C; (B), at 30°C.

[0081] [Fig.3], Mean reduction factors of population densities of different strains of Listeria monocytogenes by Carnobacterium maitaromaticum CNCM I- 5804. Error bars represent standard errors of the mean (n=3).

[0082] [Fig.4] Example of luminescence kinetics of the indicator strain Listeria monocytogenes EGDelux alone (EGDElux) or in the presence of the biopreservation strain C. maitaromaticum CNCM I-5804 (CNCM I-5804).

[0083] [Fig.5] Mean inhibition strength levels (100-LDIr) of the indicator strain L. monocytogenes EGDelux by the strain C. maitaromaticum CNCM I-5804 in the matrices selected for inoculation level D3. Error bars represent standard errors of the means (n=3).

[0084] [Fig.6] Coefficients of variation (CV) of the inhibition strengths measured at the three dilution levels. [Fig.7] Counts of L. monocytogenes in cheese production in the absence and presence of the CNCM 1-5804 strain.

[0085] [Fig.8] (A) Verification of a-Listeria monocytogenes activity of supernatants of bioprotection strains; (B) Search for potential incompatibilities between CNCM 1-5804 and other bioprotection strains; 1: control (TSBYE); 2: HOLDBAC®, 3: CNCM 1-5242 from culture stored at -80°C, 4: CNCM 1-5243 from culture stored at -80°C, 5: CNCM 1-5243 from lyophilized culture.

[0086] The present invention will be described below in more detail and with the aid of one or more examples which do not limit the invention in any way.

[0087] Example 1 - Inhibition of L. monocytogenes on agar

[0088] Materials and methods

[0089] Preparation of Camobacterium ma / taromaticum inocula

[0090] Pre-cultures of Carnobacterium ma / taromaticum (CNCM I-5242 and CNCM I-5804) were prepared from aliquots frozen at -80°C. 100 μL of these aliquots were each added to a 15 mL Falcon tube containing 10 mL of TSB-YE, then incubated at 30°C for 24 h.

[0091] Preparation of Listeria monocytogenes inocula

[0092] Five strains of Listeria monocytogenes (Scott A, PL1447, CA2165, Lal65 and SN167) are streaked onto TSA-YE and incubated for 72 h at 30°C. A liquid culture in 10 mL of TSB-YE is then produced from one colony for each strain. These cultures are incubated for 24 h at 30°C, then mixed equivolumetrically. An OD measurement is used to estimate the concentration using a pre-established ratio. 50% glycerol is added to the culture mixture to achieve a final concentration of 10%. The glycerol-coated culture mixture is then aliquoted into 1 mL cryovials and frozen at -80°C.

[0093] Double-layer growth and inhibition halo

[0094] Petri dishes containing TSA-YE agar medium are inoculated with 1.5 μL of each Carnobacterium maitaromaticum culture. Seeding is carried out in the center of each agar medium dish. These dishes are incubated for 24 h at 30°C.

[0095] On the same day, a pre-culture of the mixture of 5 strains of Listeria monocytogenes is carried out in TSB-YE medium, at 30°C. After growth, the culture is diluted in supercooled TSA-YE in order to reach an OD of approximately 0.01 (i.e. approximately 10 6 CFU / mL). 15 mL of seeded liquid agar is gently poured onto the first agar plates to create a second layer. The plates are incubated at 30°C for 24 hours.

[0096] Inhibition results on agar

[0097] The results obtained show that strain CNCM I-5804 does not exhibit an inhibition halo towards the mixture of the 5 Listeria monocytogenes strains, whereas strain CNCM I-5242 exhibits a halo (Figure 1). The same is true for strain CNCM I-5243 (results not shown). This suggests that the mechanism of action of strain CNCM I-5804 to inhibit Listeria monocytogenes is different from that of the other two strains.

[0098] Example 2 - Monitoring of L. monocytogenes in a real cheese matrix

[0099] Materials and methods

[0100] Preparation of Carnobacterium mattaromaticum inocula

[0101] Pre-cultures of Camobacterium mattaromaticum CNCM I-5804 and CNCM I-5242 were made from aliquots frozen at -80°C. 100 μL of these aliquots were each added to a 15 mL Falcon tube containing 10 mL of TSB-YE, then incubated at 30°C for 24 h.

[0102] 500 pL of pre-culture are taken to measure the optical density (OD). This measurement is used to estimate the concentration of each using a pre-established OD / (CFU / mL) ratio. The cultures are then centrifuged at 5000 g for 5 min and the pellets are taken up in a volume of 9.5 mL of sterile physiological saline. This operation is repeated a second time to wash the cultures. The washed cultures are then diluted in cascade up to 5.0.10 8 CFU / mL.

[0103] Preparation of Listeria monocytogenes inocula

[0104] Five strains of Listeria monocytogenes (Scott A, PL1447, CA2165, Lal65 and SN167) are streaked onto TSA-YE from starters. After incubation for 72 h at 30°C, a liquid culture in 10 mL of TSB-YE is made from one colony for each strain. These cultures are incubated for 24 h at 30°C, then mixed after measuring the OD. This measurement allows the concentration to be estimated using a pre-established ratio. 50% glycerol is added to the culture mixture to reach a final concentration of 10%. The glycerol-coated culture mixture is then aliquoted into 1 mL cryovials and frozen at -80°C.

[0105] Before use, the aliquot is thawed at room temperature and then centrifuged at 5000 g for 5 minutes. The supernatant is removed and the pellet is taken up in the same volume of sterile physiological saline. These steps are carried out a second time. The mixture is then diluted in cascade to a concentration of 5.0.10 5 CFU / mL in sterile physiological water.

[0106] Preparation of Saint-Nectaire samples

[0107] Cheese samples are prepared from pieces of Saint-Nectaire Dairy AOP. For the same test, the cheeses belong to the same batch. These cheeses are cut into pieces of approximately 10 g (+ / - 1.5 g) after removing the rind. Each piece is placed in an individual screw-top pillbox. A well is then dug in the center of the pieces using a Durham bell jar.

[0108] The pieces of cheese are then inoculated or not with the strains of C. maltaromaticum, according to the following methods:

[0109] - A negative control cheese, not inoculated

[0110] - A positive control cheese, not inoculated

[0111] - A cheese inoculated with 20 pL of C. maltaromaticum CNCM I-5804

[0112] - A cheese inoculated with 20 pL of C. maltaromaticum CNCM I-5242

[0113] - A cheese inoculated with 20 μL of C. maltaromaticum CNCM I-5804 and 20 μL of C. maltaromaticum CNCM I-5242

[0114] Half of these cheese pieces are incubated at 30°C and the other half at 12°C for 6 days. After these 6 days, 20 μL of the diluted Listeria monocytogenes mixture are added to all the cheeses except the negative controls. The pots containing the cheeses are then reincubated at their respective temperatures. For each temperature, there are as many pots of each modality as there are counting points.

[0115] Enumeration of Listeria monocytogenes

[0116] At each counting point, one cheese of each modality is removed from the oven. Each piece is homogenized in a Stomacher (3 X 3 min) in a bag with a side filter containing 90 mL of sterile sodium citrate buffer. The mixture is diluted in cascade (1 mL of mixture + 9 mL of sodium citrate buffer) until the appropriate dilution is reached. 100 or 200 μL of the dilutions are then spread on PALCAM selective medium in duplicate. The dishes are incubated for 24 to 48 h at 30°C.

[0117] Results: Growth of L. monocytogenes in cheese matrix The results obtained highlight the inhibitory effect of the CNCM 1-5804 strain, used alone or in mixture with the CNCM 1-5242 strain (Figure 2A, Figure 2B). At 12°C, the addition of the CNCM I-5804 strain alone allows a reduction in the quantity of L. monocytogenes of approximately 0.7 log in the cheese matrix after 27 days (Figure 2A). At 30°C, the addition of the CNCM I-5804 strain allows a reduction in the quantity of L. monocytogenes regardless of the incubation duration (Figure 2B): this reduction is very clearly greater than 1 log for all the incubation durations tested and the quantity of L. monocytogenes counted in the cheese matrix then never exceeds 1.5.10 4 CFU / g.

[0118] Furthermore, a complementary behavior of the two strains of C. mattaromaticum C CM I-5804 and CNCM I-5242 is observed when they are added as a mixture to the cheese matrix: in this case, the reduction of the L monocytogenes population is very significant and reaches 1.4 log after 27 days of incubation at 12°C, and 2.5 log after 27 days of incubation at 30°C. At 30°C and in the presence of the two strains of C. mattaromaticum, the population of L. monocytogenes reaches 1.9.10 5 CFU / g at 6 days of growth, then a continuous decline phase occurs and allows a final population level of 5.7.10 to be found 2 UFC / g after 27 days, a level lower than the initial level (8.7.10 2 CFU / g).

[0119] Example 3 - Activity spectrum ar\ \- Listeria monocytogenes of C. mattaromaticum CNCM I-5804

[0120] Materials and methods

[0121] Co-culture of C. mattaromaticum CNCM I-5804 strain with a collection of L. monocytogenes strains and enumerations

[0122] 43 strains of L monocytogenes were isolated on TSAYE agar consisting of trypticase-soy broth (TSB, bioMérieux, Marcy-L'Etoile, France) supplemented with 6 gL 1 of yeast extract (YE, Biokar Diagnostics, Paris, France) and 15 gL 1of bacteriological agar (BD), then incubated for 24 h at 30°C. After incubation, the isolates are transferred to the wells of a microplate (Corning 3370, Corning Inc., NY, USA) containing 145 pL of TSBYE each, before incubation for 24 h at 30°C. This first microplate is then replicated by transferring 5 pL from each well to a new microplate containing 145 pL of TSBYE per well. In parallel, 10 mL of TSBYE are inoculated with a starter culture of the strain C. maltaromaticum CNCM I-5804. All of these cultures (microplate and culture of the biopreservative strain) are incubated for 22 h at 30°C. On the day of the test, the microplate containing the Listeria monocytogenes strain collection is diluted by decimal microplate dilutions up to 1 / 1000. Five microliters from each well of this microplate are then transferred to a new microplate, containing 135 pL of TSBYE and 10 pL of C. maltaromaticum CNCM I-5804 culture per well.The final dilution level of the Listeria monocytogenes collection is thus 1 / 30000. This co-culture microplate is incubated for 24 h at 30°C. After incubation, the co-culture microplate is cascade diluted in Tryptone-Salt broth (TS, Biokar Diagnostics, Paris, France) up to a dilution factor of 10. 7. Each microplate allows 2.5 μL of diluted culture to be deposited on agar with PALCAM selective supplement (Biokar Diagnostics, Paris, France). The inoculated agar plates are then incubated at 37°C for 48 h before counting the L. monocytogenes colonies. This experiment is also carried out without the addition of the biopreservative strain C. maltaromaticum CNCM I-5804 in order to produce controls. The reduction factor in the density of L. monocytogenes by the strain C. maltaromaticum CNCM I-5804 is obtained by calculating the ratio of the population densities of L. monocytogenes in the presence of the strain C. maltaromaticum CNCM I-5804 to those obtained in the controls. The co-culture experiment is repeated 3 times.

[0123] Results: reduction in the densities of L. monocytogenes strains in the presence of C. maltaromatic 5804

[0124] The results show that the C. maltaromaticum CNCM I-5804 strain has the ability to reduce the population density of all L. monocytogenes strains tested (Figure 3). This property varies among strains, ranging from a reduction of a factor of 1.2 log ± 0.4 log for the CIP 7818 strain to a factor of 4.7 log ± 0.4 log for the CIP 12510 strain. Apart from these extreme values, the ability of C. maltaromaticum CNCM I-5804 to reduce L. monocytogenes populations during co-cultures in TSBYE medium ranges continuously between reduction factors of 2.1 log ± 0.2 log and 4.0 log ± 0.2 log, with an average of 2.9 log ± 0.1 log. These results show that the biopreservation strain CNCM I-5804 exhibits a broad inhibition capacity with average levels of pathogen population reduction of approximately 3 logio.

[0125] Example 4 - Anti-Z.. monocytogenes activity of C. mattaromaticumC C 1-5804 in different food matrices

[0126] The objective of this work is to determine whether the C. ma / taromaticum CNCM I-5804 strain is capable of inhibiting the growth of an indicator strain of L. monocytogenes (L monocytogenes EGDelux) in preparations derived from different food matrices of interest. These matrices include matrices of animal and plant origin, including alternatives to animal products. A recent study suggests that these alternatives constitute particularly favorable environments for the growth of different food pathogens, including L monocytogenes. These data motivate the choice of these matrices for the present study.

[0127] Materials and methods

[0128] Description of the matrices

[0129] In addition to two laboratory media serving as controls (TSBYE and TSBYE without glucose), five food matrices were selected. These matrices were of animal or plant origin: smoked trout, plant-based sausage, UHT almond milk, UHT oat drink, and UHT soy milk.

[0130] Preparation of matrices

[0131] Laboratory media consist of tryptic soy broth (TSB) (Biokar Diagnostics, Paris, France) supplemented with 6 gL 1 yeast extract (YE) (Biokar Diagnostics, Paris, France), without or with the addition of 2.5 gL 1of glucose. Liquid matrices (laboratory broths and drinks) were used as is for the tests, while solid matrices were subjected to juice preparation, by cutting under sterile conditions, addition of physiological saline and grinding in a stomacher. For the vegetable sausage matrix, 140 g were used with a physiological saline ratio of 1:1 (w / w). In the case of smoked trout, 180 g were used with a physiological saline ratio of 2:1 (w / w). The homogenized matrices are distributed in the wells of different microplates (Corning) at a rate of 135 pL per well, and stored at -80°C until the tests.

[0132] Preparation of the strains

[0133] The strain C. maltaromaticum CNCM I-5804 is streaked on tryptic soy agar with yeast extract (TSAYE) consisting of 15 gL amended TSBYE broth 1of bacteriological agar (BD), then incubated for 48 h at 30°C. An isolate is then transferred to the wells of a microplate (Corning 3370, Corning Inc., NY, USA) marked GO (generation 0) containing 150 pL of TSBYE each before incubation for 24 h at 30°C. At the end of the incubation, 10 pL from each well are transferred to the wells of new microplates (Corning 3370) marked G1 (generation 1) containing 110 pL of TSBYE per well. After 24 h of incubation at 30°C, these G1 microplates are supplemented with glycerol at a rate of 30 pL per well to reach a final concentration of 10% (v / v). These microplates containing glycerolated cultures of C. maltaromaticum CNCM I-5804 serve as working collections and are stored at -80°C until testing.

[0134] The bioluminescent strain L monocytogenes EGDelux (Riedel et al., 2007) was streaked on agar with PALCAM selective supplement (Biokar Diagnostics, Allonne, France) and incubated at 30°C for 24 h. One isolate was then transferred to 10 mL of TSBYE incubated at 30°C for 24 h. Glycerol was added to the resulting starter culture to reach a final concentration of 10% (v / v). The homogeneous mixture was distributed in 1.5 mL aliquots and frozen at -80°C until use.

[0135] Measurement of anti-Z. monocytogenes activity of C. maltaromaticum CNCM I-5804 strain in selected matrices

[0136] The measurement of the anti-Z. monocytogenes activity of C. maltaromaticum CNCM I-5804 is based on its co-culture with the bioluminescent strain L monocytogenes GGe\ . 48 hours before co-culture, a G1 microplate (generation 1) is thawed and 15 pL of cultures are transferred into the wells of a microplate (Corning 3370) containing 135 pL per well of selected matrix. This pre-culture microplate, noted G2, is incubated for 48 h at 30°C. After incubation, the G2 microplates containing C. maltaromaticum CNCM I-5804 are diluted by decimal dilution to 1 / 10,000, by transferring 15 pL of cultures into microplates noted G3) containing 120 pL per well of selected matrix. These different dilutions make it possible to evaluate the magnitudes of the inhibitory effects exerted by the biopreservation strain at different inoculum levels. The microplates corresponding to the 10-fold dilutions 2 (D2), 10 3 (D3) and 10 4(D4) are opaque white microplates (Corning 3917) suitable for reading luminescence by limiting inter-well light diffusion, and are those subject to co-cultures.

[0137] 24 hours before co-culture, L. monocytogenes EGDelux is cultured by transferring 100 pL of starter into 10 mL of TSBYE and incubating at 30 °C. This culture is washed twice before use for co-culture. Each wash is done by centrifugation at 5000 g for 5 min, removal of the supernatant and resuspension of the pellet in sterile physiological saline. This culture is then diluted 1 / 10,000 in physiological saline and 15 pL of this dilution is transferred to the wells of G3 microplates (D2, D3 and D4) immediately after preparation.

[0138] Anti-Z. monocytogenes activity was measured by luminescence kinetic monitoring. Co-cultures were incubated with axial shaking at 30°C for 36 h and their luminescence was measured every 20 min using an Infinite 200 plate reader (Tecan). The experiment was performed 3 independent replicates for each matrix.

[0139] Luminescence Disturbance Indicator (LDI)

[0140] The measurement of the inhibition of L. monocytogenes EGDelux by C. mattaromaticum CNCM I-5804 is done by calculating the luminescence disturbance indicator (LDI):

[0141] 1 y 1 LDI = - > - njt

[0142] Where n denotes the number of measurements, l the luminescence expressed in an arbitrary unit and t the time in seconds.

[0143] A derived relative measure, the luminescence inhibition indicator LDIr, is calculated as:

[0144] LDI

[0145] LDIr = 100 x

[0146] LDIc Where LDI denotes a value obtained under any condition and LDIc the value obtained in the absence of inhibition, i.e. the LDI of the bioluminescence control strain L. monocytogenes EGDelux alone. For this work, the index used is 100-LD / r. Thus, this value is 0 for the L. monocytogenes EGDelux control and a positive value indicates inhibition of luminescence.

[0147] Results :

[0148] Figure 4 shows an example of luminescence kinetics of the indicator strain L. monocytogenes EGDelux in the absence or presence of the biopreservation strain C. mattaromaticum CNCM I-5804. The level of luminescence produced by the indicator strain reflects its specific growth rate. The decrease in the luminescence level of the indicator strain compared to the control (EGDelux) when it was grown in the presence of the biopreservation strain (C. mattaromaticum CNCM I-5804) indicates an inhibition of its growth.

[0149] The relative luminescence disruption indicator (LDIr) was calculated to summarize the measured luminescence kinetics. A derived measure, 100- LDIr, provides a strength value for the luminescence inhibition exerted by the biopreservation strain. The results show that at inoculum level D3, and in all matrices tested, the C. mattaromaticum CNCM I-5804 strain exerted an inhibitory effect on the indicator strain L. monocytogenes EGDelux (Figure 5). The highest level of inhibition measured was in the glucose-free TSB control medium, where the C. maitaromaticum CNCM I-5804 strain inhibited 96.6% ± 2.4% of the indicator strain's luminescence. The levels of extinction of the indicator strain's luminescence by C.maitaromaticum CNCM I-5804 were particularly high in the matrices vegetable sausage (91.3% ± 3.5%), smoked trout (90.3% ± 3.4%) and soy drink (87.8% ± 4.9%), and comparable to the level of inhibition observed in TSBYE laboratory medium with glucose (88.3 ± 7.8%). The inhibition levels of the luminescent strain were high, although to a lesser extent, in oat drink 72.6% ± 3.1%) and almond milk (59.6% ± 3.2%).

[0150] Sensitivity of the inhibitory properties of the strain C. maitaromaticum CNCM I-5804 to the inoculum level The use of three dilution levels of the strain C. maltaromaticum C CM I-5804, namely 10 2 (D2), 10 3 (D3) and 10 4(D4) allowed to calculate, for each matrix, the coefficient of variation of the inhibition forces all dilutions combined (Figure 6). These coefficients of variation inform the sensitivity of the inhibitory activity to the inoculation level of the biopreservative strain. The results show that the inhibition properties of the C. maltaromaticum CNCM I-5804 strain vary significantly depending on the inoculum level in almond milk (99.1% variation). These properties showed intermediate levels of variation depending on the inoculum level in the oat drink (53.9%) as well as in smoked trout (38.6%). In vegetable sausage, the variability of the response (31.1%) was comparable to that observed in the TSBYE control medium (26.6%). Finally, the variability of the inhibition property depending on the inoculum level was the lowest in soy drink (20.4%), similar to that measured in TSBYE control medium without glucose (19.3%).

[0151] In conclusion, co-cultures of the indicator strain with C. maltaromaticum CNCM I-5804 show on the one hand that the biopreservation strain has a high inhibition capacity of L monocytogenes EGDelux in all the matrices retained and more particularly in matrices derived from vegetable sausage, smoked trout and soy beverage. On the other hand, inoculation of the strain C. maltaromaticum CNCM I-5804 at different levels (1 / 100, 1 / 1000 and 1 / 10000) showed that its inhibitory capacities were little affected by these levels for matrices derived from smoked trout, vegetable sausage (25% < CV < 50%), and soy beverage (CV < 25%).

[0152] Example 5 - Challenge tests in soft cheese model

[0153] The objective of this work is to evaluate the anti-Z. monocytogenes effect of a strain of C. maltaromaticum (CNCM I-5804) in a soft cheese model. These challenge tests were carried out at the UMRF (Aurillac).

[0154] Materials and methods

[0155] One production consisted of two 5 L milk vats (two 250g cheeses / vat), including one test vat with L. monocytogenes and C. maltaromaticum CNCM I-5804, and one control vat with L. monocytogenes alone without a protective culture. The milk used was pasteurized and standardized milk (MG=protein content, MG / TP=1). The cultures used for the soft paste technology were Flora Danica and the RNA culture. The surface cultures were Geotrichum candidum (LIP Jacynthe culture) and Penicillium candidum (LIP Edelweiss culture). Two productions were carried out on the same day, with the same milk.

[0156] The CNCM I-5804 strain was precultured for 24 hours at 30°C in TSB-YE medium (10 mL). The preculture was washed twice: centrifugation at 5000 g for 10 minutes and then the pellet was taken up in the same volume of physiological saline. After the second wash, the pellet was taken up in 10 mL of UHT whole milk. These 10 mL were used to inoculate a 100 mL culture of UHT whole milk, which was incubated for 48 hours at 30°C before being frozen at -20°C until use. For L. monocytogenes, a mixture of 5 strains was used, including Scott A, CA2165 Fromage-CA Wisconsin E. Ryser, SN167 (strain isolated from St-Nectaire cheese), Lal65 (strain isolated from Laguiole cheese), and PL1547. After pre-culture, the 5 strains are mixed and counted on PALCAM medium at 37°C, then the mixture is stored at -80°C.

[0157] The mixture of L. monocytogenes (50 CFU.mL 1 ) and the strain C. maitaromaticum CNCM I-5804 (10 6 -10 7CFU.mL-1) was inoculated into the milk at D-1 (start of cold maturation = T0). Flora Danica and RNA ferments were inoculated at 1% at mid-cold maturation (T8h D-1). After salting, Geotricum candidum and Penicillium candidum were sprayed on the surface.

[0158] Samples were collected at different stages of production. A 30 mL bottle of milk was collected at T0 (D-1) at the start of cold maturation, at T8h (D-1) at mid-cold maturation, and at T20h (D-1) at the end of cold maturation. To enumerate L. monocytogenes, 30 g of cheese were collected at T5d, T15d, and T40d.

[0159] Listeria monocytogenes was counted on PALCAM agar at each sample (mixture of paste and rind for cheeses). For the 10° (milk) and 10 1 (cheeses), the inoculation conditions were 1 mL on 3 boxes. The dry extracts of the paste were measured on the cheese samples at T40d.

[0160] Results

[0161] Microbiology In the control cheeses, L monocytogenes, inoculated at 1.7 log 10 CFU.mL 1 in milk, grows beyond 20 hours after renneting and its growth continues until T40j with an average of 3.5 log 10 CFU.g 1 at T5j, 4.4 logio UFC.g 1 at T 15j and 5.8 logio UFC.g 1 at T40j (Figure 7, Table 1). Table 1. L monocytogenes counts (logio CFU.g 1 ) in cheese production in the absence and presence of the CNCM I-5804 strain.

[0162] In cheeses inoculated with the bioprotective culture, the growth of L. monocytogenes was lower than that observed in control cheeses throughout ripening. An average reduction of L. monocytogenes of 1.1 log CFU.g- 1 at T5d, T15d and T40d is observed for cheeses inoculated with the CNCM I-5804 strain compared to the control.

[0163] Physical chemistry

[0164] Very good reproducibility from the point of view of the progress of the cheese technology appears through the measurements of pH (Table 2) and dry extracts of the paste (Table 3).

[0165] Table 2. pH measurements in cheese production in the absence and presence of strain CNCM 1-5804.

[0166] Table 3. Measurements of dry extracts of the paste in cheese production after 40 days of ripening.

[0167] The pH and dry extract values ​​are correct compared to the expected values ​​in soft cheese technology. Thus, the bioprotective strain tested has no significant impact on the evolution of pH during production.

[0168] Conclusion

[0169] The results of L. monocytogenes enumeration and inhibition calculations compared to the control show the effectiveness of the CNCM 1-5804 strain in cheeses, and the physicochemical analyses show that the strain is compatible with soft cheese technology.

[0170] Example 6 - Search for potential incompatibilities between CNCM 1-5804 and other bioprotective strains

[0171] The objective of this work is to verify the absence of inhibition halos indicating a phenomenon of competition by interference between a selection of bioprotective strains and C. mattaromaticum CNCM 1-5804.

[0172] Materials and methods

[0173] The emitter strains selected for this test are C. mattaromaticum CNCM 1-5242, Lactobacillus plantarum (HOLDBAC®) and C. maltaromaticum CNCM 1-5243. The indicator strain is C. maltaromaticum CNCM 1-5804, allowing compatibility with other biopreservation strains to be assessed. Listeria monocytogenes EGDe is used as a control indicator strain to verify the production of a diffusible inhibitory substance by the selected emitter strains. Ten milliliters of trypticase soy broth (TSB, 30 gL 1 ) supplemented with yeast extract (YE, 6 gL 1) (TSBYE) are inoculated using sterile ose, by transferring a sample from cultures of these strains stored at -80°C. In parallel with these inoculation conditions, another TSBYE broth is inoculated with the strain C. maltaromaticum CNCM I-5243 from freeze-dried culture (Lallemand Speciality Cultures). The inoculated broths are incubated at 30°C for 24 h.

[0174] After 24 hours of culture, a TSAYE agar (TSBYE supplemented with 12 gL 1bacteriological agar) is inoculated with the culture of C. maltaromaticum CNCM I-5804 or L. monocytogenes EGDe diluted 1 / 100. The agar plates thus prepared are placed at 4°C for 30 min. In parallel, the emitting cultures are centrifuged at 5000 g for 10 min. The culture supernatants are heat-treated for 30 min at 80°C in order to destroy any cells in suspension. Wells are pierced in the agar plates using a sterile Durham bell jar, then 25 pL of the treated supernatants are placed there. The agar plates are then incubated for 48 hours at 30°C before reading.

[0175] Results

[0176] The results reveal the presence of an inhibition halo around each deposit of culture supernatant of biopreservative strain on the agar containing L monocytogenes EGDe, confirming the production of diffusible inhibitory substances by these strains (Figure 8A). On the agar containing C. maltaromaticum CNCM I-5804, an inhibition halo is observed around the deposit of culture supernatant of the strain L piantarum (HOLDBAC®), suggesting an ability of this strain to inhibit CNCM I-5804 (Figure 8B, deposit H). However, no halo of inhibition was observed around the deposits corresponding to the supernatants of the C. maltaromaticum strains tested, suggesting the absence of major incompatibility between the C. maltaromaticum CNCM I-5242 and CNCM I-5804 strains, nor between C. maltaromaticum CNCM I-5243 and CNCM I-5804.

[0177] Conclusion

[0178] Single-layer activity tests suggest the absence of major incompatibility between the strains C. maltaromaticum CNCM 1-5242 and C. maltaromaticum CNCM I-5804, as well as between the strains C. maltaromaticum CNCM 1-5243 and C. maltaromaticum CNCM 1-5804, opening the possibility of combining these different biopreservation solutions.

Claims

Claims

1. Strain of Camobacterium mattaromaticum deposited under number CNCM I-5804.

2. Bacterial preparation comprising strain CNCM 1-5804.

3. Bacterial preparation according to claim 2, said preparation further comprising a lactic ferment and / or a ripening ferment.

4. Bacterial preparation according to claim 2 or 3, characterized in that said preparation is a protective culture of C. mattaromaticum.

5. Bacterial preparation according to any one of claims 2 to 4, characterized in that said preparation further comprises at least one strain of C. mattaromaticum different from CNCM I-5804.

6. Use of a strain according to claim 1 or a bacterial preparation according to any one of claims 2 to 5 in the preparation of a food product.

7. Use according to claim 6, for inhibiting the growth of bacteria of the genus Listeria in a food product.

8. Food product comprising the strain CNCM I-5804.

9. A method of preparing a cheese, said method comprising: - seeding with at least one strain of C. maitaromaticum according to claim 1 or a bacterial preparation according to claims 2 to 5, in milk or during the processing of milk.

10. A method for reducing microbial contamination during the manufacturing process of a fermented dairy product characterized in what it includes is the addition of a quantity of 10 2 UFC.mL 1 at 10 11 UFC.mL- 1 , including 10 6 at 10 7 UFC.mL 1, relative to the volume of the dairy product, of C. maltaromaticum according to claim 1 or a bacterial preparation according to claims 2-5.

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