WO2024133466A1

WO2024133466A1 - Novel strain of carnobacterium maltaromaticum and uses thereof

Info

Publication number: WO2024133466A1
Application number: PCT/EP2023/086967
Authority: WO
Inventors: Cécile MANGAVEL; Frédéric BORGES; Anne-Marie REVOL-JUNELLES; Marcia LEYVA-SALAS
Original assignee: Universite De Lorraine
Priority date: 2022-12-21
Filing date: 2023-12-20
Publication date: 2024-06-27
Also published as: FR3144161A1

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

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

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.

The presence of pathogenic bacteria in food is a major public health problem. In the cheese sector, 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 involving consumer health and the economic stability of industry players. As an example, it is possible to cite the withdrawal from marketing of several batches of cheese contaminated by L. monocytogenes, widely publicized in December 2015. As a result, players in the sector are actively looking for solutions to better control the Listeria danger.

Certain companies specializing in the production and marketing of microorganisms offer biopreservative/bioprotective microbial cultures with properties

For example, it is known that certain strains of Lactobacillus piantarum have anti-Listeria activity. A strain of L. piantarum is notably marketed under the HOLDBAC® brand as a bio-preservation/bioprotection ferment.

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

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

There is therefore a real need to have means to reduce the presence, and in particular to induce the decline, of L monocytogenes in food products.

In this context, the Inventors have identified a new strain of C. mattaromaticum possessing 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 makes it possible to inhibit the population of L monocytogenes. Furthermore, 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.

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

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 the number CNCM 1-5804, in accordance with the Budapest Treaty .

In co-culture with the L. monocytogenes strain \\Avn \esc&n\.e EGDelux, the strain CNCM 1-5804 induces quenching of luminescence, which demonstrates its ability to inhibit L. monocytogenes.

In a classic 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 that is different and complementary to that of the C. maitaromaticum strains. already identified.

In addition, the combination of CNCM 1-5804 with at least one other strain of C. maitaromaticum induces inhibition of L monocytogenes that is much more effective than with the strains of C. maitaromaticum used alone. This synergistic effect of strain CNCM 1-5804 with other strains of C. maltaromaticum reveals a promising potential to reduce the amount of L monocytogenes in a cheese-type food.

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

By "bacterial preparation" is meant a composition packaged for the growth of Carnobacterium maltaromaticum, said composition comprising a viable strain of C. maltaromaticum and essential components for the growth of the bacteria, such as a source of carbon and energy, a source of potassium and phosphorus, a source of calcium, a source of magnesium, lipids, proteins or amino acids.

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

Said culture medium may be a synthetic medium. By synthetic medium, according to the present invention is meant a medium into which characterized individual components are introduced and subjected to rigorous quantitative and qualitative control.

Said culture medium can also be a natural medium. By natural environment, according to the present invention is meant 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).

Said bacterial preparation may be in liquid or solid form.

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 can be from 10 ² CFU.mL- ¹ to 10 ¹¹ CFU.mL ¹ , in particular from 10 ⁵ CFU.mL ¹ to 10 ⁷ CFU.mL ¹ , more particularly from at least 10 ⁵ CFU.mL ¹ , or from at least 10 ⁶ CFU.mL ¹ , or from at least 10 ⁷ CFU.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 can be 10 ² CFU.mL- ¹ , 10 ³ CFU.mL - ¹ , 10 ⁴ CFU.mL- ¹ , 10 ⁵ CFU.mL- ¹ , 106 CFU.mL- ¹ , 10 ⁷ CFU.mL- ¹ , 10 ⁸ CFU.mL ¹ , 10 ⁹ CFU.mL ¹ , 10 ¹⁰ CFU.mL ¹ or 10 ¹¹ CFU.mL- ¹ , relative to the total volume of the bacterial preparation.

In another embodiment of the invention, said bacterial preparation contains a quantity of C. maltaromaticum, in particular of the strain CNCM I-5804, of 10 ² CFU.g ¹ to 10 ¹¹ CFU.g ¹ , in particular of 10 ⁵ CFU.g ¹ to 10 ⁷ CFU.g ¹ , of at least 10 ⁵ CFU.g- ¹ , or of at least 10 ⁶ CFU.g ¹ , or of at least 10 ⁷ CFU.g ¹ , relative to the total weight of the bacterial preparation.

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 can be 10 ² CFU.g ¹ , 10 ³ CFU.g ¹ , 10 ⁴ CFU.g ¹ , 10 ⁵ CFU.g ¹ , 10 ⁶ CFU.g- ¹ , 10 ⁷ CFU.g ¹ , 10 ⁸ CFU.g ¹ , 10 ⁹ CFU.g ¹ , 10 ¹⁰ CFU.g ¹ or 10 ¹¹ CFU.g ¹ , relative to the total weight of the bacterial preparation.

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

The term “concentrate” means a liquid containing a quantity of bacteria greater than 10 ⁵ CFU.mL ¹ , in particular greater than 10 ¹⁰ CFU.mL ¹ , in particular greater than 10 ¹¹ CFU.mL ¹ , relative to the total volume of the concentrate. .

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

By “lyophilisate” is meant the product obtained after lyophilization of a culture of C. maltaromaticum after appropriate incubation.

By "adapted incubation" is meant an incubation according to a conventional method to obtain an initial population of C. maltaromaticum close to 10 ⁷ CFU.mL ¹ , relative to the total volume of the incubation.

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 CFU.mL- ¹ of C. maltaromaticum CNCM I-5804, relative to the total volume of the bacterial preparation .

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

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

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

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 pathogenic or toxic microorganisms for humans or animals and present in said food products, in order to reduce the risk of contamination of the food product by these pathogenic or toxic microorganisms.

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

Said protective culture may be in the form of a liquid concentrate or in the form of a lyophilisate.

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

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

The inhibition test in agar medium can be easily carried out by those 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.

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

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

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

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

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

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

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

Said protective culture of the invention can be used in the production of fermented or non-fermented dairy products, in particular in the production of cheeses or fermented milks.

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 as an ingredient or constituents milk.

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

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

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

Said protective culture of the invention can also be used in the production of processed plant food products (vegetable sausages for example).

The use of the protective culture of the invention during the manufacture of food products makes it possible to inhibit bacteria of the Listeria genus which persist in a food product in small quantities and to extend the shelf life of said food product.

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

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.

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 and in particular before packaging the food product.

By way of 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 10$ CFU.mL-i to 10 CFU.mL-1, more particularly at least 10 ⁶ CFU.mL- ¹ , in particular between 10 ⁶ CFU.mL- ¹ and 10 ⁷ CFU.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 CFU.g ¹ to 10 ¹¹ CFU.g ¹ , in particular from 10 ⁵ CFU.g ¹ to 10 ⁷ CFU.g ¹ , of at least 10 ⁵ CFU. g ¹ , or at least 10 ⁶ CFU.g ¹ , or at least 10 ⁷ CFU.g ¹ , relative to the total weight of said solid food product, during the storage period of said food product.

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

Said method comprises:

- seeding 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.

According to the process of the invention, seeding 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 process of the invention, when the seeding is done on the cheese paste, it can be carried out by spraying or spraying a liquid preparation comprising the CNCM I-5804 strain onto the cheese paste.

“Inoculation during milk processing” in cheese making means inoculation during storage, pre-ripening, coagulation, draining, molding, pressing, salting, ripening, care or washing.

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

Furthermore, seeding into milk with the CNCM I-5804 strain can be carried out before or simultaneously with the seeding of lactic ferments.

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

The milk used in said process may be milk, whole or not, raw or pasteurized or having undergone heat treatment. “Pasteurized whole milk” means whole milk. having undergone heat treatment, for example at 71.5°C for 15 seconds, then quickly cooled to 4°C.

In one embodiment of the process, the milk is inoculated with the CNCM I-5804 strain at a rate of 10 ² CFU.mL- ¹ to 10 ¹¹ CFU.mL- ¹ , in particular from 10 ⁶ CFU.mL- ¹ to 10 ⁷ CFU.mL- ¹ , relative to the volume of milk.

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

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

In one embodiment, the bacteria other than CNCM I-5804 is a proteobacterium.

In one embodiment, the lactic acid bacteria other than CNCM I-5804 is chosen from the group comprising the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus and Staphylococcus.

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

In a preferred embodiment, the seeding is carried out by 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.

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

In one embodiment, the inoculated milk is incubated at a temperature of 2°C to 65°C. In one embodiment, the inoculated 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.

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

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

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

The appended figures illustrate the invention:

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

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

[Fig.3], Average 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).

[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).

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

[Fig.6] Coefficients of variation (CV) of the inhibition forces 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.

[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 freeze-dried culture .

The present invention will be described below in more detail and with the aid of one or more examples which in no way limit the invention.

Example 1 - Inhibition of L. monocytogenes on agar

Material and methods

Preparation of Camobacterium ma/taromaticum inocula

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

Preparation of Listeria monocytogenes inocula

Five strains of Listeria monocytogenes (Scott A, PL1447, CA2165, Lal65 and SN167) are streaked on TSA-YE and incubated for 72 h at 30°C. A liquid culture in 10 mL of TSB-YE is then carried out from one colony, for each strain. These cultures are incubated for 24 h at 30°C, then mixed equivolumically. A DO measurement makes it possible to estimate the concentration using a pre-established ratio. 50% glycerol is added to the culture mixture to reach a final concentration of 10%. The glycerol culture mixture is then aliquoted into 1 mL cryovials, then frozen at -80°C.

Double-layer growth and halo of inhibition

Petri dishes containing TSA-YE agar medium are inoculated with 1.5 μL of each of the Carnobacterium maitaromaticum cultures. Seeding is carried out in the center of each box of agar medium. These boxes are incubated for 24 hours at 30°C.

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 are delicately poured onto the first agar plates, so as to create a second layer. The boxes are incubated at 30°C for 24 h.

Inhibition results on agar

The results obtained show that the CNCM I-5804 strain does not present a halo of inhibition towards the mixture of the 5 strains of Listeria monocytogenes, while the strain CNCM 1-5242 presents a halo (Figure 1). The same goes for the CNCM I-5243 strain (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.

Example 2 – Monitoring L. monocytogenes in a real cheese matrix

Material and methods

Preparation of Carnobacterium mattaromaticum inocula

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

500 pL of pre-culture are taken in order to measure the optical density (OD). This measurement is used to estimate the concentration of each using a pre-established DO/(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 water. This operation is repeated a second time in order to wash the cultures. The washed cultures are then cascade diluted to 5.0 × 10 ⁸ CFU/mL.

Preparation of Listeria monocytogenes inocula

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

Before use, the aliquot is thawed at room temperature 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 water. These steps are carried out one second times. The mixture is then diluted in cascade to a concentration of 5.0 × ^{10 5} CFU/mL in sterile physiological water.

Preparation of Saint-Nectaire samples

The cheese samples are prepared from pieces of Saint-Nectaire AOP dairy. 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 cap pill bottle. A well is then dug in the center of the pieces using a Durham bell.

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

- A negative control cheese, not inoculated

- A positive control cheese, not inoculated

- A cheese inoculated with 20 μL of C. maltaromaticum CNCM I-5804

- A cheese inoculated with 20 μL of C. maltaromaticum CNCM I-5242

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

Half of these pieces of cheese are incubated at 30°C and the other at 12°C for 6 days. At the end of these 6 days, 20 μL of the diluted mixture of Listeria monocytogenes are added to all the cheeses except the negative controls. The jars 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.

Enumeration of Listeria monocytogenes

At each counting point, one cheese of each type is taken out of the proofer. Each piece is homogenized in a Stomacher (3 X 3 min) in a bag with 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. 100 or 200 μL of the dilutions are then spread on PALCAM selective medium in duplicates. The boxes are incubated for 24 to 48 hours at 30°C.

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 whatever the incubation duration (Figure 2B): this reduction is very clearly greater than 1 log for all durations. incubation tested and the quantity of L monocytogenes counted in the cheese matrix then never exceeds 1.5.10 ⁴ CFU/g.

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 population of L monocytogenes is very important 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 phase of continuous decline occurs and allows a level of final population of 5.7.10 ² CFU/g after 27 days, i.e. a level lower than the initial level (8.7.10 ² CFU/g).

Example 3 - Ar\ \- Listeria monocytogenes activity spectrum of C. mattaromaticum CNCM I-5804

Material and methods

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

43 strains of L monocytogenes are 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 ¹ of bacteriological agar (BD), then incubated for 24 hours at 30°C. After incubation, the isolates are transferred to the wells of a microplate (Corning 3370, Corning Inc., NY, USA) containing 145 μL 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 μL of TSBYE per well. In parallel, 10 mL of TSBYE are inoculated with a starter culture of the C. maltaromaticum strain CNCM I-5804. All of these cultures (microplate and culture of the biopreservative strain) are incubated for 22 hours at 30°C. On the day of the test, the microplate containing the collection of Listeria monocytogenes strains is diluted by decimal dilutions in a microplate up to 1/1000. Five microliters from each well of this microplate are then transferred to a new microplate, containing 135 µL of TSBYE and 10 µL of C. maltaromaticum CNCM I-5804 culture per well. The final dilution level of the Listeria monocytogenes collection is thus 1/30,000. 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 selective PALCAM 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 addition of 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 witnesses. The co-culture experiment is repeated 3 times.

Results: reduction in densities of L. monocytogenes strains n presence of C. maltaromatic 5804

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

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

The objective of this work is to determine whether the C. ma/taromaticum strain CNCM I-5804 is capable of inhibiting the growth of an indicator strain of L. monocytogenes (L monocytogenes EGDelux) in preparations 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.

Material and methods

Description of matrices

In addition to 2 laboratory environments serving as controls (TSBYE and TSBYE without glucose), 5 food matrices were selected. These matrices are of animal or vegetable origin: smoked trout, vegetable sausage, UHT almond milk, UHT oat drink and UHT soy milk.

Preparation of matrices

Laboratory media consist of trypticase soy broth (TSB) (Biokar Diagnostics, Paris, France) supplemented with 6 gL ¹ of yeast extract (YE) (Biokar Diagnostics, Paris, France), without or with addition of 2 .5 gL ¹ of glucose. The liquid matrices (laboratory broths and drinks) were used as is for the tests, while the solid matrices were subject to the preparation of juice, by cutting in sterile conditions, adding physiological water and grinding in a stomacher. . For the vegetable sausage matrix, 140 g was used with a physiological water proportion of 1:1 (w/w). In the case of smoked trout, 180 g was used with a physiological water proportion 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.

Preparation of strains

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

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

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

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 to the wells of a microplate (Corning 3370) containing 135 pL per well of selected matrix. This pre-culture microplate marked G2 is incubated for 48 hours at 30°C. After incubation, the G2 microplates containing C. maltaromaticum CNCM I-5804 are diluted by decimal dilution up to 1/10,000, by transferring 15 μL 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 dilutions of factors 10 ² (D2), 10 ³ (D3) and 10 ⁴ (D4) are opaque white microplates (Corning 3917) suitable for reading luminescence by limiting the diffusion of inter-well light, and are those that are the subject of co-cultures.

24 hours before co-culture, L. monocytogenes EGDelux is cultured by transferring 100 μL of starter into 10 mL of TSBYE and incubation 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 water. This culture is then diluted 1/10,000 in physiological water and 15 μL of this dilution are transferred to the wells of the G3 microplates (D2, D3 and D4) immediately after preparation.

The measurement of anti-Z.. monocytogenes activity is carried out by kinetic monitoring of luminescence. The co-cultures are incubated with axial shaking at 30°C for 36 h and their luminescence is measured every 20 min using an Infinite 200 plate reader (Tecan). The experiment was the subject of 3 independent repetitions for each matrix.

Luminescence Disturbance Indicator (LDI)

The measurement of the inhibition of L. monocytogenes EGDelux by C. mattaromaticum CNCM I-5804 is done by calculating the luminescence disturbance indicator (LDI, Luminescence Disturbance Indicator):

1 y 1 LDI = - > - n j t

Where n designates the number of measurements, l the luminescence expressed in an arbitrary unit and t the time in seconds.

A derived relative measure, the luminescence inhibition indicator LDIr, is calculated as follows:

LDI

LDIr = 100 x

LDIc Where LDI designates a value obtained under any condition and LDIc the value obtained in the absence of inhibition, that is to say 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.

Results :

Figure 4 presents an example of luminescence kinetics of the indicator strain L. monocytogenes EGDelux in the absence or presence of 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 level of luminescence of the indicator strain compared to the control (EGDelux) when it was cultivated in the presence of the biopreservation strain (C. mattaromaticum CNCM I-5804) indicates an inhibition of its growth.

The relative luminescence disturbance indicator (LDIr) was calculated in order to summarize the measured luminescence kinetics. A derived measurement, 100-LDIr, makes it possible to obtain a strength value of the inhibition of luminescence exerted by the biopreservation strain. The results show that at the D3 inoculum level, and in all the matrices tested, the C. mattaromaticum strain CNCM I-5804 exerted an inhibitory effect on the indicator strain L. monocytogenes EGDelux (Figure 5). The highest level of inhibition measured was in the TSB control medium without glucose, where the C. maitaromaticum strain CNCM I-5804 inhibited 96.6% ± 2.4% of the luminescence of the indicator strain. The levels of luminescence quenching of the indicator strain by C. maitaromaticum CNCM I-5804 were particularly high in the vegetable sausage matrices (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 the TSBYE laboratory environment 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 in almond milk (59.6% ± 3.2%). %).

Sensitivity of the inhibitory properties of the C. maitaromaticum strain CNCM I-5804 at the inoculum level The use of three dilution levels of the C. maltaromaticum strain C CM 1-5804, namely 10 ² (D2), 10 ³ (D3) and 10 ⁴ (D4) made it possible to calculate, for each matrix, the coefficient of variation in inhibition forces at all dilutions (Figure 6). These coefficients of variation provide information on the sensitivity of the inhibitory activity to the level of inoculation of the biopreservative strain. The results show that the inhibition properties of the C. maltaromaticum strain CNCM I-5804 vary significantly depending on the level of almond milk inoculum (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 as a function of the inoculum level was the lowest in soy drink (20.4%), similar to that measured in TSBYE control medium without glucose (19.3%).

In conclusion, the co-cultures of the indicator strain with C. maltaromaticum CNCM I-5804 show on the one hand that the biopreservation strain has a high capacity for inhibiting L monocytogenes EGDelux in all the matrices selected and more particularly in matrices derived from vegetable sausage, smoked trout and soy drinks. On the other hand, the inoculation of the C. maltaromaticum strain 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 the matrices derived smoked trout, vegetable sausage (25% < CV < 50%), and soy drink (CV < 25%).

Example 5 - Challenge tests in soft cheese model

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 UMRF (Aurillac).

Material and methods

A production included 2 vats of 5 L of milk (2 cheeses of 250g/vat) including 1 test vat with L monocytogenes and C. maltaromaticum CNCM I-5804, and 1 control vat with L. monocytogenes alone without protective culture. The milk used was pasteurized and standardized milk (MG=protein level, MG/TP=1). The ferments used for soft dough technology were Flora Danica and RNA ferment. The surface ferments were Geotrichum candidum (LIP Jacynthe ferment) and Penicillium candidum (LIP Edelweiss ferment). Two productions were carried out on the same day, with the same milk.

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 then reusing the pellet in the same volume of physiological water. After the second wash, the pellet is taken up in 10 mL of UHT whole milk. These 10 mL are used to inoculate a culture of 100 mL of UHT whole milk which is 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 the strains 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.

The mixture of L. monocytogenes (50 CFU.mL ¹ ) and the C. maitaromaticum strain CNCM I-5804 (10 ⁶ -10 ⁷ CFU.mL-1) was inoculated into milk on 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.

Samples were taken at different stages of manufacturing. A 30 mL bottle of milk was taken 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. In order to enumerate L. monocytogenes, 30 g of cheese were taken at T5d, T15d and T40d.

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

Results

Microbiology In control cheeses, L monocytogenes, inoculated at 1.7 logio CFU.mL ¹ in milk, grows beyond 20 hours after renneting and its growth continues until T4Od with an average of 3.5 logio CFU.g ¹ at T5d, 4.4 logio CFU.g ¹ at T 15d and 5.8 logio CFU.g ¹ at T40d (Figure 7, Table 1).

Table 1. Counts of L monocytogenes (UFC.g ¹ logo) in cheese production in the absence and presence of the CNCM I-5804 strain.

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 in L. monocytogenes of 1.1 logio CFU.g- ¹ at T5d, T15j and T40j is observed for the cheeses inoculated with the CNCM I-5804 strain compared to the control.

Physical chemistry

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 dough (Table 3).

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

Table 3. Measurements of dry extracts of the dough in cheese production after 40 days of ripening.

The pH and solids values are correct compared to the values expected in soft dough technology. Thus the bioprotective strain tested has no significant impact on the evolution of pH during production.

Conclusion

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

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

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

Material and methods

The transmitter 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 emitting strains. Ten milliliters of trypticase-soy broth (TSB, 30 gL ¹ ) supplemented with yeast extract (YE, 6 gL ¹ ) (TSBYE) are inoculated using a sterile ose, by transfer of a sample from cultures of these strains stored at -80°C. In parallel with these inoculation conditions, another TSBYE broth is inoculated with the C. maltaromaticum strain CNCM I-5243 from a freeze-dried culture (Lallemand Specialty Cultures). The inoculated broths are incubated at 30°C for 24 h.

After 24 hours of culture, TSAYE agar (TSBYE supplemented with 12 gL ¹ of bacteriological 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 drilled in the agar plates using a sterile Durham bell, then 25 μL of the treated supernatants are deposited there. The agar plates are then incubated for 48 hours at 30°C before reading.

Results

The results reveal the presence of a halo of inhibition around each deposit of biopreservative strain culture supernatant 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, a halo of inhibition is observed around the deposit of culture supernatant of the L piantarum strain (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 strains CNCM I-5242 and CNCM I-5804, nor between C. maltaromaticum CNCM I-5243 and CNCM I-5804.

Conclusion

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

[Claim 1] Strain of Camobacterium mattaromaticum deposited under the number CNCM I-5804.

[Claim 2] Bacterial preparation comprising the strain CNCM 1-5804.

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

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

[Claim 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.

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

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

[Claim 8] Food product comprising the strain CNCM I-5804.

[Claim 9] Process for preparing a cheese, said process 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 the milk or during the milk processing.

[Claim 10] Method for reducing microbial contamination during the manufacturing process of a fermented dairy product characterized in that it includes the addition of a quantity of 10 ² CFU.mL ¹ to 10 ¹¹ CFU.mL- ¹ , in particular from 10 ⁶ to 10 ⁷ CFU.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.