WO2016102853A1

WO2016102853A1 - Novel strain of lactobacillus plantarum

Info

Publication number: WO2016102853A1
Application number: PCT/FR2015/053642
Authority: WO
Inventors: Annabelle GOYON
Original assignee: Seb Sa
Priority date: 2014-12-22
Filing date: 2015-12-18
Publication date: 2016-06-30
Also published as: EP3247226A1; AU2015370758A1; FR3030571A1; CA2971981A1

Abstract

The invention relates to an isolated strain of the Lactobacillus genus, characterised by its capacity to grow in the germination medium of a seed, and to limit the growth of bacteria such as Enterobactericaceae, more specifically E. coli, or such as Bacillus spp, in the event of contamination of said seed during the germination thereof compared to germination without Lactobacillus.

Description

New strain of Lactobacillus plantarum

The practice of germination of cereals and legumes, including rice well established in Asia has become popular in the Western world, The sprouted seeds can be used in many dishes, such as salads, soups, stews, drinks and bakery products.

Sprouted seeds are considered to have exceptional nutritional value. It is also known that sprouted seeds such as sprouted or partially sprouted brown rice contain significant levels of gamma-amino butyric acid (GABA). GABA is a neurotransmitter of brain cells that plays a role in preventing certain diseases and stimulating the production of growth hormone.

Germination can be done without sophisticated equipment. The seeds are placed in water at an adequate temperature for several hours. Sprouted seeds can be stored for a few days to over a week in the refrigerator.

The germination begins with the imbibition of the seed and ceases as soon as the radicle has pierced the seed coat.

During germination, the soaking and / or preservation phases of the wet seeds may be favorable for the development of pathogenic microorganisms or alteration. Bacteria such as Escherichia coli or Enterococcus spp., Pseudomonas spp., Bacillus spp., Sphingomonas spp., Microbacterium spp. or filamentous fungi belonging in particular to the genus Aspergillus develop. Ingestion of such bacteria can be dangerous for your health. The subject of the present invention is a new isolated strain of the genus

Lactobacillus is charatérisée by its capacity to develop in the medium of germination of a seed, and its capacity to limit the development of bacteria like Enterobactericaceae and more particularly E. coli or like Bacillus spp, in case of contamination of said seed during germination compared to germination without Lactobacillus. According to one particular embodiment, the subject of the invention is an isolated strain of the genus Lactobacillus plantarum and more particularly the strain NUT-2 deposited with the CNCM on September 4, 2013 under the number CNCM 1-4799.

In a particular implementation of the invention, the strain of L. plantarum NUT-2 makes it possible to limit the growth of a strain of E. coli O157: 1-17.

In the context of the present invention, the seeds are chosen in particular from the following species: legumes such as beans, lentils, peas, soybeans, Chenopodiaceae such as quinoa or cereals such as wheat, barley, corn corn or rice.

The development of the strain according to the invention is characterized by an increase in the number of bacteria over time. This development or bacterial growth can be measured in various ways well known to those skilled in the art. Non-exhaustive examples include dry biomass measurement, counting of cells using the Thoma cell, turbidimetry, solid-state counting (incubation, incubation and enumeration), or measurement of the cellular activity (eg, consumption of a subtrate or a cellular component).

The limitation of the development or growth of bacteria, in particular E. coli, Enterobactericaceae, and / or Bacillus spp., Is defined by comparing the number of bacteria present in the medium at the end of the germination under conditions identical experimental except for the presence of the strain of the invention, and more particularly of the NUT2 strain. The limitation is here defined at the end of the germination by a reduction in the number of bacteria of at least 0.5 log, preferably 1 log in the presence of the strain of the invention, and more particularly of the NUT2 strain. relative to the number of bacteria measured in the absence of the strain of the invention and more particularly of the strain NUT2.

The present invention also relates to a composition comprising a strain according to the invention.

The strain, like the composition, according to the invention can be used in germination processes. Such a method comprises bringing into contact with the strain or a composition according to the invention, germination medium and seeds. The germination process comprises a first step of dipping the seeds in a dipping medium in the presence of the strain according to the invention at a temperature and for a duration adapted to the nature of the seed. This step can be done in the dark. In a particular implementation the first dipping step is made in the presence of the NUT-2 strain deposited with the CNCM on September 4, 2013 under the number CNCM 1-4799 - or a composition comprising said strain. This is present in an amount of bacteria comprising between 10 ⁵ to 10 ⁹ CFU / g of seed, preferably 10 ⁷ CFU / g of seed. The dipping medium is defined by those skilled in the art depending on the type of seed. It is most often constituted by water.

For brown rice, the first stage takes place in room temperature water for 16 hours.

In a particular embodiment of the germination process, the soaking step comprises an activation step during which the germination medium comprising the strain and the seeds is placed at a temperature of between about 35 ° C. and about 40 ° C. for a period of time. a duration of between about two and about four hours. The temperature can vary between 30 ° C and 45 ° C. The duration of this activation step can be between 1:30 and 4:30. This increase in temperature reduces the total soaking time by 6 hours.

For example the activation step, brown rice can be made by dipping rice grains in water at about 30 ° C-40 ° C, preferably 32 ° C, for a period of time between 2 and 4 hours. In a second step, or germination step (proper), the soaking medium is removed and the wet seeds are placed in a container which is optionally placed away from light. The seeds are rinsed with water several times during this second stage, the duration of which depends on the nature of the seeds. It is generally between 4 and 48h (about 4 to 8h for quinoa, 16h for rice or 48h for mung bean). The number of rinses is adapted according to the type of seed, it is generally between 1 and 10 rinses, preferably from 1 to 5 rinses.

Rinsing is usually done with water.

The appearance of the radicle through the seed coat indicates the end of the germination process. This can however be continued according to the wishes of the user, for example until obtaining young shoots.

Such a germination process is suitable for all types of seeds and in particular seeds selected from the following species: legumes such as beans, lentils, peas, soybean, chenopodiaceae such as quinoa or cereals such as wheat , barley, corn or rice.

For subsequent baking of the seeds, the seed germination process can be carried out directly in a seed baking device (or cooker as described for example in the patent application WO2012 / 056171). Thus, the cooker may also comprise a germination accessory comprising a removable basket (such as that described in the patent application WO2012 / 056171.

In this implementation of the invention, the seeds are placed directly in an accessory of the cooker and the accessory placed in the tank of the cooker with water and in the presence of the strain according to the invention during the step of soaking.

Then after removing the water from the tank, the wet seeds are left or not in the accessory with or without light for a period of time adapted to the seed that is germinated, ie from 4 to 48 hours and more particularly 30 hours, in particular 32 hours. From time to time, the seeds are washed. When seed germs are visible to the naked eye, a final rinse of the seeds is done. They are then poured into the bowl of the cooker. The water level is adjusted and the bean sprouting stage is started. Cooking is very fast since soaking has already softened the seeds. For germinated rice, the cooking is done at a temperature of the order of 100 ° C.

(between 90 and 110 ° C), more precisely between 101 ° C and 108 ° C. At this temperature nutrients that have multiplied during the seed germination phase are preserved.

Such a method of preparation makes it possible to obtain a preparation containing much more GABA in the sprouted rice thus cooked (up to 10 times more GABA compared to a normal non-sprouted brown rice).

Thus, according to another aspect, the subject of the invention is a process for cooking seeds comprising the following steps: a step of germination of the seeds as described above and a step of cooking the sprouted seeds obtained in the previous step .

Thus, in a particular implementation, the cooking process comprises the following steps: the step of dipping the seeds is carried out in the presence of the strain or the composition according to the invention in a dipping medium at a temperature and during a duration adapted to the nature of the seed,

During the germination stage, the moist seeds are preferably left out of the light and are regularly rinsed until the appearance of the radicle through the seeds of the seed, - The sprouts obtained previous step are cooked.

In an implementation of the cooking method described above, the soaking step comprises an activation step as described above.

In a particular implementation, at least one step or all the steps of the cooking process is carried out in a cooking device. An example of a cooking device is described in the patent application WO 2012/056171.

Thus, in the case of brown rice, the process comprises the following steps: the brown rice grains are placed directly in an accessory of the cooking device with water. The isolated strain of the genus Lactobacillus according to the invention is added and mixed with the grains. The accessory is placed back in the tank of the device. This soaking step is carried out at room temperature for 16 hours, after removing water from the container, the wet grains can be left in the accessory in the dark or outside the cooking device for a period of 24 hours at 48h in particular 32h, the grains are washed several times with water, - When the radicles are visible with the naked eye, the seeds are washed one last time with water and and are placed in the tank of the device, the water level is adjusted and Γ cooking step is started.

Cooking is very fast since soaking has already softened the rice. In addition, the cooking is at a temperature of about 100 ° C (between 90 ° C and 110 ° C). In one embodiment, the temperature is more precisely between 101 ° C and 108 ° C. At this temperature the nutrients that have multiplied during the seed germination phase are preserved.

The present invention is illustrated in a nonlimiting manner by the following examples and figures.

Fifiure 1: Bio-Preservation Capacity (Impact of inoculation of the L. plantarum NUT-2 strain at concentrations of 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of Koshihikari rice on the total flora, enterobacteria and Bacillus sp after 16 hours of germination).

Fifiure 2: Bio-Preservation Capacity (Impact of NUT-2 at concentrations of 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of Chu Cheong rice on total flora, Enterobacteria and Bacillus sp.). Fifiure 3: Impact of the presence of L. plantarum NUT2 at concentrations of 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of rice of the Koshihikari variety following contamination with E. coli O157H7 (10 ² CFU / g of rice ). Fifiure 4: Impact of the presence of L. plantarum NUT2 at concentrations of 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of rice of the Chu Cheong variety following contamination with E. coli O157H7 (10 ² CFU / g of rice).

Fifiure 5: Impact of the presence of L. plantarum NUT2 at concentrations of 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of rice of the Koshihikari variety following contamination with E. coli O157H7 (10 ¹ CFU / g of rice ).

Fifiure 6: Impact of the presence of L. plantarum NUT2 at concentrations of 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of rice of the Chu Cheong variety following contamination with E. coli O157H7 (10 ¹ CFU / g of rice).

Example 1: Isolation of the NUT-2 strain

The strain of Lactobacillus plantarum NUT-2 was isolated from the Japanese rice NIPPON BARE paddy.

The isolation of this strain was carried out after enriching the rice in MRS broth (Man, Rogosa and Sharpe) at 35 ° C. ± 2 ° C. for 24 hours under aerobic conditions and isolated on LPSM medium (L. plantarum Selective Medium). at 35 ° C ± 2 ° C for 16 to 24 hours under anaerobic conditions (AnaeroGen system, Oxoid). This strain was then stored at -80 ° C. in glycerolated stocks.

For the needs of the study this strain was isolated on MRS medium agar supplemented with L-cysteine (0.05% w / v). Petri dishes were incubated at 35 ° C ± 2 ° C for 16 to 24 hours under anaerobic conditions (AnaeroGen system, Oxoid).

EXAMPLE 2 Characterization of the NUT-2 Strain (Determination of the Minimal Inhibitory Concentration by the ETEST Diffusion Method (Biomérieux)

The determination of the lowest antibiotic concentration required to inhibit bacterial growth (Minimum Inhibitory Concentration or MIC) was performed on agar medium using the Etest ^® system. This The system relies on the use of a strip impregnated with increasing amounts of antibiotics. Ten antibiotics were tested with this method, the minimum and maximum concentrations vary among antibiotics (Table 1). These antibiotics have been selected in accordance with EFSA's recommendations ("EFSA Update" (EFSA Journal (2008) 732, 1-15).

Table 1: Etest ^® strips (BioMérieux) used for the determination of the MIC.

From colonies isolated on MRS medium supplemented with L-cysteine (0.05% w / v), an inoculum was prepared in physiological saline (NaCl 0.85% - lot 130703) at a density of about 0.5 on the MacFariand scale. 140 mm Petri dishes containing Mueiler-Hinton Agar (BioMérieux - Lot No. 1002307830 Exp 20130724) were inoculated by flooding with the prepared inoculum.

Etest ^® strips were then applied to the agar according to the supplier's recommendations (6 strips per box). After incubation at 35 ° C ± 2 ° C for 24-48 hours, symmetrical inhibition ellipses were observed.

The CM! was read directly from the scale and expressed in ng / mL at the point where the inhibition ellipse crosses the strip. In the case where the point of intersection of the inhibition ellipse and the strip was located between two graduations, the highest concentration was selected as CM!

Example 3 Results Obtained with the L Plantarum NUT-2 Strain

Three independent tests were carried out for the determination of the MIC on the 10 antibiotics tested. The test readings were therefore performed after 24 hours of growth.

The results obtained with the Lactobacillus plantarum strain NUT-2 for the antibiotics tested are shown in Table 2. The results were interpreted using the tables supplied with the Etest ^® strips (version 16029 B - 2011/01) and threshold values defined by the EFSA (European Food Safety Authority - Technical guidance- Update of the criteria used in the assessment of antibiotics to antibiotics of human or veterinary importance - Prepared by the Panel on Additives and Products or Substances used in Anima! Feed - The EFSA Journal (2008) 732, 1-15 and the EFSA Journal (2012); 10 (6): 2740).

Table 2: Interpretation of the results obtained in the determination of Minimal Inhibitory Concentrations (MIC) by the Etest ^® method of the L. plantarum NUT-2 strain. The results were interpreted according to the criteria defined by EFSA for L plantarum / L pentosus (The EFSA Journal (2008) 732, 1-15 and the EFSA Journal (2012), 10 (6): 2740).

Antibiotic C I (mg / ml) Interpretation

Ampici! Line (AM) 4.0 Resistant

Chioramphenicol (CL) 12.0 Resistant

Ciindamycin (CM) 4.0 Resistant

Erythromycin (EM) 0.38 Sensitive

Gentamycin (GM) 16.0 Sensitive

Kanamycin (KM) 32.0 Sensitive

Qinupristine / dalfopristine 1.0 Sensible

(QDA)

Streptomycin (SM) 128.0 Sensitive

Tetracycline (TC) 192.0 Resistant

Vancomycin (VA)> 256 Resistant The strain of Lactobacillus plantarum NUT-2 appeared to be sensitive to erythromycin, gentamicin, kanamycin, qinupristin / dalfopristin and streptomycin.

The strain of Lactobacillus plantarum NUT-2 is resistant to ampicillin, chloramphenicol, clindamycin, tetracycline and vancomycin. like Lactobacillus strains.

Example 4 Molecular Characterization of the NUT-2 Strain

From isolated colonies, DNA extractions are performed to confirm the identification of the strain. A PCR amplification reaction with universal primers is performed on the extracted DNA to amplify all of the 16S DNA. This region of the DNA is very conserved and the sequence makes it possible to distinguish the different bacterial species. The amplification product obtained was then sequenced. The comparison of the sequence obtained with the sequences available in the databanks makes it possible to identify the genus and the bacterial species. The sequence alignments performed in the databases have demonstrated that the NUT-2 strain has a 99% homology with the bacterial species LactobaciHus plantarum. The results of these sequence alignments are shown in Table 3. Table 3: Results of sequence alignments made from the NUT-2 lactic acid bacteria sequence. The strains whose sequences had the highest homologies with the sequence of the sample are shown in the table. The 10 sequences having the highest homologies out of a total of 100 sequences were retained. The database included more than 1,500 sequences of different species.

Rank Species Sq. Ref. % identity

bacterial

1 L. plantarum KF149794.1 99.00

2 L. plantarum KF149725.1 99.00

3 L. plantarum KF149647.1 99.00 4 L. plantarum KF149066.1 99.00

5 L. plantarum KF148981.1 99.00

6 L. plantarum KC836733.1 99.00

7 L. plantarum KC836684.1 99.00

8 L. plantarum KC836660.1 99.00

9 L. plantarum KC836659.1 99.00

10 L. plantarum KC836641.1 99.00

Analysis of the sequence coding for all of the 16S DNA made it possible to identify the NUT2 strain at the level of the species and to confirm its membership in the species Lactobacillus plantarum.

Example 5: The biopreservation capacity of NUT-2 (impact on the total flora, Enterobacteria and Bacillus spp.) Was tested at different concentrations on two rice varieties (Koshihikari and Chu Cheong).

For each test 10 grams of rice were cleaned with sterile water (Sensory Center Protocol of Taste and Food CSGA). The rice is then placed in flasks with 20 ml of water and 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of NUT-2 are added in different flasks. The control consists of 10 g of rice in 20 ml of water. The flasks are placed in the dark at 30 ° C for 16h. NUT-2, Enterobacteria and Bacillus spp. And total sprouts are counted by dilution and spreading from each vial. The results are shown in Figures 1 and 2. The results show persistence and even growth of L. plantarum NUT-2 during germination and reduction of the Enterobacteriaceae and Bacillus spp population (Table 4).

Table 4: Decreased development of Enterobacteriaceae and Bacillus sp. depending on the amount of L. plantarum NUT-2

Rate Log reduction (uninoculated rice log - inoculated rice log) inoculation

Koshihikari Chu Cheong

Enterobacteriaceae Bacillus sp. Enterobacteriaceae Bacillus sp. 10 ^E 5 0.8 1.4 2.2 2.2

10 ^E 6 3.0 4.3 3.2> 6.8

10 ^E 7>6.6>5.7>6.8> 6.8

Example 6 Impact of the Presence of L. plantarum NUT-2 following contamination with E. coli strain 0157H7 (10 ¹ and 10 ² CFU / g of rice) tested on two rice varieties (Koshihikari and Chu Cheong).

For each test 10 grams of rice were cleaned with sterile water (CSGA protocol). The rice is then placed in flasks with 20 ml of water and 10 ⁵ , 10 ⁶ or 10 ⁷ CFU / g of NUT-2 are added in different flasks. The control consists of 10 g of rice in 20 ml of water. The flasks are placed in the dark at 30 ° C for 16h. NUT-2, E. coli 0157H7 bacteria are counted by dilution and plating from each vial. The results are shown in Figures 3 to 6.

Inoculation of rice with Lactobacillus plantarum NUT-2 inhibits the development of E. coli 0157: H7 from a concentration of 10 ⁶ CFU / g.

Claims

1. Lactobacillus plantarum strain characterized in that it is the NUT-2 strain filed with the CNCM on September 4, 2013 under number CNCM 1-4799.

2. Composition characterized in that it comprises the Lactobacillus strain according to claim 1.

3. A germination method comprising a step of dipping the seeds and a germination step of said seeds, characterized in that the soaking step comprises bringing the strain of claim 1 or the composition according to claim 2 into contact with said seeds. .

4. A method of germination according to claim 3 characterized in the strain of claim 1 or the composition according to claim 2 is used in an amount of between 10 ⁵ to 10 ⁹ CFU / g of seed, preferably 10 ⁷ CFU / g of seed.

5. A method of germination according to claim 3 or 4 characterized in that the soaking step comprises an activation step during which the germination medium comprising the strain according to claim 1 or the composition according to claim 2 is placed at a temperature of from about 35 ° C to about 40 ° C for a time of from about two hours to about four hours.

6. Germination method according to one of claims 3 to 5 characterized in that the seeds are chosen in particular from the following species: legumes such as beans, lentils, peas, soybean, Chenopodiaceae such as quinoa or cereals like wheat, barley, corn or rice.

7. A process for cooking seeds characterized in that it comprises:

a seed germination step implementing a method according to one of claims 3 to 6 and A step of cooking the sprouted seeds obtained in the previous step.

The method of cooking seeds according to claim 7, wherein the step of dipping the seeds is carried out in the presence of the strain according to claim 1 or the composition according to claim 2 in a dipping medium at a temperature and during a duration adapted to the nature of the seed,

During the germination stage, the moist seeds are preferably left out of the light and are regularly rinsed until the appearance of the radicle through the seeds of the seed, The sprouted seeds obtained at the previous step are set to cook.

Cooking method according to claim 8 wherein the soaking step comprises an activation step according to claim 5.

10. Cooking method according to one of claims 7 to 9 wherein at least one of the steps is carried out in a cooker.

11. Cooking method according to one of claims 7 to 9 wherein all the steps are carried out in a cooker.