AU2015370758A1

AU2015370758A1 - Novel strain of Lactobacillus plantarum

Info

Publication number: AU2015370758A1
Application number: AU2015370758A
Authority: AU
Inventors: Annabelle GOYON
Original assignee: SEB SA
Current assignee: SEB SA
Priority date: 2014-12-22
Filing date: 2015-12-18
Publication date: 2017-07-27
Also published as: WO2016102853A1; EP3247226A1; FR3030571A1; CA2971981A1

Abstract

The invention relates to an isolated strain of the

Description

1

New Lactobacillus plantarum strain

The practice of germination of cereals and legumes and in particular of rice is well established in Asia and has become popular in the Western world. The seeds accordingly germinated may be used in numerous dishes, such as salads, soups, ragouts, beverages and bakery products.

The germinated seeds are considered to have an exceptional nutritional value. It is also known that the germinated seeds such as the germinated or partially germinated brown rice contain considerable contents of gamma-aminobutyric acid (GABA). The GABA is a neurotransmitter of the brain’s cells which contributes to the prevention of some diseases and to the stimulation of the production of the growth hormone.

The germination may take place without any sophisticated equipment. The seeds are placed in water at an adequate temperature for several hours. The germinated seeds may be preserved during a few days up to more than a week in the refrigerator.

The germination starts with the imbibition of the seed and stops as soon as the radicle has pierced the teguments of the seed.

During the germination, the phases of soaking and/or conservation of the wet seeds may be favorable to the development of pathogenic or spoilage microorganisms. It is frequent that bacteria like coliform bacteria such as Escherichia coli or Enterococcus spp., Pseudomonas spp., Bacillus spp., Sphingomonas spp., Microbacterium spp. or filamentous fungi belonging in particular to the Aspergillus type develop. The ingestion of such bacteria may turn out to be dangerous to health.

An object of the present invention is a new isolated strain of the Lactobacillus type characterized by its capacity to develop in the germination

9201793_1 (GH Matters) P106220.AU 2 medium of a seed, and its capacity to limit the development of bacteria such as the Enterobacteriaceae and more particularly E. coli or such as Bacillus spp, in the case of contamination of said seed during its germination in comparison with a germination without Lactobacillus.

According to a particular embodiment, an object of the invention is an isolated strain of the Lactobacillus plantarum type and more particularly the NUT-2 strain deposited with the CNCM on September 4th, 2013 under number CNCM I-4799.

In a particular implementation of the invention, the L. plantarum NUT-2 strain allows limiting the growth of an E. coli 0157:H7 strain.

In the context of the present invention, the seeds are chosen in particular among the following species: legumes such as common beans, lentils, peas, soya beans, chenopodiaceae such as quinoa or cereals such as wheat, barley, 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 may be measured in different manners well known to those skilled in the art. Mention may be made without limitation to: the measurement of the dry biomass, the enumeration of the cells using the Thoma cell, by turbidimetry, by counting on a solid medium (sowing, incubation and enumeration), or still by measurement of the cellular activity (for example: consumption of a substrate or of a cellular constituent).

The limitation of the development or of the growth of the bacteria and in particular of E. coli, of the Enterobacteriaceae, and/or of the Bacillus spp is defined by comparing the number of bacteria present in the medium upon completion of the germination under identical experimental conditions with the exception of the presence of the strain of the invention, and more particularly of the NUT-2 strain.

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Herein, the limitation is defined upon completion of the germination by a reduction of the number of bacteria by at least 0.5 log, preferably by 1 log in the presence of the strain of the invention, and more particularly of the NUT-2 strain in comparison with the number of bacteria measured in the absence of the strain of the invention 5 and more particularly of the NUT-2 strain.

Another object of the invention is a composition comprising a strain according to the invention. ίο The strain, as well as the composition, according to the invention may be used in germination methods. Such a method comprises bringing the strain or a composition according to the invention, together with the germination medium and the seeds. 15 The germination method includes a first step of soaking the seeds in a soaking 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 may be performed in the shade. 20 In a particular implementation, the first soaking step is done in the presence of the NUT-2 strain deposited with the CNCM on September 4th, 2013 under number CNCM I-4799 - or a composition comprising said strain. This is present in an amount of bacteria comprised between 105 to 109 UFC/g of seeds, preferably 107 UFC/g of seeds. 25

The soaking medium is defined by those skilled in the art according to the type of seed. Most often, it is constituted by water.

As regards the brown rice, the first step takes place in water at ambient 30 temperature for 16 hours.

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In a particular implementation of the germination method, the soaking step comprises an activation step during which the germination medium comprising the strain and the seeds is placed at a temperature comprised between about 35°C and about 40°C for a duration comprised between about two hours and about four 5 hours. The temperature may vary between 30°C and 45°C. The duration of this activation step may be comprised between 1h30 and 4h30.

This rise of temperature allows reducing the total soaking time by 6 hours. ίο For example, the activation step of the brown rice may be done by soaking the rice seeds in water at about 30°C-40°C, preferably 32°C, for a duration comprised between 2 and 4 hours.

In a second step, or germination step (strictly speaking), the soaking 15 medium is removed and the wet seeds are placed in a recipient which is possibly placed in the shade. The seeds are rinsed with water several times during this second step whose duration depends on the nature of the seeds. It is generally comprised between 4 and 48h (in the range of 4 to 8h for quinoa, 16h for rice or still 48h for the mungo bean). 20

The number of rinses is adapted according to the type of seed, it is generally comprised between 1 and 10 rinses, preferably from 1 to 5 rinses.

Rinsing is usually done with water. 25

The appearance of the radicle through the teguments of the seed indicates the end of the germination method. However, this may be continued depending on the desires of the user, for example until obtaining sprouts. 30 Such a germination method is adapted to all types of seeds and in particular to seeds chosen among the following species: legumes such as common beans,

9201793_1 (GH Matters) P106220.AU 5 lentils, peas, soya beans, chenopodiaceae such as quinoa or cereals such as wheat, barley, corn or rice.

For a subsequent cooking of the seeds, the method of germination of the 5 seeds may be performed directly in a device for cooking seeds (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). 10 In this implementation of the invention, the seeds are placed directly in an accessory of the cooker and the accessory is placed in the tub of the cooker with water and in the presence of the strain according to the invention during the soaking step. 15 Then, after having removed water from the tub, the wet seeds are left or not in the accessory in the shade or out the shade for a duration adapted to the seed that is being germinated, namely from 4 to 48h and more particularly 30 hours, in particular 32 hours. 20 From time to time, the seeds are washed. When the germs of the seeds become visible to the naked eye, a last rinse of the seeds is performed. Afterwards, they are poured in the tub of the cooker. The water level is adjusted and the step of cooking the germinated seeds is launched. Cooking is very quick since the soaking has already softened the seeds greatly. 25

As regards the germinated rice, cooking is done at a temperature in the range of 100°C (comprised between 90 and 110°C), more specifically between 101°C and 108°C. At this temperature, the nutrients which have multiplied during the phase of germination of the seeds are preserved. 30

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Such a preparation mode allows obtaining a preparation containing much more GABA in the germinated rice accordingly cooked (up to 10 times more GABA in comparison with a non-germinated normal brown rice).

Thus, according to another aspect, an object of the invention is a method for cooking seeds comprising the following steps: a step of germination of the seeds as described hereinabove and a step of cooking the germinated seeds obtained at the previous step.

Thus, in a particular implementation, the cooking method comprises the following steps: the step of soaking the seeds is carried out in the presence of the strain or the composition according to the invention in a soaking medium at a temperature and for a duration adapted to the nature of the seed, during the germination step, the wet seeds are preferably left in the shade and are regularly rinsed until the appearance of the radicle through the teguments of the seed, the germinated seeds obtained at the previous step are cooked.

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

In a particular implementation, at least one step or all steps of the cooking method is performed in a cooking device. An example of a cooking device is described in the patent application WO2012/056171.

Thus, as regards the brown rice, the method comprises the following steps: the brown rice seeds are placed directly in an accessory of the cooking device with water. The isolated strain of the Lactobacillus type according to the invention is added and mixed with the seeds. The accessory is placed again 9201793J (GH Matters) P106220.AU 7 in the tub of the device. This soaking step is performed at ambient temperature for 16h, after having removed water from the container, the wet seeds may be left in the accessory in the shade or outside the cooking device for a duration from 24h to 48h in particular 32h, the seeds are washed several times with water, when the radicles become visible to the naked eye, the seeds are washed for a last time with water and are placed in the tub of the device, the water level is adjusted and the cooking step is launched.

Cooking is very quick since the soaking has already softened the rice greatly. Furthermore, cooking is done at a temperature in the range of 100°C (comprised between 90°C and 110°C). In one embodiment, the temperature is more precisely set between 101°C and 108°C. At this temperature, the nutrients which have multiplied during the phase of germination of the seeds are preserved.

The present invention is illustrated in a non-limiting manner by the following examples and figures.

Figure 1: Biopreservation capacity (Impact of the inoculation of the L. plantarum NUT-2 strain at concentrations of 105, 106 or 107 UFC/g of rice of the Koshihikari variety on the total flora, the Enterobacteriaceae and the Bacillus sp. after 16 hours of germination).

Figure 2: Biopreservation capacity (impact of NUT-2 at concentrations of 105, 106 or 107 UFC/g of rice of the Chu Cheong variety on the total flora, the Enterobacteriaceae and the Bacillus sp.).

Figure 3: Impact of the presence of L. plantarum NUT-2 at concentrations of 105, 106 or 107 UFC/g of rice of the Koshihikari variety subsequently to a contamination by E. coli 0157H7 (102 UFC/g of rice).

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Figure 4: Impact of the presence of L. plantarum NUT-2 at concentrations of 105, 106 or 107 UFC/g of rice of the Chu Cheong variety subsequently to a contamination by E. coli 0157H7 (102 UFC/g of rice).

Figure 5: Impact of the presence of L. plantarum NUT-2 at concentrations of 105, 106 or 107 UFC/g of rice of the Koshihikari variety subsequently to a contamination by E. coli 0157H7 (101 UFC/g of rice).

Figure 6: Impact of the presence of L. plantarum NUT-2 at concentrations of 105, 106 or 107 UFC/g of rice of the Chu Cheong variety subsequently to a contamination by E. coli 0157H7 (101 UFC/g of rice).

Example 1: isolation of the NUT-2 strain

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

The isolation of this strain has been carried out after enrichment of the rice in MRS (Man, Rogosa and Sharpe) broth at 35°C ± 2°C for 24 hours in aerobiosis and isolation on a LPSM (L. plantarum Selective Medium) medium at 35°C ± 2°C for 16 to 24 hours in anaerobiosis (AnaerogGen, Oxoid system). Afterwards, this strain has been preserved at -80°C in glycerol stocks.

For the need of the study, this strain has been isolated on an agar medium MRS containing added L-cysteine (0.05% m/v). The Petri dishes have been incubated at 35°C ± 2°C for 16 to 24 hours in anaerobiosis (AnaeroGen, Oxoid system).

Example 2: characterization of the NUT-2 strain (determination of the Minimum Inhibitory Concentration by the diffusion method ETEST (Biomerieux)

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The determination of the lowest antibiotic concentration necessary to inhibit the growth of the bacterium (Minimum Inhibitory Concentration or MIC) has been carried out on an agar medium using the Etest® system. This system is based on the use of a test strip impregnated with increasing amounts of antibiotic. Ten 5 antibiotics have been tested with this method, the minimum and maximum concentrations vary depending on the antibiotics (Table 1). These antibiotics have been selected in accordance with the recommendations of the EFSA (« Update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance » - The EFSA Journal (2008) 732, 1 -15). 10

MIC

Table 1: Etest® (BioMerieux) test strips used for the determination of the FAMILY Antibiotic Concentration range (pg/mL) Aminosides (Aminoglycosides) Gentamicin (GM) 0.016 to 1.024 Kanamycin (KM) 0.016 to 256 Streptomycin (SM) 0.016 to 256 Glycopeptides Vancomycin (VA) 0.016 to 256 Macrolides, Lincosamides and streptogramins Clindamycin (CM) 0.016 to 256 Erythromycin (EM) Quinupristin/dalfopristin (QDA) Penicillins Ampicillin (AM) 0.016 to 256 Amphenicols Chloramphenicol (CL) 0.016 to 256 Tetracyclines Tetracycline (TC) 0.016 to 256 15 Starting from colonies isolated on an agar medium MRS containing added

L-cysteine (0.05 % m/v), an inoculum has been prepared in physiological saline water (NaCI 0.85% - batch 130703) with a density of about 0.5 on the McFarland scale. 140 mm Petri dishes containing Mueller-Hinton gelose (BioMerieux - batch 9201793_1 (GH Matters) P106220.AU 10 number 1002307830 Exp 20130724) have been sown by inundation with the prepared inoculum.

Afterwards, the Etest® test strips have been applied on the agar in 5 accordance with the recommendations of the supplier (6 test strips per dish). After incubation at 35°C ± 2°C for 24 to 48 hours, symmetrical inhibition ellipses have been observed.

The MIC has been read directly from the graduation scale and expressed in ίο pg/mL at the point where the inhibition ellipse crosses the test strip. In the case where the intersection point of the inhibition ellipse and the test strip is located between two graduations, the highest concentration has been retained as MIC.

Example 3: results obtained with the L. plantarum NUT-2 strain 15

Three independent experiments have been carried out for the determination of the MIC on the 10 tested antibiotics. Hence, the readings of the tests have been carried out after 24 hours of growth. 20 The results obtained, with the Lactobacillus plantarum NUT-2 strain, for the tested antibiotics, are indicated in Table 2. The interpretation of the results has been carried out using the tables provided with the test strips Etest® (version 16029 B - 2011/01) and the threshold values defined by the EFSA (European Food Safety Authority - Technical guidance - Update of the criteria used in the 25 assessment of bacterial resistance to antibiotics of human or veterinary importance - Prepare by the Panel on Additives and Products or Substances used in Animal Feed - The EFSA Journal (2008) 732, 1-15 and the EFSA Journal (2012);10(6):2740). 30 Table 2: Interpretation of the results obtained during the determination of

the Minimum Inhibitory Concentrations (MIC) by the Etest® method of the L. plantarum NUT-2 strain. The results have been interpreted in accordance with the 9201793_1 (GHMatters) P106220.AU 11 criteria defined by the EFSA for L plantarum / L. pentosus (The EFSA Journal (2008) 732, 1-15 and the EFSA Journal (2012);10(6):2740)

Antibiotic MIC (mg/mL) Interpretation Ampicillin (AM) 4.0 Resistant Chloramphenicol (CL) 12.0 Resistant Clindamycin (CM) 4.0 Resistant Erythromycin (EM) 0.38 Sensitive Gentamicin (GM) 16.0 Sensitive Kanamycin (KM) 32.0 Sensitive Quinupristin / dalfopristin (QDA) 1.0 Sensitive Streptomycin (SM) 128.0 Sensitive Tetracycline (TC) 192.0 Resistant Vancomycin (VA) >256 Resistant 5 The Lactobacillus plantarum NUT-2 strain has turned out to be sensitive to erythromycin, gentamicin, kanamycin, quinupristin / dalfopristin and to streptomycin.

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

Example 4: molecular characterization of the NUT-2 strain 15 Starting from isolated colonies, DNA extractions are carried out in order to confirm the identification of the strain. A PCR amplification reaction with universal primers is performed on the extracted DNA in order to amplify the totality of the DNA 16S. This region of the DNA is very well preserved and the sequence allows distinguishing the different bacterial species. Afterwards, the obtained amplification 20 product has been sequenced. The comparison of the sequence obtained with the

9201793_1 (GHMatters) P106220.AU 12 sequences available in the databases allows identifying the type and the bacterial species.

The alignments of sequences performed in the databases have allowed 5 demonstrating that the NUT-2 strain has a 99% homology with the bacterial species Lactobacillus plantarum. The results of these sequence alignments are indicated in Table 3.

Table 3: Results of the sequence alignments performed from the sequence 10 of the lactic bacterium NUT-2 strain. The strains whose sequences presented the highest homologies with the sequence of the sample appear in the table. The 10 sequences having the highest homologies out of a total of 100 sequences have been retained. The database included more than 1500 sequences of different species. 15

Rank Bacterial species Seq. Ref. % identity 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

The analysis of the coding sequence for the totality of the DNA 16S has allowed identifying the NUT-2 strain at the species and confirming its belonging to the Lactobacillus plantarum species.

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Example 5: The biopreservation capacity of NUT-2 (impact on the total flora, the Enterobacteriaceae and the Bacillus spp.) has been tested at different concentrations on two rice varieties (Koshihikari and Chu Cheong) 5 For each test, 10 grams of rice have been washed with sterile water (protocol of the Centre for Taste and Feeding Behavior CSGA). Afterwards, the rice has been placed in vials with 20 ml of water and 105, 106 or 107 UFC/g of NUT-2 have been added in different vials. The control test is constituted by 10 g of rice in 20 ml of water. The vials have been placed in the shade at 30°C for 16h. ίο NUT-2, the Enterobacteriaceae and the Bacillus spp and the total germs have been enumerated by dilution and spreading from each vial. The results are described in Figures 1 and 2.

The results show a persistence and even a growth of L. plantarum NUT-2 15 during the germination and the reduction of the population of Enterobacteriaceae and Bacillus spp (Table 4). 20

Table 4: decrease in the development of Enterobacteriaceae and Bacillus sp. according to the amount ofL. plantarum NUT-2

Inoculation rate Logarithmic reduction (log non-inoculated rice - log inoculated rice) Koshihikari Chu Cheong Enterobacteriaceae Bacillus Enterobacteriaceae Bacillus sp. sp. 10b5 0.8 1.4 2.2 2.4 10b6 3.0 4.3 3.2 >6.8 10E7 >6.6 >5.7 >6.8 >6.8

Example 6: Impact of the presence of L. plantarum NUT-2 subsequently to a contamination by the E. coli 0157H7 strain (101 and 102 UFC/g of rice) tested on two rice varieties (Koshihikari and Chu Cheong)

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For each test, 10 grams of rice have been washed with sterile water (protocol CSGA). Afterwards, the rice has been placed in vials with 20 ml of water and 105, 106 or 107 UFC/g of NUT-2 have been added in different vials. The 5 control test is constituted by 10 g of rice in 20 ml of water. The vials have been placed in the shade at 30°C for 16h. NUT-2, the E. coli 0157H7 bacteria have been enumerated by dilution and spreading from each vial. The results are described in Figures 3 to 6. 10 The inoculation of rice with Lactobacillus plantarum NUT-2 allows inhibiting the development of E. coli 0157:H7 starting from a concentration of 106 UFC/g.

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Claims

1. A Lactobacillus plantarum strain characterized in that it consists of the NUT-2 strain deposited with the CNCM on September 4th, 2013 under number CNCM I-4799.

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

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

4. The germination method according to claim 3 characterized in that the strain according to claim 1 or the composition according to claim 2 is used in an amount comprised between 105 to 109 UFC/g of seeds, preferably 107 UFC/g of seeds.

5. The germination method 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 comprised between about 35°C and about 40°C for a duration comprised between about two hours and about four hours.

6. The germination method according to any of claims 3 to 5 characterized in that the seeds are chosen in particular among the following species: legumes such as common beans, lentils, peas, soya beans, chenopodiaceae such as quinoa or cereals such as wheat, barley, corn or rice.

7. A method for cooking seeds characterized in that it comprises: a step of germination of the seeds implementing a method according to any of claims 3 to 6 and a step of cooking the germinated seeds obtained at the previous step.

8. The method for cooking seeds according to claim 7 wherein the step of soaking the seeds is carried out in the presence of the strain according to claim 1 or the composition according to claim 2 in a soaking medium at a temperature and for a duration adapted to the nature of the seed, during the germination step, the wet seeds are preferably left in the shade and are regularly rinsed until the appearance of the radicle through the teguments of the seed, the germinated seeds obtained at the previous step are cooked.

9. The cooking method according to claim 8 wherein the soaking step comprises an activation step according to claim 5.

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

11. The cooking method according to any of claims 7 to 9 wherein all steps are carried out in a cooker.