Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/25—Lactobacillus plantarum

Definitions

• 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.
• GABA gamma-amino butyric acid
• 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.
• 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.
• 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.
• the strain of L. plantarum NUT-2 makes it possible to limit the growth of a strain of E. coli O157: 1-17.
• 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.
• 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.
• the dipping medium is defined by those skilled in the art depending on the type of seed. It is most often constituted by water.
• the first stage takes place in room temperature water for 16 hours.
• 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.
• 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.
• a second step, or germination step 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.
• 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).
• the cooker may also comprise a germination accessory comprising a removable basket (such as that described in the patent application WO2012 / 056171.
• 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.
• 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.
• 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).
• 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 .
• 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,
• 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.
• the soaking step comprises an activation step as described above.
• At least one step or all the steps of the cooking process is carried out in a cooking device.
• a cooking device is described in the patent application WO 2012/056171.
• 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, -
• 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 ⁇ cooking step is started.
• Cooking is very fast since soaking has already softened the rice.
• the cooking is at a temperature of about 100 ° C (between 90 ° C and 110 ° C).
• 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.
• 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).
• the strain of Lactobacillus plantarum NUT-2 was isolated from the Japanese rice NIPPON BARE paddy.
• 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.
• 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).
• 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.
• 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!
• test readings were therefore performed after 24 hours of growth.
• 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).
• MIC Minimal Inhibitory Concentrations
• Vancomycin (VA)> 256 Resistant The strain of Lactobacillus plantarum NUT-2 appeared to be sensitive to erythromycin, gentamicin, kanamycin, qinupristin / dalfopristin and streptomycin.
• Lactobacillus plantarum NUT-2 The strain of Lactobacillus plantarum NUT-2 is resistant to ampicillin, chloramphenicol, clindamycin, tetracycline and vancomycin. like Lactobacillus strains.
• 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).
• Table 4 Decreased development of Enterobacteriaceae and Bacillus sp. depending on the amount of L. plantarum NUT-2
• 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).

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• 2014
  • 2014-12-22 FR FR1463068A patent/FR3030571A1/fr active Pending
• 2015
  • 2015-12-18 WO PCT/FR2015/053642 patent/WO2016102853A1/fr active Application Filing
  • 2015-12-18 EP EP15823683.6A patent/EP3247226A1/de not_active Withdrawn
  • 2015-12-18 CA CA2971981A patent/CA2971981A1/fr not_active Abandoned
  • 2015-12-18 AU AU2015370758A patent/AU2015370758A1/en not_active Abandoned

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