Bacterial strains having an outstanding ability to produce menaquinone

A method of preparing a fermented dairy product enriched in menaquinone, wherein:

• the method comprises:
• adding Lactococcus lactis subsp cremoris strain CNCM I-4128 or a Lactococcus lactis subsp cremoris variant thereof to a milk substrate,
• fermenting the milk substrate inoculated with the strain CNCM I-4128 or variant, and
• obtaining a fermented dairy product enriched in menaquinone as compared to the milk substrate before the strain CNCM I-4128 or variant is added to the milk substrate;
• the strain CNCM I-4128 or variant:
• i) produces at least 12 μg, of menaquinone per 100 g of milk fermented with the strain CNCM I-4128 or variant when measured by Test A, or
• ii) produces at least 260 μg of menaquinone per 100 g of freeze-dried cells when measured by Test B;
• the strain CNCM I-4128 or variant is alive or under a frozen or freeze-dried preserved state when added to the milk substrate;
• Test A comprises the following steps:
• inoculating two flasks comprising 100 mL of skimmed UHT milk, with 5×104 to 107 cfu/mL of the strain to be tested,
• incubating the inoculated flasks without stirring at a constant temperature of from 23° C. to 30° C. in a water bath,
• measuring and recording the evolution of the pH in one of the two flasks with a pH probe,
• stopping the incubation when the pH reaches 4.60+/−0.1 by cooling to 6° C. the flask in which the pH has not been measured,
• storing the cooled flask at 6° C. for from 14 hours to 20 hours,
• manually homogenizing the milk, and
• performing a chemical extraction of menaquinone and measuring the quantity of menaquinone following Protocol P;
• Test B comprises the following steps:
• culturing 1×102 to 1×107 cfu/ml of the strain to be tested for from 14 hours to 20 hours in 50 ml of M17-lactose broth medium at 30° C.,
• centrifuging 25 mL of the culture at 8000 rpm during 10 minutes,
• discarding the supernatant and resuspending the pellet with 25 mL of a tryptone salt solution containing 0.1% tryptone and 0.85% salt,
• centrifuging the resuspended culture at 8000 rpm for 10 minutes,
• discarding the supernatant and resuspending the pellet in 25 ml of reconstituted milk powder at 10% w/w,
• placing the cell suspension in a freeze dryer,
• obtaining from 2 to 4 g freeze-dried cells, and
• performing a chemical extraction of menaquinone and measuring the quantity of menaquinone following Protocol P;
• and
• Protocol P comprises the following steps:
• preparing a sample in a tube, wherein:
• when measuring the quantity of menaquinone from Test A, the sample consists of 10 mL of the fermented milk from Test A, and
• when measuring the quantity of menaquinone from Test B, the sample consists of 10 mL of a 10 times dilution in ethanol/water, 50/50 V/V, of the freeze-dried cells from Test B,
• mixing the sample with 5 ml of HCl 1N in the tube,
• heating the resulting mixture in the tube at 100° C. for 10 min in a water bath,
• adding 10 ml of isopranol to the tube,
• placing the tube in a water bath equipped with ultrasound at 22° C. (+/−3° C.) for 10 minutes,
• adding 5 ml of hexane to the tube,
• mixing the contents of the tube by vortexing for 5 minutes,
• centrifuging the resulting suspension at 4600 rpm for 5 minutes,
• harvesting the organic phase,
• centrifuging again for 5 min at 4600 rpm,
• harvesting the organic phase,
• concentrating the harvested organic phase with a speed vac system to obtain a dry product,
• rehydrating the dry product with 1 ml ethanol,
• filtrating the rehydrated product on a 0.45 μm filter to form an extract, and
• injecting the extract into a high performance liquid chromatography (HPLC) system to separate and detect menaquinone, wherein:
• the detection is performed using a fluorometer after post-column reduction of menaquinone by Zn,
• vitamin K1 is used as an internal standard for the extraction/purification steps, and
• menaquinone 4 (MK-4) is used as an external calibration for quantification.