USOO7981657B2

(12) United States Patent (10) Patent No.: US 7,981,657 B2 Garault et al. (45) Date of Patent: Jul. 19, 2011

(54) METHOD FOR OBTAINING VARIANTS OF (58) Field of Classification Search ...... 435/252.1; LACTIC ACD BACTERIA USABLE FOR 426/72, 311 PRODUCINGVITAMIN K2 AND See application file for complete search history. APPLICATION TO THE PREPARATION OF (56) Ref Cited FOOD PRODUCTS eeees e (75) Inventors: Peggy Garault, Montlhery (FR); Gaëlle FOREIGN PATENT DOCUMENTS Quere, Villebon sur Yvette (FR); Chloé E. i.e.: A 3E Beal, Paris (FR): Natalia Bomchil, JP 63-198993. A 8, 1998 Cachan (FR); Jean-Michel Faurie, Jouy JP 2000-287676 A 10, 2000 En Josas (FR); Guillaume Gobert, OTHER PUBLICATIONS Palaiseau (FR); Gérard Lipowski, Issy les Moulineaux (FR) Tsukamoto, Y. et al., “Construction of a Bacillus subtilis (natto) with High Productivity of Vitamin K (Menaquinone-7) by Analog Resis (73) Assignee: Compagnie Gervais Danone, Paris (FR) tance'. Biosc. Biotechnol. Biochem... vol. 65 No. 9 2001, pp. 2007 2015. (*) Notice: Subject to any disclaimer, the term of this Gasson, M. J., “Plasmid Complements of Streptococcus lactis NCDO 712 and Other Lactic Streptococci. After Protoplast-Induced patent is extended or adjusted under 35 Curing”, Journal of Bacteriology, vol. 154, No. 1, 1983, pp. 1-9. U.S.C. 154(b) by 90 days. Morishita, T. et al., “Production of Menaquinones by Lactic Acid Bacteria”, Journal of Dairy Science, vol. 82, No. 9, 1999, pp. 1987 (21) Appl. No.: 12/444,347 1903. XP-OOO983779. Vido, K., et al. “Roles of Thioredixon Reductase during the Aerobic (22) PCT Filed: Oct. 4, 2007 Life of Lactococcus lactis', Journal of Bacteriology, vol. 187, No. 2, Jan. 2005, pp. 601-610. XP-002430548. (86). PCT No.: PCT/EP2007/060572 Hart, J. P. et al., “Electrochemical Detection of Depressed Circulat S371 (c)(1), ing Levels of Vitamin K in Osteoporosis”,Journal of Clinical Endo (2), (4) Date: Apr. 3, 2009 crinology and Metabolism, vol. 60, No. 6, Jan. 1985, pp. 1268-1269. Primary Examiner — Ganapathirama Raghu (87) PCT Pub. No.: WO2008/040793 (74) Attorney, Agent, or Firm — Birch, Stewart, Kolasch & PCT Pub. Date: Apr. 10, 2008 Birch, LLP. (65) Prior Publication Data (57) ABSTRACT US 201O/OO47396 A1 Feb. 25, 2010 The present invention relates to the production of variants of lactic bacteria stems that produce, under standard fermenta (30) Foreign Application Priority Data tion conditions, at least about 1.2 times more K2 Vitamin than the starting lactic bacteria stems cultured in the same condi Oct. 4, 2006 (FR) ...... O6 08690 tions. The invention further relates to a method for preparing food products, including fermented products and/or fresh (51) Int. Cl. diary products, enriched with K2 Vitamin, and to the food CI2N L/12 (2006.01) products thus obtained. A2.3L I/30 (2006.01) (52) U.S. Cl...... 435/252.1; 426/72: 426/311 5 Claims, No Drawings US 7,981,657 B2 1. 2 METHOD FOR OBTAINING VARLANTS OF lation mechanisms. In fact, since the 1980s, vitamin K has LACTIC ACID BACTERIA USABLE FOR also been recognized for its role in bone metabolism (Hart et PRODUCINGVITAMIN K2 AND al., 1984; Hart et al., 1985). APPLICATION TO THE PREPARATION OF This vitamin is a cofactor in an enzymatic reaction modu FOOD PRODUCTS lating the activity of osteocalcin in the regulation of bone formation (Hauschka PV et al., 1989: Ducy Petal., 1996). Its The present invention relates to the field of food products role more precisely consists of modulating the carboxylation rich in nutrients, vitamins and/or trace elements in order to ofosteocalcin, a key protein that regulates the bone formation improve the content and qualitative and quantitative balance process. In the case of vitamin K deficiencies, this reaction of nutritional intake in humans. 10 The invention more particularly involves means of enrich does not occur, leading to an increase of the ratio of decar ing food in vitamin K. boxylated osteocalcin to carboxylated osteocalcin in the More precisely, the present invention concerns obtaining blood (Väänänen et al., 1999). variants of lactic acid bacteria strains that produce, under Demographic developments in the western countries have standard fermentation conditions, at least approximately 1.2 15 led to progressive aging of the population, associated in cor times more vitamin K2 than the initial lactic acid bacteria ollary with an increase in degenerative diseases, notably strains cultured under the same conditions. osteoporosis. As a result, osteoporosis is now recognized as a The invention also concerns a method for preparing food major public health problem. products, notably fermented products and/or fresh dairy Demographic estimates made in the 1990s have sounded products, enriched in Vitamin K2, as well as the food products the alarm by predicting a considerable increase in the inci thus obtained. dence of this disease in the next 50 years, notably in seniors. Vitamin K is a fat-soluble vitamin that occurs in two natu Therefore, it quickly became necessary and urgent to take ral forms: Vitamin K1 (or phylloquinone) and vitamin K2 (or action to prevent this disease, previously rarely screened for menaquinone) and managed late. Vitamin K1 is synthesized by plants. It is principally found 25 Now it is recognized that prevention of osteoporosis must in green vegetables (leafy vegetables) and Soybean oil. Vita begin in childhood, through optimal bone growth, and be min K1 is involved more directly in the blood coagulation continued throughout life by maintaining bone mass. It is process. known that nutritional factors play an important role in the As for vitamin K2, it is produced by the bacteria of the development and maintenance of bone reserves. Up until intestinal flora. It also appears in Small quantities in certain 30 now, the nutritional strategies envisioned or proposed to pre foods after a fermentation procedure (cheese, typical Asian vent osteoporosis relied essentially on two key factors, cal products such as Japanese miso and natto, based onfermented cium and vitamin D. However, we now know that other nutri Soy, etc.). Numerous bacteria are capable of synthesizing tional factors may be worthy of note. vitamin K2. Thus, in addition to the intestinal flora bacteria, As a result of its major role in bone formation, Vitamin K and notably the species Escherichia coli, Bacillus subtilis and 35 increasingly appears in the literature as a promising route for Bacteroides spp., certain species or Subspecies of lactic acid preserving lifelong bone health in humans. bacteria Such as Lactococcus lactis spp. lactis, Lactococcus The recommended dietary intake of vitamin K in humans lactis spp. Cremoris, Leuconostoc lactis, Leuconostoc (1.5ug/day/kg body weight) was established by considering mesenteroides and Propionibacterium sp. can be mentioned. its role in coagulation phenomena. Now, recent studies Sug The quantity of vitamin K2 synthesized by these bacteria 40 gest that these dietary recommendations are ultimately under generally varies from approximately 29 to 90 ug/L for fer estimated if one also takes into account the activity of vitamin mented milk (Morishita et al., 1999). It is important to empha K in bone metabolism (Ronden et al., 1998). size that Vitamin K2 production is most often measured on Although vitamin K requirements are still poorly under freeze-dried cell pellets and the results of these measurements stood, it remains that low intakes are associated with a low are highly heterogeneous with regard to production levels as 45 bone mass and an increased risk of fractures in adults (Hartet a function of the strains tested, which can vary by more than al., 1985; Knapen et al., 1989: Szulc et al., 1993: Booth etal, a factor of 3 (Morishita et al., 1999; Parker et al., 2003). In 2000). Moreover, intervention studies in menopausal women terms of biological activity, Vitamin K2 is especially known have shown that vitamin K reduced bone loss for this target for its action on Soft-tissue calcification. group (Shiraki et al., 2000; Braam et al., 2003). Finally, Vitamin K was initially described for its essential role in 50 animal studies Suggest that it would play a favorable role the process of blood coagulation. Thus, large deficiencies in during peak bone mass, especially in the case of synergetic Vitamin Klead to hemorrhages, with abnormal prolongation combination with vitamin D. However, studies clearly linking of the normal coagulation time, and ecchymoses. For a long Vitamin K and bone growth have only been conducted in time, it was believed that large deficiencies invitamin Kwere animals So far. Somewhat rare in adults, and that needs could be met in 55 Moreover, recent studies have introduced additional argu principle in a satisfactory manner by a varied and balanced ments in favor of the impact of vitamin Konbone metabolism diet and by means of endogenous production of the vitamin and, in particular, on the constitution and preservation of the by colon bacteria. In this regard, the people at risk are typi bone mass (Booth et al., 2000; Shiraki et al., 2000; Braam et cally: al., 2003: Hirano et Ishi, 2002). newborns, whose intestines do not have bacteria producing 60 Unlike adults, there are few data available with regard to vitamin Kat birth; the beneficial effects of vitamin K on bone metabolism in individuals with hepatic, bile or intestinal function disor children. It is only known that it is essential to optimize bone ders (liver diseases, cystic fibrosis, colitis, dysentery, mass during the growth period, in order to build a maximal etc.); and bone reserve and to protect adults from the risk of future individuals who take antibiotics for a long time. 65 osteoporosis. More recently, it was discovered that the impact of vitamin In any case, the result of all the data currently available is K on human health is not limited to its role in blood coagu that improving the vitamin K content in food products is a US 7,981,657 B2 3 4 particularly interesting and promising route for allowing the processed food products containing microorganisms be pre individual to build and maintain good bone structure. pared by using exclusively natural strains or variants of natu ral strains. In this context, there are already industrial products on the According to a first aspect, the present invention concerns food market containing a notable quantity of vitamin K. Cer a method for obtaining a natural variant of a lactic acid bac tain dairy products containing lactic acid bacteria, Such as teria producing, under standard fermentation conditions, a “Petits Gervais aux Fruits’ sold in France by the Applicant greater quantity of vitamin K2, by a factor equal to at least can particularly be mentioned. Nevertheless, it is noted that, approximately 1.2, than that produced by said lactic acid on the one hand, the vitamin K content of these products bacteria Strain cultured under the same conditions, said generally depends on the type of ferments used and, on the 10 method comprising at least: a) the culture of said lactic acid bacteria Strain under stan other hand, the Lactococcus lactis Strains conventionally dard fermentation conditions, on a selection medium induc used in dairy products do not produce a sufficient quantity of ing a modification of the cellular redox state; and vitamin K to truly fill the needs of the population, or even to b) the selection of said variant if it produces at least help alleviate possible vitamin K deficiencies. 15 approximately 1.2 times more vitamin K2 than said lactic Therefore there is a need in the current state of the art for acid bacteria strain cultured under the same conditions. food products, notably fermented products and/or fresh dairy The “variants' are, in the sense of the invention, lactic acid products, that contain vitamin K in Sufficient quantities to bacteria Strains capable of producing more vitamin K2 than contribute to satisfying requirements and, if necessary, cor the strains from which they arise. More precisely, the variants recting deficiencies, in children, adolescents, adults and the according to the present invention are capable of producing at elderly. least approximately 1.2 times more vitamin K2 than the initial In the following, the terms "vitamin K2 and “vitamin K” strains. Preferably, the quantity of vitamin K2 produced by a are used interchangeably to designate vitamin K2. variant conforming to the present invention is greater by a The present invention therefore seeks to address this need factor at least equal to approximately 1.5 than that obtained by proposing the preparation of food products, notably fer 25 by culturing the initial lactic acid bacteria strain under the mented products and/or fresh dairy products, by using new same standard fermentation conditions. It is still more pre variants of lactic acid bacteria Strains that produce quantities ferred that this factor be equal to at least approximately 1.7, of vitamin K significantly greater than those produced by the more preferentially equal to at least approximately 1.8, and strain from which they are derived. more preferentially still at least equal to approximately 1.9. Moreover, in the course of their work, the Inventors devel 30 Even more preferred values for this factor are at least equal to 2, 2.2, 2.4, 2.5, 2.7, 2.8, 2.9, and 3. oped conditions for using lactic acid bacteria that improve the The “reference' or “initial' lactic acid bacteria strains are production of vitamin K in a very perceptible manner with the strains from which the variants according to the invention regard to the conventional production conditions. Thus, for are obtained. These strains may be natural or may even be purposes of preparing food products, such as fermented prod 35 variants themselves, i.e., natural variants or mutants. ucts and/or fresh dairy products, enriched in vitamin K2, one Within the scope of the present invention, the “laboratory could advantageously use the vitamin K“overproducer vari conditions' are completely standard fermentation conditions ants that are the subject of the present invention under the well-known to the person skilled in the art. Thus, the expres conditions for use identified by the Inventors as particularly sions “laboratory conditions” and “standard fermentation favorable to the production of vitamin K that are the subject of 40 conditions” are completely synonymous here. The preferred French patent application No. 06/08690 of Oct. 4, 2006. “laboratory conditions” in the sense of the present invention Here, the term “variant covers: are the following: the strain is precultured on commercial natural variants, i.e., obtained spontaneously from a refer M17 medium (Difco TM M17) or on an equivalent medium. ence lactic acid bacteria strain by selection pressure; For the subsequent culture, inoculation is done at 1% by the natural variants that did not undergo any genetic 45 preculture. The incubation temperature is approximately 30° manipulation, but are principally obtained by mutation C. The laboratory conditions may be modified if necessary by and selection from the reference strain; and the person skilled in the art, on the basis of general knowledge mutants comprising one or more mutations in their and, possibly after routine experimentation. The culture genome, which were induced by genetic engineering, medium is an appropriate medium for culturing lactic acid i.e., by directed mutagenesis techniques, particularly by 50 bacteria Strains, notably Lactococcus spp. Strains. genetic transformation using vectors, applied to the ref According to a preferred embodiment, the lactic acid bac erence strain. teria strain is chosen from among the genera Lactococcus, Note that, in certain countries (notably in Europe), precau Leuconostoc, Enterococcus and Propionibacterium. It is par tions must be taken by food producers when they develop ticularly chosen from among the species Lactococcus lactis, products intended for human and/or animal food in which 55 Leuconostoc lactis, Leuconostoc pseudomesenteroides, Leu microorganisms are incorporated, especially living microor conostoc mesenteroides, Leuconostoc dextranicum, Entero ganisms. Indeed, genetically-modified organisms (microor coccus faecium, and Propionibacterium sp. ganisms here) (GMOs or mutants) can cause fear and appre Preferably, as is developed below, in the process according hension in consumers. This negative image of GMOs in to the invention, a selection medium is used chosen from certain countries is such that the public has a tendency to 60 among culture media containing bacitracin oran oxidant Such boycott foods containing GMOs. Thus, in a context where as peroxide. consumers continually require more transparency regarding The selection medium thus used permits inducing a modi the contents of the food products offered to them and the fication in the cell redox state. Advantageously, this modifi origin of the ingredients that these products contain, produc cation is correlated to the modification, in said variant and by ers may be motivated to offer almost exclusively, or even 65 comparison with the lactic acid bacteria strain from which it exclusively, products free of GMOs. In the context of the arose, of the expression of at least one gene chosen from present invention, it could therefore be advantageous that the among genes 1 to 27 listed in Table I below: US 7,981,657 B2 5 6 TABLE I standard experimental conditions, it can be called a vitamin K2 “overproducer variant. In particular, a variant in the sense NCBI Gene Gene accession of the present invention produces at least approximately 5.7 No. name Function of the corresponding protein No. ug, still more preferably at least approximately 5.9 g, and even more preferably at least approximately 6.1 ugand, better 1 cys D O-acetylhomoserine sulfhydrylase mg 0091 gpo gpo protein (glutathione mg 1088 still, at least approximately 6.3 ug of vitamin K2 per 100 g of peroxidase) fermented milk under Standard fermentation conditions. 3 met S- mg 1225 More preferably, a variant according to the present invention methyltetrahydropteroyltriglutamate produces at least approximately 6.5 ug, preferably at least homocysteine methyltransferase 10 4 putative NADH dehydrogenase mg 0195 approximately 7 Jug, preferably still at least approximately 7.5 5 metF MetF Protein methylene tetrahydrofolate mg 1226 ug, still more preferably at least approximately 8 Jug, even reductase more preferably 8.5 ug, even more preferably still at least 6 trxH H-type thioredoxin mg 0406 7 fr protein for regulation of mg 1023 approximately 9 ugand even better, at least approximately 9.5 iron assimilation 15 ug, and better still, at least approximately 10g of vitamin K2 8 qor quinone oxidoreductase mg 1850 9 rdc fumarate reductase flavoprotein mg 1441 per 100 g of fermented milk under standard fermentation Subunit conditions. 10 adhE alcohol-acetaldehyde dehydrogenase mg 2432 In a second aspect, the present invention concerns a natural 11 purO phosphoribosylaminoimidazole- mg 0973 variant of a lactic acid bacteria strain that can be obtained by Succinocarboxamide synthase 12 cydB cytochrome dubiquinol oxidase, mg 1863 a process such as described above, as well as biologically pure subunit II cultures and fractions of cultures of said variant, said variant 13 purE phosphoribosylaminoimidazole mg 0999 producing, under standard fermentation conditions, a quan carboxylase catalytic Subunit 14 pur(R phosphoribosylformylglycinamidine mg 0975 tity of vitamin K2 greater by a factor at least equal to approxi synthase I mately 1.2 than that produced by the initial lactic acid bacteria 15 trxA Thioredoxin mg 0779 25 strain cultured under the same conditions. 16 noxB NADH dehydrogenase mg 1734 In particular, a variant conforming to the present invention 17 cpo non-heme peroxidase chloride mg 1737 18 metK MetF Protein mg 2160 produces, under laboratory conditions (or standard fermenta S-adenosylmethionine synthase tion conditions): 19 trxB Protein TrxBl mg 1588 at least approximately 1.5 times more, preferably at least thioredoxin reductase 30 approximately 2 times more, preferably still at least 20 cysK O-acetylserine sulfhydrylase mg 1775 21 metC cystathionine beta-lyase mg 1776 approximately 3 times more, vitamin K2 than the CHN 22 metS MetF Protein mg 1764 12 ferment sold by CHR. Hansen A/S (Horsholm, DK); Methionyl-tRNA synthetase and/or 23 feoB ferrous iron transport protein B mg 0199 at least approximately 1.5 times more, preferably at least homolog 35 24 citB aconitate hydratase mg 0636 approximately 2 times more, still more preferably 25 ico isocitrate dehydrogenase mg 0637 approximately 3 times more and, advantageously, up to 26 fhuD ferrichrome ABC transporter mg 0349 at least approximately 10 times more, vitamin K2 than Substrate binding protein the model natural strain Lactococcus lactis ssp. Cremoris 27 Idh L-lactate dehydrogenase mg 1120 MG1363 filed with CBS (Baarn, NL) under number 40 CBS 36.4.89. This model strain of Lactococcus lactis Preferably, the expression of genes 1 to 27 is modified in ssp. Cremoris is perfectly well-known to the person said variant with regard to the lactic acid bacteria Strain. skilled in the art. It was described for the first time by By "modification of the gene expression' is meanthere that Gasson in 1983 (Gasson M., 1983). the gene expression considered is quantitatively modified In agreement with the definition of “natural variant” given with regard to that observed in the initial lactic acid bacteria 45 above, a variant according to the present invention is a natural strain: variant obtained by selection pressure on an appropriate cul either the expression is increased, and this will preferably ture medium. be the case for at least one gene chosen from 1 to 15: Several examples of such natural variants obtained by or the expression is decreased, and this will preferably be selection pressure are provided in the present application. the case for at least one gene chosen from 16 to 27. 50 Briefly (for more detail, see the “Examples' part below), the Preferably, the variant is selected from step b) if it pro preferred examples of natural variants and methods for duces, under standard fermentation conditions: obtaining them are the following. at least approximately 1.5 times more, preferably at least According to a first embodiment, a natural variant con approximately 2 times more, even more preferably at forming to the invention is obtained by selection pressure in a least approximately 3 times more vitamin K than fer 55 culture medium containing bacitracin. As a function of the ment CHN-12; and/or initial strains, the concentration of bacitracin in the medium at least approximately 1.5 times more, preferably at least can be, for example, at least approximately 0.4 mg/L, prefer approximately 2 times more, even more preferably at ably at least approximately 1 mg/L, still more preferably at least approximately 3 times more and advantageously least approximately 2 mg/L, and even more preferred at least up to approximately 10 times more vitamin K2 than 60 approximately 3 mg/L and in the most preferred manner of model natural strain MG 1363. all, at least approximately 4 mg/L. However, it is clear for the Advantageously, the variant is selected in step b) if it pro person skilled in the art that the concentration of bacitracinto duces, under standard fermentation conditions, at least be used to obtain natural variants conforming to the invention approximately 5.5 ug of vitamin K2 per 100 g of fermented will be determined as a function of the level of bacitracin milk. 65 resistance of the lactic acid bacteria strain used initially. If If the quantity of vitamin K2 produced by the variants is at necessary, the person skilled in the art will work with several least approximately 5.5ug per 100 g of fermented milk under different concentrations chosen as a function of the properties US 7,981,657 B2 7 8 of the initial strain. Advantageously, the person skilled in the a) the use of at least one variant and/or at least one ferment art could work with bacitracin concentration ranges. Such as described above, in an intermediate preparation of A particularly interesting natural variant essentially has the said product; and same biological properties as natural variant I-3557, filed b) obtaining said product enriched in vitamin K2. with the Collection Nationale de Culture des Microorganis- 5 The variants and/or the lactic ferment can notably be mes National Microorganism Culture Collection (CNCM, implemented by using bacteria concentrates precultured in Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex place (on the food product production site), or by using bac 15, France) on Jan. 20, 2006. teria precultured by a ferment Supplier, then packaged and By the expression “a variant Ahaving essentially the same sent to the food production site or sites. The Suppliers may 10 package the bacteria in the fresh or frozen state; alternatively, biological properties as natural variant I-3557, is meant here the bacteria may be dried or freeze-dried. The bacteria are, in that variant A is a natural variant obtained by selection pres all cases, added to the dairy product mass in a completely Sure in a medium containing bacitracin. However, the con conventional manner (like any other known lactic ferment). centration of bacitracin used in the medium to obtain variant A sixth aspect of the present invention pertains to a food A is not determinant for satisfying the present definition. The 15 product enriched in vitamin K2 that can be obtained by a determinant condition, in return, is that variant A is capable of process such as disclosed above. The invention pertains to producing, under laboratory conditions, approximately as human and/or animal food products, with an emphasis on much, preferably at least as much, Vitamin K2 as variant human food products. Advantageously, such a food product I-3557. A natural variant in the sense of the invention is enriched in vitamin K2 strengthens the bone solidity of the preferably natural variant I-3557. person who consumes it. Preferably, this person is a child. In a second embodiment, a natural variant according to the Preferably, a food product in the sense of the invention is present invention is obtained by selection pressure on a cul chosen from among fermented products, fresh fermented or ture medium containing at least one oxidant. For example, the unfermented dairy products, products based on fermented or oxidant can be chosen from among peroxide, perchloric ions, unfermented plantjuices (fruits, vegetables, grains, soy, etc.), ferrous ions, menadione, paraquat, oxygen or any other 25 and their combinations. In a more particularly preferred man appropriate oxidant compound. Preferably, the oxidant is per ner, a food product in the sense of the invention is a fermented oxide. Like with bacitracin, the peroxide concentration in the product and/or a fresh dairy product. medium is determined as a function of the initial lactic acid In the context of the invention, “fresh dairy products' des bacterial strain. For example, one or more concentrations of ignates more particularly fresh and fermented dairy products, peroxide in the following ranges can be tested: at least 30 ready for human consumption, i.e., fresh and fermented dairy approximately 20, 25, 27, 28.5 mg/L. Once again, the person foods. The present application particularly pertains to fer skilled in the art will determine one or more concentrations, mented milk and yoghurt. Said fresh and fermented dairy or even a range of concentrations, appropriate for peroxide foods can alternatively be fromage blanc or petit-Suisse. that will be tested experimentally in the usual way. The terms “fermented milk' and “yoghurt are given their Advantageously, a natural variant conforming to the inven 35 usual meanings in the dairy industry, i.e., products that are tion essentially has the same biological properties as natural intended for human consumption and that arise from lactic variant I-3558 (filed with the CNCM on Jan. 20, 2006). The acid fermentation of a dairy Substrate. These products may definition of the expression “a variant Ahaving essentially the contain secondary ingredients such as fruits, vegetables, same biological properties as natural variant I-3557 given sugar, etc. For example, refer to French Decree 88-1203 of above is applied here mutatis mutandis (peroxide instead of 40 Dec. 30, 1988 relating to fermented milk and yoghurt, pub bacitracin; variant I-3558 instead of variant I-3557). Prefer lished in the Journal Officiel de la République Française on ably, such a variant is natural variant I-3558. Dec. 31, 1988. A third aspect of the present invention pertains to the use of One can also refer to the “Codex Alimentarius' (prepared particular selection conditions to obtain natural variants of by the Codex Alimentarius Commission of the FAO and lactic bacteria Strains that, conforming to the preceding 45 WHO, and published by the Information Division of the FAO: description, produce, under standard fermentation condi see more particularly volume 12 of the Codex Alimentarius tions, at least approximately 1.2 times more vitamin K2 than “Codex Standards for milk and dairy products” and standard the initial strains cultured under the same conditions. “CODEX STANA-11 (a)-1975”). These are notably: The expression “fermented milk' is thus reserved in the the use of bacitracin resistance; and/or 50 present application for dairy products prepared from a dairy the use of resistance to an oxidant, such as peroxide. Substrate that has undergone a treatment at least equal to As indicated previously, said natural variants are advanta pasteurization, and then inoculated with microorganisms geously capable of producing at least approximately 5.5ug of belonging to one or more characteristic species for each prod vitamin K2 per 100 g of fermented milk under standard fer uct. A "fermented milk has not undergone any treatment mentation conditions. 55 removing a constitutive element of the dairy Substrate used, A fourth aspect of the present invention pertains to a lactic and particularly has not had the coagulum drained. The ferment that comprises at least one variant Such as described coagulation of “fermented milk” must not be obtained by above. means other than those that result from the activity of the According to a fifth aspect, the present invention concerns microorganisms used. a production method for a food product enriched in vitamin 60 The term “yoghurt is reserved for fermented milk K2, comprising at least: obtained, according to local and established usage, by the a) the use of at least one variant and/or at least one ferment development of specific thermophilic lactic acid bacteria Such as described above, in an intermediate preparation of called Lactobacillus bulgaricus and Streptococcus thermo said product; and philus, which must be alive in the final product, in an amount b) obtaining said product enriched in vitamin K2. 65 of at least 10 million bacteria per gram of the lacteous portion. Alternatively, a process for increasing the vitamin K2 con In certain countries, regulations allow the addition of other tent of a food product comprises at least: lactic acid bacteria in the production of yoghurt, and notably US 7,981,657 B2 9 10 the additional use of strains of Bifidobacterium and/or Lac of lactic acid bacteria (notably Lactococcus lactis) that can tobacillus acidophilus and/or Lactobacillus casei. These overproduce vitamin K2 by using bacitracin or peroxide as additional lactic acid strains are intended to confer various selection agents. properties to the final product, Such as improving the balance I-1 Protocol for Obtaining Variants Resistant to Bacitracin of intestinal flora or modulating the immune system. A preculture is made from a crystal of a natural Strain of In practice, the expression "fermented milk' is thus gen Lactococcus lactis in the presence of 2 mL conventional erally used to designate fermented milk other than yoghurt. commercial M17 culture medium (M17 medium, DifcoTM) Fermented milk products can have various names, according with the addition of 5 g/L lactose (hereinafter M17 Lac to the country, such as, for example “Kefir”, “Kumis’, medium) and hemin (20 uL/mL) (hereinafter, M17 Lac-- “Lassi”, “Dahi', “Leben”, “Filmj81k”, “VIIIi”, and “Acido 10 hemin medium). Incubation was performed with stirring at philus milk'. 30° C. In the case of fermented milk, the quantity of free lactic The preculture served to inoculate 2 mL of M17 Lac-- acid contained in the fermented dairy substrate must not be hemin Supplemented with bacitracin (4 ug/mL). The degree of inoculation was 1%. The culture was then incubated for 48 less than 0.6 g per 100 g at the time of sale to the consumer, 15 and the content of protein with regard to the lacteous portion hat 30°C. with stirring. should not be less than that of regular milk. Then, 100LL of this suspension were deposited on an M17 Finally, the name “fromage blanc' or “petit-suisse' is, in Lac agar. A paper disk soaked with 2.5 mg bacitracin was the present application, reserved for an unaged, unsalted deposited in the center of the dish. The agar was incubated for cheese, that has been fermented by only lactic acid bacteria 48 h at 30°C. The clones near the paper disk were cultured in (and no fermentation other than lactic acid fermentation). The the presence of bacitracin (4 g/mL) in 2 mL M17 Lac-- hemin. The incubation lasted 24 h at 30°C. with stirring. content in dry solids of fromage blanc can be lowered to 15 g. The cells were isolated on M17 Lac agar in the presence of or 10g per 100g of fromage blanc, according to whether their bacitracin (2 ug/mL) after an incubation of 48 h at 30°C. The fat content is greater than 20 g or at most 20g per 100 g of isolated clones were cultured on M17 Lac--hemin, and then fromage blanc, after complete desiccation. The dry solids 25 incubated for 24 h at 30° C. with stirring. This suspension content of a fromage blanc is comprised between 13 and 20%. served for creating the frozen stock. The dry solids content of a petit-Suisse is not less than 23 g per These experiments permitted the Inventors to select the 100 g of petit-Suisse. It is generally comprised between 25 natural variant Lactococcus lactis subsp. Cremoris I-3557 and 30%. Fromage blanc and petit-Suisse are generally filed with CNCM on Jan. 20, 2006. grouped under the name “fresh cheeses, used conventionally I-2 Protocol for Making a Dairy Product Example with the in the technical field of the present invention. “Bacitracin' Variant In a seventh aspect, the present invention concerns the use A preculture was done from a crystal of the strain in 2 mL of at least one variant and/or at least one ferment as described M17 Lac. above, to prepare a food product enriched in vitamin K2. 35 The preculture served to inoculate 50 mLUHT whole milk Advantageously, Such a food product enriched in Vitamin at 1%, which was incubated at 30° C. for 24 h. K2 strengthens the bone solidity of the person who consumes Table II below gives the results of the vitamin K2 assay it. Preferably, this person is a child. expressed in lug Equivalent MK-4/100 g of product, for the It is clear that the present invention is not limited by the bacitracin resistant variant and the corresponding wild-type strain. single description above. Other embodiments and advantages 40 of the invention will arise upon reading the examples below, provided purely for purposes of illustration. TABLE II Strain 1-3557 Wild-type EXAMPLES 45 Vitamin K 8.90 3.32 As a preliminary remark, it should be noted that the proto (in Ig 100 g) cols for obtaining the natural variants described below are applicable to any initial lactic acid bacteria Strain. For prac The bacitracin-resistant variant overproduces vitamin Kby tical reasons, as a function of the initial strain, the person a factor of 3 compared with the initial wild-type strain. skilled in the art may be induced to modify certain experi 50 II—Obtaining and Using Natural Variants Resistant to Per mental conditions developed by the Inventors. In any case, the oxide modifications that the person skilled in the art may introduce Lactococcus lactis respiration was discovered fairly to the procedures below will be minor and require only simple recently (Duwat et al., 2001). Sequencing the genome of a routine operations not involving any inventive step. strain of L. lactis (IL1403) confirmed the presence of genes I—Obtaining and Using Natural Variants Resistant to Baci 55 coding for the functions necessary to aerobic respiration (Bo tracin lotinet al., 2001). In fact, L. lactis has the men and cytABCD Although exposure to agents such as bacitracin or peroxide operons coding for the proteins necessary to menaquinone is known to permit selecting bacteria strains that have an synthesis and biogenesis of cytochrome D. This species also increased resistance to these agents, the link between bacitra has three genes involved in the last steps of heme synthesis cin or peroxide resistance and bacterial vitamin K2 produc 60 (hemH, hemK and hemN, which are required in the oxidation tion levels has never been established in the literature. of porphyrin for attaching the iron to the heme), but does not Within the scope of their work, the Inventors discovered in have the genes involved in the first steps of this process. a completely unexpected manner that bacteria were capable However, L. lactis can perform oxidative phosphorylation in of developing an original mechanism for resistance to certain the presence of protoporphyrinogen. agents such as bacitracin or peroxide involving an increase of 65 It was also shown that L. lactis could respire in the presence vitamin K2 production. The Inventors envisioned benefiting of oxygen and heme in the culture medium. This respiration from this discovery for purposes of obtaining natural variants permitted the cells to reach a larger biomass and the final pH US 7,981,657 B2 11 12 observed is higher than what is usually obtained. Cultures in TABLE III the presence of oxygen and/or heme permit obtaining com parable growth curves during approximately the first 6 or 7 Strain Vitamin K (Ig 100 g) hours of fermentation. Then, the consumption of glucose Wild-type 2.92 O.45 decreases in the case of cultures in the presence of oxygen and 1-3558 5.94 O.76 heme, and the production of lactate is thus reduced. This translates a metabolic shift which occurs late in the culture. L. As shown in Table III above, the variant produces approxi lactis respiration therefore occurs toward the end of the expo mately twice as much vitamin K2 as the corresponding wild nential growth phase (Duwat et al., 2001). 10 type strain. The role of L. lactis respiration is not yet known, nor is the III Genotype Characterization of Natural Variants I-3557 role of the vitamin K2 in this type of fermentative metabo and I-3558. lism. The Inventors further observed that vitamin K2 was III-1-Materials and Methods produced by L. Lactis Strains while respiration was not yet The strains were cultured for one night at 30° C. on M17 induced under the conditions tested (no heme in the medium 15 lactose medium. A commercial whole milk was inoculated by and no stirring permitting good oxygenation of the medium). means of each of these strains in an amount of 1% preculture. In the cytoplasm, the proteins had few disulfide bridges, The inoculated milk was placed in 12 mL tubes. The fermen unlike the extracellular proteins. There is a widespread enzy tations were stopped in the desired physiological State, the matic system that allows limiting the number of disulfide exponential growth phase or the slowing phase, by plunging bridges. The S–S bonds are reduced into SH functions by the tubes into liquid nitrogen. The tubes were then stored at means of an enzyme, thioredoxin. This enzyme is regenerated -80° C. until used. The preceding experiments showed that by thioredoxin reductase. Vido etal (2005) created an L. lactis Vitamin K is essentially produced in the slowing phase (data mutant trXB1 by genetic engineering. The trxB1 gene codes not shown). for thioredoxin reductase. Two-dimensional electrophoresis Extraction of Total RNA study of the proteins synthesized by this mutant showed that 25 All the samples were treated identically. it overproduced certain enzymes of the vitamin K2 synthesis These samples were thawed in the presence of RNA protect pathway, i.e., the MenB and MenD enzymes. (Qiagen ref76506) in order to prevent degradation of the In view of these data and after personal observations, the RNA. The cells from these samples were recovered by cen Inventors believed that one of the possible routes for improv 30 trifugation. ing the production of vitamin K2 by L. lactis could be to Then, the cellular RNA of each sample was isolated by induce respiration. Another route could be to try to mobilize means of a Mixer Mill MM300 cell disrupter (Qiagen) with Vitamin K2 to respond to oxidative stress. beads (Biospec Products ref 11079101Z, Zirconia/Silica The Inventors therefore sought to obtain natural variants Beads diameter 0.1 mm) in the presence of Trizol(R) (Invitro resistant to oxidative stress. It is important to note that the 35 gen ref 15596-026). The concentration and the purity ratios natural variants obtained do not show overexpression of the (230/260 and 260/280 nm) of the RNA were then measured Men operon. by spectrophotometry with the ND-1000 NanodropR spec I-11 Protocol for Obtaining Variants Resistant to Oxidative trophotometer (Nanodrop Technologies). The RNA quality Stress (RIN, ratio 16/23 S) was also measured by means of the 2100 Peroxide was chosen as an example of a usable oxidant. Of 40 Bioanalyzer expert software, version B.02.05 (Agilent course, other oxidants such as perchloric ions, ferrous ions, Technologies) and RNA 6000 Series II Nano Kits (Agilent menadione, paraquat, oxygen or any other appropriate oxi Technologies ref 5067-1511). dant compound could be used under similar conditions. Labeling of mRNA and Hybridization on DNA Chip After a preculture on M17 Lac medium, the initial natural The targets were synthesized by reverse transcription of strains were transplanted into the medium containing increas 45 mRNA using the same specific reverse primers as those used ing concentrations of peroxide (for example, a range from at for the synthesis of the PCR products spotted onto the DNA least 20 to at least approximately 25, 27, 28.5 mg/L). Cultures chips. These direct markers were made by the Eurogentec were incubated at 30°C. After 24h, since the first tubes of the company by means of the CyScribe first strand cDNA label concentration range did not show any growth, they were ing System dCTP/purification CyScribe GFX kit (Amer returned to incubate for an additional 24 h. The clones were 50 sham ref RPN6202X) and fluorescent molecules Cy3 then isolated by streaking on agar medium. A clone was dCTP/Cy5-dCTP bound to the nucleotides (Perkin Elmer selected for a peroxide concentration of 27 mg/L. The Inven refNEL576/NEL577). tors noted that beyond a peroxide concentration of 28.5 mg/L. The hybridization of the labeled targets on the DNA chip to there was no growth. the PCR products, of the Lactococcus lactis Cremoris 55 MG 1363 strain (sequence available on NCBI), was done by These experiments permitted the Inventors to select the the Eurogentec company, who produces and sells these DNA natural variant Lactococcus lactis subsp. Cremoris I-3558 chips itself. This company uses a conventional hybridization filed with CNCM on Jan. 20, 2006. and washing protocol (Eurogentec hybridization buffer—ref II-2 Protocol for Making a Dairy Product Example with the AR-HYB-01, incubation for one night at 42°C. in the Adva “Peroxide’ Variant 60 lytix Slidebooster SB800 station Implen, washing of the The selected clone was grown in whole milk for 24 h. hybridized DNA chips in a 0.2xSSC/0.1% SDS buffer for 5 Samples were then taken and frozen at -80° C. for a final minutes with stirring at ambient temperature then rinsing in a Vitamin Kassay. 0.2xSSC buffer for 5 minutes at ambient temperature with Table III below indicates the quantity of vitamin K2 pro occasional stirring then drying of the DNA chips by centrifu duced by the peroxide-resistant variant, compared to the 65 gation at 1000 rpm for 5 minutes). quantity produced by the initial strain (quantities expressed in The hybridization data were acquired by the Eurogentec ug Equivalent MK-4/100g of fermented milk). company by means of an Axon 4100A scanner and GenePix US 7,981,657 B2 13 14 Pro 5.1 software (Axon Instruments). The hybridized DNA (<0.01), with regard to the mean background noise and on the scans were then sent to the Inventors by the Eurogentec com value of the expression ratios (r22.00 and rs2.00). pany. III-2. Results Data Processing The two variants had similar results with regard to the The digital results from scanning the hybridized DNA expression of certain genes. The majority of genes concerned involved the redox state of the cell and notably in relation to chips were obtained by the Inventors by means of GenePix the Fe—S clusters. In this context, methionine and cysteine Pro 6.0 Software. metabolism are modified, as is iron transport. The expression These preliminary digital results were followed by statis of several enzymes involved in oxidative stress defense tical processing with MANGO software developed by the mechanisms was modified: thioredoxin H, thioredoxin B1, Transcriptomique GODMAP platform of the CNRS of Gif 10 NADH dehydrogenase, NADH dehydrogenases, glutathione Sur Yvette (91) So as to obtain significant expression ratios: peroxidase, etc. each spot corresponding to a gene was duplicated on the DNA In the exponential phase, certain enzymes involved in L. chips and each biological experiment was reproduced three Lactis respiration are overexpressed: fumarate reductase times, including a label Swap (dye-swap) in order to limit the 15 (fraC) and cytochrome oxidase (cydD). Likewise, purine biases inherent in incorporating fluorescent molecules bound base metabolism appears to be modified. to nucleotides, thus representing six slides for comparison. Table IV below gives the expression ratios of certain genes The differential expression ratios were selected on the and establishes a comparison between the variants and the basis of reproducibility criteria such as the adjusted p-value wild-type strain in the slowing phase. TABLE IV

Gene number in NCBI the present accession Variant Variant Protein Gene application No. I-3557 1-3558 O-acetylhomoserine cysD 1 mg 0091 4.04 3.70 sulfhydrylase gpo protein (glutathione gpo 2 mg 1088 3.16 3.73 peroxidase) 5-methyltetrahydropteroyltri- metE 3 mg 1225 2.94 3.12 glutamate homocysteine methyltransferase Putative NADH dehydrogenase — 4 mg 0195 2.66 3.77 MetF protein metF 5 mg 1226 2.44 2.64 methylene tetrahydrofolate reductase thioredoxin H-type trixH 6 mg 0406 2.37 2.63 ferric uptake regulation fur 7 mg 1023 2.16 4.91 protein quinone oxidoreductase qor 8 mg 1850 2.13 2.35 NADH dehydrogenase noxB 16 mg 1734 -2.01 -2.42 non-heme chloride peroxidase cpo 17 mg 1737 -239 -2.52 MetK protein S- metK 18 mg 2160 -351 -2.07 adenosylmethionine synthase TrxB protein trixB1 19 mg 1588 -3.62 -2.94 thioredoxin reductase O-acetylserine sulfhydrylase cysK 2O mg 1775 -3.86 -3.54 cystathionine beta-lyase metC 21 mg 1776 -502 -5.27 L-lactate dehydrogenase Idh 27 mg 1120 -10.98 -4.31

Table V below gives the expression ratios of genes in the variants by comparison with the wild-type strain in the expo nential growth phase. TABLEV

Gene number in NCBI the present accession Variant Variant Protein Gene application No. I-3557 1-3558 fumarate reductase flavoprotein frcC 9 Img 1441 6.28 6.52 Subunit alcohol-acetaldehyde dehydrogenase adhE 10 Img 1916 4.36 3.14 phosphoribosylaminoimidazole- purO 11 Img 0973 3.61 3.13 Succinocarboxainide synthase cytochrome dubiquinol oxidase, cydB 12 Img 1863 3.15 2.95 subunit 11 phosphoribosylaminoimidazole purE 13 Img 0999 2.12 2.58 carboxylase catalytic Subunit phosphoribosylformylglycinamidine pur(R 14 IImg 0975 2.29 2.42 synthase I Thioredoxin trxA 15 IImg 0779 3.07 2.19 putative NADH dehydrogenase 4 Img 0195 2.41 2.02 US 7,981,657 B2 15 TABLEV-continued

Gene number in NCBI the present accession Variant Variant Protein Gene application No. I-3557 1-3558 MetS protein metS 22 Img 1764 -2.49 -2.08 Methionyl-tRNA synthetase ferrous iron transport protein B feoB 23 Img 0199 -3.42 -2.21 homolog TrxB protein trixB1 19 Img 1588 -2.74 -2.58 thioredoxin reductase aconitate hydratase citB 24 Img 0636 -3.1 -2.88 isocitrate dehydrogenase icod 25 Img 0637 -7.32 -7.16 ferrichrome ABC transporter fhuD 26 Img 0349 -15.1 -16.80 Substrate binding protein

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