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(12) United States Patent (10) Patent No.: US 7,981,657 B2 Garault Et Al

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(12) United States Patent (10) Patent No.: US 7,981,657 B2 Garault Et Al 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.
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