Cultivation method promoting vitamin k producing by lactic acid bacteria, its application in food products manufacture

FIELD: food industry.

SUBSTANCE: biomass production envisages cultivation of at least one lactic acid bacteria strain producing vitamin K2 under conditions of resting cells. One performs at least primary cultivation of bacteria under conditions of respiration sustention for production of the primary culture or pre-culture in a medium containing at least one compound containing a haem nucleus, the end concentration being at least nearly 0.5 mcg/ml. A fat-containing milk cultural medium is inoculated with live bacterial cells in an amount of approximately 108 CFU/ml - 1011 CFU/ml. The inoculated medium is fermented during approximately 4 hours - 48 hours at a temperature of approximately 4°C - 50°C. The lactic acid bacteria are chosen from the species Lactococcus lactis, Leuconostoc lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc dextranicum, Enterococcus faecium and Propionibacterium sp.

EFFECT: one produces biomass with a level of vitamin K2 producing from approximately 30 mcg to approximately 75 mcg and more of vitamin K2 per 100 g of the fermented cultivation medium.

16 cl, 6 dwg, 5 tbl

 

The present invention relates to the field of food products enriched with nutrients, vitamins and/or trace elements to improve the composition and the qualitative and quantitative balance of nutrients consumed in the human body.

The invention relates in particular to means of fortifying foods with vitamin K.

If to speak more precisely, the present invention relates to a method of increasing the amount of vitamin K2 obtained by culturing at least one strain of lactic acid bacteria producing vitamin K2, wherein said strain is cultivated in a "dormant cells" so that the amount of vitamin K2 produced by the culture of dormant cells, exceeds by a factor equal to at least about 1.2, the amount of vitamin K2 obtained by cultivation of the specified strain under standard fermentation conditions.

In addition, the present invention relates to a biomass obtained from a culture of lactic acid bacteria producing vitamin K2, as described above.

The invention relates also to a method of producing vitamin K2, to methods of production of food products enriched with vitamin K2, in particular fermented products and/or fresh milk products and food products obtained by such method is I.

Vitamin K is a fat - soluble vitamin that occurs in nature in two forms: vitamin K1, or phylloquinone) and vitamin K2 (or menechino).

Vitamin K1 is synthesized by plants. It occurs mainly in the green parts of plants (leafy vegetables) and soybean oil.

Vitamin K1 is involved directly in the process of blood clotting.

Vitamin K2 is produced by bacteria of the intestinal flora. In small quantities it is also present in some foods subjected to fermentation (cheese, typical Asian foods containing fermented soy, such as Japanese miso (fermented soybean paste), natto (soy cheese) and others). Many bacteria can synthesize vitamin K2. Thus, in addition to bacteria of the intestinal flora, and especially such of them as Escherichia coli, Bacillus subtilis and Bacteroides spp, examples include some species or sub-species of lactic acid bacteria such as Lactococcus lactis spp.lactis, Lactococcus lactis spp.cremoris, Leuconostoc lactis, Leuconostoc mesenteroides and Propionibacterium sp. Vitamin K2 is synthesized by these bacteria, in most cases ranges from about 29 μg up to 90 µg/l fermented milk (Morishita et al., 1999). It is important to emphasize that the measurements produced amount of vitamin K2 in the majority are held in samples of lyophilized cultures of cells and that the results of these measurements showed the t significant variation in the levels of production, depending on the tested strains, among which some strains are able to produce three times more than the other (Morishita et al., 1999; Parker et al, 2003). In the framework of the biological activity of vitamin K2 known, moreover, its effect on the deposition of calcium in soft tissues.

First vitamin K has been described as a vitamin, plays an important role in the blood clotting process. Thus, a significant lack of vitamin K leads to bleeding with abnormal increase in the duration of blood clotting and hemorrhage. For a long time it was believed that significant vitamin K deficiency in adults is rare and may be successfully covered both by a varied and balanced diet, and due to the endogenous production of vitamin bacteria of the intestine. In this respect, to the risk group usually include:

- baby, intestines which does not already contain bacteria-producers of vitamin K;

- people with impaired liver function, gall bladder or intestines (as a result of hepatitis, cystic fibrosis, colitis, dysentery and others), and

individuals a long time taking antibiotics.

Recently it was revealed that the effect of vitamin K on human health is not only limited to its role in the mechanisms of blood coagulation. In fact, since the 1980s, it was recognized that vitamin K plays a role in the metabolism of KOs is Noah fabric (Hart et al., 1984; Hart et al, 1985).

This vitamin acts as a cofactor in the enzymatic reaction, which causes the activity of osteocalcin under the regulation of bone formation (P.V. Hauschka et al., 1989; P. Ducy et al., 1996). If to speak more precisely, its role is to normalize conditions carboxylation of osteocalcin is a key protein that regulates the process of bone formation. In case of deficiency of vitamin K this reaction does not occur, resulting in the blood increases the ratio decarboxylating of osteocalcin to karboksilirovannogo osteocalcin (Väänänen et al., 1999).

Demographic trends in Western countries appear in the progressive ageing of the population, the consequence of which is the increasing prevalence of degenerative pathologies, in particular osteoporosis. For this reason, osteoporosis is now seen as a major problem threatening the health of the people.

Demographic programs developed in the 1990's, first sounded the alarm about what is predicted in the next 50 years there will be a significant increase of the incidence of a specified disease, especially among older people. In this regard, it was immediately decided on the necessity and the urgency of taking measures to prevent the said pathology, which in those years was rarely detected on Anna stage and treatment which was carried out, usually in an advanced stage.

Currently, it is widely recognized that the prevention of osteoporosis should begin in childhood with control for optimal bone growth and continue throughout life by maintaining bone mass. It is known that nutritional factors play an important role in the development and maintenance of healthy bone tissue. To date, the nutrition strategy, planned or proposed for the prevention of osteoporosis was based primarily on two key factors, namely calcium and vitamin D. However, we now know that it is not less important may be other nutritional factors.

The literature increasingly it has been suggested that vitamin K due to its important role in osteogenesis can serve as a promising agent for prevention of bone health throughout life.

The recommended intake with dietary vitamin K for humans (1,5 µg/day/kg body mass) was established only with regard to its role in blood coagulation. However, recent studies suggest that this rate of consumption is understated because it does not take into account the activity of vitamin K in bone metabolism (Ronden et al., 1998).

Even if the need for vitamin K still being underestimated is authorized, but the fact remains - low rate of consumption are associated with reduced bone mass and increased risk of fractures in adult individuals (Hart et ah, 1985; Knapen et al., 1989; Szulc et al., 1993; Booth et ah, 2000). Moreover, intervention studies among women in menopause have shown that vitamin K reduces the cases of osteoporosis in this population group (Shiraki et al., 2000; Braam et al., 2003). Finally, animal studies suggest that vitamin K may play a useful role in achieving peak bone mass and that this effect is even greater extent than previously to be associated with vitamin D synergy effect. However, studies showing a clear correlation of vitamin K to bone growth, was conducted only on animals.

Moreover, recent studies have provided additional arguments in favor of the influence of vitamin K on bone metabolism, in particular in building and maintaining bone mass (Booth et al., 2000; Shiraki et al., 2000; Braam et al., 2003; Harada and Ishi, 2002).

In contrast to the action of vitamin K in adults, there is little data on the beneficial effect of vitamin K on bone metabolism in children. We only know that it is extremely important to optimize bone mass during growth in order to maximize bone reserve and prevent the Oia risk of osteoporosis in adulthood.

But in any case, based on all currently available data we can conclude that the increased content of vitamin K in foods is particularly important and promising means of achieving and maintaining the individual's normal physique.

Within this market of food products have already appeared on industrial products, which contain high amounts of vitamin K. noteworthy examples include some dairy products containing lactic acid bacteria, such as "Petits Gervaix aux Fruits (fruit baby yogurt/curd), implemented in France by the applicant. However, it should be noted that, on the one hand, the content of vitamin K in foods in most cases depends on the type of starter cultures, and on the other hand, strains of Lactococcus lactis, commonly used in dairy products, do not produce the amount of vitamin K, sufficient to meet the needs of the population, or at least some coverage of the possible lack of vitamin K.

Thus, in the existing prior art noted the need for food, particularly in fermented products and/or fresh milk products that contain vitamin K in quantities sufficient to meet the needs (and, if necessary, to cover his de is icita) children and adolescents, as well as adults and seniors.

In the context of the description, the terms "vitamin K2" and "vitamin K" are used interchangeably and refer to vitamin K2.

Therefore, the present invention aims at meeting the specified requirements at the expense of the first proposed method of food production, such as dairy products and/or fresh milk products, in which ferment, capable of producing vitamin K, are used in conditions conducive to a very tangible way to the production of vitamin K compared with the traditional manufacturing environment.

Moreover, in the course of their work, the inventors have obtained new natural alternatives natural strains of lactic acid bacteria that produce vitamin K in quantities exceeding the quantity produced of the original natural strains, of which the options were received (see "Examples"). Thus, the newly received options that "overproducing vitamin K, preferably can be used under conditions of the method of the invention is particularly favorable for the production of vitamin K.

According to the first aspect, the present invention relates to a method of increasing the amount of vitamin K2 obtained by culturing at least one of the States is mA lactic acid bacteria, producing vitamin K2, wherein said strain is cultivated under conditions of dormant cells and which includes at least:

(a) inoculation suitable for this purpose culture medium of live bacterial cells in an amount of about 108CFU/ml to 1011CFU/ml, and

(b) fermentation inoculated as described above, the environment for approximately 4 h to 48 h, preferably from about 8 h to 48 h, at a temperature from about 4°C to 50°C, preferably at a temperature from about 4°C to 40°C, so that at the end of stage (b) the amount of vitamin K2 produced by culture of dormant cells, exceeds by a factor equal to at least about 1.2, the amount of vitamin K2 obtained by cultivation of the specified strain under standard conditions the fermentation.

The expression "culture of dormant cells and culture conditions of dormant cells" are part of a common terminology in the field of engineering that applies the present invention. Therefore, the concept of "resting cells" well known to the skilled in the art specialist. In France these expressions in English are well known and in most cases are not translated into French.

"Standard conditions the fermentation is in full compliance, as svidetel what was their name, standard and well known to the skilled in the art specialist (these are referred to as the "laboratory conditions"). Preferred "standard fermentation conditions" in the context of the present invention are the following: the pre-strain is grown on the prepared agar M17 medium (agar Difco™ M17) or equivalent environment with the addition of 20 µl/ml 0.5 mg/ml of hemin in 0.1 M solution of soda. For further culturing the inoculation using 1% of the produced preculture. The incubation temperature is about 30°C. Aeration is provided by simple stirring. Fermentation condition can vary skilled in the art specialist based on their own knowledge and, of course, the results of experiments to Refine the methodology. However, it is important to follow a systematic adherence to the following three main criteria: (i) cultural environment is suitable for cultivation of strains of lactic acid bacteria, in particular strains of Lactococcus spp.; (ii) at least one compound containing a core of heme (for example, gemin, catalase or chlorophyll derivatives), is added to the environment to receive preculture and/or in the culture medium (preferably in a medium to obtain preculture, and culture with the food); (iii) the process of making preculture and/or cultivation (preferably only the cooking process preculture) is carried out under conditions of agitation.

Private variants of embodiment of the method of the invention include the following:

- at the stage of (a) cultural environment inoculums living bacterial cells in an amount of about 5×108CFU/ml to 1010CFU/ml, more preferably from about 2×109CFU/ml to 6×109CFU/ml;

- at the stage (b) the fermentation medium is carried out under standard conditions for approximately 12 h to 36 h, preferably from about 15 h to 24 h;

- at the stage (b) the fermentation medium is carried out under standard conditions at a temperature from about 15°C to 35°C, preferably from about 20°C to 30°C.

Preferably at the end of stage (b) the amount of vitamin K2 produced by culture of dormant cells, exceeds by a factor equal to at least about 1.5, the amount of vitamin K2 obtained by cultivation of the specified strain in standard fermentation conditions. The specified ratio is more preferably equal to at least about 1.7; even more preferably, at least about 1.8, even more preferably at least about 1.9. The most preferred values of the specified coefficient ablauts the values equal to about 2, about 2.2, about 2.4, about 2.5, about 2.7, about 2.8 to about 2.9 and about 3.

Preferably the level of production of vitamin K2, which is reached by means of the invention is about 30 mcg of vitamin K2/100 g of fermented milk under standard fermentation conditions. In the best case, the level of production may reach about 40 mcg of vitamin K2/100 g of fermented milk, more preferably it can reach or even exceed approximately 45 μg or 50 μg of vitamin K2/100 g of fermented milk. Thus, preferred, in particular, are the levels of production of about 55 mg, about 60 mg, about 65 mg, about 70 mg, or about 75 mcg of vitamin K2/100 g of fermented milk or even higher.

According to one variant embodiment of the invention, the strain of lactic acid bacteria producing vitamin K2, which is used in the method of the invention, is selected from the genus Lactococcus, Leuconostoc, Enterococcus and Propionibacterium. In particular, the lactic acid bacteria strain selected from the species Lactococcus lactis, Leuconostoc lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc dextranicum, Enterococcus faecium and Propionibacterium sp. Preferably, the strain of lactic acid bacteria is selected from natural variants Lactococcus lactis subsp.cremoris-producer of vitamin K2, which were obtained by the authors of the present invention during the Prov is given to them (see below the "Examples"section), such as: option I-3557, registered in the National collection of cultures of microorganisms (France) (France's Collection Nationale de Culture des Microorganismes) (CNCM, Pasteur Institute, 25, rue du Docteur Roux, 75724 Paris cedex 15, France) 20/01/2006; I-3558, registered in the CNCM 20/01/2006 and I-3626, registered in the CNCM 19/06/2006.

In the context of describing the term "variant" includes:

natural variants, i.e. variants obtained spontaneously from the standard strain of lactic acid bacteria under pressure selection; therefore, these natural options are not products of genetic engineering, and the obtained mainly by mutation and selection of the standard strain, and

mutants obtained by one or more mutations in the genome, induced genetic engineering, i.e. methods of site-directed mutagenesis, in particular, by the method of genetic transformation using vectors applicable to the standard strain.

In all cases the "options" in the context of the invention represent strains are able to produce vitamin K2. If the production version of the vitamin K2 is at least about 5.5 μg/100 g of fermented milk under standard fermentation conditions (also called the "laboratory conditions"), then that option is preferably "overproducers" strain.

It should be noted that some countries (in particular, in Europe) producers of food products prescribed to observe safety precautions when designing products intended for consumption by humans and/or animals that are introduced microorganisms, more specifically - living microorganisms, as genetically modified organisms (in this case, microorganisms), referred to as GMOs or mutant, can cause consumers apprehension and fear.

Such a negative view, which in some countries suffers the reputation of GMOs leads to the fact that consumers are boycotting food products containing GMOs. Thus, under the pressure of consumer demand for greater transparency in the designation of levels and origin of ingredients to offer them food producers will probably have to put on sale quasiequivalence, even exclusive, such products that do not contain GMOs. Therefore, in the context of the present invention, it may be preferable to manufactured food products containing microorganisms, made exclusively with natural strains or natural variants of natural strains.

According to one variant embodiment of the invention at the stage (a) is suitable for the purpose of the invention, the culture medium contains fat. It contains before occhialino, at least about 0.5% fat; more preferably, at least about 1.5% fat; most preferably, at least about 3.5% fat. In one embodiment, such an environment may contain, for example, milk cream or soy milk. It can also be normal milk or milk with a high buffer capacity. Needless to say, that can be used and combinations of these different environments. Examples of "milk with high buffer capacity" include, in particular, milk with added β-glycerol, and/or citrate and/or milk proteins, and/or any other suitable for this purpose food ingredient with buffer capacity.

For example, bold, milk typically contains about 1.5% fat; whole milk is about 3.5% fat.

In one of the preferred variants of the embodiment of the method of the invention also includes at least one phase preceding stage (a) and consisting of primary cultivation of the strain in terms of maintenance respiration in a suitable nutrient medium, containing at least one porphyrin in a final concentration of at least about 0.5 μg/ml, More preferred concentration and/or ranges of concentrations of porphyrin comprise at least about 1 μg/ml; preferably, at least about 5 μg/ml or at least about 10 mg/ml

Preferably the resulting preculture incubated at a temperature from about 4°C to 40°C, more preferably at a temperature from about 18°C to 35°C, most preferably at a temperature of about 30°C.

The incubation period of preculture can vary depending on the strain and other conditions. Preferably it is at least about 12 hours; more preferably, at least about 16 hours

The supply of oxygen preculture can be carried out by stirring or aeration.

Intermediate stage can also be carried out between a pre-stage of the primary cultivation of getting preculture and stage (a) of the method of the invention. This intermediate stage is the concentration of biomass obtained at the end of the primary stage of cultivation, for example, by centrifugation of preculture with subsequent extraction of bacterial sludge.

It should be noted that taking into account the application of the object of the present invention in the food industry in terms of implementation of the method must be (i) suitable for implementation on an industrial scale (in the framework of feasibility, yield, production costs, equipment and other) and (ii) applicable to food products (within the physical and organoleptic characteristics of the finished products (taste, Z. the groin, the consistency, appearance and others)).

The second aspect of the present invention relates to an enriched biomass is produced by culturing at least one strain of lactic acid bacteria producing vitamin K2 in terms of resting cells, as described above.

In the third aspect of the present invention mentioned above, the biomass used for the production of a food product enriched in vitamin K2.

A fourth aspect of the present invention relates to a method for producing vitamin K2, which includes at least:

(a) the implementation of the method of increasing the amount of vitamin K2 produced by culturing at least one strain of lactic acid bacteria producing vitamin K2, in accordance with the previous description, and

(b) the extract obtained in this way vitamin K2.

According to the fifth aspect of the present invention relates to a method of manufacturing a food product enriched in vitamin K2, or to methods for enrichment of food products with vitamin K2.

In the first variant embodiment of one such method includes at least:

(a) producing vitamin K2 method according to a fourth aspect of the invention;

(b) adding biogas produced in this way vitamin K2 in the specified food product or semi-finished product and

(C) receive is of the specified food product, enriched in vitamin K2.

In the second variant embodiment of the method of manufacturing a food product enriched in vitamin K2, includes at least:

(a) culturing at least one strain of lactic acid bacteria producing vitamin K2, in terms of resting cells according to the method of increasing the amount of vitamin K2 derived from cultures of the indicated strains (the first aspect of the invention);

(b) adding a biomass obtained from the culture stage (a), in the specified food product or semi-finished product and

(C) obtaining the specified food product enriched in vitamin K2.

Alternative it can provide simultaneous holding of the above-described steps (a) and (b):

(a) culturing at least one strain of lactic acid bacteria producing vitamin K2, in terms of resting cells according to the method of increasing the amount of vitamin K2 derived from cultures of the indicated strains (the first aspect of the invention), in that the food product or in its interim basis, and

(b) obtaining the specified food product enriched in vitamin K2.

In this case, the strain or strains can be used in the form of concentrates bacteria, primarily cultivated in place, i.e. at the place of production of food or bacteria, primarily cultiv is reported to the supplier of starter cultures and then packaged and delivered to the place or places of food production. Suppliers can pack up the bacteria in the liquid or frozen state; alternatively, bacteria can be subjected to drying or lyophilization. In all cases, bacteria are added to milk a lot of completely traditional way (like any other famous sourdough lactic acid bacteria). At a later stage of cultivation under conditions favorable for the production of vitamin K2, apply the modes according to the method of the invention.

Another variant embodiment of the method of enrichment of food products with vitamin K2 includes at least:

(a) adding biomass obtained according to the present invention, the food product or in its interim basis, and

(b) obtaining the specified food product enriched in vitamin K2.

In typical cases, biomass is used in the same way as traditional sourdough lactic acid bacteria.

The sixth aspect of the present invention relates to a food product enriched in vitamin K2, obtained as described above.

Alternative food product enriched in vitamin K2 method of the present invention contains the above-described biomass.

The invention relates to food products intended for human and/or LM is now, preferably to products intended for human consumption. Mainly such a food product enriched in vitamin K2, increases bone strength in an individual consuming this product. Preferably this individual is a child.

Preferably the food product in the context of the invention is selected from fermented foods, fermented or non-fermented fresh milk products, fermented or non-fermented products containing juice of plant origin (fruits, vegetables, cereals, soybean, and other), and their combinations. More preferably the food product in the context of the invention is a fermented product and/or fresh milk products.

In the context of the invention "fresh dairy products" refer, in particular, fresh and fermented dairy products ready for human consumption, i.e. fresh and fermented dairy foods. More specifically, the present invention relates to fermented milk and yoghurt. These fresh and fermented dairy products, alternatively, can be a cottage cheese (homemade cheese) or "petits-suisses" (FR. product type curds).

"Fermented milk" and "yogurt" is the standard name used in the dairy industry, i.e. products, etc is naznachenie for humans and the resulting acidification of the milk substrate due to lactic acid fermentation. These products may also contain additional ingredients such as fruits, plants, sugar, etc. Can be referenced, for example, on the standard of France No. 88-1203 (30 December 1988) on fermented milk and yogurt, published in the Official journal of the French Republic (Official Journal of the French Republic) December 31, 1988

You can also refer to the "Codex Alimentarius" (standards for food products) (composed of the Codex Alimentarius Commission under the auspices of FAO and who and published by the communication Department, FAO and available on the website http://www.codexalimentarius.net; specifically see volume 12 of the Code of Alimentarius Codex standards for milk and milk products (Codex Standards for milk and dairy products) and the standard CODEX STAN A-11 (a) - 1975").

The expression "fermented milk" means in this application dairy products manufactured at the milk substrate is subjected to processing that is equivalent, at least, pasteurization, and fermented by microorganisms belonging to the species, characteristic for the particular product. "Fermented milk" is not subjected to processing to remove some of the composite element used dairy substrate, in particular, does not undergo surgery to remove serum from the clot. Coagulation "fermented milk" should not be conducted in any other way, except by Akti the particular microorganisms used.

The term "yogurt" means fermented milk obtained as a result of life locally and globally deployed specific thermophilic lactic acid bacteria such as Lactobacillus bulgaricus (Bulgarian Bacillus) and Streptococcus thermophilus (thermophilic Streptococcus), and the concentration of living microorganisms in the finished product should be at least 10 million bacteria in 1 g of dairy product.

In some countries, food legislation is allowed to add other lactic acid bacteria in yogurt and additionally mainly to use strains of Bifidobacterium and/or Lactobacillus acidophilus and/or Lactobacillus casei. These additional strains of lactic acid are intended to give a finished product of various properties, for example to maintain the balance of intestinal flora, or to modulate the immune system.

In practice, the expression "fermented milk" in most cases is used to refer to fermented milk products, different from the yogurt. Depending on the country it may be, for example, kefir, koumiss", "Lassi" (Indian fermented milk drink), "Dahi (Indian milk product type cheese), "assurance & financial services" (the Egyptian fermented beverage type kefir), "Filmjôlk" (Finnish fermented milk drink with cereals), "Villi" (Finnish fermented milk on Itok type yogurt) or "acidophilus milk".

In the case of fermented milk, the amount of free lactic acid contained in the fermented milk substrate must be not lower than 0.6 g/100 g in the period of sale to the consumer, and the protein content provided by the dairy part of the product must not be lower than in conventional milk.

And, finally, the name "cottage cheese" or "petit-suisse" (product type home cheese) in this application refers to cheese, which is not subjected to any refining or salting and which is only subjected to fermentation with lactic bacteria (and no other fermentation, in addition to lactic acid fermentation). The amount of dry matter in cheese cottage can be reduced to 15 g or 10 g/100 g of product, in accordance with what the fat content in it can exceed 20 g or greater part of 20 g/100 g of cottage cheese after complete dehydration. The content of dry matter in cheese cottage ranges from 13% to 20%. The amount of dry matter in the "petit-suisse" is not less than 23 g/100 g of product. In most cases, the solids content in the "petit-suisse" is from 25% to 30%. The cottage cheese and "petit-suisse" are usually called "fresh cheese", i.e. without cheese ripening, used the traditional way in the field of technology to which the present invention relates.

The following figures illustrate the present invention but in no way limit the th purpose or scale.

Figure 1. Histogram showing the effect of the concentration of milk fat in the production of vitamin K2 lactic acid bacteria. Strains 1 and 2: examples of natural strains of Lactococcus lactis ssp. cremoris.

Fig 2. Examples of the kinetics of the process of producing vitamin K2 in whole milk and kinetics of the process of increasing acidity (inset top left) when using natural strains of lactic acid bacteria (strain no. 1).

Figure 3. Histogram showing the production of vitamin K2 cultures of resting cells at different temperatures. Control: cultivation under standard growth conditions.

Figure 4. Histogram showing the effect of preparation conditions of the primary culture or preculture by producing vitamin K2, depending on the strain.

Figure 5. A graph showing the influence of the initial population of bacteria natural variant I-3558 on the production of vitamin K2 in the resting phase cells conducted using ordinary milk or milk with a high buffer capacity. "Without maintenance respiration: obtaining preculture traditional method and subsequent cultivation in the conditions of dormant cells in whole milk. "Laboratory conditions keep breathing": getting preculture in terms of breathing, conducted in vitro with a nutrient medium, and further culturing under conditions in which Oaxaca cells in whole milk. "Laboratory conditions maintain breathing in milk with high buffer capacity": getting preculture in terms of respiration and subsequent cultivation in the conditions of dormant cells in milk with high buffer capacity by adding β-glycerol. "The maintenance respiration of bacteria in the fermenter": getting preculture in terms of maintenance respiration, held in the fermenter, followed by cultivation in the conditions of dormant cells in conventional milk.

6. Histogram showing the effect of viability of bacteria natural variant I-3558 on the production of vitamin K2 in the resting phase cells. VitK STZ: getting preculture in terms of maintenance respiration with processing streptozotocin and subsequent cultivation in the conditions of dormant cells in whole milk with the addition of erythromycin. VitK R+: getting preculture in terms of maintenance respiration carried out in vitro with a nutrient medium and then cultivating under conditions of dormant cells in whole milk.

It goes without saying that the present invention is not limited to the above description. Other variants of embodiments and advantages of the invention will become apparent from the examples below are purely for illustrative purposes.

Examples

Part A. preparation of natural VA is Ianto natural strains of lactic acid bacteria, capable of producing increased amounts of vitamin K

As a preliminary remark, it should be said that the methodology of the natural options, described below, applicable to any kind of source of lactic acid bacteria strain. Depending on the source of the strains used skilled in the art specialist, it may be desirable, primarily for practical reasons, changing some of the experimental conditions, developed by the authors of the invention. In any case, the changes that are skilled in the art specialist deems necessary to make the following techniques will be likely to be minor and require only simple and routine operations, not including inventive stage.

A-I - Receipt and application of natural variants that are resistant to bacitracin

Although it is known that exposure to the effects of such agents as bacitracin or peroxide, makes possible the selection of bacterial strains with increased resistance to these agents, but the relationship between resistance to bacitracin or peroxide and levels of production by bacteria of vitamin K2 to date has not been covered in the literature.

In the course of their work the authors of the invention a completely unexpected way and managed to discover that bacteria JV is able to create an original mechanism of resistance to some agents, such as bacitracin or peroxide, which includes increased production of vitamin K2. The inventors tried to apply his discovery to obtain natural variants of strains of lactic acid bacteria (mainly Lactobacillus lactis), capable of overproducing vitamin K2, using bacitracin or peroxide, for example, as agent for the selection of such options.

A-I-1 Method for obtaining variants resistant to bacitracin

Cooking preculture was conducted from a crystal of natural Lactobacillus lactis in 2 ml of traditional prepared nutrient medium M17 (M17 agar, Difco™) supplemented with 5 g/l lactose (hereinafter - M17 Lac) and hemin (20 µl/ml; hereinafter M17 Lac+geminus). Incubation was carried out under conditions of agitation at 30°C.

Preculture was used for inoculation of 2 ml of medium (Ml7 Lac+geminus) with the addition of bacitracin (4 µg/ml). The rate of inoculation was 1%. Then the culture was incubated for 48 h under conditions of stirring at 30°C.

At the next stage, 100 μl of the resulting suspension were applied to the agar medium M17 Lac. In the center of the Petri dishes were placed in the circle of paper impregnated with 2.5 mg of bacitracin. Incubation was carried out for 48 h at 30°C. Clones near the circle of paper were cultured in the presence of bacitracin (4 µg/ml) in 2 ml of medium (M17 Lac+geminus). Incubation continued for 24 h the conditions of stirring at 30°C.

Cells were distinguished on the agar M17 Lac in the presence of bacitracin (2 μg/ml) after incubation for 48 h at 30°C. the Selected clones were cultured in (M17 Lac+ geminus), and then were incubated for 24 h under conditions of stirring at 30°C. the resulting suspension was used to prepare frozen uterine yeast.

These experiments allowed the inventors to select a natural variant of Lactococcus lactis subsp.cremoris-I-3557, registered in the CNCM 20/01/2006.

A-I-2 is an example of a method of production of a dairy product using a variant resistant to bacitracin

Getting preculture was carried out on the crystal strain in 2 ml Ml7 Lac.

Preculture was used for inoculation (1%) 50 ml of whole milk, UVT (ultra-high-temperature) treatment, followed by incubation at 30°C for 24 hours

In table 1 is the result of the analysis of the amount of vitamin K2, expressed in µg/equivalents MK-4/100 g of the product produced variant resistant to bacitracin, and the corresponding wild (rekultivirovana) strain.

Table 1
StrainI-3557Wild
Vitamin K (µg/100 g)8,90 3,32

Resistant to bacitracin variant showed the ability to overproduction vitamin K by a factor of 3 compared to the original wild strain.

A-II - Receipt and application of natural variants resistant to peroxide

Breathing Lactococcus lactis has been demonstrated recently (Duwat et al., 2001). Genome sequencing strain L. lactis (IL1403) confirmed the presence of genes encoding functions required for aerobic respiration (Bolotin et al., 2001). Bacteria L. lactis really contain men and cytABCD operons encoding proteins required for the synthesis of managenone and biogenesis of cytochrome D. These bacteria also contain three genes involved in the last stages of the synthesis of heme (hemH, hemK and hemN, which are required in the oxidation of the porphyrin to connect with iron gem), but no genes involved in the early stages of this process. However, bacteria L.lactis able to perform oxidative phosphorylation in the presence of protoporphyrinogen.

It was also shown that breathing L.lactis can take place in the presence of oxygen and heme in a nutrient medium. Breathing favors the formation of cells more biomass and achieve higher ultimate pH than is usually observed. Culture in the presence of oxygen and/or heme showed comparable growth curves during the first the x about 6 or 7 hours of fermentation. After that, the consumption of glucose was decreased in the case of cultures with the presence of oxygen and heme in a nutrient medium, resulting in the reduced production of lactate. This reflects the shift in metabolism that occurs quite late in the process of cultivation. Thus, the breath of L. lactis is observed by the end of the exponential phase of growth (Duwat et al, 2001).

The role of respiration L.lactis still not as well known as the role of vitamin K2 in these bacterial species with rather enzymatic metabolism. The inventors have also observed that vitamin K2 was producyrovtsa L.lactis strains and in that time, when the breath was not induced in the tested conditions (in the absence of heme in a nutrient medium without mixing environment, contributing to its good oxygenation).

Proteins in the cytoplasm fewer disulfide bonds than in extracellular proteins. There is a widespread enzyme system, which limits the number of disulfide bridges. Features S-S-bonds are reduced to functions HS the enzyme thioredoxin. This enzyme is regenerated thioredoxin-reductase. Vido et al. (2005) created by genetic engineering mutant L.lactis, which they called trxB1. Gene trxBl encodes thioredoxin-reductase. Studies using electrophoresis of proteins synthesized by this mutant showed that he overproduce some of the Fe is cops, involved in the synthesis of vitamin K2, namely MenB and MenD enzymes.

On the basis of these data, as well as his own observations, the inventors suggested that one possible way to increase the production of vitamin K2 bacteria L.lactis may be the induction of respiration. Another way may be to try mobilization response of vitamin K2 on oxidative stress.

In this regard, the inventors attempted to distinguish natural variants that are resistant to oxidative stress.

A-II-1 - a Technique to obtain variants that are resistant to oxidative stress

As an example, suitable for use oxidizing agent was selected peroxide. Needless to say that under such conditions it is possible to use other oxidizing agents, such as giperhlorirovanie ions, iron ions, menadione, paraquat, oxygen or any other suitable oxidizing connection.

After receiving preculture on the environment Ml7 Lac natural source strains was re-inoculated by injection on the same medium but containing higher concentrations of peroxide (e.g., at least about 20 mg/l to at least about 25 mg/l, about 27 mg/l and about 28.5 mg/l). Cultures were incubated at 30°C. After 24 h not shown the growth of bacteria cultures in test tubes with nutrient from the food, containing peroxide in the said first concentration of the specified range, were incubated for further 24 h and Then cell clones were distinguished by depletion agar medium. He was selected clone with peroxide concentration of 27 mg/L. the inventors have noted no increase in peroxide concentration above 28.5 mg/L.

Thus, these experiments allowed the inventors to select a natural variant of Lactococcus lactis subsp.cremoris I-3558, registered in the CNCM 20/01/2006.

A-II-2 - Example of a method of production of a dairy product with the use of variants resistant to peroxide

The selected clone was grown in whole milk within 24 hours. Then samples and frozen at -80°C for subsequent analysis of vitamin K.

Table. II (see below) shows the quantity of vitamin K2 produced resistant to peroxide option, compared to the amount of vitamin K2 produced by the original strain (amounts expressed in ág-equivalents MK-4/100 g fermented milk).

As shown by table II (above), a variant was produced about two times more vitamin K2 than the corresponding wild strain.

A-III - Receipt and application of natural variants that are resistant to structural analogues of aromatic amino acids

Aromatic amino acids show negative feedback on the way their own synthesis in relation to the level stage that is common to processes for the synthesis of vitamin K and folate. If these amino acids are present in the medium, these processes are not activated. Therefore, the inventors attempted to resolve this negative regulation.

A-III-1 - a Technique to obtain variants that are resistant to structural analogues of aromatic amino acids

Natural L.Lactis strain were inoculated on agar medium specific chemical composition (Cocaign-Bousquet, M., et al., 1995), which did not contain any tryptophan or phenylalanine or tyrosine. The term "definite chemical composition" absolutely clear to the skilled in the art specialist. This is an environment that contains only simple and well-defined connection (for example, vitamin B9, vitamin B12, adenine, tyrosine, and others), in contrast to the synthetic environment that contains complex compounds (for example, yeast extract, casein hydrolysate, and others).

Circle of boom and for blotting was placed at the center of Petri dishes and were drenched with 80 μl of the solution, containing 50 mm the following compounds: m-ferienland, p-ferienland, m-fertilizin and phenylalaninamide. These compounds are structural analogs of aromatic amino acids. The cups were incubated at 30°C. Around the paper circles were formed zone of inhibition of growth. After 48 hours in this area appeared resistant clones. These clones were grown in a nutrient medium a specific chemical composition that does not contain aromatic amino acids. Wednesday was added a solution containing structural analogues of these amino acids. The final concentration of each of these compounds was 1 mm.

A-III-2 - Example of a method of production of a dairy product with resistance to aromatic amino acids clones

Preculture, obtained as described in section A-III-1 (above), was used for inoculation of 50 ml of medium Ml7 Lac (the amount of inoculum 1%) adding 1 ml of hemin (500 mg/l). These preculture were used for inoculation of the medium, not containing hemin, but with stirring. Was received by the latter type of culture. Wednesday M17 Lac was inoculable and was maintained at 30°C without stirring. Culture night was maintained at 30°C under conditions of agitation (250 rpm). Then these cultures were tsentrifugirovanie for 5 min at 6000g. The supernatant was removed and replaced by 50 ml of whole pier is CA. After 24 hours, the fermented milk was placed at -80°C for subsequent analysis of vitamin K2 (see below section B-VI).

Experiments with analysis of vitamin K2 has allowed the inventors to select one of the clones as a natural option that can overproductivity vitamin K2 (see below section B-VI: this is a natural variant of Lactococcus lactis subsp.cremoris I-3626 was registered in the CNCM 19/06/2006.

Part B. Selection of conditions conducive to the production of vitamin K lactic acid bacteria

B-I - Effect of fat contained in the environment

In the analyses of various products by the inventors, it was observed that the highest amount of vitamin K2 contained a product like fermented cream (Russ. sour cream). Moreover, vitamin K2 in this product was pronounced hydrophobic. Therefore, the authors of the invention has been hypothesized that the presence of fat or at least a hydrophobic environment may favor the production of vitamin K2.

In accordance with this fermentation was carried out on milk containing different concentrations of fat. Preculture for inoculation were obtained on the medium M17 Lac. Milk was inoculable 1% inoculum. The fermentation was maintained at 30°C for 24 hours. Then the samples were frozen for later analysis.

Figure 1 presents the results obtained for DV is x natural L.lactis strains subsp.cremoris (strains no.1 and no.2).

If the results obtained with one of the two study environmental strains, be considered as examples, the use of bold milk (1,5% fat) instead of skim increased production of vitamin K2 by a factor of 4. The transition from bold milk for whole milk (3.5% fat) increased production by a factor of 2.

This trend was observed in case of all studied natural strains.

However, increasing the amount of vitamin K2 produced in an environment with such a fat content, was, apparently, asymptotic, because fermentation of cream with a fat content of 40% were not given such a high amount of vitamin K2, as the fermentation of whole milk (data not shown).

B-II - effect of the rate of growth of lactic acid bacteria

During growth in milk natural L.lactis strain subsp.cremoris (natural strain no. 1) the authors of the invention have carried out the regulation of the kinetics of the production of vitamin K2.

As shown in figure 2, the production of vitamin began only when growth was slowing. The growth rate can be determined by regulation of the kinetics of increase of acidity. At the maximum slew rate of acidity bacteria has entered a phase of slow growth.

This type of behavior is relatively standard in the synthetic process is as secondary metabolites. This gives you the opportunity to explore various options for reducing the rate of growth in the sub-optimal physico-chemical conditions (pH, temperature and others), bacteriostatic compounds (antibiotics), cultivation in the conditions of dormant cells. The latter provides for inoculation the number of cells corresponding to, at least, their number reached usually at the end of the traditional fermentation. In this case, bacterial growth does not occur, the growth rate of zero.

Therefore, the inventors attempted the combined use of culture of dormant cells and temperature effects.

Whole milk was inoculable by direct inoculation with yeast, containing 1011CFU/g at a concentration of 10 g/L. Then the milk was incubated at different temperatures.

As shown in figure 3, the lower temperature had a positive effect on the production of vitamin K2. On the other hand, the cultivation of bacteria in a state of dormant cells increased the production by a factor of approximately 2:20 µg/100 g versus 10 ág/g with a standard fermentation growth.

B-III - Impact of receiving preculture in terms of maintenance respiration

Vitamin K2 is involved in the respiratory chain. Bacteria L.lactis able to breath, but the breath is observed tol is to at the end of fermentation, that is, when the metabolic flow slows down. This property can be compared with the ability observed in the kinetics of the production of vitamin K2.

It is important to note that the production of the food product while maintaining respiration difficult to implement, as it seems, on an industrial scale. Practical problems of aeration, mixing, pricing, etc. is quite difficult to solve without revision of the traditional equipment and production processes, and this will require very large investments and additional operating costs, which is unacceptable for the food industry.

On the other hand, if such a need arises, the receipt of preculture can be done in terms of maintenance respiration, without creating too many problems for manufacturers.

Within this, the inventors examined the effect of obtaining preculture in terms of breathing on the production of vitamin K2 lactic acid bacteria.

To this end, preculture were prepared on the environment M17 Lac with the addition of 20 µl/ml 0.5 mg/ml of hemin in 0.1 M solution of soda. Inoculation was carried out on 1% of preculture, the incubation temperature was 30°C. Aeration was provided by simple stirring.

First prepared by preculture was inoculable whole milk in to the Icesave inoculum 1%, as is commonly the case with traditional fermentation. However, under these conditions a positive impact on the production of vitamin K2 was not observed (data not shown).

Therefore it was decided to use preculture for the implementation of the fermentation conditions of dormant cells. Preculture were prepared as described above and were incubated overnight. Then samples of 50 ml were tsentrifugirovanie at 6000g for 5 min the Supernatant was removed and was replaced with whole milk. Milk was incubated at 30°C for 24 hours. The samples were frozen at -80°C for subsequent analysis.

As shown in figure 4, the observed behavior was different depending on the strain. Preculture made in the conditions of respiratory support, had no effect on the production of vitamin K2 natural strain no.1 L.lactis subsp.cremoris. On the other hand, it increased by a factor of 2, the amount of vitamin K2 produced by natural option I-3558.

Thus, the approach preculture obtained under conditions of respiratory support, is preferred, but, apparently, in the case of some strains, at least.

In practice, however, it is important to have fresh preculture, and frozen stuff. Furthermore, for carrying out the fermentation under the conditions of dormant cells rivers is recommended to have concentrated ferment.

B-IV - Additional results regarding the incentive effects of breathing on the production of vitamin K2

These results should be correlated with the results set forth in section A-III (see above).

As stated above in section A-III-2, culture selected natural variant I-3626 containing structural analogues of aromatic amino acids, was used for inoculation of 50 ml of culture medium M17 Lac (the amount of inoculum 1%) adding 1 ml of hemin (500 mg/l). This culture was used for inoculation of the nutrient medium containing hemin, but supported in the conditions of mixing. Was received by the latter type of culture. Wednesday M17 Lac was inoculable and was maintained at 30°C without stirring. The culture was kept over night at 30°C under conditions of agitation (250 rpm). Then these cultures were tsentrifugirovanie for 5 min at 6000g. The supernatant was removed and replaced by 50 ml of whole milk. After 24 hours, the fermented milk was placed at -80°C for subsequent analysis of vitamin K2.

Table III (below) shows the results of the production of vitamin K2 natural option I-3626 in the described conditions. Vitamin K2 is expressed in ág-equivalents MK-4/100 g of fermented milk.

Table II
StrainVitamin K (µg/100 g)
Wild2,92±0,45
I-35585,94±0,76
The method of obtaining preculture
Table III
Without stirring, without heminWith stirring, with Gemina
Vitamin K20,7725,77

These results indicate that the breath (the presence of hemin in terms of mixing) has a great influence on the production of vitamin K2. Under these conditions, the production of vitamin increased by a factor of 5. The original strain was produced 21,5 mg/100 g after pre-cultivation preculture without maintenance respiration (data not shown). When cooking preculture was conducted in the conditions of maintenance respiration, the production of vitamin fell to 10.3 ág/100 g (data not shown). Thus, in the case of the original strain of getting preculture in terms of maintenance respiration has a negative impact on the production of vitamin K2.

B-V - Effect dose inoculation, final population of bacteria and pH of milk

In terms of resting cells, described above in relation to obtaining preculture while maintaining respiration, the initial population of bacteria was increased to approximately 1010CFU/ml

Because the specified dose inoculation hardly applicable in an industrial way, the inventors studied t is offered by the influence of the initial number of cells for the production of vitamin K2 in the process of the fermentation of milk.

In addition, because the number of cells in preculture obtained under conditions of respiratory support, exceeded the number in preculture obtained by conventional method, the inventors have tried to find out whether the increase in the production of vitamin observed when using preculture obtained under conditions of respiratory support, simply increasing the dose of inoculation or specific influence of breathing process.

The inventors have also tried to determine is whether the final population of bacteria after the rest phase cells decisive role in the quantity produced of vitamin K2. Knowing that a certain number of bacteria are killed due to the lower pH during the fermentation process, along with other reasons, the inventors studied the effect of milk with high buffer capacity on the level of production of vitamin K2.

To find answers to these different questions, their tests were conducted under conditions of dormant cells in traditional milk or milk with high buffer capacity using as inoculum of preculture obtained by conventional method and / or maintain breathing, at doses of inoculation of approximately 106CFU/ml to 1010CFU/ml

Cooking preculture strain I-3558 was conducted on the environment Ml7 Lac at 30°C. experimental nth maintaining breathing this medium was supplemented with 20 μl/ml 0.5 mg/ml hemin (in 0.1 M solution of soda), and received preculture stirred during incubation that lasted all night. Then different amounts of these preculture (see below .IV) tsentrifugirovanie at 10000g for 10 minutes.

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Table IV
InoculationVolume preculture (R+)Volume preculture (R-)
100%40 ml160 ml
75%30 ml120 ml
50%20 ml80 ml
30%12 ml48 ml
20%8 ml32 ml
10%4 ml16 ml
5%2 ml8 ml
1%0.4 ml1.6 ml
0,01%0,04 ml0.16 ml

The supernatant was removed and either replaced by 40 ml of traditional whole milk (in the case of preculture R+ and R-), or β-glycerol at a final concentration of 0.075 M (only in case of preculture R+). Then the samples were incubated at 30°C for 24 hours. Selected aliquots to determine the number, after which the samples were frozen at -80°C for subsequent analysis. The results of the analysis and quantification of the vitamin before and after the rest phase of the cells presented in .V (see below). This table shows the results of the analysis of vitamin K2 and determine its quantity before (T0) and after the rest phase cells (Tf) using traditional preculture and preculture obtained under conditions maintain breathing.

Table V
DoseT0 R-Tf R-VitK R-T0 R+Tf R+VitK R+T0 R+TPTf R+TPVitK R+TP
100%E+10E+09At 44.47E+09E+09 3.40E+095.30E+0935,9
75%E+09E+0929.01E+09E+092.55E+095.80E+09
50%E+09E+0918.58E+09E+0919.821.70E+096.40E+0926.7
30%E+09E+0913.72E+09E+0916.751.02E+097.70E+09
20%E+09E+099.13E+08E+0914.096.80E+086.40E+091855
10%E+09E+09E+08E+093.40E+084.60E+09
5%E+081.10E+096.4E+08E+099.411.70E+083.60E+0913.666
1%E+082.15E+096.88E+07E+096.353.40E+07-4.20E+09
0,1%E+071.50E+096.26E+06E+096.193.40E+06-4.80E+09
R- = preculture obtained by conventional method;
R+ = preculture obtained under conditions of maintenance respiration;
TP = the fermentation of milk with high buffer capacity due to β-glycerol;
T0 = initial population of bacteria in CFU/ml of culture;
Tf = final population of bacteria in CFU/ml of culture;
VitK = concentration of vitamin K2 in the ág-equivalents MK-4/100 g of the product.

The results are presented in figure 5. Also for purposes of illustration, and presents the results obtained in the case of cooking preculture in terms of maintenance respiration, held in the fermenter, followed by a resting phase cells in traditional milk (curve "breathing in the fermenter"). The results are comparable with the results obtained in the preparation of preculture in test tubes with nutrient medium (curve respiration in laboratory conditions").

The results obtained (.V and figure 5) indicate that:

- the content of vitamin K2 was dose dependent inoculation;

the slope of the curve in the case of preculture obtained under conditions of respiratory support, was more than preculture obtained by conventional method. So, for this population is AI bacteria producing vitamin K2 was higher in the case of cooking preculture in terms of maintenance respiration. Therefore, the benefits of maintaining breathing seemed to assume a rather specific effect of the respiration process than simply increasing the dose of inoculation, which confirms the advantage of this approach;

- the production of vitamin K2 was higher in the case when the resting phase cells was carried out in milk with high buffer capacity. This can be explained by either the best survival of bacteria in this environment, because with the same initial population final population of bacteria was 2 to 6 times higher in milk with higher buffer capacity than conventional milk (see .V), or the best "extraction" of vitamin K2 in milk with high buffer capacity.

References

- Bolotin et al., 2001. Genome Research 11, 731-753.

- Duwat et al., 2001. J. Bacteriol. 183 (15), 4509-16.

- Morishita et al., 1999. J. Dairy Sci. 82, 1897-1903.

- Parker et al., 2003. Journal of Food Science 68 (7), 2325-2330.

- Vido et al, 2005. J. Bact. 187. 601-10.

Hart JP, et al., [letter]. Lancet. 1984; 2:283.

Hart JP, et al., J. Clin. Endocrinol. Metab. 1985; 60:1268-9.

- Hauschka PV, et al., Physiol. Rev. 1989; 69: 990-1047.

- Ducy P, et al., Nature. 1996; 382:448-52.

- Väänänen HK, et al., Calcif. Tissue Int. 1999; 64: S79.

- Ronden JE, et al., Biochim. Biophys. Acta. 1998; 1379: 16-22.

- Knapen MH, et al., Ann. Intern. Med. 1989 Dec 15; 111 (12):1001-5.

- Szulc P, et al., J. Clin. Invest. 1993 Apr; 91 (4):1769-74.

Booth SL, et al. Am. J. Clin. Nutr. 2000; 71: 1201-8.

- Shiraki M, et al., J. Bone Miner. Res. 2000; 15: 515-21.

- Braam LAJLM, et al., Calcif. Tissue Int. 2003 Jul; 73 (1):21-6.

- Harada J. and Ishii Y. J. Orthop.Sci. 2002; 7:364-369.

Cocaign-Bousquet M., et al. Journal of Applied Bacteriology 1995; 79, 108-116.

1. Method for the production of biomass enriched with vitamin K2 obtained by culturing at least one strain of lactic acid bacteria producing vitamin K2, selected from the species Lactococcus lactis, Leuconostoc lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc dextranicum, Enterococcus faecium and Propionibacterium sp.;
wherein said strain is cultivated under conditions of dormant cells and which includes at least:
(a) primary culturing the specified strain while maintaining breath-suitable for primary or preculture medium containing at least one compound containing a core of heme, at a final concentration of at least about 0.5 μg/ml;
(b) inoculation of lactic culture medium containing fat, live bacterial cells in an amount of about 108CFU/ml to 1011CFU/ml, and
(in) fermentation of the inoculated medium for approximately 4 h to 48 h at a temperature from about 4°C to 50°C with obtaining the target biomass level production of vitamin K2, which can reach from about 30 μg to about 75 μg and higher vitamin K2/100 g of fermented culture medium.

2. The method according to claim 1, characterized in that the lactic culture medium contains at least about 0.5% fat.

3. The method according to claim 1 or 2, characterized in that the lactic culture medium represents milk or milk with a high buffer capacity.

4. The method according to claim 1, characterized in that the specified preculture incubated at a temperature from about 4°C. to 40°C.

5. The method according to claim 1, characterized in that the oxygen supply of the specified preculture carried out by mixing or aeration.

6. The method according to claim 1, characterized in that the specified preculture persists for at least about 8 hours

7. The method according to claim 1, characterized in that the lactic acid bacteria strain selected from the following natural strains of Lactococcus lactis subsp.cremoris producing vitamin K2:
Lactococcus lactis subsp.cremoris I-3557, registered in the National collection of cultures of microorganisms (France) (France's Collection Nationale de Culture des Microorganismes) (CNCM, Pasteur Institute, 25, rue du Docteur Roux, 75724 Paris cedex 15, France) 20.01.2006;
Lactococcus lactis subsp.cremoris 1-3558 registered in the CNCM (Pasteur Institute, 25, rue du Docteur Roux, 75724 Paris cedex 15, France) 20.01.2006; and
Lactococcus lactis subsp. cremoris I-3626, registered in the CNCM (Pasteur Institute, 25, rue du Docteur Roux, 75724 Paris cedex 15, France) 19.06.2006.

8. Biomass enriched with vitamin K2 obtained by culturing at least one strain of lactic acid bacteria selected from the species Lactococcus lactis, Leuconostoc lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc dextranicum, Enterococcus faecium and Propionibacterium sp., producing vitamin K2, in terms of resting cells according to the method including at least:
(a) primary culturing is shown strain while maintaining breath-suitable for primary or preculture environment, containing at least one compound containing a core of heme, at a final concentration of at least about 0.5 μg/ml;
(b) inoculation of lactic culture medium containing fat, live bacterial cells in an amount of about 108CFU/ml to 1011CFU/ml, and
(in) fermentation of the inoculated medium for approximately 4 h to 48 h at a temperature from about 4°C to 50°C with obtaining the target biomass level production of vitamin K2, which can reach from about 30 μg to about 75 μg and higher vitamin K2/100 g of fermented culture medium.

9. The use of biomass according to claim 8 for the production of a milk product enriched in vitamin K2.

10. Method of production of a milk product enriched in vitamin K2, which includes at least:
(a) culturing at least one strain of lactic acid bacteria producing vitamin K2, selected from the species Lactococcus lactis, Leuconostoc lactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc dextranicum, Enterococcus faecium and Propionibacterium sp., in terms of resting cells according to the method including at least:
(i) primary culturing the specified strain while maintaining breath-suitable for primary or preculture medium containing at least one compound containing a core of heme, the final concentration of the, at least about 0.5 μg/ml;
(ii) inoculation of lactic culture medium containing fat, live bacterial cells in an amount of about 108CFU/ml to 1011CFU/ml, and
(iii) fermentation of the inoculated medium for approximately 4 h to 48 h at a temperature from about 4°C to 50°C with obtaining the target biomass level production of vitamin K2, which can reach from about 30 μg to about 75 μg and higher vitamin K2/100 g of fermented culture medium,
(b) adding a biomass obtained from the culture in stage (a), in the specified dairy product or semi-finished product and
(C) obtaining the specified dairy product enriched in vitamin K2.

11. The enrichment method of the dairy products with vitamin K2, comprising at least:
(a) adding biomass of claim 8 in specified dairy product or semi-finished product and
(b) obtaining the specified dairy product enriched in vitamin K2.

12. Dairy product enriched in vitamin K2, obtained by the method according to claim 10 or 11.

13. Dairy product according to item 12, characterized in that it is a fermented product or a fresh dairy product.

14. Dairy product enriched in vitamin K2-containing biomass in item 8.

15. Dairy product according to 14, characterized in that it is a fermented product is CT or fresh dairy product.

16. Application not in the medical purposes of the dairy product according to any one of the preceding p-15 to increase the strength of the bones of the consumer.



 

Same patents:

FIELD: food industry.

SUBSTANCE: method for production of Lactococcus lactis strain version producing, under standard fermentation conditions, a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times, the said method including, at least: a) inoculation of the sock bacterial strain under standard fermentation conditions in a cultural medium causing a change in the oxidation-reduction state of a cell containing bacitracin or a peroxide and b) selection of the strain version if producing a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times. Lactococcus lactis subsp.cremoris 1-3557 strain deposited in Collection CNCN of Pasteur Institute on 20.01.2006 and Lactococcus lactis subsp.cremoris 1-3558 strain deposited in Collection CNCM of Pasteur Institute on 20.01.2006 produce at least 1.2 times more vitamin K2 than the stock/parent bacterial strain inoculated under the same conditions. Additionally, the invention deals with a lactic acid starter containing at least one of the above strains and to the method for preparation of a cultured milk product containing the above strain and/or the lactic acid starter.

EFFECT: invention enables increase of vitamin K2 content in the product.

11 cl, 5 tbl

The invention relates to the microbiological industry, and specifically to a method for producing ubiquinone-10 (coenzyme ABOUT10used in medicine to treat a number of diseases, and also as a reagent for conducting research studies

FIELD: chemistry.

SUBSTANCE: disclosed is a method of cultivating Bacillus brevis strain 101 for producing gramicidin S. Submerged cultivation of a culture is carried out on a synthetic culture medium. The medium contains yeast autolysate and casein hydrolysate in concentration of 0.1 g/l and 0.2 g/l on amine nitrogen, glycerine in concentration of 20 ml/l, edible 40% lactic acid 2.0-4.0 ml/l, ammonium phosphate-chloride 0-3.4 g/l, di-substituted ammonium phosphate 0.85-4.5 g/l, mono-substituted potassium phosphate 0-1.0 g/l, magnesium sulphate heptahydrate 0.9 g/l, sodium citrate 1.0 g/l. Content of amine nitrogen in the initial medium is equal to 1.3-1.6 g/l. When concentration of amine nitrogen falls to less than 1.4 g/l, a concentrated culture solution is added to the medium until achieving concentration of 1.75 g/l. The concentrated culture solution contains glycerine, edible 40% lactic acid, di-substituted ammonium phosphate and chloride and magnesium sulphate with ratio of concentration of glycerin, lactic acid, nitrogen, phosphorus and magnesium equal to 1:(0.008-0.032):(0.027-0.036):(0-0.008):(0.002-0.008). During growth, the rate of stirring is increased from 200 to 500 rpm. pH is kept at 6.5-6.8 by adding potassium and sodium hydroxide. The process is stopped 2-6 after the onset of a stationary phase.

EFFECT: method enables reproducible production of a large amount of gramicidin S.

10 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: invention deal with a method for production of Lactobacillus delbrueckii subsp. bulgaricus strain with reduced lactic acid postproduction. The method involves exposure of Lactobacillus delbrueckii subsp. bulgaricus, mother strain possessing high texturising properties and high acid postproduction to the impact of a weak mutagen represented by ethyl methanesulfonate (EMS) and subsequent selection of strains with preset characteristics. Characterisation of selected Lactobacillus delbrueckii subsp. bulgaricus texturising strains (versions) is as follows: a) they have pH (measured by standard method) within the range of 4.25-4.55 after 14 days of storage; b) they have texturising properties (measured using viscosity determination analysis) no less than 25 Pa or a) hey reduce pH by at least 0.20 or 0.30 pH units after 14 days of storage at a temperature of 8°C; b) they have texturising properties (measured using viscosity determination analysis) no less than 25 Pa. Invention relates to application of the selected strains for production of fermented dairy products and dairy products containing Lactobacillus delbrueckii subsp. bulgaricus strain with the above characteristics.

EFFECT: invention enables production of a dairy product having more expressed texturising properties with less acid postproduction.

12 cl, 5 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to applying a microorganism of Lactobacillus genus, characterised by ability to specific binding with bacterium of mutans Streptococci group in which specific binding is characterised by: (i) thermal stability; and/or (ii) proteolytic stability; and/or (iii) calcium dependence; and/or (iv) formation at pH within 4.5 to 8.5; and/or (v) formation with saliva added for preparing an anti-caries composition for treating or preventing caries caused by mutans Streptococci different from Streptococcus mutans. The specific binding is analysed by the following procedures: (a) cultivation of said microorganism to a steady state; (b) mixing of said microorganism and bacterium of mutans Streptococci group which is cultivated to the steady state; (c) incubation of the mixture prepared at the stage (b) in the environment allowing formation of the aggregated microorganism and bacterium of mutans Streptococci group; and (d) detection of the aggregates by the presence of the pellet. A method for preventing and treating caries caused by mutans Streptococci different from Streptococcus mutans involves introduction of the microorganism of Lactobacillus genus showing the ability to specific binding with bacterium of mutans Streptococci group in which specific binding is characterised by the properties specified above (i)-(v).

EFFECT: invention provides specific binding of said microorganism of Lactobacillus genus with such strains mutans Streptococci different from Streptococcus mutans which are cariogenous dental pathogens.

24 cl, 3 dwg, 1 tbl, 27 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly protection of plants from diseases caused by phytopathogenic bacteria and phytoplasma. The disclosed agent is obtained by preparing a producer inoculum, growing the producer on a fermentative medium, drying the culture fluid, extracting a macrolide complex using lower alcohols, concentrating the extract and mixing said extract with additives and solvents in the following ratio, wt %: macrolide complex 17.0-23.5, solvents and additives - up to 100, where the producer used is a Streptomyces fradiae ARRIAM-53 strain.

EFFECT: invention increases plant protection effectiveness.

2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention concerns a method for standartisation of control seed strain of chicken cholera agents (Pasteurella multocida). The presented method involves cloning of the S- or M-form Pasteurella strain, gradual passaging by intramuscular, and then intranasal introduction in a susceptible organism of weight about 350 g; in 3 hours following after the death of every infected body, the microorganism is respectively isolated into physiological saline, and sealed by into 0.5-1.0 ml Pasteur pipettes with the Pasteurella strain isolated by the intramuscular passage being used over a period of 15-20 days as a by-product for the intranasal passage, while the Pasteurella strain isolated by the intranasal passage is used 2-3 days following the isolation procedure during the next 3 days as a reduction for producing a 10-hour (9 h at 37 and 1 h at 20°C) broth culture of encapsulated Pasteurellas.

EFFECT: invention enables producing the first-generation culture of encapsulated Pasteurellas recovered from the body with a specific microbial cell titre with these cells to be used for producing biopreparations.

15 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: dry enzymatic peptone, glucose, dialysate of baker's yeast and microbiological agar are mixed in given quantities. The obtained mixture is added to 1 litre of distilled water and boiled until complete dissolution of the agar. While hot, the mixture is poured into vials, sterilised in an autoclave and cooled to 47-55°C. The cooled medium is poured into Petri dishes and held until complete setting of the gel. Weighed zinc nanopowder in a physiological solution in amount of 0.005 mcg of zinc nanopowder per 0.1 ml of the physiological solution per Petri dish is deposited on the solid surface of the gel in aseptic conditions.

EFFECT: invention increases accuracy of results of investigating microbial contamination of air.

2 tbl, 1 ex

FIELD: veterinary science.

SUBSTANCE: invention relates to application of Bifidobacterium longum ATCC BAA-999 in production of a medicinal agent or a therapeutic nutrient composition for treatment of inflammatory bowel disease in a mammal and in production of a medicinal agent or a therapeutic nutrient composition for suppression of bowel inflammation in a mammal. The therapeutic nutrient composition is a nursery mix and fodder for pets and contains from 104 to 1012 CFU/g of Bifidobacterium longum ATCC BAA-999 to the weight of dry base.

EFFECT: invention provides for demonstration of high anti-inflammatory activity of the above probiotic strain of bifidus bacteria.

12 cl, 4 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: nutrient composition contains the nonpathogenic bacterial strain Lactococcus rhamnosus ATCC 53103 or Lactococcus rhamnosus CGMCC 1.3724 able to stimulate a systemic immune response, and the nonpathogenic bacterial strain Micrococcus varians MCV8 or Streptococcus salivarius DSM 13 084 able to have bacteriostatic and bactericidal effect on pathogens related to the onset of otitis, such as Streptococcus pneumoniae, non-typed Haemophilus influenzae and Moraxella catarrhalis The composition contains 104 to 1012 CFU/g of fresh weight of the composition of each strain. The composition additionally contains Streptococcus thermophilus and additionally contains at least one prebiotic in the amount of 0.3 to 6% of composition weight. The composition represents a baby formula or a food additive.

EFFECT: invention provides an effective protection against nasopharyngeal mucosa colonisation with pathogenic bacteria related with the onset of otitis.

7 cl, 1 tbl, 2 ex

FIELD: veterinary science.

SUBSTANCE: composition comprises a source of lipids, containing fatty acids, also n-3 long-chain polyunsaturated fatty acide (LC PUFA-LC PUFA), prebiotic fibres, such as fructo-oligosaccharides, inulin, galacto-oligosaccharides, gum arabic and sialo-oligosaccharides or their mixture and a probiotic bacterial strain, in particular, Lactobacillus paracasei CNCMI-2116 or Lactobacillus rhamnosus ATCC 53103 or Bifidobacterium lactic CNCM 1-3446 and additionally contains n-6 long-chain polyunsaturated fatty acid in specified quantities. At the same time the mixture of prebiotic fibres may contain 40% - 60% of gum arabic, 10% - 20% of inulin and 30% - 40% of fructo-oligosaccharide.

EFFECT: invention makes it possible to compensate for growth retardation without use of synthetic hormones and increasing calorie consumption.

5 dwg, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to methods of recycling household wastes. The method involves removing inorganic impurities, grinding, mixing wastes with an organic additive containing a culture of microorganisms and composting to obtain organic fertiliser. The organic additive used is compost based on poultry droppings, which is taken in amount of 300-500 kg per ton of wastes, and microbial strains Bacillus subtilis B-168, Bacillus mycoides B-691, Streptomyces sp. Ac-154, Mukor psychrophilus F-1441, Candida utilis Y-2441 in amount of 1·106-1·107 cells per millilitre per ton of poultry droppings.

EFFECT: simple process and low cost of processing household wastes.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are offered an anxiolytic and/or antidepressant drug which contains vitamin K2 in the amount of 10 mcg to 100 mcg as an active component, a food additive for the same application and an appropriate method of treating.

EFFECT: vitamin K2 (preferentially - menaquinone-4 and/or menaquinone-7) is safe to use for a long period of time and shows tranquilising action, particularly bland, antidepressant and antistress action.

4 cl, 2 dwg

FIELD: food industry.

SUBSTANCE: method for production of Lactococcus lactis strain version producing, under standard fermentation conditions, a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times, the said method including, at least: a) inoculation of the sock bacterial strain under standard fermentation conditions in a cultural medium causing a change in the oxidation-reduction state of a cell containing bacitracin or a peroxide and b) selection of the strain version if producing a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times. Lactococcus lactis subsp.cremoris 1-3557 strain deposited in Collection CNCN of Pasteur Institute on 20.01.2006 and Lactococcus lactis subsp.cremoris 1-3558 strain deposited in Collection CNCM of Pasteur Institute on 20.01.2006 produce at least 1.2 times more vitamin K2 than the stock/parent bacterial strain inoculated under the same conditions. Additionally, the invention deals with a lactic acid starter containing at least one of the above strains and to the method for preparation of a cultured milk product containing the above strain and/or the lactic acid starter.

EFFECT: invention enables increase of vitamin K2 content in the product.

11 cl, 5 tbl

FIELD: food industry.

SUBSTANCE: method for production of a food vitamin concentrate involves autolysis of concentrated viable cells of sedimentary wine yeast preliminarily suspended in potable water at a ratio of 1:1,5. Prior to suspension sedimentary yeast is washed with potable water at a ratio of 1:5. The produced autolysate is heated prior to separation from the sediment. The autolysate with sediment is maintained at a temperature of 0-(-2)°C during 3 weeks for transfer of .tartaric acid salt residues into the sediment. The autolysate clear part is collected into a vessel while the sediment is centrifuged at 5-10°C with the produced clear autolysate part added into the above vessel. The autolysate clear part is concentrated under vacuum. Water separated by way of centrifugation is delivered for extraction of residual alcohol and tartaric acid salts. Lacking Group B vitamins are added to the concentrate.

EFFECT: method allows to manufacture the target product with increased content of amino acids and Group B vitamins.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to food industry, particularly to biologically active food additives. The composition of biologically active substances based on betulin with regulated rate of releasing components for reducing degree of alcohol intoxication, preventing and relieving alcohol intoxication and alcohol withdrawal syndrome contains betulin encapsulated in edible fats or wax or mxiture thereof, amber acid - 50%, ascorbic acid - 50%, glutamic acid - 50% and/or sodium glutamate and food additives, with the following ratio of components in wt %: betulin 1-10, microcapsulated amber acid 50% 1-40, microcapsulated ascorbic acid 50% 1-40, microcapsulated glutamic acid 50% and/or sodium glutamate 50% 2-75 and food additives 1-95.

EFFECT: invention enables to obtain a novel composition, having complex prolonged effect for protecting the body upon consumption of alcohol, for preventing and relieving alcohol intoxication and alcohol withdrawal syndrome, which reduces development of pathological phenomena of alcohol withdrawal syndrome, as well as alcohol tolerance before, during and after consumption.

5 cl, 1 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: invention refers to a mutant bacterium producing higher levels of intracellular and/or extracellular folate as compared with a wild type which is sensitive to methotrexate, and has growth rate at least 0,1 µ/hour when grown on a medium which contains methotrexate 1.25 mg/l and no folate-dependent metabolites. Also, the invention refers to a composition for foodstuff or food additive enrichment containing said bacterium, and to a method of sampling said bacteria.

EFFECT: invention allows producing high-folate bacteria.

13 cl, 3 dwg, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to testosterone amplifier, to a drug which prevents, relieves and/or treats symptoms or diseases caused by testosterone deficiency and to an additive for treating menopausal disorders in males. As an active ingredient, vitamin K (vitamin K2, menaquinone-4 or menaquinone-7) 10 mcg to 100 mg is used.

EFFECT: inventions provide higher endogenous testosterone levels.

5 cl, 9 dwg, 2 ex, 2 tbl

FIELD: food industry.

SUBSTANCE: invention relates to biologically active supplements containing probiotics. The biologically active supplement for animals contains at least one probiotic and animal hydrolysate, dry brewing yeast, vitamin C, vitamin E, beta-carotene, zinc proteinate, manganese proteinate, ferric sulphate, cuprum proteinate, calcinol and sodium selenite. One proposes a fodder composition containing the biologically active supplement and one or several ingredients acceptable for animal feeding. One proposes a set containing the biologically active supplement, one or several ingredients acceptable for animal feeding, in case of necessity -one or several probiotics, one or several remedies against diarrhea as well as an instruction on blending and application of the biologically active supplement and the other components of the set. One proposes the biologically active supplement production method, a method for administration of probiotics to animals and a method for stimulation of animal health condition, in particular, stimulation of health condition of the digestive tract or animal well-doing.

EFFECT: invention allows to produce a biologically active supplement promoting the immune system strengthening, reinforcing beneficial effect of probiotics and extending the period of probiotics viability.

24 cl, 5 tbl, 4 ex

FIELD: food industry.

SUBSTANCE: invention relates to biologically active additives (BAA) production. BAA includes ascorbic acid, potato starch, calcium stearate, stearic acid, aerosil, a flavouring agent (identical to natural or natural) as well as a sweetening agent represented by fructose at the following components ratio, g per tablet weighting 0.57-3.2 g. ascorbic acid - 0.009250-0.0297; potato starch - 0.0285-0.1258; calcium stearate - 0.001-0.0116; stearic acid - 0.001-0.0116; aerosil - 0.002-0.0233; flavouring agent - 0.00114-0.0076; the rest is fructose. Alternatively the BAA sweetening agent may be represented by glucose at the following components ratio, g per tablet weighting 0.57-3.2 g: ascorbic acid - 0.00925-0.03547; potato starch - 0.0285-0.1258; calcium stearate - 0.001-0.0116; stearic acid - 0.001-0.0116; aerosil - 0.002-0.0233; flavouring agent - 0.00114-0.0076; the rest is glucose. Alternatively the BAA sweetening agent may be represented by a combination of sugar and glucose at the following components ratio, g per tablet weighting 2.6-3.2 g: ascorbic acid - 0.023-0.0297; sugar - 0.9045-1.2160, potato starch - 0.0936-0.1258; calcium stearate - 0.01305-0.0175; stearic acid - 0.01305-0.0175; aerosil - 0.0261-0.0351; flavouring agent - 0.00549-0.0076; the rest is glucose.

EFFECT: invention allows to cardinally enhance the tablets mechanical strength, improve their compressibility, ensure stable and long storage life (2 years) of BAA in the form of tablets, reduce the volume of processing deficiency due to tablet stratification in the process of production of BAA tablets.

6 cl

FIELD: medicine.

SUBSTANCE: method of intestine flora modification in animals with inflammatory disease of intestine (IDI) or subjected to risk of IDI development, includes introduction to animal of composition which includes, at least, one antioxidant. Antioxidant is selected from group, consisting of vitamin E, as general tocopherol, in amount from approximately 50 to approximately 1000 mcg, vitamin C in amount from approximately 30 to approximately 400 mcg and β-carotene in amount from approximately 0.1 to approximately 5 mcg per gram of feeding stuff consumed by animal in terms of dry substance. Also by introduction of said composition to animal realised is method of reduction of inflammation in gastro-intestinal tract of animal. Also claimed are set, composition of feeding stuff and combination of feeding stuff composition and, at least, one probiotic and prebiotic.

EFFECT: inventions ensure improvement of balance of useful and harmful bacteria in gastro-intestinal tract of animal.

35 cl, 4 tbl, 2 ex

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