RU2453593C1 - Strain lactobacillus helveticus used for making lactic acid bacillus containing products - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: strain Lactobacillus helveticus L-KS is recovered from silo, actively propagates in a nutrient medium with accumulation of an production biomass in a short 12-14-hour period of cultivation time of the high lactic acid bacillus concentration, is able to sour milk. The strain Lactobacillus helveticus L-KS exhibits acid-forming activity, antagonist activity with respect to pathogenic microorganisms and opportunistic pathogens. The strain Lactobacillus helveticus L-KS is deposited in Russian National Collection of Industrial Microorganisms, No. B-10895.

EFFECT: usability of the strain Lactobacillus helveticus L-KS in making the products containing - biologically active food additives, fermented and non-fermented foodstuff, bacterial preparations, hygienic and cosmetic agents due to high biologically and technologically significant strain properties.

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Description

The invention relates to the food, cosmetic, biotechnological and medical industries, in particular, it is used for the preparation of dairy, fermented and non-fermented foods, hygiene and cosmetics, dietary supplements and bacterial preparations.

It is known that the normal indigenous microflora of the human body plays an important role for a healthy state of health: it participates in metabolic processes, synthesizes vitamins, utilizes harmful substances, is an antagonist in relation to conditionally pathogenic and pathogenic microflora, and contributes to the formation of the human body's resistance to harmful factors .

Currently, due to adverse environmental conditions, stress, prolonged antibacterial, radiation and chemotherapy, dysbiosis resulting from past diseases, the number of people suffering from disorders of the normal microflora of the body is increasing. In this regard, for the treatment and prevention of these disorders, increasing attention is being paid to both drugs and food products containing normal human microflora, in particular lactobacilli.

The following types of lactobacilli are found in the human body: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus rhamnosus and others. Known inventions based on lactobacilli (patent No. 2259192, Russia, patent No. 2413761, Russia, patent No. 2412239, Russia, patent No. 2376366, Russia, patent No. 637297, Switzerland; patent No. 2134179, Germany; patent No. 0430642, Japan; patent No. 4645667, USA).

The quality of medicinal products and preparations is largely due to the properties of the producer strain - its specific activity and adaptability. A large assortment of biologically active and technologically advanced strains of lactobacilli makes it possible to develop new mono-and complex medical nutrition products and drugs that can provide high colonization resistance of the human intestine, restore its normal microflora and reduce the incidence of various intestinal infections.

The objective of the present invention is to obtain a strain of microorganisms of the species Lactobacillus helveticus L-KS with high biological activity, resistance to environmental conditions, with stabilizing acid formation in the entire temperature range of cell development, antagonistic activity against pathogenic and conditionally pathogenic microflora that ferment milk and multiplies by artificial nutrient media of various compositions.

The strain Lactobacillus helveticus L-KS obtained from silage, deposited in VKPM, Federal State Unitary Enterprise GosNIIgenetika, collection No. B-10895.

The inventive strain can be used in the production of fermented milk products, fermented foods, such as kvass, beer, etc., unfermented foods, such as juices, drinks, etc., starter cultures for the preparation of fermented milk products and biologically active food additives. The strain Lactobacillus helveticus L-KS can also be used for the production of hygiene and cosmetics, for example creams, lotions, napkins, soaps, powders, tampons, nourishing masks, gels, women's pads, cosmetic milk, various powders, etc.

In addition, the strain of Lactobacillus helveticus L-KS can be used in the production of bacterial preparations, in the form of suppositories, oral and other similar agents.

The technical result of the claimed invention is to obtain a strain of Lactobacillus helveticus L-KS, which can be used as a biologically active substance in dairy, fermented and non-fermented foods, starter cultures, hygiene and cosmetics, dietary supplements and bacterial preparations. Lactobacillus helveticus L-KS has biologically and technologically significant properties, such as acid-forming activity, the ability to build up microbial biomass in various nutrient media. The strain Lactobacillus helveticus L-KS is antagonistically active against pathogenic and conditionally pathogenic microorganisms.

Cultural and morphological properties of the strain

Cells are gr + thin long rods, with rounded ends, can fold into chains, do not form catalase. The strain of Lactobacillus helveticus L-KS is able to ferment milk within 12-14 hours. Separately grown colonies on a semi-liquid nutrient medium have an elongated form of colonies.

Physico-biochemical properties of the strain

Indisputable motionless facultative anaerobic. The temperature of cultivation varies in a wide range of 34-40 degrees Celsius. It does not form catalase, it does not dilute gelatin, it does not form hydrogen sulfide, it does not restore nitrates. It produces acetic and L (+) lactic acids, without gas formation, acidifying the growth medium to a pH of 4.2-3.8. It ferments glucose, galactose, fructose, lactose, attracts, mannose, melecitose, rhamnose, cellobiose, trehalose, does not ferment raffinose, arabinose, xylose. The strain fermented milk by 12-14 hours of cultivation.

The strain Lactobacillus helveticus L-KS has antagonistic activity against pathogenic and conditionally pathogenic microflora (table 1).

Table 1 Antagonistic activity of the strain L. helveticus L-KS Strain Zone of growth inhibition of test cultures, mm / M ± m Sh.sonnei Sh.flexneri E.coli S.aureus Pr.vulgaris L. helveticus L-KS 20 ± 1.4 22 ± 1.6 18 ± 2.0 25 ± 1.2 20 ± 0.6

The strain actively increases microbial mass during growth on hydrolyzate-dairy medium (GM), Bifidum medium (BF), milk (M), producing acids (table 2).

table 2 The accumulation of microbial mass (CFU / ml lg 10) and the activity of acid formation (in degrees Turner) of the strain Lactobacillus helveticus L-KS in the dynamics of development NUTRITION CULTIVATION TIME
BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU ° T CFU ° T CFU ° T GM 8.2 90 9.0 118 7.4 212 Bf 7.8 84 9.2 102 7.2 158 M 8.4 82 8.6 106 7.6 164

The strain maximally accumulates microbial mass by 12-24 hours of cultivation, reaching 8-9 microbial cells in 1 ml of lg, producing acids.

Thus, the proposed strain of Lactobacillus helveticus L-KS is technologically advanced, because it multiplies in various nutrient media with the accumulation of production biomass with a high concentration of bifidobacteria; possesses acid-forming activity, as well as antagonistic activity in relation to pathogenic and conditionally pathogenic microorganisms.

Sour-milk, fermented, non-fermented foods, hygienic, cosmetics, dietary supplements and bacterial preparations containing the Lactobacillus helveticus L-KS strain can be used in the food, medical, biotechnological and cosmetic industries.

The ability of the strain Lactobacillus helveticus L-KS to increase biomass in various nutrient media, acid-forming activity, antagonistic activity against pathogenic and conditionally pathogenic microflora, as well as other properties useful for the human body that are characteristic of B. helveticus species, make it possible to use the strain in the composition of various corrective agents for therapeutic purposes.

Example 1. The use of strain Lactobacillus helveticus L-KS in the composition of biologically active additives

1 Passage: the lyophilized strain of Lactobacillus helveticus L-KS in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at a temperature of 38-40 degrees Celsius until the formation of visible growth in the last dilutions.

2 Passage: a grown culture of 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 38-40 degrees Celsius for 12-14 hours until growth with a visible aerobiosis zone is formed.

3 Passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultivated for 12-14 hours at 38-40 degrees Celsius to obtain biomass containing live lactobacilli of at least 10 ⁸ ml.

The result is a biologically active additive, which is a liquid concentrate with a high, not less than 10 ⁸ / ml, content of lactobacilli.

Example 2. The use of strain Lactobacillus helveticus L-KS for the preparation of fermented and non-fermented foods

1 Passage: the lyophilized strain of Lactobacillus helveticus L-KS in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at 38-40 degrees Celsius until the formation of visible growth in the latest dilutions.

2 Passage: a grown culture of 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 38-40 degrees Celsius for 12-14 hours until a dense growth is formed.

3 Passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultivated for 12-14 hours at 38-40 degrees Celsius to obtain biomass with a content of live lactobacilli of at least 10 ⁸ / ml.

Getting a fermented or non-fermented food product: the grown biomass is introduced into food raw materials (milk, juices, beer, kvass, etc.) in an amount up to 10% of the volume, mixed and poured into containers. The result is a product with a concentration of live lactobacilli at least 10 ⁶ / ml

Example 3. The use of strain Lactobacillus helveticus L-KS for the preparation of fermented milk product

1 Passage: the lyophilized strain is introduced into the culture medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at 38-40 degrees Celsius until the formation of visible growth in the latest dilutions. Or dry lyophilized biomass of lactobacilli is introduced in an amount of 1-2 doses in a test tube with 30 ml of culture medium. Cultivate at a temperature of 38-40 degrees Celsius to obtain biomass containing live lactobacilli at least 10 ⁷ / ml.

2 Passage: the grown culture of the Lactobacillus helveticus L-KS strain from the last dilutions or from a test tube with grown biomass is introduced into the accumulation medium with a volume of up to 10% and cultivated at a temperature of 38-40 degrees Celsius until a biomass with a content of lactobacilli of at least 10 ⁸ / ml is obtained .

3 Passage: the resulting biomass of the strain Lactobacillus helveticus L-KS in a volume of up to 10% is introduced into sterile milk, cultivated until a clot forms (fermented milk product). The number of passages No. 2 can be increased depending on the volume of production.

Example 4. The use of strain Lactobacillus helveticus L-KS to obtain bacterial preparations

The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 38-40 degrees Celsius until the biomass of lactobacilli with the content of living cells of at least 10 ⁸ in 1 ml is obtained. Protective drying media (sucrose-gelatin medium, skim milk) are added to the resulting biomass. The resulting suspension is poured into ampoules, vials and other containers, frozen at a temperature of minus 35-45 degrees Celsius and lyophilized from a frozen state in a vacuum dryer. The resulting preparation contains at least 10 ⁷ living cells of lactobacilli. This content of lactobacilli is consistent with the scientific and technical documentation for a similar drug with lactobacilli - "Lactobacterin" - and is sufficient to use the resulting product as a probiotic drug.

Example 5. The use of strain Lactobacillus helveticus to obtain hygiene and cosmetics

The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 38 ± 1 ° C until the biomass of lactobacilli with the content of living cells of at least 10 ⁸ in 1 ml is obtained.

The resulting biomass of lactobacilli of the strain Lactobacillus helveticus is introduced in an amount of not more than 1% in the composition of soap or cream.

Claims (1)

The strain Lactobacillus helveticus VKPM B-10895 used to obtain products containing lactobacilli.