RU2460776C9 - Strain lactobacillus acidophilus used for preparing product containing lactic acid bacilli - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: strain Lactobacillus acidophilus L-KC is recovered from a healthy infant, 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 acidophilus L-KC exhibits acid-forming activity, antagonist activity with respect to pathogenic microorganisms and opportunistic pathogens. It makes it possible to use the strain Lactobacillus acidophilus L-KC in preparing the product with lactic acid bacilli - biologically active food additives, fermented and non-fermented foodstuff, bacterial preparations, toiletry and care preparations. The strain Lactobacillus acidophilus L-KC is deposited in Russian National Collection of Industrial Microorganisms, No. B-10932.

EFFECT: invention provides higher biological activity of the product containing Lactobacillus.

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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 sour-milk, fermented and non-fermented foods, hygienic and cosmetic products, biologically active additives and bacterial preparations.

It is known that the normal obligate 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 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 reuteri, Lactobacillus rhamnosus and others.

Known patents for inventions, the basis of which are 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; U.S. Patent No. 4,645,667).

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 acidophilus L-KC 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, fermenting milk and breeding on artificial nutrient media of various compositions.

The strain Lactobacillus acidophilus L-KC was obtained from a baby, deposited in VKPM, Federal State Unitary Enterprise GosNIIgenetika, collection No. B-10932.

The inventive strain can be used in the production of fermented milk products, fermented foods, for example: kvass, beer, etc., unfermented foods, for example: juices, drinks, etc., starter cultures for the preparation of fermented milk products and biologically active additives to food. The strain Lactobacillus acidophilus L-KC 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 acidophilus L-KC 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 acidophilus L-KC, 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 acidophilus L-KC 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 acidophilus L-KC is antagonistically active against pathogenic and opportunistic microorganisms.

Cultural and morphological properties of the strain

Cells are gr + straight rods with rounded ends, meet individually or form chains, motionless, do not form spores, do not form catalase, do not dilute gelatin. On dense nutrient media form small, round, with smooth edges, matte colonies. 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. They do not form pigment, catalase, hydrogen sulfide, gelatin does not thin, nitrates do not restore. It forms acetic and DL - lactic acids without gas formation, acidifying the growth medium to pH 4.2-3.4. It ferments glucose, galactose, fructose, lactose, beckons, mannose, melecitose, rhamnose, cellobiose, trehalose, variable maltose, sorbitol, does not ferment raffinose, arabinose, xylose. The strain fermented milk by 12-14 hours of cultivation.

The strain Lactobacillus acidophilus L-KC has antagonistic activity against pathogenic and conditionally pathogenic microflora (Table 1, 2).

Table 1 Antagonistic activity of the strain L. acidophilus L-KC Strain Zone of growth inhibition of test cultures mm / M ± m Kl. pneumo nia No. 4 Kl. pneumo nia No. 142 E. coli M17 E. coli 0:75 Ps. aerugi nosa L. acidophilus L-KC 32 ± 1,2 22 ± 1.8 25 ± 2.4 25 ± 1.6 28 ± 0.8

table 2 Antagonistic activity of L. acidophilus to S. aureus strains isolated from various objects of the human body Strain Staphylococcal growth suppression zone 24 hours after seeding, mm objects Nasal cavity Throat Leather Urine Intestines L. acidophilus L-KC ≥35 ≥35 ≥35 ≥35 ≥35

The strain Lactobacillus acidophilus L-KC actively increases the microbial mass when grown on hydrolyzate-milk medium (GM), Bifidum medium (BP), milk (M), producing acids (Table 3).

Table 3 The accumulation of microbial mass (CFU / ml lg 10) and the activity of acid formation (in degrees Turner) of the strain Lactobacillus acidophilus L-KC in the dynamics of development NUTRITION CULTIVATION TIME
BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU ° T CFU ° T CFU ° T GM 8.8 102 9.2 124 7.6 224 Bf 8.2 84 8.6 108 7.4 138 M 8.2 92 8.8 112 7.8 134

The strain maximally accumulates microbial mass by 12-14 hours of cultivation, reaching in 1 ml of at least log 8-9 microbial cells, producing acids.

Thus, the proposed strain of Lactobacillus acidophilus L-KC is technologically advanced, because it multiplies in various nutrient media with the accumulation of industrial biomass with a high concentration of lactobacilli; 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 acidophilus L-KS strain can be used in the food, medical, biotechnological and cosmetic industries.

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

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

1 passage: a lyophilized strain of Lactobacillus acidophilus L-KC 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 cultured for 12-14 hours at 38-40 degrees Celsius to obtain biomass with a content of 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 acidophilus L-KC for the preparation of fermented and non-fermented foods

1 passage: a lyophilized strain of Lactobacillus acidophilus L-KC 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.

2nd 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 with a well-visible zone of aerobiosis is formed.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultured 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 acidophilus L-KC for the preparation of fermented milk product

1 passage: 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 last 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.

2nd passage: a grown culture of the Lactobacillus acidophilus L-KC 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 acidophilus L-KC 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 acidophilus L-KC 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 bacterial preparation.

Example 5. The use of strain Lactobacillus acidophilus 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 acidophilus is introduced in an amount of not more than 1% in the composition of soap or cream.

The clinical efficacy of Lactobacillus acidophilus was studied as part of a liquid dietary supplement, Normoflorin L. The studies were carried out on the basis of the Gastroenterological Center of the Municipal State University named after N.P. Ogareva ". Under observation were 20 patients aged 32 to 54 years with exacerbation of chronic biliary-dependent pancreatitis in the acute stage. Patients received 20 ml of Normoflorin L dietary supplement in the morning against the background of the main treatment. The course of treatment was 30 days. The results of evaluating the clinical efficacy of treating patients showed that the inclusion of a dietary supplement based on Lactobacillus acidophilus in the standard therapy of chronic pancreatitis significantly increased the effectiveness of treatment. Significantly reduced the manifestations of disorders of the gastrointestinal tract: bloating, rumbling in the abdomen, diarrhea and unstable stool. There was stabilization of body weight, the disappearance of signs of trophic disorders and hypovitaminosis. Lactobacillus acidophilus supplementation did not cause any side effects.

Claims (1)

Strain Lactobacillus acidophilus VKPM No. B-10932 used to obtain products containing lactobacilli.