UA64347A - Lactobacillus acidophilus bacteria strain used in producing bacterial preparations and functional diary foods - Google Patents

Abstract

Lactobacillus acidophilus bacteria strain is used in producing bacterial preparations and functional diary foods. The strain is produced by a method of protoplast and regeneration of base strain L.acidophilus VKPM V-7771, isolated of arbitrarily ripened milk. The strain reveals a high biological activity and has a low limit of acid-forming which enables to obtain functional products with a prolonged term of storage.

Description

Description of the invention

The invention relates to biotechnology and is a strain of acidophilic bacteria used in the production of 2 biologically active bacterial preparations and functional products.

The creation of bacterial preparations involving biologically active strains of lactic acid bacteria and the development of functional dairy products based on them is an urgent task of modern biotechnology.

The need for the production of this group of products is caused by the deterioration of the ecological situation, the quality of drinking water and the growth of dysbacteriosis in people who used antibiotics in the treatment of various diseases. 70 The main criteria for the selection of biologically active cultures promising for use in the composition of functional products are natural origin, resistance to the products of the metabolism of the macroorganism, antagonistic and cholesterase activity, and the ability to adhere. From a technological point of view, physico-chemical properties and organoleptic indicators of finished products using active microflora are important. Currently, there is a lot of information about the use of acidophilic rods in the technologies of 79 fermented dairy products and special purpose products with medicinal properties.

The strain Iasiorasis acidorpiyis is known: TsMPM 8-3324, which was obtained by selection of the original strain

And assyurasiyiv asiaorpiyz BP-27 - a representative of the normal flora of the reproductive system of a healthy adult woman. The strain is resistant to antibiotics, has antagonistic and adhesive activity (Pat. BO 1491884, C12M 1/20, AbIK 35/74, 1989). However, the strain has a narrow range of application - only in the form of suppositories for the treatment of disorders of the microbiocenosis of the reproductive system.

A known strain of lactic acid bacteria I asiorasiyshz acidorpyyz SMOSM 1-1225 was selected for its ability to adhere to intestinal epithelial cells and competitively displace harmful bacteria that cause intestinal diarrhea, immunomodulation and/or decrease fecal enzyme activity. The strain can be used as a preventive and therapeutic agent against disorders of the gastrointestinal tract of humans or animals. It can be produced in the form of a composition of frozen or dried biologically pure culture and a food carrier" (Pat. OA 26430, С12М 1/20, AbIK 35/74, 1999). This invention does not provide for the selection of cultures based on antagonistic and cholesterase activity, resistance to aggressive compounds of the digestive system: bile, phenol, common salt.

The closest to the claimed strain is the bacterial strain I asioraciis acidorpishz (Mob) VKPM B-5097, intended for use in the production of fermented milk products (Pat. ZO 1708831, C12M 1/20, A2ZS v 9m27, 1992) - a prototype. This strain was isolated from human milk and classified as production-valuable. -"f

The strain has high proteolytic and antagonistic activity. Milk coagulates at the optimal temperature of 36-397C and the introduction of 295 starters in 4.5-5 hours, 495 starters in 3 hours, the limit of acid formation is more than i. 19077 (pH 3.7x0.1). The disadvantage of the known strain is a large limit of acid formation, which negatively affects the shelf life of the fermented milk product. In addition, the invention lacks data on the adhesive properties of the strain and the ability to reduce the cholesterol content in the fermented milk product, which significantly narrows its functional characteristics.

The task of the invention is to obtain a biologically active strain /. asidorp5, which has a low limit of acid formation, which makes it possible to obtain a functional product with a long shelf life. "

The proposed strain H. asidorpishz 21 was obtained by the method of protoplastization and regeneration from the original Z strain of H. asidorpishz VKPM B-7771, isolated from arbitrarily fermented milk. The strain H. asidorpiiv 21 was investigated for the main morphological, physiological and biochemical properties (Brief identifier of bacteria

From" Bergy, 1982; L.A. Bannikov Selection of lactic acid bacteria and their application. in the dairy industry, 1975). The strain is deposited in the depository of the National Collection of Industrial Microorganisms 1MV (Ukraine) under number 1MV B-7057.

The strain is characterized by the following properties. b Cultural and morphological characteristics of the strain. "" Gram-positive, immobile rods with sizes of 0.9-1.2 and 3.0-9.5 μm are located in the form of individual cells or in chains of 2-6 members. The cell structure is not granular. The strain grows on the surface of agar with hydrolyzed milk, forming strand-like colonies of white color, deep colonies have the shape of pieces of cotton wool 1.5-2.0 mm in

ChK" 50 in diameter. In the hydrolyzed broth, it forms a homogeneous turbidity and finely dispersed sediment. "mch. Physiological and biochemical properties of the strain.

The strain is a facultative anaerobe. The optimal growth temperature is 37-12C. When adding 395 cultures to milk at this temperature, the lactic activity is 7 hours. Creates a homogeneous dense clot without separation of whey with a pleasant sour-milk taste. Accumulation of cells in 10956 skimmed milk is (1.0-2.0).108 CFU/ml, in hydrolyzed broth with glucose and lactose (1905 each) - (1.2-2.5).109

R» CFU/ml. The claimed strain differs favorably from the known one in its low acid formation limit - 150-107T (Table 1), which allows obtaining a fermented dairy product of guaranteed quality with an extended shelf life. The strain grows in broth with pH 8.3 and in milk with 0.595 phenol, acid-resistant - 6895 cells survive at pH 3.

In addition, the strain is resistant to aggressive compounds of the digestive system: it grows in hydrolyzed broth with 590 mass and 2095 bile.

Ferments - glucose, lactose. Fructose and xylose - slowly. Does not ferment galactose, mannitol, sorbitol, rhamnose, glycerol, sucrose, inositol, mannose, raffinose, maltose. The strain coagulates and restores litmus milk, db Does not form CO" from citrate and ammonia from arginine. The proteolytic activity of the strain in skimmed milk, determined as the amount of tyrosinantryptophan (0.29 in the control), is 8.01 mgoo. In the production nutrient medium, the strain accumulates up to 121 mg7o of free amino acids (HPLC method). When growing in milk and in a liquid medium of MRS, in which the source of cholesterol was, respectively, milk fat and egg yolk, the strain utilizes 25 and 15956 of cholesterol, respectively. The cholesterase activity of the strain was studied by IP mygo according to a modified method (see N.F. Kigel, O.M. Rozhanska, O.V. Naumenko, "Visnyk agrarnoi nauki, 2002).

This level of activity indicates a high potential ability of the strain to reduce the level of cholesterol in human blood.

Antagonistic property. Antagonistic activity was determined by the "well" method (N.S. Yegorov, 1986).

The result was evaluated as positive if the size of the zone of growth retardation of the test culture around the well was more than 7/0 LOmm. Conditionally pathogenic strains of Ezspegispia soii 0111 K-59 and 113 were used as test cultures; Rgoiviz University of Idagov 2029; etharpiosossiz azgeiz 209. Sterile skimmed milk with acidity adjusted to pH 3.8 and 4.8 with a sterile lactic acid solution and probiotic strain I B. sazei were used as controls

Zpigoga isolated from the drink "Husky.

The H. asidorpyiz 21 strain showed high antagonistic activity to the used test cultures of pathogenic /5 and conditionally pathogenic microorganisms. Zones of growth retardation ranged from 14 to 24 mm. The spectrum of action of the B. sazei Zpigoia used as an additional control was slightly different from the claimed strain. In particular, it did not suppress the growth of the pathogenic strain Rgoieivs ucidagiz 2029. Strain GI. acidorpyiz 21 exceeds this probiotic culture in terms of inhibitory effect (Table 2). Cultivation of the strain in skimmed milk stimulates antagonistic activity not directly related to acid-forming capacity.

Genetic features of the strain. The minimum inhibitory concentration of kanamycin, neomycin, levomycetin, streptomycin, erythromycin and ampicillin was established, which is, respectively: 100; 100; 10; 10; 5; 0O, bmkg/ml. The strain is phage resistant.

Adhesive properties of the strain. According to the invention, the adhesion of the strain to the cells of the human laryngeal adenocarcinoma HEr-2 culture was determined (according to the method of N.F. Kigel, O.V. Naumenko, N.M. Zubkova, "Bulletin of Agrarian Science", 2003). For comparison, the original strain I. asidorpiyiz VKPM 8-7771 was used. The claimed strain is characterized by a high degree of adhesiveness - the adhesiveness index is 9.89-1.05, which is 23.3-27.5 times greater than that of the original strain.

Thus, the proposed strain shows high biological activity and has a low limit of acid formation and is recommended for the preparation of bacterial concentrates for functional food and dairy products.

Example 1. Preparation of laboratory sourdough I! .asidorpychv5 21. -

195 g of pure culture of G. asidorpiyiv 21 is added to 100 ml of 1095 sterile skimmed milk and cultured at a temperature of 37"C until a clot forms, after which it is cooled and used for the preparation of laboratory or industrial sourdough. For this, sterilized milk with a temperature of 377C -

INTRODUCING 395 primary laboratory starter and cultured at this temperature until a clot is formed within 7 hours. The number of viable cells of I..asidorpyshizv 21 in the starter is 1.5.108 CFU/ml, titrated acidity 68"T, pH-4.65.

Example 2. Preparation of a bacterial concentrate using the strain G..acidorpiiv 21.

In 7 liters of tap water, 700 g of dry skimmed milk are reconstituted, 490 g of peptone, 210 g of sodium citric acid, 350 g of sodium acetic acid, 14 g of MazO, 35 g of My5O, 700 g of lactose, 7 O ml of Tween 80 are added. of a 4090% sodium hydroxide solution, sterilized at 1217 for 30 minutes and cooled to a temperature of 5576. i" Separately prepare and add sterile solutions to the base: 700 g of glucose dissolved in 2 l of distilled water,

35 g of ascorbic acid - in 400 ml of water, sterilized at 1057C for 15 minutes, and 1.4 l of yeast 5 autolysate. After adding all the components, the active acidity of the medium should be at the level of pH 6.5. IN

1.2 liters of inoculums of strains I.asidorpiiv 21, 5.vaiimagiiz vvr are added to the prepared medium. (peptorpiyiv5 1» VKPM B8-7331 and |. sazei vvr. savei IMV B-7017 with a cell concentration of each of at least 109 CFU/ml.

Accumulation of the bacterial mass is carried out at 377"C for 12 hours with periodic neutralization (95) of the medium with a 25906 aqueous ammonia solution to an active acidity of pH 6.5. After the end of the 1" 50 K process, the culture liquid is cooled to 15"C, neutralized to pH 6 ,8 and separate the biomass on a supercentrifuge at 15,000 rpm. The resulting biomass is mixed in a ratio of 1:2 with a sterile protective medium; 1 liter of which is prepared from separate sterile solutions based on the calculation: 200 g of sucrose dissolved in 400 ml of distilled water and sterilized at 1057C for 15 minutes; 50 g of sodium citric acid - in 100 ml of distilled water and 50 g of dry skimmed milk - in 500 ml of tap water, sterilized at 1217C for 30 minutes.

The cell suspension is poured into sterile cuvettes in a layer of no more than 1 mm, frozen at minus 40°C for 16 hours and dried in a KO-30 sublimation dryer at a temperature from -257°C to 30°C with a mass fraction of moisture in the dry concentrate not more than 590; drying time - 18 hours. 1g of dry bacterial concentrate contains 2.0-1011 cells of lactobacilli, including 1.0:109 cells of acidophilic bacilli, the activity of the concentrate is 7 hours. The bacterial concentrate exhibits high antimicrobial activity in relation to intestinal 60 bacilli, Proteus and Staphylococcus.Its minimum bactericidal dose is 1Omg, and bacteriostatic - 0.01mg.

This level of antagonism is sufficient to ensure the elimination of unwanted microflora from the intestines and the sanitary level of the product during its production.

Example 3. Preparation of a functional dairy product using strain I..asidorpiyz 21.

Milk with a fat content of 2.595 is pasteurized at a temperature of 867C with a holding time of 20 minutes, cooled to 65, the fermentation temperature is 37"C, and a bacterial concentrate is added in the amount of bg/t, prepared according to example 2. Fermentation is carried out for 7 hours. The titrated acidity of the finished product is 70 "T,

active - 4, bod.pH The number of viable cells of the acidophilic bacillus in the product reaches 1.0 108 CFU/ml, the total number of bacteria per gram of the product is 1.310? cells The product has a 4095% lower cholesterol level and contains 3595% less lactose and almost 2 times more free amino acids compared to the original milk. The product shows antagonistic activity in relation to bacteria of the group of Escherichia coli - the growth retardation zone of EvzsPegispia soii 113 - (241) mm, Rgoieiv ucidagiz 2029 - (1452) mm, Earpiosossiv atzgeiz 209 - (15542) mm, Epieg osossiv sioaseae - (2453) mm , Vasiiiyiv5 zibBiyv - (1651) mm.

The product has a pure sour-milk taste, a dense and sufficiently viscous consistency that does not deteriorate during storage. Active acidity decreases by only 0.2 units within 14 days. pH, level of active microflora 70 practically does not change from the initial one (Fig. 1). The shelf life of the product at a temperature from 0 to 6" is 14 days from the end of the technological process. The fermented product made according to example 3 is a functional product, it is recommended to use it for preventive purposes to normalize intestinal microflora, cholesterol metabolism and improve digestion processes.

Table 1

Dynamics of accumulation of titrated ("T") and active (pH units) acidity during development in milk

Term, day Strains of acidophilic bacteria vBato dtkoi 138510 39 k0y 100410 4 vho 170410 3.80 120410 4,BBOM 190 210 zt ho

BECAUSE I

Table 2

Antagonistic activity against pathogenic and conditionally pathogenic microorganisms

Strain The size of zones of growth retardation of test cultures, mm -

Euspegisnia soii 0111 K-59 Euspegispia soii 113 Rgoieiv myYidagiz2029 Ziarpyosossyv atsgeiv 209 h;E 11116116 h 101611 (Se)

Note: Ku - sterile skimmed milk with pH 3.8; Ko - sterile skimmed milk with a pH of 4.8.

Claims (1)

The formula of the invention is the strain of bacteria I asiorasiyiz asidorpishe IMV B - 7057, which is used in the production of bacterial preparations and functional dairy products. (about) Cheka" (95) Cheka" what 60 b5