RU2731731C1 - Bacterial strain lactobacillus acidophilus k 1901 used as starter of direct application for preparation of fermented milk products - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and dairy industry. Strain Lactobacillus acidophilus K 1901 is isolated from Yakut kumiss, has high growth activity and is used as a starter of direct introduction for preparation of fermented milk products. Strain Lactobacillus acidophilus K 1901 is deposited in the State collection of microorganisms of the normal microflora (SCNM) of the Federal Budgetary Institution of Science of the G. N. Gabrichevskiy Moscow Research Institute of Epidemiology and Microbiology of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor) under collection number No. 1275.

EFFECT: disclosed to use strain has a fast set of biomass during cultivation, high antagonist activity, high capacity for synthesis of enzyme β-galactosidase and shortened lag phase during milk ripening compared to similar strains of the given type.

1 cl, 3 tbl, 4 ex

Description

The invention relates to biotechnology and the dairy industry and is a new strain of Lactobacillus acidophilus K 1901, isolated from the Yakut kumis, which has high growth activity and is used as a direct ferment for the preparation of fermented milk products.

The provision of food products that have a high biological value and are capable of providing a therapeutic and prophylactic effect, playing the role of functional food products, is an important aspect of the food industry. Fermented milk products occupy a special place among the functional food products, including dietary products, which ensure the possibility of their use by persons suffering from intolerance to dairy products due to a deficiency in the body of lactate dehydrogenase.

Fermented food production is based on the use of starter cultures; For example, for the production of lactic acid fermentation products, starter cultures of lactic acid bacteria are used. It is the composition and properties of microorganism strains included in the starter cultures that make it possible to obtain a wide range of products with specified parameters and characteristic features. Recently, new starter cultures of lactic acid bacteria are being developed with industrially important properties: providing microbiological safety and having organoleptic, technological, nutritional and / or medical advantages. Examples are lactic acid bacteria, which produce antimicrobial agents, polysaccharides, aromatic compounds, vitamins, beneficial enzymes, and probiotic properties. Crops should ensure reproducibility of the technological process, stable quality of finished products; in addition, cultures should be resistant to bacteriophages, have a high survival rate, provide the fermentable dairy product with the necessary properties, and also allow more flexibility in production (use different strains with different properties and the ability to combine them depending on the quality of raw materials).

To meet these requirements, Direct Vat Set (DVS) / Direct Vat Inoculation (DVI) starter cultures are used, which are produced in the form of frozen granules or in lyophilized form. To ensure the high quality of the direct starter cultures, the crop strains must have a high survival rate during the production of the direct starter cultures.

The main copyright companies in the study area are CHR HANSEN (Denmark), BRIGHT DAIRY & FOOD (China), MESH (Japan), DUPONT NUTRITION BIOSCIENCES (Denmark), GERVAIS DANONE (France), NESTEC (Switzerland).

Abroad, mutated strains of the Bulgarian bacillus are often used in the composition of direct starter cultures in order to eliminate the effect of "secondary souring" as a result of lactose fermentation (RF patent No. 2232816, RF patent No. 2446209).

The use of mutated strains as components of direct ferments has some drawbacks: so nonspecific mutagenesis leads both to the acquisition of the required properties by the strain, and to the appearance of nonspecific mutations, which leads to the instability of the genome and the gradual degeneration of this strain, which, in turn, strongly limits the duration of its use.

The objective of the invention is to obtain a new strain, which is not a genetically modified culture, which provides for the production of fermented milk products, as well as having high efficiency in the process of obtaining a dry lyophilized microbial mass for the manufacture of starter cultures of direct application.

The problem of the invention is solved by breeding a new strain of Lactobacillus acidophilus K 1901 (registration number SCNM No. 1275), which meets modern requirements for the components of fermented milk products, and has a high biochemical activity.

The present invention relates to a strain of lactic acid bacteria Lactobacillus acidophilus K 1901, which for a number of useful properties can be used as a component of a complex starter culture of direct application for the production of fermented milk therapeutic and prophylactic food.

The technical result consists in the fact that the strain proposed for use has a fast biomass gain during cultivation, high antagonistic activity, high ability to synthesize the β-galactosidase enzyme and a shortened lag phase when fermenting milk in comparison with similar strains of this type, and fermented milk products based on it have acidity (110-120 ° T), which lasts for 3 days of storage, as well as a dense clot. When storing fermented milk products using this strain, serum is not formed.

The proposed strain Lactobacillus acidophilus K 1901 is new, not described in the scientific and patent literature.

The Lactobacillus acidophilus K 1901 strain was isolated from the Yakutsk kumis. The strain Lactobacillus acidophilus K 1901 was deposited in the State Collection of Microorganisms of Normal Microflora (SCNM) of the Federal Budgetary Institution of Science of the Moscow Research Institute of Epidemiology and Microbiology named after V.I. G.N. Gabrichevsky Rospotrebnadzor under collection number 1275.

1. Study of the anatomical and morphological properties of the strain.

The study of colonies in a semi-liquid medium was carried out during 12 passages of daily cultures. In a semi-liquid medium for the cultivation and isolation of bifidobacteria (BS), the form of single colonies is a ball with a diameter of 1-3 mm, the culture grows throughout the volume of the medium without a zone of aerobiosis, transparent apigmented round colonies (S-shape) with a creamy consistency grow on the surface of the dense nutrient medium ... In the micropreparation Gram +, motionless, non-spore-forming, straight long sticks with rounded ends, forming chains of 2-6 cells.

2. Identification of the studied strain.

Bruker Daltonik MALDI Biotyper was used to identify the studied strain by cell wall proteins. For this, single colonies of a 24-hour culture of the third generation, grown on the surface of MRS-4 under anaerobic conditions, were used.

According to MALDI data, the Lactobacillus acidophilus K 1901 strain was identified as Lactobacillus acidophilus with a high degree of confidence (Score 1.865). Genetic identification of the strain was also confirmed by 16SpRNA analysis.

3. Study of the technological properties of the strain.

To determine the characteristics of the strain, cultivation was carried out at a maximum temperature of 50 ° C. (The optimum development temperature for the strain is 37-40 ° C, the minimum is 25 ° C, the maximum is 45-50 ° C). A pronounced growth was observed throughout the entire thickness of the medium without a zone of aerobiosis, in a semi-liquid BS medium at high temperatures. This culture grows in the thickness of semi-liquid nutrient media and on the surface of solid media in the presence of oxygen, i.e. the strain belongs to aerotolerant microorganisms (microaerophiles).

4. Study of the proteolytic activity of culture.

The study of proteolytic properties, the accumulation of titratable acidity in milk was carried out on sterile 12% dry skim milk (SOM) prepared in the laboratory of nutrient media at the N.N. G.N. Gabrichevsky. (COM in accordance with GOST 33629-2015 "CANNED MILK FOOD. DRY MILK. TECHNICAL CONDITIONS", supplier "Agroindustrial Union" Alev ").

The acidity of fermented milk products was determined in conventional units - Turner degrees (T) (GOST 3624-67).

Determination: In a flask or beaker of 100-150 ml, measure 10 ml of the test product with a pipette. The product residues on the pipette walls are washed off with 20 ml of distilled water, 3 drops of 1% phenolphthalein solution are added and titrated with 0.1 N NaOH solution until a pale pink color appears, which does not disappear within 1 minute. The amount of alkali consumed for titration is multiplied by 10 in terms of 100 ml of the product.

By 16 o'clock the strain ferments milk with the formation of a uniform creamy clot of pleasant taste without syneresis. 10 microbial cells are accumulated in 1 ml of fermented sterile milk by 12 hours of cultivation. Does not form catalase, does not liquefy gelatin. The data on the accumulation of titratable acidity are presented in Table 1.

As can be seen from the data presented in the table, the claimed strain of lactobacilli exhibits stable proteolytic properties.

5. Study of the antibiotic sensitivity of the strain.

The antibiotic sensitivity of the strain was studied using a model of 15 antimicrobial agents used in medical practice. The study was carried out by the disk-diffusion method in the interpretation of N.R. Nazyrova. et al., 2008, recommended by MU 2.3.2.2789-10. Discs manufactured by HiMedia were used in the work to determine the sensitivity to antimicrobial drugs. The data obtained are presented in table 2.

antibiotics, the content of which is controlled in food, and which are important in controlling the spread of antibiotic resistance.

Lactobacillus acidophilus K 1901 is sensitive to streptomycin, ciprofloxacin, kanamycin, gentamycin, polymyxin, nalidixic acid, erythromycin, vancomycin, imipenem; intermediate-resistant to ampicillin, lincomycin, chloramphenicol, tetracycline; resistant to penicillin, cephalotin.

6. Study of the enzymatic activity of the strain

When studying the enzymatic properties of the strain, the data of two test systems API-50 CHL and ANAEROTEST-23 were compared. According to the results of studying the enzymatic properties of the lactobacillus strain using two test systems, it was found that the claimed strain ferments glucose, lactose, maltose, xylose, D- arabinose, esculin, salicin, cellobiose, mannose, trehalose, melezitose, raffinose, urea, slowly utilizes sucrose, gentiobiose, N-acetylglucosamine, galactose, fructose, rhamnose, has a high ability to synthesize the enzyme β - galactosidine.

The optimum cultivation temperature is 37-40 ° C. Skim milk, Blaurock's medium, GM - medium, MRS, MRS-2, MRS-4 and others are used as media for the cultivation of the strain. The isolated antagonistically active strain is phenotypically apathogenic, i.e. does not possess hemolytic, proteolytic, leticinase, hyaluronidase, antilysozyme, DNA and RNA activity. The strain is stored as a freeze-dried culture in a protective sucrose-gelatin medium at a temperature of 4 ± 2 ° C for at least 12 months.

The strain is not genetically modified, has the status of GRAS (generally recognized as safe), refers to microorganisms that are non-pathogenic to humans, according to the classification of microorganisms given in the sanitary rules SP 1.3.2322-08 and SP 1.3.2518-09. Working with the strain does not require special precautions.

7. Study of the antagonistic activity of the strain.

The study of the antagonistic activity of the strain was carried out according to MU 2.3.2.2789-10 Guidelines for the sanitary and epidemiological assessment of the safety and functional potential of probiotic microorganisms used for food production. The prototype strain was the Lactobacillus acidophilus K3Sh24 VKPM B 3190 strain deposited in the All-Russian collection of industrial microorganisms. The results are shown in Table 3.

The presented table shows that the claimed strain exhibits a higher antagonistic activity against the main pathogenic microorganisms that cause food poisoning.

To confirm the high fermentation activity of the claimed strain in comparison with the prototype strain, which was the Lactobacillus acidophilus K3Sh24 VKPM B 3190 strain deposited in the All-Russian collection of industrial microorganisms, lyophilizates of each strain in a volume of 0.1% were introduced into reconstituted milk powder. In this case, the observed period of the lag phase in the claimed strain was significantly shorter (2.5 hours) than that of the prototype strain (3.5-4 hours), which is an important condition for the industrial production of fermented milk products.

Similarly, the rate of biomass accumulation in the inventive strain and in the prototype strain was studied, which also showed that the optical density of the culture of the inventive strain after 12 hours of cultivation in a nutrient medium based on COM hydrolyzate reached 20 OD, while in the prototype strain it was 12 OD., I.e. was 1.7 times higher.

The use of the strain is illustrated by the following examples.

Example 1. To prepare a production starter culture of direct application, at the first stage, an inoculum was obtained for inoculation into a starter culture; for this, an ampoule of a lyophilized culture of the strain was resuspended, the resulting suspension was introduced into mare, goat or cow's whole or reconstituted skim milk powder and incubated at 37 ° C 24 hours.

The resulting culture was used as a seed in a fermenter. The inoculum was introduced in an amount of 5-7% of the milk volume. Incubation time: 5-7 hours.

Example 2. The use of the strain in the composition of a dry complex starter culture of direct introduction for the preparation of yogurt.

For the preparation of yoghurt, normalized milk with a fat content of 3.2%, heated to a temperature of 42 ° C, was used. The lyophilisate of the Lactobacillus acidophilus K 1901 strain was introduced in an amount of 0.1%, together with the lyophilisate of the Streptococcus thermophillus T-5367 strain (deposited at the State Committee for National Science and Technology of the Federal State Budgetary Scientific Institution of the N.N. Gabrichevsky MNIIEM under collection number 1266) also in the amount of 0.1%. After adding the milk was thoroughly mixed and incubated for 5-6 hours at 42 ° C until the pH of the fermented milk product reached 4.5. After the end of incubation, the fermented milk product was cooled to a temperature of 2-8 ° C. The shelf life of the product received is at least 14 days.

Example 3. The use of the strain as part of a dry complex leaven of direct application for the preparation of sour cream.

To prepare sour cream, cream with a fat mass fraction of 10, 15, 20, 25% was used, obtained by separating natural or dry reconstituted milk by adding whole skim milk or buttermilk. Next, pasteurization was carried out at 86 ° C for 5 minutes. They were homogenized and cooled to a temperature of 38 ° C, and the starter culture was added in an amount of 5-10% by weight of the cream. Non-mucus-forming lactic acid bacteria of the species Streptococcus cremoris (the strain was purchased at VNIMI) and Lactobacillus acidophilus K 1901 in a ratio of 1: 9 were used as a starter culture. The leavened cream was stirred for 15 minutes and left alone to ferment. The cream was fermented until a curd formed and acidity reached 60 ° T, then cooled to 17 ° C. Filling in containers is carried out before fermentation of the product or after fermentation.

Example 4. Obtaining dry lyophilized microbial mass.

To obtain a dry lyophilized microbial mass, liquid biomass grown in a hydrolyzate milk medium was concentrated by centrifugation, 10-12% vol. concentrated protective environment based on sucrose and gelatin and 12-15% vol. reconstituted 12% milk powder and freeze-drying was carried out.

The resulting lyophilisate can be used as a component of a complex starter culture of direct introduction for the production of fermented milk products.

Thus, the properties of the inventive strain indicate the advisability of its use as a component of a direct ferment.

Claims (1)

The bacterial strain Lactobacillus acidophilus K 1901, deposited in the State Committee for National Statistics of the Federal State Budgetary Scientific Institution “MNIIEM named after G.N. Gabrichevsky Rospotrebnadzor "under the collection number 1275 and used as a direct ferment for the preparation of fermented milk products.