RU2476591C1 - Strain of enterococcus hirae used for production of fermented milk products - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of Enterococcus hirae "И"-29 with high antagonistic activity, deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under registration number of RNCIM B-10088, and can be used in production, for example, of fermented milk products such as kefir, cottage cheese or fermented baked milk.

EFFECT: invention enables to obtain a strain with high antagonistic activity and a high rate of milk fermentation.

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Description

The invention relates to biotechnology and the dairy industry and is a new strain of mesophilic streptococcus, intended for the production of dairy products and bacterial preparations.

Known strains of Streptococcus citrovorus, Streptococcus paracitrovorus, Streptococcus diacetilactis, with high resistance to growth inhibitors, the ability to stably form a viscous, milk clot, have good compatibility with other strains of lactobacilli and pronounced physiological and biochemical properties.

The disadvantage of these strains is the lower marginal acidity and rate of fermentation of milk.

Known strain of Enterococcus durans RSH (VKPM B-8717), patent No. 2273662. Since 1982, E. durans has been used in the dairy industry as a component of starter cultures, as well as a probiotic in medicine (Rocha JM, Malcata FX, On the microbiological profile of traditional Portuguese sourdough. J. Food Prot. 62 (12): 1416-29 , 1999).

It is also shown that E. durans in a number of indicators (resistance to bile and acid shock, in vitro cholesterol assimilation) is superior to known probiotic microorganisms (Dora IA Pereira and Glenn R., Gibson Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Humman Gut // Applied and Environmental Microbiology, vol. 68, No. 9, p. 489-4693, 2002).

The disadvantage of this strain is the lower marginal acidity and rate of fermentation of milk.

The objective of the invention is the creation of a fermented milk product using lactobacilli on accessible nutrient media with high antagonistic activity.

The problem is solved by using nutrient media of dense and liquid consistency: sterile skim milk, hydrolyzed milk (pH 6.8-7.0).

The local strain Enterococcus hirae VKPM B-10088, isolated in vivo from the microflora of kefir fungus.

The identification of the strain was carried out according to the method described in the book of L. A. Bannikov. Breeding of lactic acid bacteria and their use in the dairy industry - M., "Food Industry", 1975.

The strain is deposited in the All-Russian Collection of Industrial Microorganisms and has a number VKPM B-10088.

The strain Enterococcus hirae VKPM B-10090 has the following characteristics:

1. Cultural and morphological:

- optional anaerobic;

- gram-positive cocci;

- a value of 0.3-0.02 microns;

- motionless;

- located in pairs or in short chains;

- do not form endospores;

On the surface of the agar medium (hydrolyzed milk) after 24 hours at a temperature of 37 ° C form:

- round colonies,

- size 1-1.5 mm (spot);

- the nature of the contour of the edge is even;

- the relief is flat;

- surface - smooth, matte;

- White colour;

- structure - fine-grained;

- the consistency is soft.

The study of the cultural or morphological properties of the selected strain of lactic acid microorganisms was carried out on a dense nutrient medium - agarized milk.

2. Physiological and biochemical:

- milk fermentation rate - 9 hours.

- the limit of acid formation is 100 ° T.

- the strain ferments glucose, galactose, lactose, sucrose, maltose, raffinose, glycerin, dextrin.

- grows in milk at a temperature of 40-45 ° C.

- grows in hydrolyzed milk containing NaCl (2%, 3%, 4%, 6.5%).

- grows in hydrolyzed milk with bile content (20%, 30%, 40%).

- grows in milk containing methylene blue (0.0005%, 0.005%, 0.01%, 0.1%).

- withstands heating for 30 minutes at a temperature of 55 ° C, 60 ° C.

The product synthesized by the strain is lactic acid.

3. Antibiotic activity:

- the diameter of the zone of inhibition of growth in relation to the representative of the pathogenic microflora Staphylococcus aureus was 24 mm;

- the diameter of the zone of growth inhibition in relation to the representative of the opportunistic microflora of Escherichia coli was 26 mm.

Determination of the antibiotic activity of the Enterococcus hirae strain VKPM B-10088 was performed on solid nutrient medium (MPA) by agar diffusion.

The method, conditions and composition of the media for long-term storage of the strain - lyophilization; at a temperature of 4 ° C; MPG environments Rogosa; milk with chalk; milk with yeast autolysate, glucose, litmus, liver extract and CaCO ₃ ; agarized milk cheese whey.

The method, conditions and composition of the media for the propagation of the strain at a temperature of 30-37 ° C environment: agarized milk cheese whey; skim milk, unsalted cheese whey with a content of at least 4.0% lactose.

Optimum conditions and composition of the medium for fermentation - temperature 30-37 ° C; environments: cow's milk; unsalted cheese whey with a content of at least 4.0% lactose, skimmed cow's milk.

The proposed new Enterococcus hirae strain VKPM B-10088, isolated from the microflora of kefir fungus, is well adapted to local conditions, easy to cultivate and store, can be used in the production of dairy products, is not zoo and phytopathogenic, and is not dangerous for other reasons.

Due to its pronounced biological and antibiotic properties (the ability to form a viscous milk clot, the diameter of the zone of inhibition of growth of Staphylococcus aureus is 24 mm, Escherichia coli is 26 mm), the strain is of industrial value for the dairy industry.

Example 1. The use of the strain Enterococcus hirae VKPM B-10088 in the production of kefir.

Milk is weighed, sorted and filtered, subjected to primary processing. It must be fresh, natural and meet the requirements of the standard.

In the production of kefir, milk is cooled to a fermentation temperature of 20-25 ° C. Already pasteurized milk is poured into the fermenter tank and introduced into the pre-prepared yeast - 5-6% of the mass of milk. The starter culture is also enriched with Enterococcus hirae strain VKPM B-10088. After thorough mixing, the contents of the tank are left alone, ensuring a constant temperature with a water jacket. The milk is fermented to obtain a dense clot with an acidity of 90-100 ° T. The duration of ripening is 10-12 hours at a temperature not lower than 20 ° C. Upon reaching the necessary acidity and viscosity of the clot, the circulation of water in the jacket is stopped, ice water with a temperature of 1-2 ° C is supplied to the interstitial space and a stirrer is turned on to stir the clot and more quickly cool it to the ripening temperature. After reaching a clot temperature of 12-16 ° C, the supply of cooling water is stopped and it is left alone for 4-6 hours for the maturation and development of yeast. Then the contents of the tank are cooled to a temperature of 8-10 ° C to complete ripening.

Example 2. The use of strain Enterococcus hirae VKPM B-10088 in the production of cottage cheese.

Curd production in the traditional way. The technological process consists of the following operations: acceptance and preparation, milk separation, normalization, pasteurization, cooling, fermentation and fermentation of normalized milk, cutting the clot, separating whey and pouring the clot, self-pressing and pressing the clot, cooling, packaging, packaging, storage and transportation of cottage cheese .

Dairy raw materials intended for the production of cottage cheese are cleaned on milk separators or filtered through three layers of gauze or other filter cloth. The purified milk is heated to 37 ± 2 ° C and separated on cream separators. In the manufacture of cottage cheese, fatty, bold and peasant milk is normalized by fat, taking into account the mass fraction of protein in whole milk, so that a finished product with a given content of fat and moisture is obtained. Skimmed or normalized milk is pasteurized at a temperature of 78 ± 2 ° C with an exposure of 15-20 s in plate or tubular pasteurization-cooling units or in capacitive devices. After pasteurization, the milk is cooled to the fermentation temperature.

The starter culture is prepared on pure cultures of mesophilic lactic streptococci, including the Enterococcus hirae VKPM B-10088 culture. Before adding to the milk, the surface layer of the starter culture is carefully removed with a clean, disinfected bucket and removed. Then the leaven is mixed to a homogeneous consistency with a clean whorl (when prepared in starter baths) or a mixer and poured into the prepared milk in an amount of 5% of the total mass. Duration of milk fermentation - 6 hours

An aqueous solution of calcium chloride (mass fraction of calcium chloride 30-40%) is added to milk after fermentation: 400 g per 1000 kg of fermented milk. The preparation and preparation of a solution of calcium chloride is carried out in accordance with the Instructions for technological control at dairy enterprises. After adding a salt solution to fermented milk, a 1% solution of rennet is introduced at the rate of 1 g of the preparation with an activity of 100,000 ME per 1000 kg of milk.

Rennet or pepsin is added to milk in the form of a 1% aqueous solution prepared in boiled water and cooled to 36 ± 3 ° C. The enzyme solution is introduced into milk with constant stirring. After 10-15 minutes after making the enzyme solution, stirring is completed and milk is left at rest until a dense clot with an acidity of 61 ± 5 ° T for cottage cheese of 9% and 18% fat content, 65 ± 5 ° T for peasant and 71 ± 5 ° T for low-fat cottage cheese.

To process the clot, use manual lyres, in which a thin stainless steel wire serves as knives. With such wire knives, the clot is cut into cubes measuring 2 × 2 × 2 cm. The clot is first cut along the length of the bath into horizontal layers, and then into vertical and vertical lengths. After this treatment, the clot is left for 40-60 minutes to separate serum and increase acidity. The separated serum is drained from the bath. The clot after draining the whey is poured into coarse calico or dacron bags measuring 40 × 80 cm. The bags are filled about 70%, which is 7-9 kg of cottage cheese. Then the bags are tied and stacked one on top of another in a self-pressing bath, a press trolley or a UPT unit for pressing and cooling the curd.

In order to accelerate the separation of serum, as well as in case of poor excretion of serum, the clot is heated by supplying steam or hot water to the space between the cottage cheese baths. For uniform heating, the upper layers of the clot are moved with a wooden or metal plate (shovel) from one wall of the bath to another. The clot is heated to 40 ± 2 ° C for 30-40 minutes for cottage cheese of 9% and 18% fat content, 35 ± 2 ° C for 20-40 minutes for peasant and 36 ± 2 ° C for 15- 20 minutes for low-fat cottage cheese.

Self-pressing of cottage cheese lasts at least 1 hour. Pressing continues until cottage cheese with a mass fraction of moisture is provided for by the regulatory documentation. For cottage cheese of 18% fat, it is 65%; 9% fat - 73; peasant - 74.5; dining room - 76; low-fat - 80; for diet fruit 11% fat - 64, 9% fat - 66, 4% fat - 77 and nonfat - 79% moisture. When developing low-fat cottage cheese, dehydration of the clot can be carried out on a cottage cheese separator. After separation and pressing, the curd is cooled. Cottage cheese is cooled in bags located on the shelves of the refrigerator or in carts or tubs. Cottage cheese is cooled to a temperature of 12 ± 2 ° C and sent for packaging and labeling.

Example 3. The use of strain Enterococcus hirae VKPM B-10088 in the production of fermented baked milk.

Normalized milk with a fat content of 4% is subjected to heat treatment at 100 ° C without exposure, cooled to 38 ° C, add fermentation for fermented baked milk in an amount of 2%, activated biomass of bifidobacteria in milk in an amount of 0.1% and Enterococcus hirae strain VKPM B-10088 on sterile milk in an amount of 0.5%. Fermentation is carried out at a temperature of 37 ° C until titratable acidity of 65 ° T is achieved. Then the product is slowly cooled to 6 ° C, after which they are bottled.

Claims (1)

The strain Enterococcus hirae VKPM B-10088 used for the preparation of fermented milk products.