RU2847002C1 - Lactococcus lactis strain for use in production of brine cheese - Google Patents

Abstract

FIELD: biotechnology; microbiology; food industry.

SUBSTANCE: disclosed is a bacterial strain Lactococcus lactis 18 VKPM V-15100, characterized by growth in a dairy product with NaCl content 3±1 wt. %, giving the product a pure fermented milk taste.

EFFECT: invention allows to use the said strain in the brine cheese production technology.

1 cl, 3 ex

Description

Field of technology to which the invention relates

The invention relates to the field of biotechnology, microbiology and the food industry, namely to a new bacterial strain of Lactococcus lactis 18 VKPM B-15100, characterized by growth in a dairy product with a NaCl content of 3±1 wt.%, imparting a pure fermented milk taste to the resulting product, which allows the strain to be used in the technology for producing brine cheeses.

State of the art

Cheese is one of the most valuable food products in terms of protein and milk fat content [Gorbatova K.K., Gunkova P.I. Chemistry and Physics of Milk and Dairy Products. – 2012]. Brined cheeses are a product kept in brine after the first phase of fermentation and cheese forming. In Russia, GOST 33959-2016 "Brine Cheeses. Specifications" is in force, regulating the names and characteristics of finished products. The most common types of cheese are: Ossetian, feta cheese, Karachay, and Georgian. After the technological stage of forming and pressing the heads of cheese, the cheese is salted in brine containing 18 to 25% NaCl. The duration of the salting stage is from 4 to 6 days, depending on the type of cheese and the concentration of NaCl in the brine [Gorbatova K.K., Gunkova P.I. Chemistry and Physics of Milk and Dairy Products. – 2012]. After the cheese is aged in brine, it undergoes ripening, a process that involves biochemical processes, including microbial ones. The salinity of the final product ranges from 2 to 7% NaCl, depending on the cheese variety [GOST 33959-2016 "Brine Cheeses. Technical Conditions"].

The strain Lactococcus lactis subsp . lactis biovar. Diacetylactis (CNCM No. 1-1962) is known for imparting flavor and aroma to dairy-based products, specifically, imparting a malt flavor and aroma to a fermented dairy product (RU 2593719, published 10.08.2019). However, to obtain a dairy product with the specified flavor and aroma, the original milk source is pre-enriched with an amino acid, at least one of L-phenylalanine, L-leucine, L-isoleucine, or L-valine.

A mixed culture of two or more microbial strains is known for improving the flavor and aroma of fermented products, including cheese (WO 0200845, January 3, 2002). The strains used are selected from the group consisting of Lactococcus, Lactobacillus, Propionibacteria, Streptococcus, Staphylococcus, Bifidobacterium, Penicillium, Brevibacterium, Arthrobacter, Corynebacterium, Saccharomyces, and Debaromyces . However, the strains were cultured in milk supplemented with 0.5% yeast extract without NaCl, which precludes the applicability of the strains to brine cheese production.

The lactic acid bacteria strains Lactococcus lactis subsp. lactis diacetylactis CNCM No. I-4404 and Lactococcus lactis subsp. lactis diacetylactis CNCM No. I-4405 are known and are used to produce a fermented milk product with a creamy taste and aroma or to obtain 3,4-dihydroxy-3,4-dimethyl-2,5-hexanedione, which is responsible for the creamy-buttery taste (RU 2593944, published 10.08.2016). However, there is no information on the possibility of using these strains in the production of brine cheeses.

The bacterial strain Lactococcus lactis subsp. Lactis VKPM B-8558, used in the production of dairy products, and the method for obtaining a starter culture of the strain Lactococcus lactis subsp. Lactis VKPM B-8558 (RU 2295563, published 20.03.2007) are known. The strain Lactococcus lactis subsp. Lactis VKPM B-8558 can be used in the production of dairy products. A distinctive feature of the strain is the ability to synthesize exopolysaccharides in quantities that improve the rheological properties of the product without the addition of food additives, and the ability to enhance the functional properties of products obtained using this strain of lactic acid bacteria. However, there is no information on the applicability of this strain in the technology of brine cheese production.

The closest strain to the claimed invention (the prototype) is Lactococcus lactis 12/16/PSH VKPM B-14320 , used in the production of fermented milk products (RU 2803266, published 11.09.2023). Lactococcus lactis 12/16/PSH VKPM B-14320 can be used to produce fermented milk products characterized by a pure fermented milk taste, balanced acidity, and enhanced rheological properties without the addition of additional components that increase the viscosity of the finished product. However, the examples of the invention do not contain information on the applicability of Lactococcus lactis 12/16/PSH VKPM B-14320 in the production of cheeses, including brine cheeses.

Thus, the technical problem solved by the claimed invention is the need to overcome the shortcomings inherent in analogues and the prototype by obtaining a bacterial strain applicable in the technology of producing brine cheeses.

Disclosure of invention

The technical result of the claimed invention is a new bacterial strain , Lactococcus lactis 18 VKPM B-15100, characterized by a titratable acidity index of 30°T to 50°T after 9-11 hours of cultivation in a dairy product with a mass fraction of NaCl of 3±1% at a temperature of +32±4°C. The strain can be used for the production of brine cheeses, the resulting fermentation product having a pure fermented milk flavor.

The strain is deposited in the All-Russian Collection of Industrial Microorganisms of the Federal State Unitary Enterprise GosNIIGenetika (VKPM, Moscow, 1st Dorozhny Prospekt, Bldg. 1) under registration number VKPM B-15101. The strain was identified based on 16S rRNA gene sequence analysis and MALDI-TOF MS, as well as its cultural, morphological, and physiological-biochemical characteristics.

Cultural and morphological characteristics

The colonies are light beige, rounded, with smooth edges and a convex profile, shiny, 1-2 mm in diameter, have a uniform structure and soft consistency, and do not produce pigment. The cells are cocci, arranged singly, in pairs, and in chains of varying lengths. They are nonmotile, do not form spores, and stain positively with Gram staining.

Physiological and biochemical characteristics

The strain is catalase- and oxidase-negative. Cultivation is carried out on a medium of the following composition (g/l): casein peptone 10.0; meat extract 8.0; yeast extract 4.0; glucose 20.0; lactose 5.0; dipotassium phosphate 2.0; Tween-80 1.0; diammonium citrate 2.0; sodium acetate 5.0; magnesium sulfate 0.2; manganese sulfate 0.04; agar 14.0. Cultivation is carried out at a temperature of +32±2 °C under anaerobic conditions.

Implementation of the invention

The possibility of implementing the claimed invention is shown, but not limited, in examples of specific implementation.

Any type of milk can be used as a dairy product (milk source): cow's, sheep's, goat's, buffalo's, or any mixture thereof. The milk source can be ultra-high-temperature-treated (UHT), pasteurized, or sterilized milk. The milk source can be whole milk, skim milk, or skim milk. Furthermore, the milk source can be natural milk, recombinant milk, milk containing vegetable fat, or any mixtures thereof.

Example 1. Method for isolating and culturing the Lactococcus lactis 18 VKPM B-15100 strain. The strain was isolated from the invertebrate Molgula griffithsii , inhabiting the waters of the Kandalaksha Gulf of the White Sea. Isolation method: disintegrated body material of the invertebrate was resuspended in sterile Ringer's solution (Merck, Germany) and distributed over the surface of a dense nutrient medium of the following composition (g/l): casein peptone 10.0; meat extract 8.0; yeast extract 4.0; glucose 20.0; lactose 5.0; sodium chloride 25; dipotassium phosphate 2.0; Tween-80 1.0; diammonium citrate 2.0; sodium acetate 5.0; magnesium sulfate 0.2; manganese sulfate 0.04; Agar 14.0. Cultivation was carried out under anaerobic conditions at a temperature of +32±2°C for 2 days. The conditions in which this strain was detected are characterized by high salinity and low ambient temperatures. These factors are important when using the strain as a starter culture for the production of brine cheeses, as they ripen at low temperatures and high salinity.

Example 2. Cultivation of the Lactococcus lactis 18 VKPM B-15100 strain in skim milk with a mass fraction of NaCl of 3%. A sterile stock solution of 30% NaCl was added to the milk source sterilized at 1 excess atm. for 15 minutes in a mass ratio of 1:9. Then, the strain inoculum was added in an amount of at least 1*10 ⁶ CFU/ml in the final fermentation mixture. Cultivation was carried out at a temperature of +32 ± 2 °C for 10 hours. The acidity of the final product was 60 ± 5 °T. The final product was characterized by a dense clot, a light sour milk aroma and a clean sour milk taste.

Example 3. Long-term collection preservation of the Lactococcus lactis strain 18 VKPM B-15100. To increase the biomass of the strain, a dense nutrient medium of the following composition (g/l) was used: casein peptone 10.0; meat extract 8.0; yeast extract 4.0; glucose 20.0; lactose 5.0; dipotassium phosphate 2.0; Tween-80 1.0; diammonium citrate 2.0; sodium acetate 5.0; magnesium sulfate 0.2; manganese sulfate 0.04; agar 14.0. Cultivation was carried out at a temperature of +32±2°C under anaerobic conditions for 2 days. The resulting biomass was then washed from the Petri dishes with sterile Ringer's solution (Merck, Germany), and glycerol was added to the resulting solution to a final concentration of 10%. The resulting mixture was then poured into cryotubes and stored in a low-temperature freezer at -70±4°C.

Claims (1)

Lactococcus lactis strain 18 VKPM B-15100, intended for use in the production of brine cheeses.