RU2244002C1 - Strain of microorganism lactobacillus lactis for preparing curd from milk - Google Patents

Abstract

FIELD: biotechnology, microbiology, dairy industry.

SUBSTANCE: the strain of microorganism Lactobacillus lactis VKPM B-8354 is prepared without using mutagens and genetic methods and shows resistance against broad spectrum of lactophages. The strain ferments effectively milk from different trading sorts, with broad range of fatness and different methods of thermal treatment. Individual specific properties of the strain allows its applying as a monostrain ferment. Curd obtained with applying the strain L. lactis VKPM B-8354 shows good organoleptic qualities, nonacid taste and homogenous consistence. The strain is suitable especially for plants with small volume of manufacture but with varied assortment.

EFFECT: valuable properties of strain.

3 tbl, 2 dwg, 5 ex

Description

The bacteria Lactococcus lactis have long been used as starter cultures for the production of lactic acid products and, in particular, cottage cheese from milk. Depending on the composition, the starter culture can be divided into monostrain (mono-starter cultures), when only one strain is used to prepare the starter culture, and poly-strain (poly-starter culture), when several strains are included in the starter culture.

One of the problems arising from the fermentation of milk with bacterial starter cultures of L. lactis is phagolysis, i.e. lysis of bacteria under the action of lactophages (bacteria viruses L. lactis). In this case, the starter culture is spoiled and the desired product cannot be obtained. In order to avoid a similar situation, phage-resistant strains are used in industry. However, it is not possible to completely overcome the problem of phagolysis in this way, since In addition to phages that enter the reservoir for fermenting milk from the outside, phages can exist in the cells of the bacteria themselves in a latent (latent) form and do not appear under normal conditions. Under stress associated with an increase in temperature during the process, the release of free phage and the selection of phagosensitive bacterial mutants are possible. The species of bacteria L. lactis is characterized by high variability of properties [2] and a large variety of lactophages [3, 4]. Thus, it is desirable that the strain used in industry has resistance to all or at least the major known phages.

In the case of multi-strain starter cultures, if infection of at least one of the strains used in the starter culture occurs, the strain supporting the growth of lactophages is excluded from the starter culture and replaced by another [5].

In this regard, the challenge is to obtain new phage-resistant strains, expanding the arsenal of existing ones, and to have a collection of strains that can be used at any time to replace the phagolysed in both monostrain and poly-strain starter cultures. New strains can be obtained by selection of spontaneous mutants [6], as well as after treatment with mutagens [7]. There are attempts to create genetic engineering variants [5].

When choosing strains for starter cultures, the most important and decisive production characteristics are the efficiency of fermentation by milk bacteria and the organoleptic properties of the final product [1, p.274]. When making yeast for cottage cheese, strains are introduced that tell the product good taste and smell, forming clots that easily separate serum [1, p.276]. These characteristics are provided either by the combination of the properties of the strains included in the polysacre, or by the properties of one strain, if it is used as a mono-ferment.

At present, in the industry, mainly polysacastes are used [7, 1, p.275,], however, there is interest in the use of mono ferments. Their use contributes to the prolonged use of specialized strains [8], as well as to a decrease in the diversity of phages in production [9].

In the world practice of studying the bacteria L. lactis with the aim of using them as a mono-ferment, the work of E. Durmaz and T. Klaenhammer is known [5]. In this work, a phage-resistant strain of L. lactis was constructed by genetic engineering methods. This strain is considered by us as the closest analogue. It is resistant to the following bacteriophages: φ31 (type P335), φ37.7 (derivative φ31), u136, and a composition of production phages containing 10 independent lysates [5]. However, this strain has insufficient milk fermentation efficiency, which the authors determined by the accumulation of lactic acid and evaluated on the basis of changes in pH. The measured pH did not fall below 5.1.

The objective of the invention: to obtain, without the use of mutagens and genetic manipulations, a strain of Lactococcus lactis that is resistant to a wide range of lactophages, which can be used as a mono-ferment for effective fermentation of milk.

The problem is solved by obtaining a strain of Lactococcus lactis TB2, deposited in the All-Russian collection of industrial microorganisms under the number VKPM B-8354.

The inventive strain is natural. It is isolated from self-fermented cottage cheese.

Characterization of the strain of Lactococcus lactis VKPM B-8354.

1. Morphological characteristics of the strain VKPM B-8354. The cells are oval, motionless, located in the form of diplococci or short chains, gram-positive, do not form endospores, facultative anaerobes. Grow on organic media. On the surface of agar with hydrolyzed milk and synthetic media M21 and M02 (Table 1), white, convex, smooth colonies with smooth edges form after 24 hours at 28-30 ° C.

2. The species affiliation of the VKPM B-8354 strain was determined based on the GC composition and the level of DNA-DNA hybridization [10]. Earlier [11], the HC was determined for the composition of the lactophage sensitive strain VKPM B-6886, equal to 36.3 mol.%. The GC composition of the claimed strain is 36.7 mol.%. The error of determination is 0.2-0.3%. Since this indicator for the species Lactococcus lactis can vary within 35-37 mol.%, Therefore, the strain VKPM B-8354 can be assigned to the species Lactococcus lactis. This conclusion is confirmed by a high level of DNA-DNA hybridization, equal to 93%, with the VKPM B-8548 strain, which is a fusidine-resistant mutant of the VKPM B-6886 strain sensitive to lactophages.

3. The inventive strain VKPM B-8354 obtained without the use of mutagens and genetic manipulations and is resistant to the following bacteriophages: subgroup B1-U33A, U88A and U50A; subgroup B2-U13A, U14, U15A, U27A, U55A, U80A, U87A, P001, as well as lactophages R10, R103 and C7.

4. The strain VKPM B-8354 efficiently (up to pH 4.6-4.8) ferments milk of various fat content (from 1.5% to 20%), different brands and methods of heat treatment. It allows you to get cottage cheese as a final product of homogeneous consistency and non-acidic taste, with pleasant organoleptic properties. The yield of finished cottage cheese from 263 to 750 grams per liter of milk.

List of figures illustrating the invention.

Figure 1. Growth curve of the claimed strain.

1- VKPM B-8354; 2 - VKPM B-6920.

The graphs show the average results of measurements in three independent experiments.

Figure 2. Change in pH with the growth of bacteria of VKPM B-8354 and VKPM B-6920 strains in milk of various fat content, manufacturer and heat treatment.

1-1 - strain VKPM B-8354; Milk "Village House" (sterilized) 6%;

1-2 - strain VKPM B-6920; Milk "Village House" (sterilized) 6%;

2-1 - strain VKPM B-8354; Holy World milk (pasteurized) 3.2%;

2-2 - strain VKPM B-6920; Holy World milk (pasteurized) 3.2%;

3-1 - strain VKPM B-8354; milk "Lianozovsky" (sterilized) 1.5%;

3-2 - strain VKPM B-6920; Lianozovsky milk (sterilized) 1.5%.

The invention is confirmed by the following examples.

Example 1. The resistance of the claimed strain to lactophages.

In order to check the strain VKPM B-8354 for resistance to various lactophages, we used phages obtained from the collection of NPO Uglich [4]. It is shown that they belong to group B according to Bradley's classification and that among them, part belongs to subgroup B1, and the other part to subgroup B2 according to Ackerman's classification. The following phages were taken for the phage resistance test: subgroup B1 - U33A, U88A and U50A; B2 subgroup - U13A, U14, U15A, U27A, U55A, U80A, U87A, P001, as well as the lactophages R10, R103 and C7 stored in VKPM (RN-738, RN-736, RN-730). Bacteria of the VKPM B-8354 strain were grown on Petri dishes with M02 agar medium (Table 1). The grown bacteria were collected in a loop (~ 0.5 loop) in 0.3 ml of physiological saline and resuspended. Then the bacteria were mixed with M02 agar medium diluted with physiological saline to an agar concentration of 0.7%, and plates with M02 medium were poured with a thin layer of this suspension of bacteria. After the upper layer hardens, drops with a phage suspension (phage titer 10 ⁷ -10 ⁸ ) were applied to its surface. Petri dishes were incubated overnight at 30 ° C. No lysis spots were found on the lawn of VKPM B-8354 strain, while the growth of all phages was observed on the control lawn with bacteria of the phagosensitive strain VKPM B-8548.

Thus, the claimed strain VKPM B-8354 is resistant to all of the above lactophages, which is a wide range of phages common in dairy enterprises.

Example 2. The accumulation of biomass in the colonies and the weak dependence of the growth of the claimed strain on the component composition of the medium.

The level of biomass accumulation was estimated by the area of the colonies, calculated on the basis of their scanned image using the Total Lab v201 program. Bacteria of the claimed strain VKPM B-8354 and taken for control production phage-resistant strain VKPM B-6920 were scattered to separate colonies on nutrient media M02 and M03 (table 1) and incubated at 30 ° C for 24 hours. The results of a computer assessment of the area of the colonies and their average value on this Petri dish, expressed in mm ² , are presented in table. 2. After 24 hours of incubation, the average area of the colonies of the control strain, compared with the claimed, was only 66% and, therefore, was less by 34%.

When using M03 medium, in which there is no yeast extract, which is a source of vitamins, the average colony area of the claimed strain after 24 hours of growth is less than 2% on M02 medium, while it is 74% less in the control strain.

These data indicate a significant dependence of the growth of the control strain and the relative independence of the growth of the claimed strain from a fortified supplement.

Example 3. The time of generation of the inventive strain.

To determine the time of generation of the inventive strain VKPM B-8354 and the control strain VKPM B-6920 used a biophotometer company BONET-MAURY-JOUAN. From bacteria freshly grown on M02 medium (Table 1), a thick suspension was prepared in physiological saline. Cells were well resuspended and pelleted by centrifugation (Eppendorf). The washed cells were again resuspended in 80 μl and diluted 10 times in physiological saline. A 100 μl bacterial suspension was added to the biophotometer cuvettes containing 6 ml of M22 medium (Table 1) and the initial optical density was measured. Further, each hour, the value of the optical density that changes during cell growth was noted. Figure 1 presents graphs on the basis of which the time of generation of the inventive strain VKPM B-8354 and the control strain VKPM B-6920, i.e. the time required for cells to double. Its value is determined as corresponding to a doubling of the optical density of the bacterial suspension during the period of their most intensive logarithmic growth. It is equal to 35 and 51 minutes, respectively. Therefore, the generation time of the claimed strain is shorter than the generation time of the control strain by 16 minutes.

Example. 4. The change in pH during the fermentation of milk of various fat content of the claimed strain.

The acidification of milk during the growth of the inventive strain VKPM B-8354 and the control strain VKPM B-6920 was characterized by a change in pH. For the experiment, milk of various fat content, brands and heat treatment was used. Before the experiment, each of 6 liters of milk: “Village house” (sterilized) 6%; Holy World (pasteurized) 3.2%; "Lianozovsky" (sterilized) 1.5%, was poured into a liter jar, closed, aged 20 minutes in a boiling water bath and cooled to room temperature. Ferment was prepared by inoculating 10 ml of milk (10% milk powder) with bacteria taken from a separate colony from M02 medium. After adding 1% sourdough, milk cans were placed in a thermostat at 30 ° C and the pH value was measured in samples (2 ml) taken at 0, 4, 6, 9, 12, 14, 15, and 28 hours after the start of the experiment. The results of pH measurement are presented in figure 2. It can be seen from them that the inventive strain VKPM B-8354 acidifies any type of milk used more efficiently than the control strain VKPM B-6920. A method of producing a milk-protein clot involves adjusting the acidity of milk to a pH of 4.6-4.8 [12]. The inventive strain reaches this level in 9 hours of incubation in the conditions of our experience. A milk clot forms between 8 and 9 hours and has a dense texture. The control strain under the same conditions cannot independently acidify milk to the required pH value, however, when using appropriate technological schemes [12] it can be used in production.

The results allow us to conclude that it is possible to use the claimed strain as a mono-ferment for the effective fermentation of milk of various fat contents (from 1.5% to 20%), brands ("Village House", "Holy World" "Lianozovsky") and a thermal method processing (sterilization or pasteurization).

Example 5. Obtaining cottage cheese using the inventive strain.

For the preparation of cottage cheese used a milk clot obtained as described in example 4. As a control was taken strain VKPM B-6920. Glass liter jars containing a milk clot were heated in a boiling water bath for 20 minutes until the whey separated from the clot. This was followed by slow cooling of the product to room temperature and separation of the whey by discarding it on gauze. The finished curd was weighed and characterized organoleptically. The weighing results are presented in table 3. The yield of cottage cheese when using the inventive strain was higher in all three variants compared with the control. The maximum weight of cottage cheese obtained by VKPM strain V-8354 from 20% cream reached 750 g of the finished product per liter.

Organoleptic showed that the cottage cheese obtained using the inventive strain has a non-acidic pleasant taste and has a homogeneous consistency.

The inventive strain of Lactococcus lactis VKPM B-8354 obtained without the use of chemical mutagens and is not genetically modified. It is resistant to a wide range of lactophages common in the dairy industry. The strain effectively fermented milk of various brands, a wide range of fat content and a different method of heat treatment. The individual characteristics of the strain make it possible to use it as a monostrain yeast. Cottage cheese obtained using the inventive strain has good organoleptic qualities. Such properties of cottage cheese as non-acidic taste and homogeneous consistency are in demand in modern conditions, because necessary for the preparation of various curd mixtures, including dessert. The strain is especially convenient for enterprises with a small volume of production, but a diverse assortment.

Used sources of information.

1. Stepanenko P.P. Microbiology of milk and dairy products:

Textbook for high schools. - Sergiev Posad: LLC "All for You - Moscow Region". 1999.

2. Journal of Bacteriology 2000, 182 (9): 2481-91.

3. Canadian Journal of Microbiology 1992, 38: 875-882.

4. Biotechnology 1992, 6: 40-45.

5. Applied And Environmental Microbiology 1995, 61 (4): 1266-1273.

6. USSR author's certificate 938893.

7. Copyright certificate of the USSR 1486141.

8. Applied And Environmental Microbiology 1993, 59: 365-372.

9. Journal of Dairy Science 1981, 64: 2270-2277.

10. Microbiology 1997, 66 (2): 233-236.

11. FEMS Microbiology Review 2001, 25: 39-67.

12. Copyright certificate of the USSR 404459.

Claims (1)

The bacterial strain Lactococcus lactis VKPM B-8354 to obtain cottage cheese from milk.

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