Strain of microorganism lactobacillus lactis for preparing curd from milk

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

 

Bacteria Lactococcus lactis long used as starter cultures for obtaining lactic acid products and, in particular, cheese from the milk. Ferment, depending on the composition can be divided into monstermovie (monozukuri), when for the preparation of starter culture used only one strain, and polyline (politikasi), when the composition of the starter culture composed of several strains.

One of the problems arising from the fermentation of milk with a bacterial starter cultures of L. lactis is paralysis, i.e. lysis of the bacteria under the action of lactation (viruses, bacteria L. lactis). In this case, the leaven spoiled and desired product cannot be obtained. In order to avoid similar situations in the industry use phagostable strains. However, to fully overcome the problem of paralysis this fails, because in addition to phages fall into tanks for fermentation of milk from outside, phages may exist in the cells of the bacteria in the latent (hidden) form, and should not occur under normal conditions. Under conditions of stress associated with temperature rise in the process, possible free phage and selection focustitle bacterial mutants. Type of bacteria L. lactis has a high variability of the properties of [2] and a large variety of lactation [3, 4]. Thus, it is desirable that used in the industry the items the strain was resistant to all, or at least the main well-known phages.

In the case of polyline starters, if contamination of at least one of the strains used in the sourdough occurs, the strain of supporting the growth of lactation, is excluded from the starter and replaced by another [5].

In this regard, the task is to get new phagostable strains, expanding Arsenal already, and have a collection of strains that can be used to replace the affected paralysis in monochromatic and polyline starters. New strains can be obtained by selection of spontaneous mutants [6], and after treatment with mutagens [7]. There are attempts to create genetically engineered variants [5].

When selecting strains for starters the most important and decisive production characteristics are efficiency fermenting bacteria of milk and organoleptic properties of the final product [1, s]. In the preparation of starter culture for cheese impose strains, indicating the product has a good taste and smell, forming clots, easily separating serum [1, s]. These characteristics are provided or a combination of the properties of the strains included in politiques or properties of the same strain, if it is used as monozukuri.

Currently in industry mainly use politikasi [7, 1, s,], however, there is interest in the use of the of monotechnic. Their use promotes long-term use of specialized strains [8], as well as reducing the diversity of phage production [9].

In world practice, the study of bacteria L. lactis with the aim of using them as monozukuri famous work E. Durmaz and So Klaenhammer [5]. In this work, genetic engineering methods designed phagostable strain L. lactis. This strain is considered as the closest analogue. It is resistant to the following bacteriophages: ϕ31 (view N), ϕ37.7 (derived ϕ31), u136 and composition of the production of phages containing 10 independent lysates [5]. However, this strain has a lack of efficiency of fermentation of milk, which the authors was determined by the accumulation of lactic acid and evaluated based on the change of pH. Measured pH value did not fall below 5,1.

The task of the invention to obtain without the use of mutagens and genetic manipulation resistant to a wide range of lactation strain Lactococcus lactis, which can be used as monozukuri for effective fermentation of milk.

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

The inventive strain is natural. He separated from Samokhvalova cheese.

Characteristics ø the Amma Lactococcus lactis VKPM B-8354.

1. Morphological characteristics of the strain VKPM B-8354. Cells oval, stationary, are in the form of diplococci or short chains, gram-positive, endospore not forming, facultative anaerobes. Grow on organic media. On the surface of the agar with hydrolyzed milk, and synthetic environments M21 and m (table 1) after 24 hours at 28-30°To form a white, convex, smooth colonies with smooth edges.

2. Species strain VKPM B-8354 was determined on the basis HZ-composition and DNA-DNA hybridization [10]. Previously [11] was identified HZ composition sensitive to lactation strain VKPM B-6886 equal 36,3 mol.%. HZ-composition of the inventive strain equal to 36.7 mol.%. The detection error is 0.2-0.3%. Since this indicator for the species Lactococcus lactis may vary within 35-37 mol.%, it is, therefore, the strain VKPM B-8354 can be attributed to the species Lactococcus lactis. This conclusion is confirmed by the high level of DNA-DNA hybridization, equal to 93%, with the strain VKPM B-8548, which is positionality mutant sensitive to lactation strain VKPM B-6886.

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

4. The strain VKPM B-8354 effectively (to a pH of 4.6 and 4.8) suasive the milk of different fat content (from 1.5% to 20%), different brands and methods of heat treatment. It allows you to get as a final product cheese homogeneous texture and sour taste, with a pleasant organoleptic properties. The yield of cheese from 263 to 750 grams of liters of milk.

List of figures illustrating the invention.

Figure 1. Growth curve of the proposed strain.

1 - PMBC-8354; 2 - PMBC-6920.

The graph shows the average results of measurements in three independent experiments.

Figure 2. The change in pH during the growth of bacteria strains In PMBC-8354 and VKPM B-6920 in milk of different fat, manufacturer and thermal processing.

1-1 - strain VKPM B-8354; milk House in the village" (sterilized) 6%;

1-2 - strain VKPM B-6920; milk House in the village" (sterilized) 6%;

2-1 - strain VKPM B-8354; milk, "Holy Peace" (pasteurized) 3,2%;

2-2 - strain VKPM B-6920; milk, "Holy Peace" (pasteurized) 3,2%;

3-1 - strain VKPM B-8354; milk Lianozovsky (sterilized) 1,5%;

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

The invention is confirmed by the following examples.

Example 1. The stability of the proposed strain to lactation.

To verify the strain VKPM B-8354 resistance to various lactation we have used phage obtained from the collection of the NGO "Uglich" [4]. It is shown that they belong to the group In to the ossification Bradley and that among them part belongs to the subgroup B1, and another part of the subgroup B2 classification Ackerman. To test frostiest were taken the following phage: subgroup B1 - U33A, U88A and U50A; subgroup B2 - U13A, U14, U15A, U27A, U55A, U80A, U87A, R and lactifuge R10, R103 and C7 stored in PMBC (PH 738, PH-736, PH-730). Bacteria strain VKPM B-8354 were grown on Petri dishes with agar medium m (table 1). The grown bacteria were collected by loop (~0,5 loops) in 0.3 ml of saline solution and was resuspendable. Next, bacteria were mixed with agar medium m, diluted with saline to a concentration of agar 0.7 percent, and a thin layer of this suspension of bacteria was poured cups with medium m. After the top layer hardens on its surface inflicted drops with ragovoy suspension (titer phage 107-108). Petri dishes were incubated overnight at 30°C. On the lawn of strain VKPM B-8354 was not detected spots lysis, whereas the control lawn with bacteria focustitle strain VKPM B-8548 growth was observed in all phages.

Thus, the claimed strain VKPM B-8354 is resistant to all the above lactation, representing a wide range of phages that are distributed in the dairy industry.

Example 2. Biomass accumulation in the colonies and the weak dependence of the growth of the claimed strain from component composition environment.

The level of accumulation of biomass is canevali on space colonies, calculated on the basis of the scanned image using Total Lab v201. Bacteria of the proposed strain VKPM B-8354 and taken to control the production of phagostable strain VKPM B-6920 subcultured to single colonies on nutrient media m and M03 (table 1) and incubated at 30°With 24 hours. The results of the computer estimate the area of the colonies and their average value on the Petri dish, expressed in mm2presented in table. 2. After 24 hours of incubation, the average size of colonies in the control strain, compared with declare, was only 66% and, therefore, was less than 34%.

When using environment M03, in which there is no yeast extract, which is a source of vitamins, the average size of the colonies declare strain after 24 hours of growth less than that in the medium m 2%, whereas the control strain is less than 74%.

These data indicate a significant correlation between the growth of the control strain and the relative independence of the growth of the claimed strain from fortified supplements.

Example 3. The generation time of the proposed strain.

To determine the generation time of the proposed strain VKPM B-8354 and the control strain VKPM B-6920 used biophotometer company BONET-MAURY-JOUAN. From viewership on the environment m (table 1) b is Cheri was prepared in saline dense suspension. Cells were well resuspendable and precipitated by centrifugation (Eppendorf). The washed cells were again resuspendable in 80 ml and were diluted 10 times in saline. The suspension of bacteria in 100 μl was added to the cuvette biophotometer containing 6 ml of medium M22 (table 1) and measured the initial optical density. Further, every hour noted the value changing in the process of cell growth optical density. Figure 1 shows the graphs on the basis of which is determined by the generation time of the proposed strain VKPM B-8354 and the control strain VKPM B-6920, i.e. the time necessary for the cells to double. It is defined as a corresponding increase in the optical density of bacterial suspension twice in the period of most intensive of logarithmic growth. It is respectively 35 and 51 minutes. Therefore, the generation time of the proposed strain shorter than the time of generation of a control strain for 16 minutes.

Example. 4. The change of pH during fermentation of milk with different fat content of the inventive strain.

Acidification of milk in the growth process of the proposed strain VKPM B-8354 and the control strain VKPM B-6920 characterized by change in pH. For experiment used the milk of different fat content, trademarks and heat treatment. Before the beginning of the experiment, each of the 6 liters of milk: "house in the trees is e (sterilized) 6%; "Holy Peace" (pasteurized) 3,2%; "Lianozovo" (sterilized) of 1.5%, was transferred into a quart jar, closed, and maintained for 20 minutes in a boiling water bath and cooled to room temperature. Leaven was preparing, sowing 10 ml of milk (10% skimmed milk) bacteria, extracted from individual colonies from Wednesday m. After adding 1% of the leaven of the cans of milk were placed in a thermostat at 30°and the pH value was measured in samples (2 ml), extracted through 0, 4, 6, 9, 12, 14, 15 and 28 hours after the start of the experiment. The results of the pH measurements are presented in figure 2. They show that the claimed strain VKPM B-8354 acidic any type of used milk more efficiently than the control strain VKPM B-6920. The method of obtaining milk-protein clot envisages increase in the acidity of milk to a pH of 4.6 to 4.8 [12]. The inventive strain reaches this level after 9 hours of incubation under the conditions of our experience. Clot milk is formed between 8 and 9 o'clock and has a firm consistency. The control strain under the same conditions cannot zamisliti milk to the required pH value, however, when using the appropriate technological schemes [12] can be used in production.

The obtained results allow to conclude about the possibility of using the proposed strain as monozukuri for effective fermentation of milk with different fat content (1,5% to 20%), trade marks ("the House in the village", "Holy Peace" Lianozovsky) and method of heat treatment (sterilization or pasteurization).

Example 5. Obtaining curd with use of the claimed strain.

To prepare the cheese used a bunch of milk, obtained as described in example 4. As the control was taken as the strain VKPM B-6920. Glass quart jar containing milk curd, warmed up in boiling water bath for 20 minutes to separate the serum from the clot. Followed by slow cooling the product to room temperature and separating the serum by dropping it on the gauze. The finished cheese was weighed and characterized its out of line. The weighing results are presented in table 3. The output of cheese when using the inventive strain was higher in all three variants compared to the control. The maximum weight of the curd obtained by the strain VKPM B-8354 from 20% cream, 750 g of finished product per litre.

The organoleptic evaluation showed that the cheese obtained using the proposed strain, has a pleasant sour taste and has a homogeneous consistency.

The inventive strain Lactococcus lactis VKPM B-8354 obtained without the use of chemical mutagens and are not genetically modified. It is resistant to a wide range of lactation, the soap is common in the dairy industry. Strain effectively skachivat milk from different brands, wide range of fat content and different methods of heat treatment. The individual characteristics of the strain, allow to use it as monstermovie ferment. The cheese obtained using the proposed strain, has good organoleptic quality. Such properties of cheese as sour taste and homogeneous consistency demanded in modern conditions, as required for the preparation of different cheese blends, including dessert. The strain is especially useful for companies with small production volume, but a diverse range.

Used sources of information.

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

Textbook for high schools. - Sergiev Posad: LLC "All for You - the suburbs". 1999.

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

3. Canadian Journal 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. USSR author's certificate 1486141.

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

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

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

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

12. USSR author's certificate 404459.

Bacterial strain Lactococcus lactis VKPM B-8354 to get cheese from the milk.



 

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