RU2776653C1 - Method for obtaining bacterial starter culture - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A method for obtaining a bacterial starter culture of lactic acid bacteria of the Lactobacillus sakei species is proposed, including cultivation of Lactobacillus sakei LSK-45, Lactobacillus sakei DSM 20017, Lactobacillus sakei LSK-104 or Lactobacillus sakei LSK-103 strains on a nutrient medium from a decoction based on rice flour prepared at the rate of 55-75 g of rice flour per 1 liter of water, and containing peptone in the amount of 7-10 g/dm³.

EFFECT: invention provides for the creation of a bacterial starter culture of Lactobacillus sakei bacteria, which does not contain lactose and has increased biochemical activity.

1 cl, 3 dwg, 3 tbl, 4 ex

Description

The invention relates to the microbiological and food industries and can be used for the production of food products enriched with lactic acid bacteria.

A method for the production of a bacterial concentrate is known, involving the cultivation of a symbiotic starter from kefir fungal starter and thermophilic lactobacilli Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus helveticus in a ratio of 1:0.5:0.5:1 on a nutrient medium based on curd whey, rye flour and growth components at a temperature of (30±2)°C for 8-10 hours. After the starter is obtained, the biomass is separated to obtain a liquid bacterial concentrate. In another variant, for the preparation of a direct starter culture, the resulting liquid bacterial concentrate is mixed with a protective medium and frozen at a temperature not exceeding minus 20 ° C (see Patent RU No. 2524435, IPC A23C 9/12, A23C 9/127, C12P 1/00, published on July 27, 2014, Bulletin No. 21).

The disadvantage of this method is the use of curd whey containing lactose as part of the nutrient medium, the use of which is unacceptable for people suffering from lactase deficiency.

Closest to the proposed invention in terms of technical essence and the achieved result is a method for the production of a bacterial preparation of bifidobacteria, which involves the use of rice water as the basis of the nutrient medium with the addition of growth components. In the manufacture of a bacterial preparation, a strain of Bifidobacterium longum DK-100 is used (see Khamagaeva I.S., Khazagaeva S.N., Maradudina I.P. Optimization of the nutrient medium for obtaining a hypoallergenic biological product // Bulletin of the ESSTUU. 2017. No. 3 (66) pp. 93-96).

The disadvantages of this method are the complexity of the preparation of rice water and the use only for the cultivation of bifidobacteria.

It is known that at present in most non-European populations, including among the population of Asia, Africa, Latin America and representatives of northern peoples, lactase deficiency is widespread. In Russia, a condition associated with a decrease in the level of the lactase enzyme occurs in 16-18% of the adult population. Ignoring this condition can lead to serious medical and social consequences. In this regard, there is a growing demand for the production of food ingredients, including bacterial starters, which are produced using raw materials that do not contain dairy ingredients. Therefore, the use of plant-derived components for the production of bacterial preparations is of great scientific interest.

An analysis of patent documents on a given topic showed that the main disadvantages of similar production methods are the laboriousness of manufacturing a nutrient medium, the use of curd whey containing lactose as the basis for the medium.

A feature of the invention is that the nutrient medium used in the proposed method for producing bacterial starter is based on rice flour, while it does not contain milk-containing components and lactose. Thus, bacterial starter can be used in the nutrition of people suffering from lactase deficiency and used for the production of products intended for this group of consumers.

The main technical objective of the invention is to obtain a bacterial ferment of lactic acid bacteria of the species Lactobacillus sakei that does not contain lactose.

The technical result provided by the implementation of the invention is to obtain a bacterial starter of lactic acid bacteria Lactobacillus sakei with a high number of bacterial cells, low labor intensity of manufacture.

The specified technical result is achieved by the fact that in the method for obtaining a bacterial starter, including the preparation of a nutrient medium, the introduction of an inoculum, cell growth, separation of the bacterial mass from the culture medium, mixing it with a protective medium, bottling, freezing, according to the invention , a culture of lactic acid bacteria is used as an inoculum Lactobacillus sakei strains Lactobacillus sakei LSK-45, Lactobacillus sakei DSM 20017, Lactobacillus sakei LSK-104 or Lactobacillus sakei LSK-103, and a decoction based on rice flour is used as a nutrient medium, prepared at the rate of 55-75 g of rice flour per 1 liter water, as part of the nutrient medium, peptone is used in an amount of 7-10 g / dm ³ .

Distinctive features of the claimed invention are:

- new conditions for the cultivation of lactic acid bacteria, namely the use as an inoculum of a culture of lactic acid bacteria strains of Lactobacillus sakei LSK-45, Lactobacillus sakei DSM 20017, Lactobacillus sakei LSK-104, or Lactobacillus sakei LSK-103;

- the use of lactose-free rice flour as the basis of the nutrient medium, which makes it possible to obtain a bacterial starter for the production of lactose-free food products.

At the first stage of research, the growth of Lactobacillus sakei LSK-45 bacteria (VKPM collection number B-8896) was studied on three types of nutrient media: a) MRS medium recommended for the cultivation of lactic acid bacteria, based on rice flour.

The following components were added to the media based on rice water and rice flour:

glucose - 15 g,

magnesium chloride - 0.3 g,

trisubstituted sodium citrate - 1 g,

potassium phosphate monosubstituted - 0.5 g,

ascorbic acid - 0.1 g,

peptone - 5 g,

rice broth / broth based on rice flour - up to 1 liter.

It is known that amino acids, minerals and vitamins are necessary for better growth and development of Lactobacillus sakei , and magnesium and manganese ions have a great influence on growth. It is known that rice and rice flour are rich in manganese; therefore, manganese-containing salts were not added to nutrient media based on rice water and rice flour. Peptone was added to the bacterial medium as a source of amino acids. As a result, the results presented in Table 1 were obtained.

Table 1 - Growth data for two strains of Lactobacillus sakei Bacterial strains Nutrient media The number of viable bacterial cells, log CFU/cm ³ after 24 hours of cultivation pH of the nutrient medium, units Lactobacillus sakei LSK-45 MRS 9 4.59 Rice-based medium 6.77 5.42 Rice flour medium 7.3 4.9

Thus, it was found that the medium based on rice flour provides better growth of microorganisms of the species Lactobacillus sakei compared to the medium based on rice water. In this regard, a nutrient medium based on rice flour was used for further research.

For further research, an experiment planning matrix was designed, shown in Table 2. The content of peptone (x ₁ ) and the content of rice flour (x ₂ ) were chosen as factors, the number of viable bacterial cells served as the optimization parameter (y). When planning the experiment, the method of the central compositional experiment was used (Yudin Yu.V. Organization and mathematical planning of the experiment: a textbook / Yu.V. Yudin, M.V. Maisuradze, F.V. Vodolazsky. - Yekaterinburg: Publishing house of Ural. -ta, 2018. - 124 p.).

Table 2 - Experiment design matrix Experience number Factor levels Factor Level Values The number of bacterial cells, log CFU / cm ³ (y) x _{1 x2} Peptone content, g / dm ³ The content of rice flour, g / dm ³ one -one -one 0 25 ₁ 2 0 -one 5 25 at ₂ 3 +1 -one ten 25 ₃ four -one 0 0 fifty at ₄ 5 0 0 5 fifty at ₅ 6 +1 0 ten fifty at ₆ 7 -one +1 0 75 at ₇ eight 0 +1 5 75 at ₈ 9 +1 +1 ten 75 at ₉

The response surface and contour plot show surface level lines dividing the response surface into 10 zones labeled 1 to 10 (see FIG. 1). Zone 1 corresponds to the number of bacterial cells from 8.461 to 9.048 log CFU/cm ³ ; zone 2 - values 7.875-8.461 log CFU/cm ³ ; zone 3 - values 7.288-7.875 log CFU/cm ³ ; zone 4 - values 6.701-7.288 log CFU/cm ³ ; zone 5 - values 6.115-6.701 log CFU/cm ³ ; zone 6 - values 5.528-6.115 log CFU/cm ³ ; zone 7 - values 4.941-5.528 log CFU/cm ³ ; zone 8 - values 4.355-4.941 log CFU/cm ³ ; zone 9 - values 3.768-4.355 log CFU/cm ³ ; zone 10 - values 3.181-3.768 log CFU/cm ³ .

The results obtained indicate that for Lactobacillus sakei LSK-45, the best bacterial growth is obtained using 7-10 g/dm ³ peptone and 55-75 g/dm ³ rice flour (response surface zone 1). Both peptone and rice flour stimulate the growth of Lactobacillus sakei .

At the next stage of experimental studies, the growth of the following bacterial strains is studied when bacterial inoculum is introduced in an amount of 3-7% of the volume of the nutrient medium:

- Lactobacillus sakei LSK-45 (VKPM collection number B-8896);

- Lactobacillus sakei DSM 20017 (VKPM collection number B-10905);

- Lactobacillus sakei LSK-104 (VKPM collection number B-8936);

- Lactobacillus sakei LSK-103 (VKPM collection number B-8932).

At the same time, a culture of lactic acid bacteria of the species Lactobacillus sakei was used as an inoculum, the cultivation of which is carried out on a semi-liquid MRS medium for (20 ± 4) hours. The content of viable cells in the inoculum is at least 1⋅108 CFU/cm3.

The results are shown in FIG. 2 and 3.

Thus, it was found that the optimal duration of cultivation is (24-32) h at a temperature of (37±1)°C.

It is known that liquid bacterial sourdough is not shelf stable. The decrease in the number of cells in liquid sourdough is due to the acid reaction of the environment, high humidity, and the absence of protective substances. It is possible to increase the shelf life of a bacterial preparation using cryopreservation. In this regard, at the next stage of research, freezing of bacteria is carried out. Before freezing, the nutrient medium is centrifuged, the resulting cell suspension is mixed with the protective medium in a ratio of 2:1. The mixture is frozen at a temperature of at least minus 18°C.

It was found that after freezing, the number of bacterial cells is: for Lactobacillus sakei LSK-45 strain - 7⋅10 ⁸ CFU/cm ³ , for Lactobacillus sakei DSM 20017 strain - 1⋅10 ⁹ CFU/cm ³ ; for Lactobacillus sakei LSK-104 strain - 3⋅10 ⁹ CFU/cm ³ , for Lactobacillus sakei LSK-103 strain - 2⋅10 ⁹ CFU/cm ³ .

At the next stage, the qualitative characteristics of the bacterial starter of lactic acid bacteria are studied. As an inoculum in the production of sourdough, a culture of lactic acid bacteria of the species Lactobacillus sakei is used, the cultivation of which is carried out on a semi-liquid MRS medium for 20 ± 4 hours. The results are presented in table 3.

Table 3 - Qualitative characteristics of the bacterial starter culture of lactic acid bacteria Lactobacillus sakei Name of indicator Indicator value liquid frozen Consistency and appearance Dense suspension of bacterial cells Color White to dark cream Taste and smell Clean, slightly sour, without foreign flavors Active acidity (pH), units 6.5±0.5 Temperature upon release from the enterprise, °C, no more 4±2°C not less than minus 12°С The number of bacteria, CFU in 1 cm ³ , not less than 1⋅10 ⁸ micropicture Gram-positive rods arranged singly, in pairs or in short chains The volume of the product (cm ³ ), in which are not allowed:
BGKP (coliforms) ten Staphylococci S. aureus ten Pathogenic microorganisms (including salmonella) 100 Yeast and molds, CFU/cm3 ^, no more 5 in total

The proposed method for obtaining a bacterial starter of lactic acid bacteria of the species Lactobacillus sakei is carried out as follows.

The method for obtaining a bacterial starter is illustrated by graphs, where in Fig. 1 shows a response surface reflecting the expected growth of the Lactobacillus sakei LSK-45 strain - the graph shows the effect of two factors - the content of peptone and the content of rice flour on the number of viable cells of lactic acid bacteria (log CFU / cm ³ ; figure 2 shows the change in the number of viable cells of four strains of bacteria of the species Lactobacillus sakei on a medium based on rice flour, figure 3 shows the change in pH during the cultivation of four strains of bacteria of the species Lactobacillus sakei on a medium based on rice flour.

A medium based on rice flour is used to obtain liquid and frozen sourdough bacteria of the species Lactobacillus sakei . Pre-rice flour (55-75 g/dm ³ ) is mixed with water and brought to a boil. The following components are used to prepare the nutrient medium:

Glucose - 15 g,

magnesium chloride - 0.3 g,

trisubstituted sodium citrate - 1 g,

potassium phosphate monosubstituted - 0.5 g,

ascorbic acid - 0.1 g,

peptone - (7-10) g / dm ³ ,

decoction based on rice flour - up to 1 liter.

The nutrient medium is sterilized at (120±1)°C with an exposure of 30 minutes and cooled to a temperature of 37°C. As an inoculum, a culture of lactic acid bacteria strains of Lactobacillus sakei LSK-45, Lactobacillus sakei DSM 20017, Lactobacillus sakei LSK-104, Lactobacillus sakei LSK-103 is used, the cultivation of which is carried out on a semi-liquid MRS medium for (20 ± 4) h, which is added to amount of 5% of the volume of the nutrient medium. The growth of bacterial cells is carried out for (24-32) hours. The resulting bacterial mass is cooled to (4 ± 2) ° C, separated from the culture medium by centrifugation, mixed with a protective medium in a ratio of 2: 1, poured into vials, frozen at a temperature not less than minus 18°С.

Upon receipt of a frozen starter, a protective medium of the following composition is used: sucrose - 10%, sodium citrate - 2%, distilled water - the rest. The protective medium is sterilized at a temperature of (121±1)°C for 30 minutes, then the medium is cooled and stored until use.

Examples confirming the possibility of implementing a method for obtaining a bacterial starter culture:

Example 1

Preparation of liquid bacterial starter.

When obtaining a liquid bacterial starter culture of lactic acid bacteria, a nutrient medium based on rice flour is used. To prepare the nutrient medium, rice flour in the amount of 55 g/dm ³ is mixed with water and brought to a boil. Prepare a nutrient medium of the following composition:

glucose - 15 g,

magnesium chloride - 0.3 g,

trisubstituted sodium citrate - 1 g,

potassium phosphate monosubstituted - 0.5 g,

ascorbic acid - 0.1 g,

peptone - 10 g,

decoction based on rice flour - up to 1 l (rice flour content 55 g / dm ³ ).

The prepared medium is sterilized at (121±1)°C for 30 minutes and cooled to 37°C. Then, an inoculum of lactic acid bacteria containing a strain of Lactobacillus sakei LSK-45 in the amount of 3% is added to the medium and the cells are grown for 32 hours. At the end of the process, the bacterial mass is cooled to 4°C. Then the cells are separated from the culture medium by centrifugation and poured into vials. Vials with bacterial starter are closed with sterile stoppers and rolled up with metal caps. Liquid bacterial starter of lactic acid bacteria is stored for up to 3 months at a temperature of (4±2)°C.

Example 2

Preparation of frozen bacterial sourdough.

When obtaining a liquid bacterial starter culture of lactic acid bacteria, a nutrient medium based on rice flour is used. To prepare the nutrient medium, rice flour in the amount of 75 g/dm ³ is mixed with water and brought to a boil. Prepare a nutrient medium of the following composition:

glucose - 15 g,

magnesium chloride - 0.3 g,

trisubstituted sodium citrate - 1 g,

potassium phosphate monosubstituted - 0.5 g,

ascorbic acid - 0.1 g,

peptone - 7 g,

decoction based on rice flour - up to 1 l (rice flour content 75 g / dm ³ ).

The prepared medium is sterilized at (121±1)°C for 30 minutes and cooled to (37±1)°C. Then, an inoculum of lactic acid bacteria containing a strain of Lactobacillus sakei DSM 20017 in an amount of 5% is added to the medium and cells are grown for 24 hours. At the end of the process, the bacterial mass is cooled to 4°C.

The resulting bacterial starter is mixed, the cells are separated from the culture medium by centrifugation, a protective medium is added in a ratio of 2:1. The following components are used in the environment:

Sucrose - 10%;

Sodium citrate trisubstituted - 2%;

Distilled water - the rest.

The resulting mixture is poured under sterile conditions into 2 ml glass vials and frozen at a temperature of at least minus 18°C. The vials are closed with sterile stoppers and rolled up with metal caps. Frozen sourdough can be stored for up to 6 months at a temperature of at least minus 12°C.

Example 3

Preparation of liquid bacterial starter.

When obtaining a liquid bacterial starter culture of lactic acid bacteria, a nutrient medium based on rice flour is used. To prepare the nutrient medium, rice flour in the amount of 65 g/dm ³ is mixed with water and brought to a boil. Prepare a nutrient medium of the following composition:

glucose - 15 g,

magnesium chloride - 0.3 g,

trisubstituted sodium citrate - 1 g,

potassium phosphate monosubstituted - 0.5 g,

ascorbic acid - 0.1 g,

peptone - 10 g,

decoction based on rice flour - up to 1 l (rice flour content 65 g / dm ³ ).

The prepared medium is sterilized at (121±1)°C for 30 minutes and cooled to 37°C. Then, an inoculum of lactic acid bacteria containing a strain of Lactobacillus sakei LSK-103 in the amount of 7% is introduced into the medium and the cells are grown for 28 hours. At the end of the process, the bacterial mass is cooled to 4°C. Then the cells are separated from the culture medium by centrifugation and poured into vials. Vials with bacterial starter are closed with sterile stoppers and rolled up with metal caps. Liquid bacterial starter of lactic acid bacteria is stored for up to 3 months at a temperature of (4±2)°C.

Example 4

Preparation of frozen bacterial sourdough.

When obtaining a liquid bacterial starter culture of lactic acid bacteria, a nutrient medium based on rice flour is used. To prepare the nutrient medium, rice flour in the amount of 75 g/dm ³ is mixed with water and brought to a boil. Prepare a nutrient medium of the following composition:

glucose - 15 g,

magnesium chloride - 0.3 g,

trisubstituted sodium citrate - 1 g,

potassium phosphate monosubstituted - 0.5 g,

ascorbic acid - 0.1 g,

peptone - 7 g,

decoction based on rice flour - up to 1 l (rice flour content 75 g / dm ³ ).

The prepared medium is sterilized at (121±1)°C for 30 minutes and cooled to 37±1°C. Then, an inoculum of lactic acid bacteria containing a strain of Lactobacillus sakei LSK-104 in the amount of 3% is added to the medium and the cells are grown for 24 hours. At the end of the process, the bacterial mass is cooled to 4°C.

The resulting bacterial starter is mixed, the cells are separated from the culture medium by centrifugation, a protective medium is added in a ratio of 2:1. The following components are used in the environment:

Sucrose - 10%;

Sodium citrate trisubstituted - 2%;

Distilled water - the rest.

The resulting mixture is poured under sterile conditions into 2 ml glass vials and frozen at a temperature of at least minus 18°C. The vials are closed with sterile stoppers and rolled up with metal caps. Frozen sourdough can be stored for up to 6 months at a temperature of at least minus 12°C.

The proposed invention "Method for obtaining bacterial starter" in comparison with the prototype (see Khamagaeva I.S., Khazagaeva S.N., Maradudina I.P. Optimization of the nutrient medium for obtaining a hypoallergenic biological product // Bulletin of the ESSTUU. 2017. No. 3 (66 ) S. 93-96) has the following advantages:

- new conditions for the cultivation of lactic acid bacteria, namely the use as an inoculum of a culture of lactic acid bacteria strains of Lactobacillus sakei LSK-45, Lactobacillus sakei DSM 20017, Lactobacillus sakei LSK-104, or Lactobacillus sakei LSK-103;

- the use of lactose-free rice flour as the basis of the nutrient medium, which makes it possible to obtain a bacterial starter that can be used for the production of lactose-free food products.

- preparation of a decoction based on rice flour is a less labor-intensive process compared to the preparation of rice decoction based on rice grains.

Claims (1)

A method for producing a bacterial starter of lactic acid bacteria of the species Lactobacillus sakei , which involves preparing a nutrient medium, introducing an inoculum, growing cells, separating the bacterial mass from the culture medium, mixing it with a protective medium, bottling, freezing, characterized in that a culture of lactic acid bacteria Lactobacillus is used as an inoculum sakei strains of Lactobacillus sakei LSK-45, Lactobacillus sakei DSM 20017, Lactobacillus sakei LSK-104 or Lactobacillus sakei LSK-103, and a decoction based on rice flour is used as a nutrient medium, prepared at the rate of 55-75 g of rice flour per 1 liter of water , as part of the nutrient medium, peptone is used in the amount of 7-10 g / dm ³ .

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