Method for production of bacterial concentrate and its application as probiotic biologically active food additive

FIELD: food industry.

SUBSTANCE: inventions group relates to biotechnology and may be used for preparing bacterial preparations applicable as probiotic biologically active additives. The bacterial concentrate production method envisages preparation of a nutritional medium, sterilisation and cooling. Inoculum introduction, cell propagation, bacterial mass separation from culture liquid, dispensing and closuring. Into the nutritional medium composition one introduces cedar or linseed oil of fish or ringed seal fat in an amount of 1-1.5% of the medium weight; the inoculum is represented by a Bifidobacterium longum DK-100 strain. The produced bacterial concentrate is used as a probiotic biologically active food additive.

EFFECT: inventions group allows to recover the digestive tract microflora.

2 cl, 3 tbl, 3 dwg, 5 ex

The proposed group of inventions relates to biotechnology and can be used for the preparation of bacterial preparations used as probiotic biologically active additives to food.

A method of obtaining a food product, including the restoration of skimmed milk powder in normalized milk with sugar, heat treatment, cooling, seeding environment yoghurt sourdough probiotic lactic acid bacteria, fermentation, cooling, cooking the juice part with polyunsaturated ω-3 fatty acids and/or free amino acid, mix with yoghurt part in a 1:1 ratio, dispersion and packing (see EN NO. 2282995, A23C 9/12, A23L 1/30 17.12.2004).

The disadvantage of this method is the complexity of the process of obtaining and using only ω-3 polyunsaturated fatty acids in relatively small amounts.

The closest way to the claimed group of inventions on the totality of symptoms is the method of obtaining a bacterial preparation for treatment and prevention of hypercholesterolemia, comprising preparing culture media, sterilization, cooling, introducing an inoculum of Lactobacillus helveticus GCNM 147, increase biomass, branch biomass from the culture fluid, filling, capping, labeling, storage (see EN No. 2072692, A61K 38/46, C12N 1/20, C12R 1/225, 21.01.1997.

The disadvantage of this method is the high acid-forming ability of L. helveticus and not high enough cholesteroldegrading microbial activity, which reduces consumer and probiotic properties.

Thus, in the production of bacterial concentrates the main task is the selection of culture conditions for production of concentrates with high cholesteroldegrading, probiotic and consumer properties.

The technical result that ensures the implementation of the present invention is to improve consumer properties and level of degradation of cholesterol.

This technical result in the implementation of the invention is achieved in that in the method of obtaining a bacterial concentrate, comprising preparing culture media, sterilization, cooling, making inoculum, increase biomass, branch biomass from the culture fluid, bottling, corking, according to the invention in a nutrient medium composition contribute 1-1,5% by weight of the cedar environment or flax oil, or fish or seal oil and sowing carry inoculum of a strain of Bifidobacterium Bifidobacterium longum DK-100.

This technical result is also achieved by the application of the bacterial concentrate, obtained by the claimed method, as probiotic the tion of biologically active food supplements.

Distinctive features of the proposed method are entering into the composition of the nutrient medium cedar or flax oil, or fish or seal oil, the choice of their optimal dose and the use of Bifidobacterium longum DK-100, with marked high cholesteroldegrading activity, consumer and probiotic properties of the concentrate.

In addition, the distinguishing feature of the claimed invention is the use of bacterial concentrate obtained by the proposed method, as probiotic biologically active food supplements.

For the implementation of the proposed method experimental studies were performed.

At the first stage of the research we investigated the effects of polyunsaturated fatty acids on the growth of biomass and the number of viable cells of Bifidobacterium. For this purpose, the inoculum of pure cultures were made in nutrient medium on the basis of cheese whey with the addition of cedar or flax oil, or fish or seal oil in the amount of 0.5-1.5%. The accumulation of biomass of microorganisms was performed by periodic cultivation at 36±1°C. the Growth of the cultures was assessed by the change in optical density at λ=450 nm photocolorimeter. The titer of viable cells of Bifidobacterium was determined by the number of CFU/cm³when seeding cell suspension on Wednesday the MK. For control of bacterial taken the concentrate of the appropriate strain of bacteria without the addition of vegetable oil or animal fat.

The results are shown in table 1 and Fig.1, 2.

As the data presented in table 1, the introduction of pine and flax oil, fish and seal oil in a nutrient medium accelerates the buildup of biomass and the growth of bifidobacteria in the medium compared with the control. The maximum growth of bifidobacteria was observed when the concentration of pine and flax oil, fish and seal oil in the amount of 1.5% (Fig.1 and 2).

These strains possess probiotic properties, but the most intensive growth of biomass of bifidobacteria and the highest number of viable cells is observed when the strain Century longum DK-100 (Fig.1 and 2).

In the next phase of studies investigated the effects of polyunsaturated fatty acids cedar and flax oil, fish and seal fat cholesteroldegrading properties of bifidobacteria.

As a source of cholesterol used purified serum. Cultivation was carried out for 24 hours with double neutralization. During this period followed dynamicauxiliary in a nutrient medium.

The results are shown in table 2 and Fig.3.

As can be seen from the table.2, there is a high destruction of cholesterol in the cultivation process all strains of probiotic microorganisms. The cholesterol levels in the medium were determined by enzymatic method (see BALABINA M. D. Methods for the determination of cholesterol/M. D. BALABINA, centuries SLEPYSHEVA, A. C. KOZLOV//Hepatology-2004.-T6, No. 6.- S. 73-75; THE 9398-267-23548172-2002), based on the introduction into a nutrient medium in a person's blood.

Most cholesteroldegrading activity possesses Century Longum DK-100, which metabolizes 68,09% total cholesterol when making cedar oil, 74,39% - when making linseed oil, 79,07% - when adding fish oil and 77,03% - when making seal oil, respectively (Fig.3).

Thus, the obtained experimental data allow to conclude that the introduction of pine and flax oil, and fish and seal oil containing polyunsaturated fatty acids, increases cholesterolcholesterol properties and the number of viable cells of Bifidobacterium, which testifies to the growth of probiotic properties.

A comparative analysis of the assimilation of cholesterol are presented in table 3 (SHENDEROV B. A. Medical and microbial ecology and functional nutrition. Volume II: Socio economic and clinical consequences of the imbalance of the microbial ecology of humans and animals.- M: Publishing house of the GRANT,1998.-416 C.).

The obtained results show that the cultivation of bacteria in a nutrient medium with the addition of 1.5% cedar or Flaxseed oil, fish or seal oil increases cholesterolcholesterol ability of bifidobacteria. Most high cholesterolcholesterol activity was observed in cultures with the addition of fish oil.

It should be noted that the BAA on the basis of probiotic microorganisms and cedar and Flaxseed oil, fish or seal oil in the literature was not found.

On the basis of the conducted researches optimal dose make cedar or Flaxseed oil, fish or seal oil in the amount of 1-1,5% of the volume of the nutrient medium, stimulating strong growth and viability of bifidobacteria. Also found that these bacterial concentrate the drugs have a high consumer properties: consistency homogeneous, without separation of serum; the taste and smell clean, sour taste of the respective added vegetable oil or animal fat; high holesterinesterzy activity and a high number of viable cells of Bifidobacterium.

Summarizing the obtained results, we can conclude that bifidobacteria bacterial concentrates with cedar or flax oil, fish or seal oil have a high biochemical high cholesterolcholesterol activity.

The inventive method is as follows.

As a nutrient medium for cultivation of bifidobacteria use clarified cheese whey, which is added growth components: a buffer salt, ascorbic acid, peptone, agar. Set the pH of the medium within (7±0,1). Then sterilized at 121°C for 30 minutes, cooled to a temperature of cultivation 36±1°C, make a 1-1,5% by weight of the cedar environment or flax oil, or fish or seal oil and 5% inoculum of the leaven of Bifidobacterium longum DK-100. Conduct capacity cells of bifidobacteria within 20-24 hours at double the neutralization environment through 10 hours to maintain the pH at an optimum level. Then separate the bacterial mass from the culture fluid. The obtained cell suspension is poured in aseptic condition is the conditions in sterile vials 12 cm ³, sealed, cooled to (4±2)°C.

The obtained liquid bacterial concentrate is used as a probiotic biologically active additives to food.

Example 1.As a nutrient medium for cultivation of bifidobacteria use clarified cheese whey, which is added growth components: a buffer salt, ascorbic acid, peptone, agar. Set the pH of the medium within (7±0,1). Then sterilized at 121°C for 30 minutes, cooled to a temperature of cultivation 36°C, contribute 1% cedar oil and 5% inoculum of the leaven of Bifidobacterium longum DK-100. Conduct capacity cells of bifidobacteria within 24 hours at double the neutralization environment through 10 hours to maintain the pH at an optimum level. Then separate the bacterial mass from the culture fluid. The obtained cell suspension is poured aseptically into sterile vials 12 cm³, sealed, cooled to (4±2)°C.

Example 2.As a nutrient medium for cultivation of bifidobacteria use clarified cheese whey, which is added growth components: a buffer salt, ascorbic acid, peptone, agar. Set the pH of the medium within (7±0,1). Then sterilized at 121°C for 30 minutes, cooled to a temperature of cultivation 36°C, make % linseed oil and 5% inoculum of the leaven of Bifidobacterium longum DK-100. Conduct capacity cells of bifidobacteria within 24 hours at double the neutralization environment through 10 hours to maintain the pH at an optimum level. Then separate the bacterial mass from the culture fluid. The obtained cell suspension is poured aseptically into sterile vials 12 cm³, sealed, cooled to (4±2)°C.

Example 3.As a nutrient medium for cultivation of bifidobacteria use clarified cheese whey, which is added growth components: a buffer salt, ascorbic acid, peptone, agar. Set the pH of the medium within (7±0,1). Then sterilized at 121°C for 30 minutes, cooled to a temperature of cultivation 36°C, contribute 1% fish oil and 5% inoculum of the leaven of Bifidobacterium longum DK-100. Conduct capacity cells of bifidobacteria within 24 hours at double the neutralization environment through 10 hours to maintain the pH at an optimum level. Then separate the bacterial mass from the culture fluid. The obtained cell suspension is poured aseptically into sterile vials 12 cm³, sealed, cooled to (4±2)°C.

Example 4.As a nutrient medium for cultivation of bifidobacteria use clarified cheese whey, which is added growth components: a buffer salt, as erbenova acid, peptone, agar. Set the pH of the medium within (7±0,1). Then sterilized at 121°C for 30 minutes, cooled to a temperature of cultivation 36°C, contribute 1% of seal fat and 5% inoculum of the leaven of Bifidobacterium longum DK-100. Conduct capacity cells of bifidobacteria within 24 hours at double the neutralization environment through 10 hours to maintain the pH at an optimum level. Then separate the bacterial mass from the culture fluid. The obtained cell suspension is poured aseptically into sterile vials 12 cm³, sealed, cooled to (4±2)°C.

Example 5.The use of liquid bacterial concentrate obtained according to examples 1-4, as probiotic biologically active additives to food.

The obtained liquid bacterial concentrates on examples 1-4 are used as probiotic dietary supplements to the diet to restore the microflora of the gastrointestinal tract, boost immunity and protect the body from cardiovascular diseases.

It is recommended to take an adult to 3 times a day, one teaspoon at meal times in four weeks.

1. A method of obtaining a bacterial concentrate, providing for the preparation of culture media, sterilization, cooling, making inoculum, the capacity of the cells, separating the bacteria is raise the weight from the culture fluid, the bottling, corking, characterized in that the composition of the nutrient medium contribute cedar or Flaxseed oil or fish or seal fat in the amount of 1-1,5% by weight of the medium as inoculum used Bifidobacterium Bifidobacterium longum DK-100.

2. The application of the bacterial concentrate obtained by the method according to p. 1 as probiotic biologically active food supplements.