RU2250259C1 - Method for broth production for cultivation of bifidobacterium and lactobacillus - Google Patents

Abstract

FIELD: biotechnology, microbiology, in particular broth production for cultivation of bifidobacteria and lactobacillus.

SUBSTANCE: claimed method includes water-based broth production. Water used for broth production is pretreated by non-filtered modulated ultraviolet light containing in radiant flux not less than 30 % of ultraviolet quantum with wave length less than 350 nm, for period sufficient to increase water intrinsic energy by not less than 2 times. Method also includes measurement of water intrinsic energy variation before treatment and during whole treatment process.

EFFECT: microorganisms with activated cultural properties; decreased cultivation time.

5 tbl, 2 ex

Description

The invention relates to the use of ultraviolet radiation in biotechnology, can be used in the microbiological, food industry and relates to methods for preparing culture media for the cultivation of bifidobacteria and lactobacilli.

A known method of producing biomass of microorganisms, namely, that the biomass is grown in a nutrient medium of the claimed composition, in which a complex preparation of gibbersib is used as a cell growth stimulator, obtained on the basis of the culture fluid generated during the cultivation of Fusarium moniliforma in the amount of 1 · 10 ^-12 -1 · 10 ^-60 % (RF Patent No. 2093570, IPC 6 C 12 N 1/20, publ. 10/20/1997, bull. No. 29).

Known technical solution, which provides for the preparation of a nutrient medium for the cultivation and isolation of bifidobacteria of the claimed composition based on distilled water (Patent No. 2203944, IPC 6 C 12 N 1/20, publ. 10.05.2003, bull. No. 13).

A disadvantage of the known technical solutions is the insufficiently high yield and the considerable duration of growing biomass.

The technical problem to which the present invention is directed is to obtain maximum results on the intensification of the recovery process of bifidobacteria and lactobacilli cultures from an inactive state and increase the yield of microorganism biomass while reducing their growing time by using active water in the preparation of nutrient medium compositions.

The stated technical problem is solved in that in a method for preparing a nutrient medium for culturing bifidobacteria and lactobacilli, providing for the preparation of a nutrient medium based on water, according to the invention, the water for preparing the nutrient medium is pre-treated with unfiltered modulated ultraviolet light with a content of not less than 30% ultraviolet quanta with a wavelength shorter than 350 nm, for the time required to increase the internal energy of water n e less than 2 times. An additional difference of the method lies in the fact that they measure the parameters characterizing the change in the internal energy of the water, before water treatment and during the entire processing process.

When processing water as mentioned in the method, an increase in the energy of the volume of the liquid being processed occurs, followed by an increase in the internal energy of the hydraulic medium of self-oscillating microculture systems. When changing (decreasing) the radiation power of an external source to maintain the maximum level of energy flow into the medium being processed, the internal energy of the self-oscillating system of the hydraulic medium tends to restore equilibrium with the level of environmental energy. In this case, the internal energy of the hydraulic environment of the self-oscillating system is released, the energy levels in the interatomic bonds are redistributed. Thus, the released energy of the hydraulic medium when interacting with microcultures leads to an increase in energy in self-oscillating systems of microcultures, which helps to increase their cultural properties. It was experimentally established that the use of activated water in the composition of nutrient media contributed to the exit from the state of suspended animation of the largest number of cells and activation of their vital activity hundreds of times.

The method is as follows.

The water intended for the preparation of a nutrient medium is treated with unfiltered modulated light of ultraviolet radiation with a content of at least 30% ultraviolet quanta with a wavelength shorter than 350 nm in the radiant flux. Through experimental studies, it was found that the most optimal is the use of ultraviolet quanta with a wavelength of 190-250 nm. The modulation of the flow is carried out according to the laws of quantum mechanics, describing the phenomenon of superposition in liquid media (“Open Physics.” A complete interactive physics course. Version 2.5 edited by Professor MIPT S. M. Kozell). It is advisable to modulate ultraviolet radiation, for example, by the frequency of (any) one of the known biological rhythms of an object that will be exposed to activated water, the modulation can be amplitude or pulsed. Processing is carried out for the time necessary to increase the internal energy of water by at least 2 times. In this case, the measurements of the parameters characterizing the change in the internal energy of the water are carried out before the water treatment and during the entire processing process by known methods using a viscometer. As a water treatment device equipped with a source of ultraviolet radiation, a known device for the same purposes can be used, or, for example, a water treatment device, the design of which is described in application No. 2003107495 with a priority of March 19, 2003 for the invention "Water treatment device".

For example, if the prepared volume of water for activation is 3000 ml and a working wavelength of 230 nm is selected, 30% of the lamp power, the accompanying radiation is visible and infrared light; 500 W radiation source. Fixed light exposure is 240 minutes.

Next, activated water (with modified internal energy) is used to prepare a nutrient medium for the cultivation of bifidobacteria and lactobacilli. The use of activated water in the composition of nutrient media for the cultivation of bifidobacteria and lactobacilli contributes to the exit from the state of suspended animation of the largest number of cells and activation of their vital activity hundreds of times.

The method is illustrated by the following examples.

Example 1. For activation, a culture strain of the species Lactobacillus casei from the collection of the SSC RF IMBP RAS was used. On the basis of water treated with ultraviolet radiation in the manner mentioned in the method, a standard MPC culture medium was prepared for the cultivation of lactobacilli. The culture was initially in a lyophilized state. The culture was rehydrated and 10 times triturated in a nutrient medium. Incubated 48 hours at 37 ° C.

Survival was assessed by the number of colonies grown. The data are presented in tables 1-4.

Table 1
The survival of lactobacilli in a nutrient medium prepared by the proposed method Medium MPC 0.2%, prepared on the basis of water not treated with ultraviolet 1 day growth 2 days growth Breeding 6 5 × 10 ⁸ ± 1 × 10 ⁶ 10 ⁸ ± 1 × 10 ⁶ 7 1.4 × 10 ⁹ ± 1 × 10 ⁷ 10 ⁹ ± 1 × 10 ⁷ 8 no growth no growth Medium MPC 0.2% based on ultraviolet treated water Breeding 6 Drain growth Drain growth 7 1 × 10 ⁹ ± 1 × 10 ⁷ 2 × l0 ⁹ ± 1 × 10 ⁷ 8 3 × 10 ¹⁰ ± 1 × 10 ⁸ 3 × 10 ¹⁰ ± 1 × 10 ⁸

Also, using the nutrient medium prepared according to the proposed method, the strain of lactobacilli Lactobacillus casei K-25 stored in the ampoule for 13 years was successfully revived from the lyophilic state (see table 2).

table 2
The revival of the strain Lactobacillus casei K-25 using a nutrient medium prepared according to the proposed method Medium MPC 0.2%, prepared on the basis of water not treated with ultraviolet Breeding 5 No growth 6 Medium MPC 0.2% based on ultraviolet treated water Breeding 5 drain growth 6 3 × 10 ¹⁰

Moreover, the number of living cells in the colonies in a nutrient medium prepared by the proposed method was larger in size than those grown on ordinary media (tables 3 and 4).

Table 3
The number of living cells in colonies in growth media prepared by the proposed method (CFU * / ml) (Experiment 1) Colonies isolated from medium MPC prepared on unirradiated water Breeding 5 drain growth 6 2.8 × 10 ⁸ 7 6 × 10 ⁸ Colonies isolated from medium MPC prepared on irradiated water Breeding 5 drain growth 6 3 × 10 ⁹ 7 7 × 10 ⁹ * CFU - colony forming units. Table 4
The number of living cells in colonies in growth media prepared by the proposed method (CFU) (Experiment 2) Colonies isolated from medium MPC prepared on unirradiated water Breeding 6 2.0 × 10 ⁹ 7 8.0 × 10 ⁹ Colonies isolated from medium MPC prepared on irradiated water Breeding 6 3.6 × 10 ⁹ 7 1.4 × 10 ¹⁰

The data in the tables indicate an increase in the number of viable colonies of lactobacilli during their cultivation in growth media prepared by the proposed method, approximately 100 times. This trend appeared on the first day of growth and continued on the next day. At the same time, cultures grown on the mentioned media did not reproduce their properties the next time they were passage to ordinary media.

Example 2. For activation, a culture strain of the species Bifidobacterium longum from the collection of the SSC RF, IMPP RAS was used. Based on water treated with ultraviolet radiation in the manner mentioned in the method, Bactofock standard nutrient medium was prepared for culturing bifidobacteria. The culture was initially in a lyophilized state. The culture was rehydrated and 10 times triturated in a nutrient medium. Incubated 48 hours at 37 ° C. Survival was assessed by the number of grown colonies (see table 5).

Table 5
The survival characteristic of bifidobacteria in a nutrient medium prepared according to the proposed method (CFU) Medium Bactofok 0.2% prepared on the basis of water not treated with ultraviolet light. 1 day growth Breeding 2 6.4 × 10 ⁴ 3 3.0 × 10 ⁵ 4 6 × 10 ⁵ 5 1.2 × 10 ⁷ Bactofok medium 0.2%, prepared on the basis of ultraviolet treated water Breeding 2 Drain growth 3 8 × 10 ⁵ 4 1.4 × 10 ⁶ 5 1.8 × 10 ⁷ 6 6 × 10 ⁷

The above examples indicate the activation of the cultural properties of microorganisms. In the process of restoring cultures of lactobacilli and bifidobacteria of the genus Lactobacillus and Bifidobacteria, which are part of many fermented milk products, probiotics and bioadditives, from an inactive state (lyophilized, dry) using nutrient media prepared according to the proposed method, the number of colony forming cells increases by approximately several logarithms ( 100 times). These processes are accompanied by an increase in biomass in the colonies of approximately 10 times. During the cultivation of lactobacilli in the mentioned media, bacteriocin production increases.

The proposed method can be used in the microbiological and food industries in the production of fermented milk products in processes associated with the restoration of industrial strains of microorganisms from a lyophilized state, the preparation of starter cultures and the manufacture of fermented milk products.

Claims (2)

1. A method of preparing a nutrient medium for the cultivation of bifidobacteria and lactobacilli, providing for the preparation of a nutrient medium based on water, characterized in that the water for preparing the nutrient medium is pre-treated with unfiltered modulated ultraviolet light with a content of at least 30% ultraviolet quanta with a wavelength shorter than 350 nm, for the time required to increase the internal energy of water by at least 2 times. 2. The method of preparation of a nutrient medium for the cultivation of bifidobacteria and lactobacilli according to claim 1, characterized in that the measurement of parameters characterizing the change in the internal energy of the water is carried out before water treatment and during the entire processing process.