KR970002254B1 - Medium composition for cultivation of bifidobacteria - Google Patents

Abstract

Culture medium for mass culture of bifido bacterium is composed of 50.0- 99.0 wt.% of water, 1.0-4.0 wt.% of trypton, 0.1-1.0 wt.% of yeast extract, 1.0-4.0 wt.% of lactose or glucose, 0.1-1.0 wt.% of casamino acid, and 0.01-0.3 wt.% of cysteine as an antioxidant at pH 6.0-7.5. For an example, the mixture of 10-60g of trypton, 1-10g of yeast extract, 10-40 wt.% of lactose, 0.1-1.0 wt.% of cysteine is melted in 1,000ml of water, and 0.01-0.3 wt.% of cysteine, is adjusted with sodium hydroxide to pH 6.5-7.5, sterilized under high pressure at 121 deg.C for 15 min, cooled to give a culture medium, which is inoculated with bifidobacterium bifidum and static fermented at 35-40 deg.C for 9-12 hr.

Description

Culture medium composition for mass culture of Bifidobacteria

The present invention relates to a culture medium composition for mass cultivation of bifidobacteria, and to a culture medium composition in which a large number of bifidobacteria can be obtained at the time of culture and recovery of cells is easy.

Bifidobacteria is a gram-positive anaerobic agar-free bacillus that breaks down glucose and produces lactic acid and acetic acid in a ratio of 2: 3, and varies in various forms such as club, curved, Y-shaped and V-shaped according to the species and culture conditions. It shows form. These Bifidobacteria are rarely found in milk, but are known to play an important role in the intestinal flora of infants and infants and to play an important role in inhibiting the growth of intestinal pathogens. Bifidobacteria with these characteristics are known to require a special essential growth factor (or growth factor: bifidus factor) for proliferation, and bifidobacterium bifidum is a sugar containing acetylglucosamine present in human oil. It is known that the essential growth factor I) and the substances present in the oil, milk, pancreatic extract and casein hydrolysate are essential growth factor II.

Although many efforts have been made to utilize Bifidobacteria in dairy production, there have been many difficulties due to the difficult growth conditions and unique physiological characteristics.

In 1951 J. B. HASSINEN et al. Proposed a method for culturing Bifidobacteria in a medium containing lactose, buffers, minerals, ammonium salts, cysteine, vitamins, biotin and calcium pantothenate.

In addition, J. Dairy Sci. 71, 3214-3221 (1988) describes a method for culturing bifidobacteria in a synthetic medium containing inorganic salts, vitamins, nitrogen and carbon sources.

As such, Bifidobacteria is a strict anaerobic bacterium, which not only minimizes the effect of oxygen upon cultivation, but also requires very complex nutritional requirements, resulting in a complex and expensive synthetic medium for the cultivation of Bifidobacteria. come.

However, such a synthetic medium is a scientifically rational medium for lactic acid bacteria, but since it is necessary to formulate a certain amount of a number of substances, the composition and preparation are very difficult, expensive, and not practical, and are not used much except for special studies.

On the other hand, in order to use Bifidobacteria for dairy production, a large inoculation amount of about 10-20% (V / V) is required. This is to prevent the killing and contamination of bacteria by promoting acid production rate and shortening the lag phase. In addition, in the case of dairy products in which other kinds of lactic acid bacteria and Bifidobacteria are mixed together, Bifidobacteria are gradually killed by the lactic acid produced by other lactic acid bacteria or dissolved oxygen in the product while the dairy products are distributed to consumers. Compared to other types of lactic acid bacteria, it is necessary to contain a high concentration of Bifidobacteria in the product, and one of the methods for this is to concentrate the Bifidobacteria at a high concentration and mix them into the product.

In order to obtain such a high concentration of Bifidobacteria, it is possible to use a method of concentrating the cells by centrifugation after culturing Bifidobacteria in large quantities as in general lactic acid bacteria.

In general, in order to obtain a high concentration of lactic acid bacteria used as a raw material for spawning or formal preparation for dairy production, it shows a high bacterial growth during culture, easy recovery of the bacteria, and economical medium with economic advantages while maintaining high vitality of the lactic acid bacteria. Should be developed first.

The present inventors, while improving the above-mentioned problems, in particular, while studying a medium composition capable of mass culturing Bifidobacteria, which have recently been highly regarded for their suitability and health promotion effects, the composition and preparation are simple and economical. It was found that a new culture medium composition which is advantageous in and exhibits a high cell count in culture and easy recovery of cells is found.

The present invention is 50.0 to 99.9 parts by weight of water, tryptone 1.0 to 4.0 parts by weight, yeast extract 0.1 to 1.0 parts by weight, lactose or glucose 1.0 to 4.0 parts by weight, casamino acid 0.1 to 1.0 parts by weight and cysteine 0.01 to 0.3 as an antioxidant It relates to a Bifidobacteria culture medium composition consisting of parts by weight.

Hereinafter, the present invention will be described in detail.

Culture medium composition of the present invention is composed of a mixture of tryptone, yeast extract, glucose, system and casamino acid (casamino acid), the mixture is adjusted to pH 6.0 ~ 7.5 and prepared by sterilization for 15 minutes at 121 ℃.

By using the culture medium composition of the present invention, Bifidobacteria such as Bifidobacterium bifidum were cultured at 33 to 40 ° C. for 9 to 12 hours, and centrifuged as a result of bacterial growth than when cultured in skim milk powder or whey. It was good and the recovery of the cells was easy, it was possible to obtain a high concentration of cells.

The new culture medium composition for mass cultivation of such Bifidobacteria was named TYG medium, and the results of the effect of this culture medium are summarized in Table 1.

After culturing at 35 ~ 40 ℃ for 9 ~ 12 hours in each culture medium, dilute several times with sterilized anaerobic saline solution and spread it on BL agar medium, and cultivate for 36 ~ 48 hours at 35 ~ 40 ℃ anaerobic thermostat. The number of colonies of Bifidobacteria displayed was counted.

After recovering the microbial cells from each culture medium at 35 to 40 ° C. for 9 to 12 hours using a centrifuge, the cells were diluted several times with sterilized anaerobic saline and plated on BL agar medium. Colonies of Bifidobacteria were counted for 36-48 hours in an anaerobic thermostat at 40 ° C.

Cultured Dairy Products Journal, 1991, November. p. 4-6 Reference

Cultured Dairy Products Journal, 1991, November. p. See 32-33

As can be seen from the above table, when TYG medium was used, the number of viable cells after culture was higher than that of 10% skim milk powder or 10% whey, and the number of viable cells after centrifugation was about 30 to 100 times higher. . This is a result showing that TYG medium is better than the skim milk powder or whey which has been generally used for mass cultivation of lactic acid bacteria and is very advantageous in terms of cell recovery.

In addition, the TYG medium of the present invention is very advantageous in terms of bacterial growth, but in terms of economy and ease of production of the medium, compared to BL medium or MRS medium, which is a widely used medium for growing Bifidobacteria. appear.

As a result of the above, it can be seen that for the mass cultivation of Bifidobacteria, using TYG medium is more suitable than any other medium.

Lactic acid bacteria that can be cultured by the TYG medium of the present invention include Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium infantis Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium thermophilum, Bifidobacterium sudobacterium Bifidobacterium gum pseudolongum).

In addition, sugars usable in the culture medium composition of the present invention include lactose, glucose, fructose, sucrose and galactose.

In addition, the culture medium composition of the present invention preferably contains an antioxidant, such antioxidants are the system, hydrochloride and ascorbic acid, which is a system.

The following examples are intended to illustrate the invention, but are not intended to limit the invention.

In the following example, the measurement of viable cell number was diluted several times with a sterilized anaerobic saline solution, and then smeared with 30 to 300 cells in BL agar medium and 36 to 35 ° C. in an anaerobic thermostat. This was done by counting the number of lactic acid bacteria colonies that appeared after 48 hours incubation.

Example 1

Tryptone 10 ~ 60g, yeast extract 1 ~ 10, lactose 10 ~ 40g and cysteine 0.1 ~ 1g are dissolved in 1000ml of water, pH adjusted to 6.5 ~ 7.5 using NaOH, and autoclaved at 121 ℃ for 15 minutes. After cooling to 37 ℃ was used as a culture medium.

The culture medium was inoculated with Bifidobacterium bifidum (CHR. HANSEN'S Lab.) And cultured at 35-40 ° C. for 9-12 hours. The viable cell count at the end of the culture is 1.7 × 10 cfu / ml and pH was 4.6.

Example 2

Lactose, glucose, sucrose, and galactose were 10 ~ 40 g based on tryptone 10 ~ 40g, yeast extract 1 ~ 10g in order to determine the proliferative effect of the Bifidobacteria by the sugar components of the medium component of Example 1 40 g each was added.

After dissolving them in 1000ml of water, respectively, the pH was adjusted to 6.5 ~ 7.5 and autoclaved at 121 ° C. for 15 minutes was used as a culture medium. Bifidobacterium bifidum was inoculated in each of these culture mediums, and cultured at 35-40 ° C. for 9-12 hours. The viable cell count and pH at the end of the culture are shown in Table 2 below.

From the above results, since glucose showed the highest viable cell count of 5.1 × 10 cfu / ml compared to other sugars, it was found that it is preferable to use glucose as a carbon source of Bifidobacteria culture medium instead of lactose.

Example 3

The following experiment was conducted to investigate the effects of the Bifidobacteria proliferation. The following substances expected to promote the growth of Bifidobacteria by using the culture medium of Example 2 (tryptone 10-40g, yeast extract 1-10g, glucose 10-40g, system 0.1-3g) as the basic medium composition Each was added and dissolved in 1000 ml of water. The pH was adjusted to 6.5-7.5 and autoclaved at 121 ° C. for 15 minutes and used as a culture medium. These culture mediums were inoculated with Bifidobacterium bifidum, respectively, and cultured at 35-40 ° C. for 9-12 hours. After the incubation was completed, the number of viable bacteria and pH was compared to determine the effect of the Bifidobacteria proliferation promoting agent. The results are shown in Table 3 below.

Control group: A medium containing only 10-40 g of tryptone, 1-10 g of yeast extract, 10-40 g of glucose, and 0.1-3 g of the system as a medium without adding the Bifidobacterial growth promoting substance.

From the above results, the effect of Bifidobacteria on the growth-promoting substance was found to be very useful for the growth of Bifidobacteria by casamino acid, which showed 8.7 × 10cfu / ml, compared to the number of viable bacteria 4.0 × 10cfu / ml in the control group. Could.

Example 4

In order to determine the growth of Bifidobacteria according to the change in the content of tryptone in TYG medium composition of Example 3, the following experiment was carried out. The content of the other components in the TYG medium bath component was added in the same manner as in Example 3, and only the content of tryptone was changed as follows to investigate the appropriate content. The results are shown in Table 4 below.

As can be seen from the above results, when tryptone was included in the range of 1-10 g / 1000 ml in TYG medium, the growth of Bifidobacteria was insufficient, but when 10 g / 1000 ml or more was added to the medium, it showed good growth. In addition, even if the tryptone content was changed within the range of 10 ~ 100g / 1000ml there was almost no difference in the growth of Bifidobacteria.

Example 5

In order to select the content of yeast extract in the composition of TYG medium of Example 3, the other ingredients were added in the same manner as in Example 3, and only the yeast extract was added as follows, followed by the change of the content of yeast extract Proliferation changes were compared. The results are shown in Table 5 below.

As can be seen from the results of Table 5, even if the content of the yeast extract TYG medium contained more than 1g / 1000ml, the growth of the Bifidobacteria did not show a difference almost good.

Example 6

In order to determine the content of glucose in the TYG medium composition of Example 3 was carried out the following experiment. The content of the other ingredients in the medium component was added in the same manner as in Example 3, only the content of glucose was added in a range of 1 ~ 100g to compare the growth of the Bifidobacteria by each content. The results are shown in Table 6 below.

As can be seen from the above results, when the content of glucose contained in the TYG medium is 1 ~ 10g / 1000ml, the growth of Bifidobacteria was weak, but when it is included in the medium of 10g / 1000ml or more it shows that good growth there was. In addition, there was little difference in the growth of Bifidobacteria due to the change of glucose content within the range of 10 ~ 100g / 1000ml.

Example 7

When the TYG culture medium of Example 3 was used for the culture medium of Bifidobacteria, the following experiment was conducted to determine whether the initial pH of the medium affects the growth and vitality of the bacteria. After adjusting the initial pH of the culture medium of Example 3 to 5.5, 6.0, 6.5, 7.0 and 8.0, and inoculated Bifidobacterium bifidum, and incubated for 9-12 hours at 35-40 ℃ and the number of viable bacteria and pH was measured. The results are shown in Table 7 below.

Example 8

Bifidobacteria were inoculated in the TYG culture medium as described above and incubated for 9 to 12 hours at 35-40 ° C., and the cells were concentrated using a centrifuge. The results are shown in Table 8 below.

In addition, the results of the above example, as shown in the comparative experiments with the conventional culture medium listed in Table 1, compared with the conventional culture medium obtained good results in bacterial growth and bacterial recovery of Bifidobacteria.

As can be seen from the results of this embodiment, when using the TYG culture medium composition according to the present invention is not only about 30 to 100 times superior to the conventional culture medium in the culture and recovery of the Bifidobacteria as well as of the present invention The culture medium composition is easy and economical to manufacture and is well suited for mass cultivation of Bifidobacteria.

Claims (2)

50.0 to 99.9 parts by weight of water, 1.0 to 4.0 parts by weight of tryptone, 0.1 to 1.0 parts by weight of yeast extract, 1.0 to 4.0 parts by weight of lactose or glucose, 0.1 to 1.0 parts by weight of casamino acid and 0.01 to 0.3 parts by weight of the system as a homeostatic agent. Culture medium composition of the Bifidobacteria made. The culture medium composition according to claim 1, wherein the pH is adjusted to 6.0 to 7.5.