JPH07265066A - Peptone for culture medium for culturing microorganism - Google Patents

Abstract

PURPOSE:To obtain a peptone, comprising an enzymic hydrolyzate of a protein derived from royal jelly and useful for promoting the growth of bifidus bacteria and lactic acid bacteria without adversely affecting the flavor of the resultant product. CONSTITUTION:This peptone for a culture medium for culturing a microorganism is obtained by carrying out the ultrafiltering or micro-filtering treatment of a raw royal jelly, heating the resultant composition containing a protein at about 10-70 deg.C, regulating the pH to <=3 or >=7 and carrying out the enzymic reaction. The obtained hydrolyzate is used as the peptone and added in an amount of >=0.01% to a culture medium for culturing the microorganism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptone for a microbial culture medium comprising a protein hydrolyzate obtained by treating a protein derived from royal jelly with an enzyme. This peptone for a microorganism culture medium has an excellent effect on the growth promotion of bifidobacteria or lactic acid bacteria.

[0002]

BACKGROUND ART Peptone, which has been conventionally used as a nitrogen source for a medium for culturing microorganisms, hydrolyzes proteins such as milk casein, meat, heart muscle, gelatin and soybean protein with enzymes such as trypsin, pepsin, papain and pancreatic juice. It is prepared by disassembling. However, even if these peptones are used as the nitrogen source, the growth may not be sufficient for lactic acid bacteria and bifidobacteria, which have high auxotrophy. Therefore, it is practiced to add trace amounts of components such as vitamins having a fermentation promoting effect to the medium, but peptone and various vitamins are added to the reduced skim milk medium of 10 to 12% concentration that is generally used when lactic acid bacteria are cultured. There are also lactic acid bacteria that do not grow well even when such substances are added.

[0003]

For these reasons,
Yeast extract rich in various vitamins and other fermentation promoting substances is used as a growth promoting substance for lactic acid bacteria.
However, since yeast extract is expensive and is accompanied by yeast odor, there is a problem that only a very small amount can be added to the medium so as not to affect the flavor of the product.
Therefore, the present invention, than a hydrolyzate such as casein and soybean protein, is effective in promoting the growth or fermentation of bifidobacteria and lactic acid bacteria, and does not adversely affect the flavor of the product microorganisms treated with an enzyme protein derived from royal jelly It is an object to provide peptone for a culture medium.
Another object of the present invention is to effectively utilize resources by using a conventionally-discarded protein derived from royal jelly as a peptone for a microorganism culture medium.

[0004]

DISCLOSURE OF THE INVENTION The present invention is a peptone for a microorganism culture medium, which comprises an enzymatic hydrolyzate of a protein derived from royal jelly. The protein from royal jelly is
A protein obtained by subjecting raw royal jelly to an ultrafiltration membrane (hereinafter referred to as UF membrane) or a microfiltration membrane (hereinafter referred to as MF membrane) in the process of producing a prepared royal jelly can be used. When producing a prepared royal jelly, this protein was added to the natural royal jelly with alcohol to improve fluidity, and then the U with a molecular weight cut-off of about 20,000 was used.
It is a residue recovered on the concentration side when treated with an F membrane or an MF membrane having a pore size of 5 μm or less. The protein-containing solution thus obtained has a low pH of around 4.6 and has an alcohol content of about 7
% Of the protein-containing composition. Also, protein is contained in about 70% of the total solid content, and is in an insoluble state.

When the obtained protein-containing composition is hydrolyzed with an enzyme, heating (10 to 10
Then, the pH is adjusted to 3 or less or 7 or more and the enzymatic reaction is performed. The enzyme that can be used is not particularly limited, and an enzyme that is usually used when hydrolyzing a protein can be used. For example, if it is a plant-derived enzyme, it is papain or bromelain, and if it is an animal-derived enzyme,
Trypsin and chymotrypsin. Further, in the case of a microorganism-derived enzyme, Protease M (manufactured by Amano Pharmaceutical Co., Ltd.)
Orientase 20A (manufactured by Hankyu Kyoei Bussan Co., Ltd.) and the like. One or more of these enzymes are selected and used. The amount of enzyme added is 150-40 per gram of protein.
It is advisable to carry out the reaction for about 22 hours according to the optimum temperature and optimum pH of the enzyme used in about 0 unit. Moreover, the enzymatic reaction can be stopped by heating at 85 ° C. for about 20 minutes. The protein hydrolyzate thus obtained is used by adding 0.01% or more to a microorganism culture medium. In addition, vitamins and the like may be added to the medium as needed. Further, the protein hydrolyzate prepared as described above can be dried according to a usual method to obtain peptone for a powdery medium. This peptone for a powdery medium has good solubility in water and is also soluble in an ethanol solution.

The present invention will be described in detail below with reference to Examples, Comparative Examples and Experimental Examples. (% Represents% by weight.)

【Example】

Example 1 A raw royal jelly solution dissolved in alcohol to a solid content of 20% was treated with a UF membrane having a molecular weight cut off of 20,000 to obtain a solution containing 6% of protein. 200 g of this royal jelly protein-containing solution (pH 4.6) was adjusted to pH 3.0 with 1N hydrochloric acid. After heating this solution to 45 ° C, 180 units of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added per 1 g of protein, and at 45 ° C
The enzymatic reaction was performed for 22 hours. Then, after heating at 85 ° C. for 20 minutes to inactivate the enzyme, it was cooled to room temperature and the pH was adjusted to 6.8 with 10N sodium hydroxide. Finally, the hydrolyzate was freeze-dried to obtain 11 g of culture medium peptone.

Example 2 300 g of the royal jelly protein solution obtained in the same manner as in Example 1 was adjusted to pH 3.0 with 1.2 N hydrochloric acid. 45 this solution
After heating to 0 ° C., 360 units of protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added per 1 g of protein, and the enzyme reaction was carried out at 45 ° C. for 22 hours. Then, heat at 85 ° C for 20 minutes to inactivate the enzyme, then cool to room temperature and adjust the pH to 6.8 with 10N sodium hydroxide.
Adjusted to. Finally, the hydrolyzate was freeze-dried to obtain 16 g of culture medium peptone.

Example 3 300 g of the royal jelly protein solution obtained in the same manner as in Example 1 was adjusted to pH 7.0 with 10N sodium hydroxide. After heating this solution to 45 ° C., 360 units of protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added per 1 g of protein, and the enzyme reaction was carried out at 45 ° C. for 22 hours. Then, after heating at 85 ° C. for 20 minutes to inactivate the enzyme, it was cooled to room temperature and the pH was adjusted to 6.8 with 10N sodium hydroxide. Finally, this hydrolyzate was freeze-dried to obtain 17 g of culture medium peptone.

The method for preparing the peptone for the medium used in the experimental examples will be described below. It is shown as a comparative example.

[Comparative example]

Comparative Example 1 Raw royal jelly containing 14.5% protein (pH 4.3) 10
After adjusting 0 g to pH 3.0 with 1N hydrochloric acid, add purified water and add 20 g.
It was quantified to 0 g. After heating this solution to 45 ° C., 180 units of protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added per 1 g of protein, and the enzyme reaction was carried out at 45 ° C. for 22 hours. And 20 at 85 ° C
Heat for minutes to inactivate the enzyme, then cool to room temperature and
The pH was adjusted to 6.8 with N sodium hydroxide. Finally, the hydrolyzate was freeze-dried to obtain 32 g of culture medium peptone.

Comparative Example 2 300 g of a 5% casein solution was adjusted to pH 3.0 with 1N hydrochloric acid.
After heating this solution to 45 ° C., 180 units of protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added per 1 g of casein, and the enzyme reaction was carried out at 45 ° C. for 22 hours. Then, after heating at 85 ° C. for 20 minutes to inactivate the enzyme, it was cooled to room temperature and the pH was adjusted to 6.8 with 10N sodium hydroxide. Finally, this hydrolyzate was freeze-dried to obtain 14 g of culture medium peptone.

Comparative Example 3 300 g of a 5% soybean protein solution was adjusted to pH 3.0 with 1N hydrochloric acid. After heating this solution to 45 ° C, add 180 units of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) per 1g of soybean protein, and add 45 ° C.
The enzyme reaction was performed for 22 hours. Then, after heating at 85 ° C. for 20 minutes to inactivate the enzyme, it was cooled to room temperature and the pH was adjusted to 6.8 with 10N sodium hydroxide. Finally, the hydrolyzate was freeze-dried to obtain 15 g of culture medium peptone.

Comparative Example 4 300 g of a 5% soybean protein solution was adjusted to pH 3.0 with 1N hydrochloric acid. After heating this solution to 45 ° C, add 360 units of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) per 1 g of soybean protein, and add 45 ° C.
The enzyme reaction was performed for 22 hours. Then, after heating at 85 ° C. for 20 minutes to inactivate the enzyme, it was cooled to room temperature and the pH was adjusted to 6.8 with 10N sodium hydroxide. Finally, the hydrolyzate was freeze-dried to obtain 15 g of culture medium peptone.

With respect to the peptone for the medium obtained in Examples and Comparative Examples, this effect was confirmed using Bifidobacteria and lactic acid bacteria. The test example is shown below.

[Test example]

Test Example 1 Using the peptone for a medium obtained in Example 1 and Comparative Example 1, a growth test of bifidobacteria was performed. Bifidobacteria used in the test were Bifidobacteria longum ( Bifid
obacterium longum) SBT-2928 strain (FERM P-10657) and Bifidobacterium longum (Bifidobacterium lo
ngum ) SBT-2933R strain (FERM P-8743) mixture in the medium
0.3% was inoculated and cultured at 37 ° C. The composition of the media used was 11.53% skim milk powder and peptone for the media.
It was 0.5% and purified water 87.97%. As a result, when the medium peptone of Comparative Example 1 was used, the pH after culturing for 18 hours was
Whereas the acidity at 4.96 was 0.68, when the medium peptone of Example 1 was used, the pH after 16-hour culture was 4.71, the acidity was 0.77, and the growth of Bifidobacterium was obtained in Example 1. The culture medium was better. Therefore, the hydrolyzate of the protein solution obtained by subjecting the royal jelly to the UF membrane had a higher growth promoting effect than the hydrolyzate of raw royal jelly.

Test Example 2 Using the culture medium peptone obtained in Example 3, a growth test of bifidobacteria was carried out by varying the addition amount. Bifidobacteria used in the test are Bifidobacterium longum
( Bifidobacterium longum ) SBT-2928 strain (FERM P-10657)
And Bifidobacteri longum
um longum ) SBT-2933R strain (FERM P-8743),
The medium was inoculated with 0.3% and cultured at 37 ° C for 16 hours. The composition of the medium used was (1) skim milk powder 11.03%, medium peptone 1.0%, sodium ascorbate 0.03% and purified water 87.94%, (2) skim milk powder 11.53%, medium peptone 0.5%, ascorbic acid. Sodium 0.03% and purified water 87.94%, (3) skim milk powder 12.03%, sodium ascorbate 0.03% and purified water 87.94%. The acidity after culturing is shown in Table 1.

[0015]

[Table 1] (Unit:%) ─────────────────────────── Medium (1) 1.34 Medium (2) 0.94 Medium (3) 0.38 (without addition of peptone for medium) ─────────────────────────── As is clear from Table 1, the peptone for medium of the present invention is It can be seen that the larger the amount added, the better the results for the growth of microorganisms.

Test Example 3 Using the peptone for a medium obtained in Example 3, growth tests of various lactic acid bacteria were carried out depending on the addition amount. The lactic acid bacteria used in the test were Streptococcus thermophilus ( Stre
ptococcus thermophilus ) SBT-1021A strain (FERM P-1065
8), Lactobacillus
acidophilus) SBT-2062 strain (FERM P-10730), Lactobacillus bulgaricus
SBT-2118B strain (FERM P-13955) and Lactobacillus casei SBT-2300 strain (FERM P-132
At 45), 0.3% of the medium was inoculated and cultured at 37 ° C for 16 hours. The composition of the medium used is (1) skim milk powder
11.03%, medium peptone 1.0% and purified water 87.97%,
(2) Nonfat dry milk 11.53%, medium peptone 0.5% and purified water 87.97%, (3) Nonfat dry milk 12.03% and purified water 87.97%
Met. The acidity after culturing is shown in Table 2.

[0017]

[Table 2] (Unit:%) ──────────────────────────────────── SBT-1021A SBT- 2062 SBT-2118B SBT-2300 ──────────────────────────────────── Medium (1) 1.05 1.57 1.52 0.83 Medium (2) 0.98 1.31 1.45 0.62 Medium (3) 0.91 0.85 1.20 0.33 (without peptone added) ───────────────────────────── ──────── From Table 2, it can be seen that the effect of the peptone for the medium of the present invention increases with increasing content of any of the lactic acid bacteria.

Test Example 4 Using the culture medium peptone obtained in Example 3 and Comparative Examples 2 to 3, a growth test of Bifidobacterium was performed. Bifidobacterium used in the test was Bifidobacterium longum ( Bi
fidobacterium longum ) SBT-2928 strain (FERM P-10657) and Bifidobacterium longum ( Bifidobacterium longum )
longum ) SBT-2933R strain (FERM P-8743) was inoculated into the medium at 0.3% and cultured at 37 ° C. for 16 hours. The composition of each medium used was skim milk powder 11.53%, medium peptone 0.5%, sodium ascorbate 0.03% and purified water 87.94%. Table 3 shows the acidity, pH, and viable cell count after culturing.

[0019]

[Table 3] ──────────────────────────────────── Example 3 Comparative Example 2 Comparative Example 3 Comparative Example Example 4 ──────────────────────────────────── Acidity (%) 1.40 1.28 1.24 1.30 pH 4.35 4.30 4.29 4.31 SBT-2928 viable cell count (cfu / ml) 1.9 × 10 ⁹ 7.8 × 10 ⁸ 7.4 × 10 ⁸ 9.2 × 10 ⁸ SBT-2933R viable cell count (cfu / ml) 2.4 × 10 ⁹ 1.0 × 10 ⁹ 9.9 × 10 ⁸ 1.0 × 10 ⁹ ──────────────────────────────────── From Table 3, for the medium of the present invention It can be seen that peptone shows a higher effect on the growth of Bifidobacterium than conventional peptone for culture medium.

Test Example 5 Using the culture medium peptone obtained in Example 3 and Comparative Examples 2 to 3, growth tests of various lactic acid bacteria were conducted. The lactic acid bacteria used in the test were Streptococcus thermophilus ( Strepto
coccus thermophilus ) SBT-1021A strain (FERM P-10658)
, Lactobacillus a
cidophilus) SBT-2062 strain (FERM P-10730), Lactobacillus bulgaricus SB
T-2118B strain (FERM P-13955) and Lactobacillus casei SBT-2300 strain (FERM P-1324)
In step 5), 0.3% of the medium was inoculated and cultured at 37 ° C for 16 hours. The composition of the medium used was skim milk powder 11.53%, medium peptone 0.5%, and purified water 87.97%. Table 4 shows the acidity, pH, and viable cell count of each lactic acid bacterium after culturing.

[0021]

[Table 4] ──────────────────────────────────── Example 3 Comparative Example 2 Comparative Example 3 Comparative Example Example 4 ──────────────────────────────────── Acidity (%) 1.10 1.04 0.94 0.96 SBT-1021A pH 4.10 4.16 4.25 4.23 Viable cell count (cfu / ml) 4.2 × 10 ⁸ 2.2 × 10 ⁸ 3.0 × 10 ⁸ 1.8 × 10 ⁸ ──────────────────────── ────────────── Acidity (%) 1.30 1.10 1.20 1.20 SBT-2062 pH 3.95 4.12 3.98 4.01 Viable cell count (cfu / ml) 9.2 × 10 ⁸ 4.4 × 10 ⁸ 7.6 × 10 ⁸ 7.8 × 10 ⁸ ──────────────────────────────────── Acidity (%) 1.58 1.50 1.42 1.46 SBT- 2118B pH 3.77 3.71 3.67 3.68 Viable cell count (cfu / ml) 3.4 × 10 ⁸ 2.8 × 10 ⁸ 1.0 × 10 ⁸ 1.0 × 10 ⁸ ───────────────────── ────────────── ─ acidity (%) 0.98 0.76 0.80 0.84 SBT -2300 pH 4.54 4.65 4.50 4.49 number of living bacteria (cfu / ml) 4.1 × 10 9 2.9 × 10 9 2.6 × 10 9 2.6 × 10 9 ───────── ─────────────────────────── As is clear from Table 4, the hydrolyzate obtained by enzymatically treating the royal jelly-derived protein of the present invention was used. It was confirmed that the medium used had a higher acidity of the lactic acid bacterium culture than the medium using casein hydrolyzate or soybean protein hydrolyzate, and the viable cell count of lactic acid bacteria was 2-3 times higher. it was high.

[0022]

The protein hydrolyzate obtained by treating the royal jelly-derived protein of the present invention with an enzyme has a higher growth-promoting effect on microorganisms than the hydrolyzate of casein or soybean protein, and therefore is a medium for culturing microorganisms. By adding peptone as a peptone, the effect of accelerating the growth or fermentation of bacteria can be obtained. In addition, the protein derived from royal jelly that can be used as a raw material has been conventionally discarded, and it is possible to effectively utilize resources.

Claims (2)

[Claims] 1. A peptone for a microorganism culture medium, which comprises an enzymatic hydrolyzate of a protein derived from royal jelly. 2. The peptone for a microorganism culture medium according to claim 1, wherein the protein derived from royal jelly is obtained by subjecting royal jelly to a membrane treatment.