JPH02203782A - Method for producing koji containing galactooligosaccharides - Google Patents

Abstract

PURPOSE:To obtain a collecting source of galactooligosaccharide, feed or material for processed food by culturing a specific fungus capable of producing beta-galactosidase in a medium containing lactose and bean-curd refuse. CONSTITUTION:A fungus belonging to the genus Aspergillus or Penicillium, capable of producing beta-galactosidase is cultured in a medium consisting essentially of bean-cured refuse blended with lactose. Consequently, bean-curd refuse of waste can be effectively used.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial Application Field The present invention relates to a method for producing koji containing galactooligosaccharide. More specifically, the present invention relates to a method for producing koji containing galactooligosaccharides, which comprises culturing β-galactosidase-producing bacteria belonging to the genus Aspergillus or Penicillium in a solid medium containing lactose and okara.

Okara is a general term for the squeezed lees that is produced as a by-product when manufacturing soy protein products such as tofu and soy milk.It is produced in extremely large quantities in Japan, and except for some used for processed foods and feed, there is a large amount of okara. amount is wasted.

On the other hand, galacto-oligosaccharides are useful as growth-promoting factors for bacteria belonging to the genus Bifidobacterium that control useful actions in the intestines (Japanese Patent Application Laid-open No. 104885/1985), and feed containing this is useful for animal feed. It is known to be effective in preventing diarrhea and loose stools and in weight gain (Japanese Patent Application Laid-Open No. 138147/1983). Therefore, the galactooligosaccharide-containing koji produced according to the present invention can be used as a source of galactooligosaccharide in the pharmaceutical and food industries, and can also be used as a raw material for feed and processed foods.

(2) Conventional technology As a method for producing galactooligosaccharide, yeast Sporobolomyces singularis is grown in a liquid medium containing 10% lactose.
s) for 3 to 4 days with aeration and agitation to produce a tri- to tetrasaccharide galactooligosaccharide mixture (Canadian J
internal of chemistry, 42.1
341-1344 (1964)), a microorganism belonging to the genus Aspergillus or the genus Turkoderma and capable of producing 6'-galactosyllactose from lactose was cultured with shaking in a liquid medium containing 15% lactose for 5 to 15 days, 6゛-
Method for producing only galactosyllactose (Unexamined Japanese Patent Publication No. 1983
2-79791), a method for producing galactooligosaccharides in which 1 to 3 molecules of galactose are bound to lactose by treating a lactose-containing solution with β-galactosidase produced by Aspergillus oryzae at 37°C for 5 hours (Japanese Patent Application Laid-Open No. 1983-1997) -1048
No. 85) and the like are publicly known.

For information on how to culture microorganisms using okara as a medium,
A method for producing seasonings by culturing microorganisms belonging to Aspergillus (Japanese Patent Application Laid-open No. 170363/1983), a method for producing amazake-like foods by cultivating G. chinensis (Japanese Patent Publication No. 58/1986)
71848), and a method for producing Okara koji by growing koji mold on Okara to which an acid has been added to suppress the growth of germs (JP-A-62-195279).

However, there has been no known example of using okara together with lactose as a medium raw material for the purpose of producing galactooligosaccharides.

(3) Problems to be Solved by the Invention Galactooligosaccharide is a highly useful substance as a growth promoting factor for Bifidobacterium.

Conventionally, methods for producing galactooligosaccharides are known, as described above, by liquid culture of microorganisms in a lactose-containing liquid medium and by treatment of a lactose-containing solution with β-galactosidase. However, the liquid culture method requires a very long time, and the enzyme treatment method requires the preparation of expensive β-galactosidase in advance.

Therefore, the present inventors ■ Shorten the culture process for galactooligosaccharide production.

■ Utilizing industrial waste Sukara as a galacto-oligosaccharide production medium.

■ To provide flavorful galactooligosaccharide-containing koji that can be used as a raw material for processed foods.

For this purpose, we investigated various ways to change the conventional liquid culture method to a solid culture method.

(4) Structure of the Invention The present invention relates to a method for producing a galactooligosaccharide-containing koji characterized by culturing β-galactosidase-producing bacteria belonging to the genus Aspergillus or the genus Penicillium in a solid medium containing r-lactose and okara. It is.

In the present invention, the solid medium component may be okara alone, but some of it may be rice bran, rice hulls, soybean flour, wheat germ, etc.
Ordinary solid medium components such as rice germ and cracked wheat can also be substituted. Originally, okara was a general term for squeezed lees produced as a by-product from the production process of soybean protein products such as tofu and soy milk, and oil-containing okara is a by-product of the tofu manufacturing process made from whole soybeans and soy milk made from defatted soybeans. There are two types of defatted okara that are produced as a by-product from the manufacturing process, and in the present invention,
You may use any of them.

The appropriate amount of lactose to be contained in the medium is usually about 20 to 40 g per 100 g of the solid medium. Furthermore, by adding small amounts of inorganic salts and fats and oils to the okara-containing solid medium, the productivity of β-galactosidase and the productivity of galactooligosaccharide can be significantly improved. Examples of the inorganic salts to be added include NaHzPOt, NaJP
O4, KIIzPOa, K2HPO,, N114112
P04, (NH4)zHPO4, Ca (HzPOt)
! , CaHPO,.

Ca: +(POt)z, Mg(HzPOa)zx M
gHPO<, Mgx (Pot) z, Ca5Oa,
CaC/! , Ca(NOt)z, and other anhydrous or hydrated salts can be used alone or in combination. The amount of these inorganic salts added is slightly more than that of Type 1-c, but it is usually about 1 to 5 g per 100 g of solid medium containing Okara. Also 1 other than lactose! By adding lactose, for example, fructose, galactose, etc., to the medium in an amount ranging from about the same amount as lactose to about 172%, it is possible to produce galactooligosaccharide-containing koji that has excellent flavor and texture as a raw material for processed foods.

The microorganism used in the present invention may be any microorganism that belongs to the genus Aspergillus or the genus Penicillium and has the ability to produce β-galactosidase.

Representative strains thereof include, for example, Aspergillus oryzae IAM 2630, Aspergillus oryzae u-
8 (M Koken Bacteria No. 7378), Aspergillus sonii IAM 2703, Aspergillus tamari IAM
2502, Penicillium camemberti IF0585
5, Penicillium roqueforte IFO4622, Penicillium multicolor IF07817, etc.

These microorganisms usually produce conidia when grown on a potato dextrose agar medium or a malt extract agar medium at 25 to 30°C for 13 to 5 days, and the conidia can be suitably used as seeds. I can do it.

According to the present invention, inorganic salts and the like are appropriately added to the above-mentioned solid medium containing lactose and okara, inoculated with β-galactosidase-producing bacteria belonging to the genus Aspergillus or the genus Penicillium, preferably conidia thereof, and usually 20-3
By culturing at 5°C for about 2 days, it is possible to produce koji with a significantly high galactooligosaccharide content. In addition,
The galactooligosaccharides contained in the koji obtained in this way are
It is the same as the galactooligosaccharide obtained by treating an aqueous lactose solution with β-galactosidase produced by Aspergillus oryzae (Japanese Patent Application Laid-Open No. 104885/1985), that is, it is a mixture of galactooligosaccharides in which 1 to 3 molecules of galactose are bound to lactose. It was confirmed that there is.

Examples are shown below to specifically explain embodiments of the present invention.

Example 1 (Production of galactooligosaccharide content) 11ml
Prepare 60 capacity test tubes, add 2g of okara to each test tube,
Lactose 0.6 g, monosodium phosphate dihydrate 0.0
4 g, and 0.5n+j of water! A medium consisting of the following was added and sterilized in an autoclave at 121°C for 17 minutes.

One platinum loopful of conidia of Aspergillus oryzae strain U-8 was inoculated into each medium, and cultured stationary at 30°C under aerobic conditions. Before culturing, and on the 1st, 2nd, 3rd, 4th, and 5th days after the start of culture, 10 test tubes of koji (
Solid cultures) were collected as one group. Galactooligosaccharide was separated and purified from each piece of the 6 groups by the method described later to obtain a white powder. The yield of white powder is as shown in Table 1, and the production amount of galactooligosaccharide reached its maximum on the second day of culture and gradually decreased thereafter.

Table 1 (Separation and Purification of Galactooligosaccharides) Each piece (approximately 25 g) of the 6 groups was suspended in 0.02N hydrochloric acid of 80mj+, and after adjusting to pH 2.0, it was left at 4°C overnight. Next, the suspension was centrifuged (9000 rpm, 10 minutes) and the supernatant was collected. 0.02 of 40ml of residue
It was resuspended in N-hydrochloric acid and centrifuged to obtain a supernatant, which was added to the previous supernatant. The supernatant liquid was adjusted to pH 7.0,
The precipitated insoluble matter was removed by centrifugation and then freeze-dried. This dried product was dissolved in about 10 ml of water and dialyzed against 500 ml of water as an external liquid. Dialysis was performed by exchanging the external solution three times, and galactooligosaccharides were recovered in the external solution fraction. The external liquid fractions were combined and passed through an activated carbon column (3 x 7 cm) to adsorb galactooligosaccharides. Fill the column with 150r of water
Elute and remove monosaccharides by washing with Al, then 300mj of 5% ethanol! After washing with water to elute and remove unreacted lactose, 300 m/g of 50% ethanol was passed through the column to elute galactooligosaccharides. The eluate was concentrated under reduced pressure and then lyophilized to obtain a white powder.

The obtained white powder was analyzed by the method described below, and it was confirmed that it was a fairly pure galacto-oligosaccharide mixture, except for containing 3 to 5% protein as an impurity. The yield of white powder from each piece is shown in Table 1 above.

(Analysis of galactooligosaccharide white powder) Galactooligosaccharide (3-5 sugar mixture) obtained by treating lactose with β-galactosidase produced by Aspergillus oryzae was used as a standard product, and the galactooligosaccharide white powder of the present invention was subjected to thin layer chromatography. It was analyzed in A cellulose thin layer plate (Avicel) was used as the fixed layer, and the developing solvent was n-butanol:viridine:water (6:4:
3, V/V) was used. After development, various color development methods were used to search for developed components, and the results showed two spots of Rfo, 44 and 0.37, indicating the presence of a trace amount of protein, and Rfo, 10 contained in the standard product. Class 3Ii and Rf
4~5tJ of O,05! Spots identical to the mixture were detected, and no other spots were detected.

The results are shown in Table 2.

Table 2 The white powder of the present invention had a protein content analysis value of 3.5 to 5.5% by Lowry method, and it was confirmed that everything else was galacto-oligosaccharides.

Example 2 20 g of okara, 6 g of milk 1M, 0.4 g of l-sodium phosphate dihydrate, and 5II+1 water were uniformly mixed, and the mixture was dispensed in equal amounts into 10 test tubes with a capacity of 77 mff, and placed in an autoclave ( The cells were sterilized at 121° C. for 17 minutes and used as a culture medium.

One platinum loopful of conidia of Aspergillus oryzae JAM 2630 strain was inoculated into each medium, and cultured aerobically for 2 days at 30°C. 24.5 g of koji (total for 10 test tubes)
I got it. In the same manner as in Example 1, galacto-oligo was separated from this koji as a white powder. Yield: 0.93g
Met.

Example 3 In the same manner as in Example 2, koji was produced using the microorganisms described below, and the galacto-oligosaccharide from China was isolated as a white powder in the same manner as in Example 1. The results are shown in Table 3.

(Used microorganisms) Aspergillus sonii IAM 2703 Aspergillus tamarii IAM 2502 Penicillium multicolor IFO7817 Penicillium Roquefort 7-IF
O4622 Penicillium camemberti IP05B5
5 Table 3 Example 4 20 g of okara, 6 g of lactose, and 5 ml of water were used as a basic medium, and 3 g of galactose was added thereto and mixed uniformly.
Place in a 0nl Erlenmeyer flask and autoclave (12
Five culture media were prepared and sterilized at 1°C for 17 minutes. on the other hand,
Similarly, 5 mediums were prepared in which 5 g of fructose was added instead of galactose. Aspergillus oryzae U in galactose-supplemented medium and fructose-supplemented medium.
-8, Aspergillus sonii IAM 2703, Aspergillus tamarii IAM 2502, Penicillium
Two platinum loopfuls of conidia of Roqueforte IF04622 and Penicillium multicolor rF07817 were each inoculated, and statically cultured at 30°C for 2 days under aerobic conditions.

After the culture was completed, the galacto-oligosaccharide from China was separated as a white powder using the same method as in Example 1 (however, twice the amount of activated carbon was used). The yield of koji and the weight of separated galactooligosaccharide (white powder) are shown in Table 4. Each node obtained has a glossy brown color, the growth of aerial mycelia is suppressed to a low level, and it emits a pronounced aroma with a slight alcohol odor, has no astringent taste, is slightly sweet, and has a moist texture. It had a delicious texture.

(Effects of the Invention) According to the present invention, by culturing β-galactosidase-producing bacteria belonging to the genus Aspergillus or Penicillium in a solid medium containing okara and lactose,
Lactose in the medium can be reduced by 3 to 5 tJ in one day of culture! J! It is possible to efficiently convert this into galactooligosaccharide and produce koji containing a significant amount of galactooligosaccharide.

This koji is useful as a source of galacto-oligosaccharide, which acts as a growth factor for Bifidobacterium, and is also useful as a material for processed foods because of its extremely excellent flavor and texture.

Claims (1)

[Claims] (1) A method for producing koji containing galactooligosaccharides, which comprises culturing β-galactosidase-producing bacteria belonging to the genus Aspergillus or Penicillium in a solid medium containing lactose and okara.