JPS61271999A - Production of galactooligosaccharide - Google Patents

Abstract

PURPOSE:To increase the content of a galactooligosaccharide, by reacting a microorganism of the genus Klyveromyces with a saccharide mixture containing at least one of glucose, galactose and lactose. CONSTITUTION:A galactooligosaccharide is purified from a saccharide mixture containing a galactooligosaccharide expressed by the formula (Gal is galactose residue; Glu is glucose residue; n is 1 or 2; the linking form of the saccharide to saccharide is mainly beta-1,4 bond), lactose which is a raw material therefor and glucose and galactose which are hydrolysis products of the lactose. In the process, a microoragnism belonging to the genus Klyveromyces, e.g., Klyveromyces fragilis IFO 0288, is reacted therewith. Thus, the lactose, glucose and galactose can be selectively assimilated and fermented to afford only the aimed galactooligosaccharide in high purity.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for purifying galactooligosaccharide, and more specifically, it relates to a method for purifying galactooligosaccharide, and more specifically, it uses microorganisms or processed products thereof to purify galactooligosaccharide with the general formula Gal
-(Gal) n-Glu (Gal is a galactose residue, Glu is a glucose residue, n is 1 or 2, and the binding mode between sugars is mainly β-1,4 bond.) Relating to a method for refining sugar.

[Prior Art 1] In recent years, galacto-oligosaccharides have been recognized as effective as growth factors for bifidobacteria, which are useful bacteria in the intestine, and various methods for producing them have been investigated.

A general method for producing galactooligosaccharides is the direct fermentation method (T etratose), in which specific microorganisms are cultured using lactose as a raw material, and galactooligosaccharides are accumulated in the culture.
letlron, 1960. vol, 9. p12s
~129, Can, J. Chemistry,
1964. vol, 42. p134i ~1344,
JP-A-56-115796> is known. Also,
An enzymatic method (edited by Eiji Shima [Food Industry and Enzymes J, p. 68, Asako Shoten) is also known, in which lactose is reacted with a specific β-galactosidase.

In particular, galactooligosaccharides represented by the general formula Gal - (Gal) n -Glu (Gal is 43 galactose residues, Glu is glucose residue, and n is an integer from 1 to 4) exhibit excellent activity in vivo. (Japanese Patent Publication No. 58-20266), and the present inventors have also already proposed a method for producing this type of galacto-oligosaccharide (Japanese Patent Application No. 59-108547, Japanese Patent Application No. 76767-1982). ).

[Problems to be Solved by the Invention] However, in order to produce galactooligosaccharides with a good yield using the above method, it is necessary to produce galactooligosaccharides at a high yield when 20 to 50% of galactooligosaccharides are produced, that is, when the raw material lactose and its decomposition products are used. Some glucose and galactose are about 50 to 80
It is necessary to stop the culture or stop the enzymatic reaction when about 50% of the enzyme is present. Therefore, in order to obtain only galactooligosaccharides that are truly effective as bifidobacteria growth factors, rough 1
~-su and its decomposition products glucose and galactose must be removed.

Well-known methods for removing sugars other than the target sugar include adsorption with activated carbon or column chromatography using molecular sieves, but these column chromatography methods require the use of large amounts of expensive ethanol. It is unsuitable for industrial production because it has drawbacks such as being difficult to process, cannot process large quantities, and takes too much time.

Therefore, when galactooligosaccharide is actually used as a growth factor for Bifidobacterium, it is currently necessary to use a mixture of glucose, galactose, and lactose, which are less effective as growth factors for Bifidobacterium in addition to galactooligosaccharide. .

The present invention solves the drawbacks of the above-mentioned prior art, and particularly the general formula % (1) (where Gal is a galactose residue, Glu is a glucose residue, n is 1 or 2, and the binding mode between sugars is mainly β-1,4 bond), lactose, its raw material, and glucose, a decomposition product of lactose,
The object of the present invention is to provide a method for purifying only galactooligosaccharides in large quantities and at low cost from a sugar mixture containing galactose.

The present inventors used various microorganisms to act on a sugar mixture containing lactose, glucose, galactose, and galactooligosaccharide obtained from lactose as a raw material by a direct fermentation method or an enzyme method. As a result of repeated research in search of microorganisms that can assimilate or ferment lactose, glucose, and galactose, but cannot assimilate or ferment the product galactooligosaccharide, we found that Kluveromyces (K 1y
ver.

found that microorganisms belonging to the genus S. myces can selectively assimilate and ferment lactose, glucose, and galactose other than the galactooligosaccharide represented by the formula ([), and thereby only the galactooligosaccharide described above can be obtained with high purity, This led to the present invention.

In other words, the method for purifying galactooligosaccharides according to the present invention uses at least 1f1 sugars among glucose, galactose, and lactose, and the general formula % (1) (where Gal is a galactose residue and Glu is glucose A microorganism belonging to the genus Klyveroiyces is applied to a sugar mixture containing a galactooligosaccharide represented by a residue, n is 1 or 2, and the binding mode between sugars is mainly β-1,4 bond. 1J is characterized by increasing the content of the galactooligosaccharides in the sugar composition.

The present invention will be explained in detail below.

(a) Sugar mixture to be purified The present invention specifically relates to the production of the galactooligosaccharide represented by the formula (1) (hereinafter simply referred to as galactooligosaccharide) from lactose or a lactose-containing substance.
The purpose is to remove lactose mixed in a 1J sugar mixture containing galactooligosaccharides, as well as glucose and galactose that are decomposition products of lactose.
The product to be purified is mainly a sugar mixture produced by these methods. Showa 59
-108547, Japanese Patent Application No. 60-76767>, or those obtained using the transfer reaction of β-galactosidase obtained from molds, yeasts, bacteria, etc., and are unpurified or other Includes primary reports using methods. However, the product to be purified is not limited to these products, and the scope of the present invention is applicable as long as it is a sugar mixture containing at least one type of sugar from glucose, galactose, and lactose, and the galactooligosaccharide. It is.

(b) Organism The microorganism that acts on the above-mentioned sugar mixture is not particularly limited as long as it belongs to the genus Kryveromyces, but specifically Kryveromyces fragilis (KIyveros
yces fragilis) l FQ-0288
can be mentioned. Although this microorganism itself is known,
The effect of this microorganism in the present invention was discovered for the first time by the present inventors.

As a method for culturing the above-mentioned microorganisms used in the present invention, known microorganism culturing methods can be employed. For example, p+-+ of the medium
3-9, cultivation! ! The culture can be carried out at a temperature of 20 to 40°C and a culture period of 1 to 5 days, and may be carried out by either stirring culture or static culture.

(c) Method of using microorganisms on sugar mixtures In the present invention, methods for using microorganisms on sugar mixtures include a method of culturing the microorganisms in a sugar mixture (hereinafter referred to as method A), a method of culturing the microorganisms in the sugar mixture, and a method of culturing the microorganisms in the sugar mixture or Any method of reacting the treated product with the sugar mixture (hereinafter referred to as method B) can be employed.

In method A, a sugar mixture containing galactooligosaccharides is supplemented with an inorganic nitrogen source such as (NH4)2804, NI''zCQ, an organic nitrogen source such as peptone, or yeast extract as a nitrogen source,
The above-mentioned microorganism is inoculated and cultured in a material to which other mineral sources, trace amounts of useful nutrients, etc. are appropriately added.

In method B, a suitable sugar such as glucose, galactose, sucrose, lactose, maltose, etc. is used as a carbon source, and (NH4)2804, N84 is used as a nitrogen source.
Bacterial cells obtained by inoculating the above-mentioned microorganisms into a medium to which an inorganic nitrogen source such as C11, an organic nitrogen source such as peptone or yeast extract, other mineral sources, trace amount useful nutrients, etc. have been appropriately added and Jalling. Alternatively, the treated product is reacted with a sugar mixture containing galactooligosaccharide. Examples of bacterial cells or processed products include live bacterial cells, frozen bacterial cells, dried bacterial cells, freeze-dried bacterial cells, and other types of bacterial cells such as polyacrylamide gel,
Examples include immobilized bacterial cells and immobilized proliferated bacterial cells prepared by a generally known method and encapsulated in calcium alginate gel or the like. The conditions for reacting the above-mentioned bacterial cells or their processed product with the sugar mixture are not particularly limited, but typically the temperature is 2.
0-40°C, reaction time 0.2-5 days, pl-13-9
It is within the scope of.

Effect 1 In the present invention, when the microorganism is allowed to act on the sugar mixture, the galactooligosaccharide is not assimilated and produced, but lactose, glucose, and galactose other than the galactooligosaccharide are selectively assimilated and fermented. In particular, lactose, glucose, galactose, etc. obtained by direct fermentation or enzymatic methods using lactose as a raw material.
When this is applied to a sugar mixture containing galactooligosaccharide as the main sugar component, lactose, glucose, and galactose are removed and a high-temperature galactooligosaccharide is obtained.

[Example 1] Hereinafter, the present invention will be explained in detail based on Examples.
The present invention is not limited to these.

In the following examples, the constant boiling of saccharides was performed using high-performance liquid chromatography (pump: Hitachi model 655, detector: Showa Denko 5E-31, column: 1 ichrosorb).
-N f-1z (5 μm > Cica Mar
(manufactured by K.K., the solvent was acetonitrile:water-65:35, the flow rate was 0.7st/5in), and the peak area was determined.

(1) MY
Cryptococcus laurentii variety laurentii r (C
ryptococcus 1aurantii var.
, Iaurentii) OKN-4 (Feiko Kenzonoyori No. 1629) was inoculated with a platinum loop and cultured with rotary shaking at 30°C for 6 days! ! Cultured in a container. The obtained culture was centrifuged using a centrifuge (8000 rpm, 10 minutes) to obtain a supernatant.

A high performance liquid chromatograph of this supernatant liquid is shown in FIG. The composition of this supernatant liquid is lactose 3.2 fl! fi1%
, galactooligosaccharide 5.0% by weight, total sugar content is a
, og was included.

Table 1 Culture composition Rough 1~-S 100gN 14
Ct
2gMlt! extract
0.2 9KH2PO40,8g Na2HPO4・12H200,3QMgSo4
・7H200.02g Water 1e
9He,.

(2) Into a sterilized 500 ml Erlenmeyer flask containing 100 μl of a medium with the composition shown in Table 2, a loopful of the 0KN-4 strain, which had been precultured on a MY agar slant medium for 2 days, was inoculated. The cells were cultured at 30°C for 2 days in a rotary shaking incubator. The obtained culture was centrifuged using a centrifuge (8000 rps, 10 minutes) to collect bacterial cells. The recovered bacterial cells were washed twice with pure water.

I) 50 mff1 of 10% lactose adjusted to f-15.5 was added to the washed bacterial cells, and the mixture was reacted at 65°C for 5 hours. This reaction solution contained 10 total sugars, and its sugar composition was 37% by weight of galacto-oligosaccharides, 4% by weight of lactose, and 4% by weight of lactose.
4% by weight, monosaccharides (glucose, galactose >1911
ffi%.

Lactose 5g (NH4)2804 0.5 0KH2Po
4 0. I QMIJSO4・71-
120 0.059 yeast extract
0.19 water
1tpH6,0 (3) The galactooligosaccharide produced in (1) and (2) above has a melting point of 229.5 to 230.5°C and a molecular weight of 504, and is O-β-D- expressed by the following formula (■). It was confirmed that it was galactopyranosyl-(1→4)-〇-β-D-galactopyranosyl-(1→4)-〇-glucose.

Example 2 100 mQ of a solution adjusted to pi-16.0 was added to the mixture containing the galactooligosaccharide obtained in (1) of Implementation @ 1 in a 500 me Erlenmeyer flask, and after sterilization, the mixture was preliminarily inoculated with MY agar slant medium. The precultured Klyveromyces fragilis ■FQ-0288 strain was inoculated and cultured with rotary shaking at 30°C for 3 days. The obtained culture was subjected to centrifugal force 111i (8000 rpm, 10 minutes) using a centrifuge.
A supernatant liquid was obtained. A high performance liquid chromatograph of this liquid is shown in FIG. The sugar composition of this supernatant liquid contained 98% by weight of galactooligosaccharides and 2% by weight of other sugars, and 3.90% of galactooligosaccharides were contained in 1q.

Example 3 In a 500a+Q Erlenmeyer flask, medium 100 II containing lactose 2.5g yeast extract 0.2Q! Put it in and die! 1m11. Klyveromyces fragilis strain IFO-0288, which had been precultured on a MY agar slant medium, was inoculated and cultured with rotary shaking at 30°C for 2 days.

The obtained culture was centrifuged using centrifugal valve 1IltIl.
1 liter (8000 rpm-110 minutes) and collected the national body.

The collected bacterial cells were washed twice with water. Example 1
1 of the mixture containing galactooligosaccharide obtained in (1) of
) H6, O:l! 1001ffi (7) was added and reacted at 30° C. for 2 days with stirring. The reaction solution was centrifuged (8000 rB, 10 minutes) to obtain a supernatant. Figure 3 shows the fast wave profile of this liquid.

The sugar composition of this supernatant is galactooligosaccharide 96i1i
@%, and other sugars were 41i1%.

Example 4 The same procedure as in Example 2 was carried out using 100 at of the mixture obtained in Example 1 (2) but containing lactooligosaccharide.

The sugar composition of the obtained supernatant was 94 m-% galactooligosaccharides.
, other sugars 61i 11%.

[Brief explanation of drawings]

Figure 1 is a high-speed graph showing the sugar composition of the supernatant obtained in Example 1 (1), and Figure 2 is a high-speed graph showing the sugar composition of the supernatant obtained in Example 2. The chromatograph shown in FIG. 3 is a high-speed chromatograph showing the sugar composition of the supernatant obtained in Example 3. Patent applicant: Nisshin Jyoto Co., Ltd. Figure 1 Figure 2 Na-Yojiki 4 Figure 3

Claims (3)

[Claims] (1) At least one type of sugar among glucose, galactose, and lactose, and the general formula Gal-(Gal)n
- A sugar containing a galactooligosaccharide represented by Glu (Gal is a galactose residue, Glu is a glucose residue, n is 1 or 2, and the binding mode between sugars is mainly β-1,4 bond). The mixture contains Kluveromyces (Kl
A method for purifying galactooligosaccharide, which comprises increasing the content of the above galactooligosaccharide in the sugar composition by using a microorganism belonging to the genus P. yveromyces. (2) The method for purifying galactooligosaccharide according to claim (1), wherein a microorganism belonging to the genus Krivellomyces is cultured in a sugar mixture containing the galactooligosaccharide. (3) The method for purifying galactooligosaccharide according to claim (1), in which cells of a microorganism belonging to the genus Krivellomyces or a treated product thereof are reacted with the galactooligosaccharide mixture.