JPH0317839B2 - - Google Patents

Description

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

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

A common method for producing galactooligosaccharides is the direct fermentation method, in which specific microorganisms are cultured using lactose as a raw material, and galactooligosaccharides are accumulated in the culture (Tetrahedron, 1960, vol.9, p125-129,
Can.J.Chemistry, 1964, vol.42, p1341-1344,
JP-A-56-115796) is known. In addition, an enzymatic method in which specific β-galactosidase acts on lactose (Eiji Ichishima, ed., "Food Industry and Enzymes", No. 68)
Page, Asakura Shoten) is also known.

In particular, the general formula Gal-(Gal)n-Glu (where Gal
is a galactose residue, Glu is a glucose residue, n
is an integer from 1 to 4) is known to exhibit excellent activity in vivo (Japanese Patent Publication No. 58-20266). (Japanese Patent Application No. 59-108547 (Patent Application No. 251896), Patent Application No. 60-76767)
No. 236790)).

[Problems to be solved by the invention] However, in order to produce galactooligosaccharides with a good yield using the above method, galactooligosaccharides must be
It is necessary to stop the culture or stop the enzyme reaction when 20 to 50% of the lactose is produced, that is, when the raw material lactose and its decomposition products glucose and galactose are present at about 50 to 80%. Therefore, in order to obtain only galactooligosaccharide that is truly effective as a Bifidobacteria growth factor, lactose and its decomposition products glucose and galactose must be removed.

As a method for removing sugars other than the target sugar,
Column chromatography methods using activated carbon adsorption or molecular sieves are well known, but these column chromatography methods use large amounts of expensive ethanol, cannot process large volumes, and are time consuming. It is unsuitable for industrial production due to drawbacks such as too much heat.

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 Bifidobacterium growth factors, in addition to galactooligosaccharide.

The present invention solves the drawbacks of the above-mentioned prior art, and in particular, the general formula Gal-(Gal)n-Glu...() (where Gal is a galactose sugar residue, Glu is a glucose residue, n is 1 or 2, and sugar The binding mode of sugars is mainly β-1,4 bonds. The purpose of the present invention is to provide a method for purifying a large amount of methane at a low cost.

[Means for Solving the Problems] The present inventors applied various microorganisms to a sugar mixture containing lactose, glucose, galactose, and galactooligosaccharides obtained by direct fermentation or enzymatic methods using lactose as a raw material. 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 galactooligosaccharides, the microorganisms belonging to the genus Kluyveromyces were discovered ( It was discovered that lactose, glucose, and galactose other than the galactooligosaccharides represented by the formula can be selectively assimilated and fermented, thereby making it possible to obtain only the above galactooligosaccharides with high purity, leading to the present invention.

That is, the method for purifying galactooligosaccharides according to the present invention uses at least one type of sugar among glucose, galactose, and lactose, and the general formula Gal-(Gal)n-Glu...() (where Gal is a galactose residue, Glu is a glucose residue, n is 1 or 2, and the binding mode between sugars includes β-1,4 bonds. It is characterized by increasing the content of the galactooligosaccharides in the sugar composition by using microorganisms belonging to the genus Kluyveromyces that can ferment but cannot assimilate or ferment the galactooligosaccharides.

The present invention will be explained in detail below.

(a) Sugar mixture to be purified The present invention specifically relates to the production of a galactooligosaccharide represented by the above formula (2) (hereinafter simply referred to as galactooligosaccharide) from lactose or a lactose-containing substance.
In producing this, the purpose is to remove lactose mixed in the sugar mixture containing galactooligosaccharides, as well as glucose and galactose, which are decomposition products of lactose. For example, the method for producing galactooligosaccharides using microorganisms belonging to the Cryptococcus genus that the present inventors previously proposed (patent application
59-108547, Japanese Patent Application No. 60-76767), or β obtained from mold, yeast, bacteria, etc.
- Those obtained using the transfer reaction of galactosidase, etc., and include unpurified ones or those that have been primarily purified by other methods. However, the products to be purified are not limited to these products;
The scope of the present invention is any sugar mixture containing at least one type of sugar among glucose, galactose, and lactose, and the galactooligosaccharide.

(b) Microorganisms to be used The microorganisms to be used to act on the above sugar mixture are those belonging to the genus Krivellomyces and can assimilate or ferment glucose, galactose, and lactose, but are not particularly limited as long as they cannot assimilate or ferment the above galactooligosaccharides. However, a specific example is Kluyveromyces fragilis IFO-0288. Although this microorganism itself is known, the action 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, the pH of the medium may be 3 to 9, the culture temperature may be 20 to 40°C, and the culture period may be 1 to 5 days, and either agitation culture or static culture may be used.

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

In method A, a sugar mixture containing galactooligosaccharides is supplemented with nitrogen sources such as inorganic nitrogen sources such as (NH ₄ ) ₂ SO ₄ and NH ₄ Cl, organic nitrogen sources such as peptone and yeast extract, other mineral sources, and trace amounts of useful nutrients. The above-mentioned microorganisms are inoculated and cultured in a material to which a source and the like are appropriately added.

In method B, suitable sugars such as glucose, galactose, sucrose, lactose, and maltose are used as carbon sources, and inorganic nitrogen sources such as (NH ₄ ) ₂ SO ₄ and NH ₄ Cl, peptone, yeast extract, etc. are used as nitrogen sources. The above-mentioned microorganisms are inoculated into a medium to which an organic nitrogen source, other mineral sources, trace amounts of useful nutrients, etc. have been appropriately added, and the microorganisms or their processed products obtained by inoculating and culturing the cells, and sugars containing galactooligosaccharides. react with the mixture. Bacterial cells or their processed products include live bacterial cells, frozen bacterial cells, dried bacterial cells, freeze-dried bacterial cells, and other commonly known methods in which the bacterial cells are encapsulated in polyacrylamide gel, calcium alginate gel, etc. Examples include immobilized bacterial cells and immobilized proliferated bacterial cells. The conditions for reacting the above-mentioned bacterial cells or their treated product with the sugar mixture are not particularly limited, but typically range from a temperature of 20 to 40°C, a reaction time of 0.2 to 5 days, and a pH of 3 to 9.

[Action] In the present invention, when the microorganism is allowed to act on the sugar mixture, the galactooligosaccharides are not assimilated and fermented, but lactose, glucose, and galactose other than the galactooligosaccharides are selectively assimilated and fermented, so particularly When applied to a sugar mixture whose main sugar components are lactose, glycose, galactose, and galactooligosaccharides obtained by direct fermentation or enzymatic methods using lactose as a raw material, lactose, glucose, and galactose are removed, resulting in a high concentration. of galactooligosaccharide is obtained.

[Examples] The present invention will be specifically described below based on Examples, but the present invention is not limited thereto.

The quantitative determination of sugars in the following examples was performed using high-performance liquid chromatography (the pump was manufactured by Hitachi, Ltd.).
Model 655, detector is Showa Denko SE-31, column is
Lichrosorb-NH ₂ (5 μm) manufactured by Cica Merk, the solvent used was acetotrile:water = 65:35, the flow rate was 0.7
ml/min) and determined from the peak area.

Example 1 Method for preparing a sugar mixture containing galacto-oligosaccharides (1) A 500 ml Erlenmeyer flask was filled with 100 ml of a medium having the composition shown in Table 1, sterilized, and precultured on a MY agar slant medium for 2 days. Cryptococcus laurentii var.laurentii (Cryptococcus laurentii var.laurentii)
A platinum loop of OKN-4 (Feikoken Bibori No. 7629) was inoculated and cultured in a rotary shaking incubator at 30°C for 6 days. 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 was 3.2% by weight of lactose, 5.0% by weight of galactooligosaccharide, and 8.0g of total sugar.

Table 1 Culture composition Lactose 100g NH ₄ Cl 2g Yeast extract 0.2g KH ₂ PO ₄ 0.8g Na ₂ HPO ₄・12H ₂ O 0.3g MgSO ₄・7H ₂ O 0.02g Water 1 PH 6.0 (2) 500ml Erlenmeyer flask Add 100 ml of a medium with the composition shown in Table 2 to sterilize it, and add the above OKN which had been pre-cultured on MY agar slant medium for 2 days.
-4 strain was inoculated with a platinum loop and cultured at 30°C for 2 days in a rotary shaking incubator. Centrifuge the obtained culture using a centrifuge (8000 rpm, 10 minutes)
was carried out, and the bacterial cells were collected. The recovered bacterial cells were washed twice with pure water. This washed bacterial body has a pH of
Add 50ml of 10% lactose adjusted to 5.5,
The reaction was carried out at 65°C for 5 hours. In this reaction solution,
It contained 10g of total sugar, and its sugar composition was 37% by weight of galactooligosaccharides, 44% by weight of lactose, and 19% by weight of monosaccharides (glucose, glass talk).

Table 2 Culture composition Lactose 5g (NH ₄ ) ₂ SO ₄ 0.5g KH ₂ PO ₄ 0.1g MgSO ₄・7H ₂ O 0.05g Yeast extract 0.1g Water 1 PH 6.0 (3) In (1) and (2) above The produced galactooligosaccharide is O-β-D-galactopyranosyl-(1→4)-O-β-D-galactopyrano with a melting point of 229.5 to 230.5°C and a molecular weight of 504. It was confirmed that it was syl-(1→4)-D-glucose.

Example 2 100 ml of the mixture containing galactooligosaccharides obtained in Example 1 (1), adjusted to pH 6.0, was placed in a 500 ml Erlenmeyer flask, sterilized, and precultured on MY agar slant medium. Klyveromyces fragilis IFO-0288 strain was inoculated and cultured with rotary shaking at 30°C for 3 days. The obtained culture was centrifuged using a centrifuge (8000 rpm, 10 minutes) to obtain a supernatant. A high performance liquid chromatograph of this liquid is shown in FIG. The sugar composition of this supernatant was 98% by weight of galactooligosaccharide and 2% by weight of other sugars, and 3.9 g of galactooligosaccharide was obtained.

Example 3 100 ml of a medium containing 2.5 g of lactose and 0.2 g of yeast extract was placed in a 500 ml Erlenmeyer flask, and after sterilization,
Klyveromyces fragilis IFO-0288 strain, which had been precultured on MY agar slant medium, was inoculated and cultured with rotary shaking at 30°C for 2 days. The obtained culture was centrifuged using a centrifuge (8000 rpm, 10
minutes) and collected the bacterial cells. The collected bacterial cells were washed twice with water. 100 ml of the mixture containing galactooligosaccharide obtained in Example 1 (1) adjusted to pH 6.0 was added to the washed bacterial cells, and the mixture was reacted at 30° C. for 2 days with stirring. Centrifuge the reaction solution (8000 rpm, 10
) to obtain a supernatant. A high performance liquid chromatograph of this liquid is shown in FIG. The sugar composition of this supernatant was 96% by weight of galactooligosaccharides and 4% by weight of other sugars.
It was in weight%.

Example 4 The same procedure as in Example 2 was carried out using 100 ml of the mixture containing galactooligosaccharides obtained in Example 1 (2). The sugar composition of the obtained supernatant is galactooligosaccharides.
94% by weight, and 6% by weight of other sugars.

[Brief explanation of the drawing]

Figure 1 is a high-speed chromatograph showing the sugar composition of the supernatant obtained in Example 1 (1), and Figure 2 is Example 2.
Figure 3 is a high-speed chromatograph showing the sugar composition of the supernatant obtained in Example 3.

Claims (1)

[Scope of Claims] 1. At least one type of sugar selected from glucose, glasstose, and lactose, and a compound having the general formula Gal-
(Gal) n-Glu (Gal is a glasstose residue,
Glu is a glucose residue, n is 1 or 2, and the binding mode between sugars includes a β-1,4 bond. ) to a sugar mixture containing a galactooligosaccharide represented by
It is characterized by increasing the content of the galactooligosaccharide in the sugar composition by acting with a microextract belonging to the genus Klyveromyces, which can assimilate or ferment glucose, galactose, and lactose, but cannot assimilate or ferment the galactooligosaccharide. A method for purifying galactooligosaccharides. 2. The microorganism belonging to the genus Krivellomyces,
The method for purifying galactooligosaccharide according to claim 1, wherein the galactooligosaccharide is cultured in a sugar mixture containing the galactooligosaccharide. 3. The method for purifying galactooligosaccharide according to claim 1, wherein the cells of the microorganism belonging to the genus Krivellomyces or a treated product thereof are reacted with the galactooligosaccharide mixture.