JPH09140393A - Production of galactosylglycerols - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound useful as a food, a chemical, a medicine, etc., by treating a mixture of glycerol and a galactose compound with a β-galactosidase in the presence of a yeast belonging to the genus Saccharomyces industrially and advantageously. SOLUTION: A mixture of glycerol and a galactose compound (e.g. lactose) is treated with a cell of a bacterium capable of producing β-galactosidase [e.g. Cryptococcus laurentii var. laurentii OKN-4 (FERM P-7629), etc.] or a β- galactosidase produced by the bacterium at 40 deg.C for 6 hours to produce the objective galactosylglycerol of the formula (Gal is a galactose residue; Gly is glycerol; (n) is 1-2) and useful a food, a chemical, a feed, a medicine, etc., efficiently and inexpensively.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing galactosyl glycerol which can be used in foods, chemical products, feeds, pharmaceuticals and the like. Galactosyl glycerols are
In particular, it is expected to be used as a sweetener, a sweetness improver, and a moisturizer in the fields of foods, chemical products and feeds. Further, galactosyl glycerols can serve as substrates for glyceroglycolipid synthesis available in the pharmaceutical field. This glyceroglycolipid is widely distributed from animals and plants to microorganisms, and elucidating the physiological role of glyceroglycolipid will lead to its application to medicine.

[0002]

2. Description of the Related Art As a reaction for adding a sugar to glycerol, there is an example in which Kaplum et al . Synthesize organically using an orthoester and a halogen derivative of sugar ( Bioorg.Khi.
m. , 1 (11), 1675-6 (1975)). However, organic synthesis has a low yield and is industrially inexpensive and difficult to produce in large quantities. Further, there is a problem in safety in food production. Further, as an example of enzymatically transferring galactose to glycerol, there is an example in which galactanase is allowed to act by using arabinogalactan which is a polysaccharide as a sugar donor. (JP-A-2-308798). However, this method has problems that the solubility of the polysaccharide is low and it cannot be used in a large amount, and the unreacted polysaccharide remains, making it difficult to purify the target product. Also Crevisi-La Planticola G
There is also an example in which -17 β-galactosidase is allowed to act (JP-A-6-339376). However, this method has the problems that the temperature stability of the enzyme is low and the yield is as low as several%, and it does not meet the demand for commercialization of industrial production of galactosylglycerols.

[0003]

The problem to be solved by the present invention is to provide a microorganism which produces β-galactosidase having a galactose transfer activity from a galactose compound such as lactose and glycerol, or The β-galactosidase prepared from the microorganism is used in combination with yeast of the genus Saccharomyces to efficiently produce galactosylglycerols that can be used in foods, chemical products, feeds, pharmaceuticals and the like.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION As a result of intensive studies to solve the above problems, the present inventors have found that a mixture of glycerol and a galactose compound has a galactose transfer activity in the presence of Saccharomyces yeast. Β-galactosidase-producing microbial cells or β-prepared from the microorganism
By the action of galactosidase, glycero-
Succeeded in efficiently transferring the galactose residue to the amino acid, and completed the method for producing galactosyl glycerol.

The β-galactosidase-producing microorganism that can be used in the present invention is not particularly limited as long as it is a β-galactosidase-producing microorganism, and for example, Cryptococcus lorente variety variety-lenti OKN-4. ( Cryptococcus laurentii var. Laurentii
OKN-4; FERM P-7629) strain and the like. This bacterium is cultivated in a medium using lactose as a carbon source, and the microbial cells obtained are those obtained by immobilizing the microbial cells by a conventional immobilization method such as polyacrylamide, calcium alginate, and carrageenan. It can be used in a method for producing glycerols.

The β-galactosidase which can be used in the present invention may be any β-galactosidase as long as it has a glycosyl transfer activity, and a microbial cell producing β-galactosidase and a common bacterial cell therefrom. A crude enzyme or a purified enzyme prepared by the method of crushing bacterial cells used in 1. can be used. β-galactosidase (3.2.1.2
3) is an enzyme that recognizes and cleaves β-galactoside in which galactose is β-linked, but it shows a glycosyl transfer reaction depending on the reaction conditions. For example, Cryptococcus Lorenty Variety Lorenty OKN-4 ( C
ryptococcus laurentii var. laurentii OKN-4; FE
Β-galactosidase A prepared from RM P-7629)
(Japanese Patent Publication No. 6-2057, Japanese Patent No. 1882111), β-galactosidase prepared from yeast belonging to Lipomyces (JP-A-1-112978), β-galactosida prepared from Rhodotorula minuta IFO879. Ze (JP-A-2-72890), β-1,4 galactosidase II (manufactured by Daiwa Kasei Co., Ltd.) prepared by Bacillus circulans and the like can be particularly preferably used.

The yeast of the genus Saccharomyces that can be used in the present invention does not consume galactose compounds and glycerol which are reaction substrates and galactosylglycerols which is a target product, and is produced when lactose is decomposed or galactosylglycerols are produced. Monosaccharide (gluco-
And galactose) may be used. Commercially available baker's yeast is preferable from the viewpoint of easy availability and price. Saccharomyces yeast, when using lactose, which is one of the substrates of the reaction,
Decomposition of lactose or galactosylglycero-
It often consumes only monosaccharides (glucose, galactose) that are produced during the production of lees. Therefore, glycerol and the raw material lactose are the only acceptors that transfer the galactose residue of lactose. Also in this reaction,
A galactose compound such as 4′-galactosyl lactose produced by transferring a galactose residue to lactose, which is one of the acceptors, can serve as a donor for the galactose transfer reaction to glycerol in this reaction. Therefore, as a result of this combined use of the yeasts of the genus Saccharomyces, a high-yield production method of galactosylglycerols, which could not be achieved by the conventional method, was completed.

As the galactose compound which is one of the raw materials used in the present invention, any galactose compound which can donate a galactose residue to glycerol may be used, but the general formula (βGal) n-Glc (Gal Is a galactose residue, Glc is a glucose residue, and n = 1 to 3)
Those represented by are preferred. For example, lactose or 4'-
Galactosyl lactose and the like can be preferably used.

The reaction system used in the present invention is not particularly limited as long as it does not inactivate the β-galactosidase used in the present invention and can survive the yeast of the genus Saccharomyces. Preferably, the pH of the reaction solution is adjusted to 3-6, 30-60
It is preferred to react at ° C.

The composition of the raw materials is preferably 1:10 to 10: 1 (w / v) of glycerol and lactose. Further, it is preferable that the concentration of lactose is 1% to 40% (w / v).

In the reaction, the enzyme solution may be used as it is in a batch method, but it is desirable to immobilize the bacterial cells or the enzyme in order to recycle the enzyme. There is no particular limitation on the immobilization method, and those entrapped and immobilized in calcium alginate, carrageenan, etc., carriers such as cellulose, agarose, polyacrylamide gel, etc., the enzyme with a cross-linking agent such as glutaraldehyde. A method in which a treated product, an ion-exchange carrier such as DEAE-cellulose, or the like is ion-bonded or physically adsorbed to chitosan beads or the like is used.
It is more desirable from the standpoint of production efficiency to fill the column and perform continuous production than to produce the immobilized carrier by the batch method.

According to the present invention, galactosylglycerol (Gal-Gly, in which Gal is a galactose residue, Gly is a galactose residue, which is a galactose residue transferred to glycerol by a transglycosylation reaction).
Indicates glycerol) and digalactosylglycerol (Gal-Gal-Gly) in which the galactose residue is further transferred is produced when the reaction time is long. In the present invention, the galactosylglycerol refers to this galactosylglycerol and digalactosylglycerol. Therefore,
At the end of the reaction (the production rate of galactosyl glycerols is 30 to 99% with respect to the raw material glasstose compound),
Galactosyl glycerols, a small amount of galactose remaining without being completely consumed in Saccharomyces yeast, glycerol as a raw material, lactose, and a sugar solution containing sugars such as 4'-galactosyl lactose and Become.

The sugar solution can be decolorized, desalted, filtered, and concentrated to give a syrup, or can be freeze-dried or spray-dried to give a powder product. In addition, by treating this sugar solution with activated carbon column chromatography, ion exchange column chromatography, gel filtration and the like in advance,
It is possible to produce a product having a high content of galactosyl glycerols.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0015]

Example 1 Glycerol 10% (W / V) and lactose 1% (W / V) were dissolved in water to 100 ml, and 0.1 ml of baker's yeast and β were dissolved in a mixed solution of 2 ml. -Galactosidase (β-galactosidase A, prepared according to "Novel β-galactosidase A and its production method (Japanese Patent Publication No. 6-2057, Patent No. 1882111)") was added at 2 units, and 6 units at 40 ° C. Let it work for hours. The analysis was performed by high performance liquid chromatography (hereinafter referred to as HPLC). GL-C610 manufactured by Hitachi Chemical Co., Ltd. is used for the HPLC column, and the analysis is
Water was used as the solvent, and the flow rate was 1 ml / min and the column temperature was 60 ° C. RI detector is used for detection, D-2500 Chrom
It was analyzed by ato-Integrator (Hitachi Ltd.).

As a result, as understood from FIG. 1, 0
The peak of lactose as a raw material disappeared at 12.2 min, which was observed in time, and a new peak (hereinafter referred to as peak A) appeared at 13.5 min. This peak A appears at a position different from the elution time of monosaccharides such as glucose and galactose similarly analyzed as a control,
It was presumed to be a substance produced by transfer of sugar to glycerol.

Comparative Example 1 Bacterial yeast was not added and only β-galactosidase was reacted in the same manner as in Example 1 for analysis. The results are shown in Table 1. It was found that the reaction efficiency was dramatically improved by using baker's yeast together.

Table 1 Glycero-lactose-span yeast production rate Example 1 10% 1% 0.1g 70% Reference Example 1 10% 1% 0.0g 30%

The production rate indicates the ratio (molar ratio) of the production amount of peak A to the raw material lactose.

Example 2 Lactose and glycerol were dissolved in water so that each of them was 10% (W / V) to make 100 ml, and 5 g of baker's yeast was added thereto.
And β-galactosidase A (20 u) were added at 50 ° C for 2
The reaction was performed for 2 hours. After completion of the reaction, aniline: acetic acid = 5:
10 ml of a solution mixed with 2 (v / v) was added and reacted at 80 ° C. for 4 hours to remove the aniline derivative of the reducing sugar produced by ether extraction. This aqueous phase is divided into activated carbon column and BI
Sequentially apply to O-GEL P-2 gel filtration column and then use peak A
307 mg (purity 98.5%) was obtained.

1 ml of a 1% aqueous solution of peak A obtained above
And 1 ml of 2N hydrochloric acid solution were mixed and boiled for 1 hour. AgCO ₃ powder was added to this solution until carbon dioxide gas was not generated, the reaction solution was neutralized, and filtered through a 0.45 μm filter. When this aqueous solution was analyzed by HPLC of Example 1, the peak of peak A disappeared, and glycerol and galactose were detected (FIG. 2). This molar ratio was 1: 1. Therefore, it was found that the structure of peak A is a substance in which one residue of galactose is bound to glycerol.

10 mM aqueous solution of peak A obtained above 2
Various glycosidase 10u was added to 00 μl, and the temperature was 40 ° C.
For 5 hours. This reaction solution was similarly analyzed by HPLC. The thus-obtained chromatogram was compared with a sample to which no enzyme was added and standard glycerol, galactose, glucose, etc., to reduce peak A and the glycerol and galactose pigments. -The appearance of burrs was examined to confirm the presence or absence of decomposition.

Table 2 shows the results. As a result, α-galactosidase, α- and β-glucosidase were not decomposed, and three types of β-galactosidase were decomposed,
The structure of this substance of peak A was found to be galactosylglycerol represented by the following formula 1 in which galactose is β-bonded. (ΒGal) n -Gly (Formula 1) (where Gal is a galactose residue, Gly is glycerol, and n =
1 and 2 are shown. )

[0024] Table 2 Enzyme name (origin) peak - click A alpha-Ca Bu Rakutoshita Bu - peptidase (Moritierella vinacea) - β- mosquito Bu Rakutoshita Bu - peptidase (A. oryzae) + beta-Ca Bu Rakutoshita Bu - peptidase (E. coli) + beta-Ca Bu Rakutoshita Bu - K- lactis + α-Glucosidase-Yeast-β-Glucosidase-C ( Cai.saccharolyticum ) -Inverter-Y (Yeast)-(+ is the peak A decomposes, -is the peak Indicates that A does not decompose)

Example 3 Glycerol 10% (W / V) and lactose 10% (W / V) were dissolved in water to make 100 ml, and the mixture was 100 ml.
In addition, immobilized yeast cells (2.5 g of baker's yeast and calcium alginate (“Production method of galactooligosaccharide (Patent No. 16
No. 28521) ”, 20 g (11un
its) was added and reacted at 40 ° C. for 22 hours. The analysis was performed by HPLC as in Example 1.

As a result, 2.34 g of galactosyl glycerol was obtained. At this time, 31.5% of the galactose contained in the raw material lactose was transferred to glycerol.

Example 4 Glycerol 10% (W / V), lactose 1-10% (W / V)
The mixture was dissolved in water to 100 ml, and 2 ml of the mixture was added with 0.1 g of baker's yeast and 2 units of β-galactosidase in the same manner as in Example 1 and allowed to act at 40 ° C for up to 6 hours. The analysis was performed by HPLC.

As a result, the raw material lactose was 1% and 2%.
%, The raw material lactose was 4% after 4 hours, and the raw material lactose 6% after 6 hours, the lactose disappeared and galactosyl glycerol was produced. At this time,
70% of the galactose derived from the raw material lactose was transferred to glycerol. Raw material lactose 8% and 10%
In the case of 1%, 7 times more glycosyl transfer product was obtained (Fig. 3). At this time, about 50% of galactose, which is a constituent sugar of the raw material lactose, was transferred to glycerol.

[0029]

As described above, according to the present invention, it is possible to efficiently provide a large amount of galactosyl glycerols. The galactosylglycerols produced by the present invention can be used for foods, chemical products, feeds, pharmaceuticals and the like. Especially in the fields of foods, chemical products and feeds, applications as sweeteners, sweetness improvers and moisturizers are expected. Further, it can be used as a substrate for glyceroglycolipid synthesis which can be used in the field of medicine. Glyceroglycolipids are widely distributed from animals and plants to microorganisms, and elucidating the physiological role of glyceroglycolipids leads to application to medicines.

[Brief description of the drawings]

FIG. 1 is a diagram obtained by HPLC analysis of a solution treated with a standard substance and β-galactosidase. 1 (a) to 1 (d) are standard substances such as galactose, glucose, and lacto-.
2 is a chromatogram of sucrose and glycerol. (E) ~
(G) is a chromatogram of the solution combined with β-galactosidase after 0 hours, 2 hours, and 6 hours.

FIG. 2 is a diagram obtained by subjecting peak A to acid hydrolysis, and analyzing the type and constituent ratio of the constituent sugars by HPLC. FIG. 2A shows peak A.
(B) is a sample after the reaction of peak A. As a control in (c), glycerol: galactose = 1: 1 (each 10 mM) was similarly analyzed.

FIG. 3 Glycerol 10% (W / V), lactose 1-1
Baker's yeast and β-galactosidase were added to a 0% (W / V) aqueous solution.
The figure which plotted the peak A (galactosyl glycerol) produced | generated for every hour by analyzing the reaction liquid which was made to act in combination with ZEZ by HPLC.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Osamu Ozawa 4-9-11 Toyosu, Koto-ku, Tokyo Nisshin Sugar Co., Ltd. Research Department (72) Inventor Yoshiaki Deho 4--9-11 Toyosu, Koto-ku, Tokyo Nisshin Sugar Co., Ltd.

Claims (4)

[Claims] 1. A mixture of glycerol and a galactose compound is treated with a microbial cell producing β-galactosidase in the presence of a yeast of the genus Saccharomyces or β-galactosidase prepared from the microorganism. To produce galactosyl glycerol. 2. A galactosyl glycerol is represented by the following formula 1
The method according to claim 1, wherein (βGal) n-Gly (Formula 1) (where Gal is a galactose residue, Gly is glycerol, and n =
1 and 2 are shown. ) 3. The method according to claim 1, wherein the galactose compound is lactose. 4. The method according to claim 2, wherein the galactose compound is lactose.