JPS61236790A - Production of galactooligosaccharide - Google Patents

Abstract

PURPOSE:To obtain the titled substance useful as a growth factor for Lactobacillus bifidus readily in high yield, by cultivating a microorganism of the genus Cryptococcus capable of producing galactooligosaccharide from lactose, and reacting the above-mentioned culture with the lactose. CONSTITUTION:A microorganism, having he ability to produce galacto oligosaccharide from lactose (substance containing the same), and belonging to the genus Cryptococcus, e.g. Cryptococcus laurentii variety laurentii OKN-4 (FERM-P No.7629), is cultivated in a culture medium containing lactose (sub stance containing the same) as a carbon source, generally at 20-40 deg.C and 3-9pH for 1-3 days, and the resultant culture or a treated material thereof is reacted with the lactose (substance containing the same) under conditions of preferably 30-60 deg.C and 3-6pH to afford the aimed substance. The concentra tion of the lactose (substance containing the same) to be used for reaction with the above-mentioned culture is preferably 2.5-30wt%.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing galactooligosaccharides, and more particularly to a method for producing galactooligosaccharides using a culture of microorganisms or a processed product thereof.

In addition, in the Suimei Sho, "galactooligosaccharide" means an oligosaccharide containing a galactose residue.

[Prior art] In recent years, galactooligosaccharides have attracted attention as bifidobacteria growth factors, and the In method involves producing them from lactose or lactose-containing substances through a transfer reaction using β-galactosidase derived from molds, yeast, bacteria, etc. A method is known in which galactooligosaccharides are produced in the medium by culturing microorganisms capable of producing galactooligosaccharides in a medium containing lactose (-Eiji Shima, ed., "Food Industry and Enzymes", Vol. 68 pages, including W1 opening).

[Problems to be solved by the invention] However, the above-mentioned conventional methods have problems in practical application, such as the number of types of oligosaccharides produced being about 10 or so, and the yield of oligosaccharides being low. Ta.

Therefore, the present invention aims to provide a method for producing galactooligosaccharide, which is considered to be useful as a bifidobacterial growth factor, with a high yield, high selectivity for the main component, and easy purification. There is.

[Means for Solving the Problems] The present inventors conducted a wide search for microorganisms in the natural world in search of microorganisms with a strong ability to produce galactooligosaccharides, which are considered to be useful as growth factors for Fidus bacteria. As a result, Cryptococcus (CryptOcOcctls)
It has been found that galactooligosaccharides can be produced with good selectivity in the culture by culturing microorganisms of the genus ) in lactose or lactose-containing substances (Japanese Patent Application No. 108,547/1983). Furthermore, as a result of conducting research in search of an efficient WJJ production method for galacto-oligo grapes in terms of practical use, we found that by reacting a culture of Cryptococcus microorganisms or a processed product thereof with lactose or a lactose-containing substance. , galactooligosaccharide is produced in a suspended state in the reaction solution, and furthermore, the main component of the produced galactooligosaccharide is O-β-D-galactopyranosyl-(1→
4) -〇-β-D-galactopyranosyl(1→4)-
It was recognized that it is 〇-glucose, and the present invention was developed.

That is, the method for producing galactooligosaccharide according to the present invention comprises a step of culturing a microorganism belonging to the genus Cryptococcus that has the ability to produce galactooligosaccharide from lactose or a lactose-containing material, and a culture obtained by the above microorganism culturing step. Alternatively, the method is characterized by including a step of reacting the treated product with lactose or a lactose-containing substance to produce galactooligosaccharide.

The present invention is based on the discovery that Cryptococcus microorganisms have the ability to produce galactooligosaccharides even under non-culture conditions. This is the manufacturing method.

The present invention will be explained in detail below.

(b) Microorganisms used in the present invention have the ability to produce galactooligosaccharides, and are species belonging to the genus Cryptococcus. For example, Cryptococcus laurentii
Variety Laurentii (Cryptococcus)
1aurentii var, Iaurentii
) QKN-4 (hereinafter referred to as 0KN-4) has the above characteristics and produces galactooligosaccharides,
This is a bacterial species discovered by the present inventors in the soil of Nasu District, Tochigi Prefecture, and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Institute Bacterial Deposit No. 7629.

0KN-4 has the following mycological properties.

In addition, tests for various mycological properties described below are conducted by ■J, L.
odder: The Yeast (1970) ■ Hiroshi Iizuka, Shoji Goto; Nomoto's classification and identification method (1969) ■ Yoji Hase; Classification and identification of microorganisms (1975), and the classification method is based on J, Loddcr: TheY C
A5t (1970).

<Mycological properties of 0KN-4> (a) Growth status in each medium MY liquid medium: Cultured at 25°C for 3 days, cell morphology was spherical,
Oval shape, elongated size: (3,0~5.3) x (4,0~5.3)μ Multipolar budding, 1slet-shaped film formation medium forms a smiley precipitate MY agar medium: 25℃ 1 When cultured for months, the colonies are pale orange to yellow-brown, shiny, soft, and viscous.

Slide culture: Hyphae and pseudohyphae are not formed in potato dextrose medium.

(b) Formation of ascospores: Not detected on normal sporulation medium. I can't stand it.

(C) Formation of extruded spores: Recognized in MY agar flat culture No.

(d) Physiological properties (1) Optimal growth conditions: pH 6-7, temperature 30°C (■Growth range: pH 3-9, temperature 20-40°C (3) Assimilation of nitrate: Not assimilated (4) Decomposition of fat No two decompositions ■ Decomposition of urea Two decompositions ■ Liquefaction of gelatin: No liquefaction ■ Formation of carotenoids: No formation or very little formation ■ Formation of organic acids: No formation ■ Formation of starch-like polysaccharides: Formation of aω Vitamin requirement: Does not grow in vitamin-deficient medium (10 Decomposition of arbutin: Decomposes (■ Cycloheximide resistance: Does not grow (Growth at 337°C: Grows IX) Growth in 50% glucose yeast extract medium: Growth (e) Assimilation of each carbon source 1) D-arabinose +2) L-arabinose +3> D-lipose
+4) D-xylose +
5) D-glucose +6) D-galactose +7) L-rhamnose
+8) L-sorbose
+9) Maltose 110) Sucrose +11) Lactose
+12〉Melibiose +
13) Cellobiose +1 Ridrevalose +15) Raffinose
+16) Melezitose +
17) α-Methyl-D-glucoside +18) Soluble starch ±19) Inulin
-20) Ethanol
+21) Atonito +22) Erythrite +23) Inojito
+24) D-Mannit
+25) D-Sorvit +26) Zulcit +27) Glycerin
+28) DL-lactate
-29) Kohaku I! salt 130
) Citrate -31) Salicin
+ (+: Assimilates well; M: Assimilation is doubtful; m: Does not assimilate) In addition, it has no fermentability for sugars.

Based on the above mycological properties, this strain was identified as belonging to Cryptococcus laurentii variety laurentii.

OKN-4 is an example of the microorganism used in the present invention, and all microorganisms belonging to the genus Cryptococcus and having the ability to produce galactooligosaccharides, as well as its natural and artificial mutant strains, can be used in the present invention. It can be used in

(0) Cultivation Step For culturing the microorganisms capable of producing galactooligosaccharides used in the present invention, a commonly used solid medium or liquid medium is used. In the culture medium used in the present invention, a carbon source that can be assimilated by microorganisms can be used as a carbon source, but preferably lactose or a substance containing lactose as a component, such as whole milk or fatty milk. , sorbit, and roughchit. Nitrogen sources include nitrogen compounds such as yeast extract, casein, corn stew liquor, soybean flour, cottonseed flour, wheat gluten, peptone, meat extract, etc.
)2 Inorganic nitrogen compounds such as SO4, NH4Ct, and urea can be appropriately used, and sodium salts, potassium salts, magnesium salts, phosphates, etc. can be used as inorganic salts. Furthermore, vitamins and trace metal salts can be added to improve the growth of the bacteria used.

The concentration of the carbon source ranges from 1 to 15% by weight, and the culture! ! The temperature is 20 to 40°C, the DH of the culture solution is within the range of 3 to 9, and the culture time is about 1 to 3 days. The culture can be carried out by static culture, aeration and agitation, or shaking culture.

(c) Galactooligosaccharide production step In the galactooligosaccharide production step, the culture obtained in the above culture step can be used as it is, but a treated culture can also be used.

The processed culture product used in the present invention refers to microbial cells obtained by centrifugation, gravel filtration, or other separation means from the culture solution after culturing the above-mentioned microorganisms, and frozen cells obtained by freezing the microbial cells. Microbial cells, dried microbial cells obtained by drying means, residual microbial cells obtained by destroying microbial cells by physical means, dried products thereof, water-soluble substances extracted from microbial cells and bacterial cell residue by extraction means, and microbial cells. It means immobilized bacterial cells or immobilized proliferating bacterial cells that have been immobilized by known immobilization means.

The amount of the culture or its treated product used in the present invention can be changed as appropriate depending on the ability of the culture or its treated product to produce galactooligosaccharides.

The concentration of lactose or lactose-containing substance used in the galactooligosaccharide production step of the present invention is 1 to 40% by weight as lactose.
, preferably from 2.5 to 30% by weight.

The reaction conditions for the culture or its processed product and lactose or lactose-containing material may be changed as appropriate within the range that produces galacto-oligosaccharides, but the temperature is 20 to 70°C, preferably 30 to 65°C, and the temperature of the reaction solution is I)H is within the range of I)H2 to 8, preferably pH 3 to 6, and the reaction time varies depending on the conditions. If the reaction time is increased, the galactooligosaccharide once produced will decompose, so it is preferable to select the time at which the yield of galactooligosaccharide is maximized. Furthermore, it is also preferable to add a trace amount of inorganic salts as a reaction stabilizer.

After the reaction has been completed, the reaction solution is subjected to separation means such as centrifugation or filtration with diatomaceous earth to remove solid content, if necessary. The resulting supernatant or filtrate has little coloring,
Since the salt concentration is low, subsequent purification operations such as decolorization and desalting are easy.

When purifying the main component galactooligosaccharide represented by formula (1) above, the reaction solution is further subjected to chromatography such as gel filtration or activated carbon adsorption.

[Examples] The present invention will be specifically explained below using Examples, but the present invention is not limited thereto.

In the examples, the total sugar m is expressed in terms of liglucose using the phenol-III acid method, and lactose and galacto-oligosaccharides were determined by 8-wave body chromatography (pump: Hitachi Model 655, detector: Showa Denko 5E- 31, column is L 1chrosorb-NH2 (5111)
Manufactured by C1Ca Mark, solvent is acetonitrile:water
65:35), the flow rate was 0.7 rI9. /1
n) was performed and determined from the peak area.

Example 1 A 500 mQ Erlenmeyer flask was filled with 10011ft of a sterilized culture medium having the composition shown in Table 1, and the above OKN-4 <Feikoken Bacterial Serial Number 7629> was pre-cultured on a MY agar slant medium for 2 days. No.) was inoculated into a platinum loop and cultured by shaking in a rotary shaking incubator at 30°C for 2 days. After the culture is completed, centrifugation is performed using a centrifuge (aoo).

rpl) L, bacterial cells were collected. The collected bacterial cells were washed twice with 50 mQ of distilled water and then suspended in 501ε water.

Add 6% lactose solution (pl-1s, 5)s to this bacterial cell suspension.
.

IIIQ was added and the reaction was carried out at 65°C for 24 hours. At 6 hours, the sugar composition of the reaction solution was examined by high-performance liquid chromatography, and as shown in Figure 1, 19% of monosaccharides, 44% of lactose, and 24% of galactooligosaccharide GO-1 were contained in 3 g of total sugar.
%, and other galactooligosaccharides 13%.

FIG. 2 shows the change in sugar composition in the reaction solution over time.

Table 1 Medium composition Lactose so gNH<C
1! 2 QKH2PO40,
8g Na2 PO4・121-12 0
0.3 QMQ SOs ・7H2
00,02! JM9 mother extract 3
g water
Isolated 1FL pH 6,0 GO-1 is a white needle-like crystal having the following properties, and is 0-β-D-galactopyranosyl-(1→4)- represented by the above formula (1). It was confirmed that it was 〇-β-D-galactopyranosyl(1→4)-D-glucose.

<Physical and chemical properties of galactooligosaccharide GO-1> (1) Elemental analysis values: C: 42.47% H: 6.30% (
2) Molecule ffi: 504 The molecular weight was determined from the mass spectra of the fully methylated product and the fully acetylated product.

(3) Ratio of constituent sugars Nigelcose: Galactose = 1:2 Glucose-B-T QSt Wako
(manufactured by Wako Pure Chemical Industries, Ltd.), galactose was quantified using F-kit lactose/galactose (manufactured by Bering Mannheim Yamanouchi Pharmaceutical), and the ratio was determined.

(4> Ratio of reducing sugars to total sugars: Total sugars direducing sugars = 3:1 Total sugars were measured by the phenol-sulfuric acid method, and reducing sugars were measured using the Somogyi-Nelson (3oa+ogyi-Ne) method.
1son) method.

■Melting point: 229.5~230.5℃ (■Specific rotation =
[0 houses +61°→+39°〈D ultraviolet absorption spectrum: No specific absorption.

■Infrared absorption spectrum: Infrared absorption spectrum by KBr method is as shown in FIG.

(9) Solubility in solvent: Easily soluble in water, acetone, alcohol, Nitroform, insoluble in benzene, contains water Poorly soluble in alcohol.

0 [I) Color reaction Aniline, phthalate reaction and ammonia A. Silver nitrate reaction is positive and ninhydrin reaction and ferric chloride reaction are negative. Ru.

GO Basic, acidic, neutral distinction: Neutral.

(121 binding mode: i) When the sodium borohydride reduction product was hydrolyzed using 1N HCg and this hydrolyzate was analyzed by thin layer chromatography, galactose and nruvit were detected. The reducing end is thereby glucose.

11) By methylation analysis ■2,3,4.6-thitra-O-methyl-1,5-di-O-acetylgalactitol■2,3.6-tri-O-methyl-1,4,5- Three types of alditol acetates were detected: tri-O-acetylgalactitol 2,3.6-tri-O-methyl-1,4,S-tri-O-acetylglucitol.

1ii) Analysis of the nuclear magnetic resonance spectrum of the fully methylated product revealed that α-amma 1264.77 (1t-1, J = 3.601-1z
, H-1(α) )6 4.29(1t1.J
= 6.90 Hz, f-1-
1'(β) ) δ 4,58 (1'H, J=
6.75 Hz, l-1-1" (β)) β-anomer: δ 4,14 (1H, J= 7.50)-1z
, l-1-1(β)) δ 4,34 (I H, J
-6,90Hz, H-1'(β))64.59
(1H, J = 6.75 Hz%H-1"(β)
) From the results of 11) and 1ii) above, it was found that the binding mode between sugar and sugar is β.

Example 2 The washed bacterial cells obtained in the same manner as in Example 1 were freeze-dried to obtain 4.8 g of dry matter. This freeze-dried bacterial cell 4.8Q
% Lactose (DH6,0) 10G (J plus 2 at 40℃
The reaction was allowed to proceed for 4 hours. After completion of the reaction, the amount of oligosaccharides produced was analyzed by high performance liquid chromatography, and the sugar composition was 18% monosaccharides, 42% lactose, 25% galactooligosaccharide GO-1, and 15% other galactooligosaccharides.

Example 3 A bacterial cell suspension obtained by suspending the washed Kokutai 1k (J (wet weight)) in distilled water obtained in the same manner as in Example 1 was processed using a Manton Goring bacterial cell crusher to 560ka/C12. So 5
Processed twice. The crushed bacterial cells were centrifuged using a centrifuge (8000 rpm), and the precipitated residue of the crushed bacterial cells was collected. The collected bacterial cell residue was washed with triple distilled water and then freeze-dried to obtain 100 g of crushed bacterial cell residue. 3゜2g of this crushed residue was mixed with 10% lactose (pH 5.5) 100
g and reacted at 60°C for 6 hours. After the completion of the reaction, the amount of oligosaccharides produced was analyzed by high performance liquid chromatography, and the sugar composition was 14.0% monosaccharides and 952.4% milk.
%, galactooligosaccharide GO-1 was 23.0%, and other galactooligosaccharides were 10.6%.

Example 4 A crushed bacterial cell product (lyophilized product) obtained in the same manner as in Example 3 was added to 25 at of Macllvaine.
butter (DI-16,0), and after adding Zymolyase 2O-T (manufactured by Seikagaku Corporation) to an enzyme concentration of 0.01%, the mixture was reacted at 30° C. for 3 hours. After the reaction is complete, the reaction solution is centrifuged (10,000 rpm).
20 minutes) to obtain a supernatant.

0.2 g of lactose was added to this supernatant io at, and 60 g of lactose was added.
The reaction was carried out for 2 hours at ℃, p)l 6.0, and the oligosaccharides in the reaction solution were analyzed by high performance liquid chromatography. The composition of the reaction solution was 3.7% monosaccharides, 80.3% lactose, and galactooligosaccharides. GO-1 was 12.9% and other galactooligosaccharides were 3.1%.

Example 5 The washed bacterial cells obtained in Example 1 (dry ffi 5.4o) were suspended in water, mixed with 120 mff1 of 3% sodium alginate solution, and then added to a 10% calcium chloride solution using a syringe. Granular gel (1-21-φ) 1
20 (J(PJlffi>) was obtained. After washing thoroughly with water,
l) 2.5% lactose solution of H5,0 5.011 to 1 g of gel
11 was added, and the mixture was reacted at 60°C for 5 hours. After the reaction, the sugar composition was found to be 8.7% monosaccharides and 72.2% lactose.
, galactooligosaccharide GO-1 was 14.7% and other galactooligosaccharides were 4.3%.

[Effects of the Invention] As explained above, according to the method for producing a galactooligosaccharide of the present invention, the galactooligosaccharide represented by the above formula (1), which is useful as a growth factor for Bifidobacterium, can be obtained in good yield and with high selectivity. Moreover, unlike the method of accumulating galactooligosaccharides in the culture while culturing microorganisms, the reaction time is shorter in the galactooligosaccharide production process, and various nutrients and salts for culturing microorganisms are removed in advance. This has the advantage that there is little coloring, the salt concentration is low, and subsequent purification operations are extremely easy.

In addition, since bacterial cells can be immobilized, this method has great effects in terms of reaction operation and practical application.

[Brief explanation of drawings]

Figure 1 is a high-speed liquid flow graph showing the sugar composition of the reaction solution obtained in Example 1, Figure 2 is a graph showing changes in its main components over time, and Figure 3 is a graph showing the sugar composition of the reaction solution obtained in Example 1. Go-
1 is a graph showing an infrared absorption spectrum of No. 1.

Claims (3)

[Claims] (1) Cyptococcus has the ability to produce galactooligosaccharides from lactose or lactose-containing substances.
a step of culturing a microorganism belonging to the genus C. ccus); and a step of reacting the culture obtained by the microorganism culturing step or a processed product thereof with lactose or a lactose-containing substance to produce galactooligosaccharide. Characteristic method for producing galactooligosaccharide. (2) The microorganism is Cryptococcus var. laurentii (Cryptococcus laurentii).
s laurentii var. laurentii
) A method for producing a galactooligosaccharide according to claim (1), which is OKN-4. (3) The above galactooligosaccharide is O-β-D-galactopyranosyl-(1→
4) -O-β-D-galactopyranosyl-(1→4)-
A method for producing a galactooligosaccharide according to claim 1 or 7, which contains D-glucose.