JPH05130885A - Production of sugar syrup having high lactosylfructoside content by microorganism - Google Patents

Abstract

PURPOSE:To increase the content of lactosylfructoside in sugar syrup by adding a specific microorganism in the production of lactosylfructoside from sucrose and lactose, thereby consuming the by-produced glucose, etc. CONSTITUTION:A microbial strain belonging to genus Candida or Pichia, capable of assimilating glucose and fructose and incapable of assimilating lactosylfructoside, lactose and sucrose (e.g. Candida cantarellii IFO-1261) is added in the treatment of sucrose and lactose with beta-fructofuranosidase or levansucrase. The glucose and fructose are consumed and the content of lactosylfructoside in the reactional liquid is increased by this process. The reaction is preferably carried out at 30-40 deg.C and pH5-8 for 12-14hr.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to lactosylfructoside (0-β- _D -galactopyranosyl-), which is useful as a bifidobacterial growth substance and an anti-caries low-calorie sweetener.
(1-4) -0-α- _D -glucopyranosyl- (1-
2) -β- _D -fructofuranoside, lactosucrose), and more particularly to a method for producing a lactosylfructoside-rich sugar solution using a microorganism.

[0002]

2. Description of the Related Art Lactosylfructoside is a trisaccharide in which fructose is bound to the glucose residue of lactose.
It is effective as an anti-cariogenic sugar and a growth substance for Bifidobacterium, which is a useful enteric bacterium for humans.

Presently, Baltis subtilis
L. subtilis levansucrase (J. Biochem., 90, 521-526 (19).
81)), (JP-A-55-118369) and a method for synthesizing lactosylfructoside from lactose and sucrose using Levansucrase derived from a bacterium belonging to the genus Aerobacterium (JP-A-55-118369). Has been reported.

In addition, Arthrobacter
cter sp. K-1) (Agric. Biol.
Chem. , 54, 913-919 (1990)) and a method for synthesizing lactosylfructoside from lactose and sucrose using β-fructofuranosidase.

However, in these methods, several kinds of oligosaccharides other than lactosylfructoside are produced, and the yield is low, so that it is extremely difficult to separate only lactosylfructoside and it is not practical.

[0006]

The present inventors have conducted a search for microorganisms for the purpose of producing lactosylfructoside by the microorganisms, and found that the β-fructofuranosidase produced by Bacillus microorganisms has a high fructose residue. It has been found that it has group transfer ability and produces a large amount of lactosylfructoside (Japanese Patent Application No. 2-332716).

However, the yield of lactosylfructoside is about 35% even when the above method is used, and sucrose and lactose which are raw materials and glucose and fructose which are decomposition products of sucrose remain in the reaction solution. is doing. Further, when glucose, which is a decomposition product of sucrose, accumulates in the reaction solution, the production rate of lactosylfructoside also decreases.

As a method for removing saccharides other than lactosylfructoside in the reaction solution, there are known methods such as column chromatography using molecular sieve such as adsorption by activated carbon and gel filtration. However, these methods by column chromatography are not suitable for industrial production because expensive ethanol is used in a large amount or cannot be processed in a large amount.

[0009]

[Means for Solving the Problems] When the lactosylfructoside is produced by a fermentation method or an enzymatic method using sucrose and lactose as raw materials, the inventors of the present invention acted on a microorganism so that the content rate of the lactosylfructoside was In order to establish a method for increasing the glucose content, we searched for microorganisms that can utilize glucose and fructose but not the products lactosylfructoside and the raw materials lactose and sucrose. As a result, Candida
The inventors have found that microorganisms belonging to the genus da) or the genus Pichia are effective, and have reached the present invention.

That is, in the method for producing lactosylfructoside according to the present invention, when the lactosylfructoside is produced, a microorganism belonging to the genus Candida or the genus Pichia is allowed to act on glucose and fructose. It is characterized in that the content rate of lactosylfructoside can be increased at low cost by assimilating.

The present invention will be described in detail below.

Microorganisms to be used Microorganisms to be used are microorganisms belonging to the genus Candida or the genus Pichia and are capable of assimilating glucose and fructose. There is no particular limitation as long as it is a microorganism that cannot be assimilated, but Candida canterary (Candid)
a cantarellii) IFO-1261 strain, Candida ka
rawawiii) IFO-10286 strain, Pichia acaciae IF
O-1681 strain, Pichia Dispora (Pichi
a dispora) IFO-0035 strain, which utilizes glucose and fructose but does not assimilate lactose, sucrose and lactosylfructoside.

These microorganisms are known per se, but the action of these microorganisms in the present invention was first discovered by the present inventors.

As a method for culturing the above-mentioned microorganism used in the present invention, a known method for culturing a microorganism can be adopted. For example, the pH of the medium is 4 to 8, the culturing temperature is 20 to 40 ° C., and the culturing period is 1 to 4 days. Either stirring culture or static culturing can be performed.

Method for Acting on Microorganisms In the present invention, a method for effecting microorganisms is to use sucrose and lactose with Bacillus megaterium (Bac).
Hillus megaterium) IFO-134
Among the above microorganisms, when β-fructofuranosidase of 98 strain is allowed to act to produce lactosylfructoside,
At least one strain of viable cells is added at the same time, and the reaction is performed while consuming glucose as a by-product to produce lactosylfructoside in high yield. The reaction is 30-40 ° C, PH5
~ 8, 12-12 hours.

[0016] According to this method, lactose cannot be removed from the reaction solution, but glucose by-produced when the fructose residue of sucrose is transferred to lactose is immediately removed from the reaction system. Is transferred only to lactose, and the yield of the product lactosylfructoside is improved. In addition, there are few transfer products other than lactosylfructoside, and lactosylfructoside is efficiently produced.

[0017]

【Example】

<Example 1> Production of bacterial cells (MY medium) Malt extract 3 g Yeast extract 3 g Peptone 5 g Glucose 10 g Water 1000 ml Medium 21 (2 liters) having the above composition was put in a small jar fermenter and pre-cultured in the same medium in advance. Candida Canterary
tarellii) IFO-1261 strain or Candida kara
waiwii) IFO-10286 strain, Pichia acaciae IFO-
1681 strain, Pichia Dispora
dispora) IFO-0035 strain culture medium 50 ml
Was inoculated and aerobically cultured at 30 ° C. for 16 hours. After the culture was completed, the cells were collected from 21 (2 liters) of the culture solution by centrifugation (8000 rpm, 10 minutes). The collected cells were washed twice with distilled water and then wet weight 3
5.4 g, 47.2 g, 48.5 g and 53.5 g of bacterial cells were obtained. Example 2 Production of Lactosylfructoside by Enzymatic Reaction (1) 200 g of sucrose and 200 g of lactose were dissolved in a 30 mM phosphate buffer (pH 6.0) to give 11 (1 liter). Bacillus megaterium IFO-1
100 units of purified β-fructofuranosidase from strain 3498 and Candida canary
a cantarellii) 25 g of wet cells of IFO-1261 strain was added and reacted at 30 ° C. for 16 hours. After completion of the reaction, cells were removed by centrifugation (8000 rpm, 10 minutes) to obtain a supernatant.

When lactosylfructoside produced by high performance liquid chromatography was measured, it was 201 g.
Of lactosyl fructoside was produced, accounting for 74% of the total amount of sugar in the reaction solution, and glucose and fructose were scarcely observed.

Example 3 Production of Lactosylfructoside by Enzymatic Reaction (2) 200 g of sucrose and 200 g of lactose were dissolved in 30 mM phosphate buffer (pH 6.0) to make 11 (1 liter). Bacillus megaterium IFO-1
100 units of purified β-fructofuranosidase of strain 3498 and Pichia acaciae.
siae) 25 g of wet cells of IFO-1681 strain was added and reacted at 30 ° C. for 16 hours. After completion of the reaction, cells were removed by centrifugation (8000 rpm, 10 minutes) to obtain a supernatant.

The lactosylfructoside produced by high performance liquid chromatography was measured to be 204 g.
Lactosyl fructoside was produced, occupying 75% of the total amount of sugar in the reaction solution, and glucose and fructose were hardly found.

Example 4 Production of lactosylfructoside by cell reaction 150 g of sucrose and 150 g of lactose were dissolved in 30 mM phosphate buffer (pH 6.0) to give 11 (1 liter). 29 g of wet bacterium of Bacillus megaterium IFO-13498 strain having β-fructofuranosidase and Candida karawaiie (Cand)
ida karawaiwii) IFO-10286
25 g of wet bacterial cells of the strain was added and reacted at 30 ° C. for 16 hours. After completion of the reaction, cells were removed by centrifugation (8000 rpm, 10 minutes) to obtain a supernatant.

The amount of lactosylfructoside produced by high performance liquid chromatography was measured to be 148 g.
Lactosyl fructoside was produced and accounted for 73% of the total sugar amount in the reaction solution, and glucose and fructose were scarcely observed.

Example 5 Production of Lactosylfructoside by Culture Lactose 100 g Sucrose 100 g Yeast Extract 1 g KH ₂ PO ₄ 1.5 g Na ₂ HPO ₄ · 12H ₂ O 8 g Water 1000 ml pH 7.0 The medium 21 (2 liters) having the above composition was placed in a small jar fermenter and separately precultured, 50 ml of a culture solution of Bacillus megaterium IFO-13498 strain and Pichia dispora.
Add 35 g of wet cells of the IFO-0035 strain, and add 1 at 30 ° C.
Aeration culture was performed for 8 hours. The culture solution 21 (2 liters) was centrifuged (15000 rpm) to remove the cells, and a supernatant was obtained.

The amount of lactosylfructoside produced by high performance liquid chromatography was measured to be 192 g.
Of lactosyl fructoside was produced, accounting for 70% of the total sugar amount in the reaction solution, and glucose and fructose were scarcely observed.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takayuki Fukushima 4-9-11 Toyosu, Koto-ku, Tokyo Nisshin Sugar Co., Ltd. (72) Inventor Yoshihiko Tsujita 4-9-11 Toyosu, Koto-ku, Tokyo Research department of new sugar stock company

Claims (1)

[Claims] 1. When lactosylfructoside is produced by reacting sucrose and lactose with β-fructofuranosidase or levansucrose, glucose and fructose can be assimilated, but lactosylfructoside and lactose, Sucrose cannot be assimilated but can belong to the genus Candida or Phichia (P.
Lactosylfructoside by a microorganism characterized in that at least one strain among the microorganisms belonging to the genus ichia) is allowed to act and the glucose and fructose produced as by-products are consumed to increase the content of lactosylfructoside in the reaction solution. Method for producing high content sugar liquid.