JPS61289856A - Production of sweetener containing galacto-oligosaccharide - Google Patents

Abstract

PURPOSE:To obtain the titled sweetener having low lactose content, by treating a mixture of lactose and galacto-oligosaccharide with a specific beta-galactosidase. CONSTITUTION:A sugar mixture containing lactose and 1-40wt% galacto- oligosaccharide of formula (Gal is galactose residue; Glu is glucose residue; n is 1 or 2; the bond between the sugars is mainly beta-1, 4-bond) is added with a beta-galactose originated from a microorganism belonging to Kluyveromyces genus (e.g. Kluyveromyces lactis) and is made to react at 5.5-8.0pH and 5-50 deg.C for 1-3hr to decompose the lactose component into glucose and lactose. A sweetener containing a galacto-oligosaccharide having a lactose content of <=10wt% can be produced by this process.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a sweetener containing a galactooligosaccharide, and more particularly, to a method for producing a sweetener containing a galactooligosaccharide, and more specifically, a sweetener having the general formula Qal-(Gal) n-Glu
(However, Qal is a galactose residue, G is a glucose residue, n is 1 or 2, and the binding mode between sugars is mainly β-1,4 bond.)↑1 Concerning the manufacturing method of taste plates.

[Prior art] In recent years, galacto-oligosaccharides have been recognized as effective growth factors for Bifidobacterium, which is a beneficial intestinal bacterium, and
The use of sugar mixtures containing ~A oligosaccharides as sweeteners has been studied.

A method for producing a sugar mixture containing galactooligosaccharides is the direct fermentation method (Tet
rahetiron, 1960. vol, 9. p1
25-129, Can, J. Chemistry,
1964. vol, 42. p. 1341-1344, JP-A-115796), and an enzymatic method in which a specific β-galactosidase is applied to rough 1--se (ed. Eiji Shima, "Food Industry and Enzymes", p. 68, Asakura Shoten) are known. ing. In particular, galac 1 represented by the general formula Qal-(Gat)n-Glu (Gal is a galactose residue, Glu is a glucose residue, and n is an integer from 1 to 4)
~ It is known that oligosaccharides exhibit excellent stickiness in vivo (Japanese Patent Publication No. 58-20266), and the present inventors have also already proposed a method for producing this type of galacto-oligosaccharides. (Patent application No. 59-108547, Patent application No. 76767-Sho 60>.

[Problem to be Solved by the Invention 1] However, in the above method, if the reaction time is too long, the galactooligosaccharide once produced will decompose, so in order to produce galactooligosaccharide with good yield, it is necessary to It is necessary to stop the culture or stop the enzyme reaction when about 30 to 70% of a certain lactose remains. Therefore, sugar mixtures containing galactooligosaccharides produced by these methods inevitably contained a large amount of lactose.

This type of sugar mixture can be used as a flavoring agent by itself, but the large amount of lactose it contains has low solubility in water, so if it is concentrated and left to stand, the lactose will precipitate, making it difficult to handle. At the same time, there was a problem that the product value decreased. It also had the disadvantage that it felt rough on the tongue when eaten, causing discomfort, and the overall sweetness was insufficient due to the low sweetness of lactose. Furthermore, there are physiologically unfavorable aspects, such as lactose-containing substances causing diarrhea in people.

Therefore, a sugar mixture containing galactooligosaccharides has a sweetness of 1
When used as No. 11, it is expected to develop a product with as little rough 1~-s content as possible, and the purpose of the present invention is to provide a method for producing such lamiri.

Means for Solving Problem E] As a result of intensive research by the present inventors on a method for producing a sweetener containing rough 1-oligosaccharide, although the amount of rough 1-hose is small,
It was discovered that β-galactosidase derived from a microorganism belonging to the genus Klyveromyces hardly decomposes galactooligosaccharide, but selectively acts on lactose to decompose it into glucose and galactose, and the present invention reached.

That is, 11 containing the galactooligosaccharide according to the present invention
The manufacturing method for the taste test is to use lactose and the general formula 0% (1) (where Qal is a galactose residue, G1 (1 is a glucose residue, n is 1 or 2, and the binding mode between sugars is mainly β). The content of lactose in the sugar composition is reduced by allowing β-galactosidase derived from a microorganism belonging to the genus Cryperomyces to act on a sugar mixture containing a galactooligosaccharide represented by -1,4 bond. It is something to do.

The present invention will be explained in detail below.

(a) Raw sugar mixture The present invention provides that the galactooligosaccharide represented by the formula (1) (hereinafter simply referred to as galactooligosaccharide) hardly decomposes,
Since lactose is selectively decomposed, the raw sugar mixture is not particularly limited as long as it contains these sugar components. Furthermore, the present invention is not only applicable to sugar mixtures produced when the above-mentioned galactooligosaccharides are produced from lactose or lactose-containing materials, but also to
It can also be applied to sugar mixtures containing galacto-oligosaccharides in which lactose is produced or mixed due to other factors. The m degree of the sugar mixture is preferably in the range of 1 to 40% by weight, more preferably in the range of 3 to 30% by weight.

The raw sugar mixture used in the present invention is, for example, Cryptococcus (Cryptococcus), as previously proposed by the present inventors.
microorganisms belonging to the genus cOcclls) can act on lactose or lactose-containing substances to produce 1q (
Patent application No. 59-108547, Patent application No. 6O-76767)
.

Alternatively, it can also be obtained using a transfer reaction using β-galactosidase obtained from filamentous fungi, yeast, bacteria, etc. (For example, in Japanese Patent Publication No. 58-20266, A method is described for treating lactose- or lactose-containing substances with β-galactosidase). Sugar mixtures obtained by these methods generally contain approximately 30 to 80% by weight of rough]-s based on the total sugar.

(b) β-galactosidase The β-galactosidase that acts on the sugar mixture is not particularly limited as long as it is derived from a microorganism belonging to the genus Klyperomyces, but specifically, β-galactosidase is derived from Klyveromyces lactis.
derived from β-galactosidase (trade name: Ma×1lact
; Manufactured by Gist-Brocades, trade name: LactaSe GODO-YNL; Joint Sake Refining) can be used. Although these enzymes themselves are known, the selective action of these enzymes in the present invention was discovered for the first time by the present inventors.

The β-galactosidase used in the present invention can be immobilized by a known immobilization method and can also be used as an immobilized enzyme.

(C) Conditions for producing sweeteners In order to cause the β-galactosidase to act on the raw sugar mixture, β-galactosidase can be added as is in a conventional manner and carried out in a batch process, or it can be immobilized as an immobilized enzyme. It may also be carried out using a floor flow system. As the operating conditions for β-galactosidase, usual conditions using this type of enzyme can be adopted. That is, l)H is
Although it varies depending on other conditions, it is generally in the range of 5.5 to 8.0, preferably 6.0 to 7.0. The working temperature is 5
・The temperature is ~50°C, preferably 25-35°C, and the working temperature varies depending on the amount of enzyme used, but the process should be carried out until the lactose content in the resulting sugar mixture is 10% by weight or less based on the total sugar. is preferable, and is usually 1 hour and 1 to 3 days.

Preferably, 1q of lactose is reduced to 1q in this way.
The sugar mixture containing galacto-oligosaccharides reduced to 0% by weight or less is subjected to purification operations such as decolorization using known activated carbon and desalting using ion exchange resins, as necessary, to obtain a colorless and transparent liquid.
4 Seasonings.

[Function] In the present invention, when the β-rough 1-sidase is made to act on the sugar mixture containing lactose and the galactooligosaccharide, the β-galactosidase selectively reacts with the lactose component without almost decomposing the galactooligosaccharide. It decomposes it into glucose and galactose, and as a result, the lactose content in the sugar mixture decreases.

As shown in Comparative Example 1, the β-galactosidase is produced using the general formula % formula % (a1 is a galactose residue, Glu is a glucose residue, n
is converted into the oligosaccharides represented by 1 to 3), but these A ribosaccharides are in small amounts, and the galactinyl sugars originally present are
- This is preferable because it does not affect oligosaccharides in any way, but rather increases the total breakdown of oligosaccharides containing galactose residues.

The sugar mixture in which the lactonis content is reduced to 10% by weight or less based on the total sugar by the method of the present invention has a sugar content of 50% by weight.
Even when concentrated to the above concentration, lactose precipitation is not observed, and the tongue does not feel rough when eating or drinking. Also,
The U intensity increases by about 1.5 times or more compared to untreated products that do not apply the method of the present invention, and the quality of sweetness becomes smooth and supple.

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

18 = The standard method for sugars in the following Examples and Comparative Examples is high performance liquid chromatography (the pump is Hitachi Model 655,
The detector is Showa Denko 5E-31, and the column is L 1ch.
osorb -N +-12 (5qm) Cica
Made by Merc, solvent is acetonitrile:water-65:35
using a flow rate of 0.7 ml/min,
It was determined from the peak area.

Example 1 Preparation of thick sugar mixture (+) 500ml! 100ml of medium with the composition shown in Table 1 in an Erlenmeyer flask! MY
Cryptococcus laurentii var. laurentii (Cry.
ptococcus 1aurentii Var, 1
aUrentii ) OKN-4 (Microtechnical Laboratory No. 76
No. 29) was inoculated into a platinum loop and cultured at 30°C for 6 days in a rotary shaking incubator. The obtained culture was centrifuged at 111f in a centrifuge (8000 rpm, 10 minutes).
and add 1 [liquid].

A high performance liquid chromatograph of this supernatant liquid is shown in FIG. The composition of this supernatant was 3.0% by weight of lactose, 5.0% by weight of galactooligosaccharide, and 8.0 g of total sugar.

Table 1 Culture composition Lactose 100 (INH4C
1! 2 (+yeast extract
0.2gKH2PO40.8! J Na 2 HPO4・12H200,3QM(J 80
4 ・7H200.02g water
1QpLI
6.0 (2> A loopful of the 0KN-4 strain, which had been precultured on a MY agar slant medium for 2 days, was added to a sterilized 500 m Erlenmeyer flask containing medium 1001119 with the composition shown in Table 2. The cells were inoculated and cultured in a rotary shaking incubator for 2 days at 30°C.The resulting culture was centrifuged using a centrifuge (8000 ppm, 10
), and the bacterial cells were collected. The recovered bacterial cells were washed twice with pure water. 01.15.
50ml of 10% lactose adjusted to 5! In addition, 6
The reaction was carried out at 5°C for 5 hours. This reaction solution contained 10 g in total, and its sugar composition was: galac I-Δligosaccharide 2
5% by weight, lactose 44% by weight, central axis (glucose,
Galactose) was 19%.

Table 2 Culture composition Rough 1~-5g (N+-14) 2804 0.5 QKt
-12P04 0,1 aM (IS
04 ・71-(200.05g yeast extract
o, i g water
1e1) H6,0 (3) The galac]-A oligosaccharide produced in (1) and (2) above has a melting point of 229.5 to 230.5°C and is represented by the following formula (II) with molecular part 504. 0-β-D-
It was confirmed that it was galactopyranosyl-(1→4)-0-β-D-galactopyranosyl-(1→4)-D-glucose.

Example 2 After adjusting 1000 q of the supernatant containing galacto-oligosaccharide obtained in Example 1 (1) to pH 6.0, β-galact1-sidase derived from Kuliveromyces lactis (trade name L actase-GODO-) was added. YNL: Godo Sake Refining Co., Ltd.) 5o was added and reacted at 30°C for 3 hours.

After the reaction was completed, the enzyme was inactivated by placing it in a boiling water bath for 5 minutes. A high performance liquid chromatograph of this reaction solution is shown in FIG. The sugar composition in the reaction solution was 33.3% by weight of monosaccharides (glucose, galactose), 5.5% by weight of lactose, and 61.2% by weight of galactooligosaccharide, and the total sugar content was 8% by weight.
This reaction solution was decolorized with activated carbon and desalted using an ion exchange resin, and then concentrated in vacuo.
A 0% (W/W) liquid material 1009 was obtained. This allows j
Table 3 shows the results of comparing the purified product of the present invention with a comparative product that was purified and concentrated without treatment with β-galactosidase.

Table 3 Example 3 After adjusting the reaction solution 1000j7 containing galac 1 to oligosaccharide obtained in Example 1 (2) to pt-16.0, β-galac 1-sidase derived from Kluveromyces lactis ( Product name Maxilact: Glsj-B
rocades) was added thereto, and the mixture was reacted at 30° C. for 3 hours, and then placed in a boiling water bath for 5 minutes to avoid stopping the reaction. The sugar composition of this reaction solution is monosaccharide (
glucose, galactose) 49%, lactose 6%
It contained Φ% and 25% by weight of galactooligosaccharide.

This reaction solution was purified in the same manner as in Example 2, and then concentrated in vacuo.
A liquid product of 0% (W/W > 150!I+ was added to (9). This liquid product did not form a white precipitate for more than a month even when left at room temperature, and had a smooth and mild sweet taste.

Comparative example 1 30% lactose solution (adjusted to pl-16, o) 10
0g, β-galactosidase derived from Culiveromyces lactis (trade name 1 actase GOD O
-YNL; 0.3g of Godo Sake Refining) was added and heated to 30°C.
Allowed time to react. Then, the reaction was stopped by placing it in a boiling water bath for 5 minutes. A high performance liquid chromatograph of this reaction solution is shown in FIG. This product contained 57% by weight of monosaccharides (glucose, galactose), 8.5% by weight of lactose, and 34.5% by weight of oligosaccharides higher than disaccharides, based on the total sugars.

Comparative Example 2 Same as Example 1 (1) (lactose 1.0
g1 Liquid containing 1.0 g of galactooligosaccharide 10 InQ
(rlH6,0> to 7 supergills 71st! (A
β- derived from Spergillus Qryzae)
Galactosidase (trade name: Oryzazyme; manufactured by Yakult)
10 mg was added and reacted at 30°C. After 1 hour, the reaction was stopped by heating in a boiling water bath for 3 minutes. High performance liquid chromatographs of the liquid before and after the reaction are shown in Figures 4 and 5, respectively. Galactooligosaccharides are decomposed by this β-galactosidase, and after the reaction, the 115
It became below.

[Brief explanation of the drawing]

FIG. 1 is a high-speed chromatograph showing the sugar composition of the supernatant obtained in Example 1 (1), FIG. 2 is a high-speed chromatograph showing the sugar composition of the supernatant obtained in Example 2, Figure 3 is a high-speed chromatograph showing the sugar composition of the supernatant liquid obtained in Comparative Example 1, and Figures 4 and 5 are high-speed chromatographs showing the sugar composition of the liquid before and after the reaction, respectively, in Comparative Example 2. It is a graph. Patent applicant: Nisshin Sugar Co., Ltd. Figure 1 Figure 2 Figure 3 Iconography Figure 4 Body 2 inches Figure 5

Claims (1)

[Claims] Lactose and the general formula Gal-(Gal)n-Glu(
However, 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. ) and a galactooligosaccharide represented by Klyveromyces (Klyvero
1. A method for producing a sweetener containing galactooligosaccharides, which comprises reducing the content of lactose in the sugar composition by activating β-galactosidase derived from a microorganism belonging to the genus S. myces.