JPS6349983B2 - - Google Patents

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 specifically, to a method for producing a sweetener containing a galactooligosaccharide, and more specifically, to a method for producing a sweetener containing a galactooligosaccharide, and more specifically,
A sweetener containing a galactooligosaccharide represented by Glu (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). Regarding manufacturing methods. [Prior Art] In recent years, galactooligosaccharides have been recognized to be effective as growth factors for Bifidobacteria, which are useful bacteria in the intestines, and the use of sugar mixtures containing galactooligosaccharides as sweeteners has been studied. As a method for producing sugar mixtures containing galactooligosaccharides, there is a 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), and an enzymatic method in which a specific β-galactosidase acts on lactose (ed. "Food Industry and Enzymes" edited by Eiji Shima, p. 68, Asakura Shoten) are known. In particular, the general formula Gal−
(Gal) n-Glu (Gal is a galactose residue,
It is known that galactooligosaccharides represented by Glu (Glu is a glucose residue and n is an integer from 1 to 4) exhibit excellent activity in vivo (Japanese Patent Publication No. 58-20266), and the present inventors also In addition, a method for producing this type of galacto-oligosaccharide has already been proposed (Japanese Patent Application No. 108547/1983, Patent Application No. 76767/1983). [Problems to be solved by the invention] However, in the above method, if the reaction time is too long, the galactooligosaccharides that have been produced will decompose, so it is necessary to use the raw materials to produce galactooligosaccharides in good yield. Some lactose is about 30
It is necessary to stop the culture or stop the enzyme reaction when ~70% remains. Therefore, a large amount of lactose was inevitably present in the sugar mixture containing galactooligosaccharide produced by these methods. This type of sugar mixture can be used as a sweetener 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. 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, it also has physiologically unfavorable aspects, such as causing diarrhea in people with lactose intolerance. Therefore, when using a sugar mixture containing galactooligosaccharide as a sweetener, it is expected to develop a product with as little lactose as possible.
The present invention aims to provide a method for producing such a sweetener. [Means for Solving the Problems] As a result of intensive research into a method for producing a sweetener containing galactooligosaccharide with a low lactose content, the present inventors discovered that Kluyveromyces
The present inventors have discovered that β-galactosidase derived from a microorganism belonging to the genus hardly decomposes galactooligosaccharide, but selectively acts on lactose to decompose it into glucose and galactose. That is, the method for producing a sweetener containing a galactooligosaccharide according to the present invention uses lactose and the general formula Gal-(Gal)n-Glu...() (where Gal is a galactose residue, Glu is a glucose residue, and n is 1 or 2, the binding mode between sugars and sugars is mainly β-1,4 bonds.) A sugar mixture containing galacto-oligosaccharides represented by It is characterized by reducing the content of lactose in it. The present invention will be explained in detail below. (a) Raw material sugar mixture In the present invention, since the galactooligosaccharide represented by the above formula (2) (hereinafter simply referred to as galactooligosaccharide) is hardly decomposed and lactose is selectively decomposed, the raw material sugar mixture is It is not particularly limited as long as it contains a sugar component of. Furthermore, the present invention is not only applicable to a sugar mixture obtained when the above-mentioned galactooligosaccharide is produced from lactose or a lactose-containing material, but also lactose is produced in a sugar mixture containing galactooligosaccharide due to other factors. Alternatively, it can also be applied to a sugar mixture obtained by mixing. The concentration of the sugar mixture is preferably in the range of 1 to 40% by weight, and 3 to 30% by weight.
The range is more preferable. The raw sugar mixture used in the present invention can be obtained, for example, by allowing a microorganism belonging to the genus Cryptococcus to act on lactose or a lactose-containing material, as previously proposed by the present inventors (patent application). Showa 59-108547, special request show
60−76767). Alternatively, it can also be obtained using a transfer reaction using β-galactosidase obtained from filamentous fungi, yeast, bacteria, etc. (for example,
Japanese Patent Publication No. 58-20266 describes a method for treating lactose or lactose-containing substances with β-galactosidase produced by Aspergillus oryzae). The sugar mixture obtained by these methods generally contains lactose in an amount of about 30 to 80% by weight 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 Kluyveromyces, but specifically β-galactosidase derived from Kluyveromyces lactis ( Product name Maxilact; Gist−Brocades
Lactase GODO-YNL, manufactured by Godo Sake Refining Co., Ltd., trade name: Lactase GODO-YNL, 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 it is according to a conventional method and carried out in a batch process, or it can be carried out as an immobilized enzyme through fixed bed distribution. It can also be done using a formula. As the operating conditions for β-galactosidase, usual conditions using this type of enzyme can be adopted. That is, P.H.
is generally 5.5, although it depends on other conditions.
-8.0, preferably 6.0-7.0. The action temperature is 5 to 50°C, preferably 25 to 35°C, and the action time varies depending on the amount of enzyme used, but the lactose content in the resulting sugar mixture is 10% by weight or less based on the total sugar. It is preferable to carry out the process until the temperature reaches the desired temperature, and usually for 1 hour to 3 days. The thus obtained sugar mixture containing galactooligosaccharide with a lactose content of preferably 10% by weight or less is subjected to purification operations such as decolorization with known activated carbon and desalting with an ion exchange resin, as necessary, to make it colorless. A clear liquid sweetener. [Function] In the present invention, when the β-galactosidase is made to act on a sugar mixture containing lactose and the galactooligosaccharide, the β-galactosidase selectively acts on the lactose component without almost decomposing the galactooligosaccharide. This is broken down into glucose and galactose, thereby reducing the lactose content in the sugar mixture. As shown in Comparative Example 1, the β-galactosidase is produced by converting a part of lactose into the general formula
Gal-Gal, Gal-(Gal) _o -Gul (Gal is a galactose residue, Glu is a glucose residue, n is 1 to
3), but these oligosaccharides are in small amounts and do not have any effect on the galacto-oligosaccharides originally present.
Rather, it is preferable because the total amount of oligosaccharides containing galactose residues increases. When the lactose content in the sugar mixture is reduced to 10% by weight or less based on the total sugar by the method of the present invention, no precipitation of lactose is observed even when the sugar content is concentrated to a concentration of 50% by weight or more. I never feel any roughness on my tongue. In addition, the sweetness level is increased by about 1.5 times or more compared to the untreated product that does not apply the method of the present invention, and the quality of the sweetness is smooth and refreshing. [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 and Comparative Examples was performed using high-performance liquid chromatography (pump: Hitachi model 655, detector: Showa Denko SE-31, column: Lichrosorb-NH ₂ (5 μm), manufactured by Cica Merk, The solvent used was acetonitrile:water = 65:35, and the flow rate was
0.7 ml/min) and determined from the peak area. Example 1 Preparation of raw sugar mixture (1) 100 ml of a medium having the composition shown in Table 1 was placed in a 500 ml Erlenmeyer flask, sterilized, and Cryptococcus lauren precultured on MY agar slant medium for 2 days. Cryptococcus laurentii var.laurentii
One 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 3.0% 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 and 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 10 g of total sugar, and its sugar composition was 25% by weight galactooligosaccharides, 44% by weight lactose, and 19% by weight monosaccharides (glucose, galactose). 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 After adjusting 1000 g of the supernatant containing galactooligosaccharides obtained in Example 1 (1) to pH 6.0, β-galactosidase derived from Krivellomyces lactis (trade name Lactase-GODO-YNL; Godo Sake) was added. purification)
5 g was added and reacted at 30°C for 3 hours. After the reaction, 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) and 5.5% by weight of lactose.
Galacto-oligosaccharide was 61.2% by weight, and sugar content was 80g as total sugar. This reaction solution was decolorized with activated carbon and desalted using an ion exchange resin, and then concentrated in vacuo to form a 60% (W-W) liquid.
I got g. The product of the present invention thus obtained is β-
Table 3 shows the results of comparison with a comparative product that was purified and concentrated without being treated with galactosidase.

[Table] Example 3 After adjusting 1000 g of the reaction solution containing the galactooligosaccharide obtained in (2) of Example 1 to pH 6.0, β-galactosidase derived from Kuliveromyces lactis (trade name: Maxilcat; manufactured by Gist-Brocades) was added. After adding 5 g of the product (manufactured by M.D.), the mixture was reacted at 30° C. for 3 hours, and then placed in a boiling water bath for 5 minutes to stop the reaction. The sugar composition of this reaction solution is monosaccharides (glucose,
Galactose) 49% by weight, lactose 6% by weight,
It contained 25% by weight of galactooligosaccharides. This reaction solution was purified in the same manner as in Example 2, and then concentrated in vacuo to obtain 150 g of a 60% (W/W) liquid. This liquid did not form a white precipitate even after being left at room temperature for more than a month, and had a smooth and refreshing sweet taste. Comparative example 1 100g of 30% lactose solution (adjusted to PH6.0)
β-galactosidase derived from Kriverominus lactis (trade name: Lactase GODO-YNL;
0.3g of Godo Sake Refining) was added and reacted at 30°C for 3 hours. 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 Lactose 1.0 obtained in the same manner as in Example 1 (1)
g, 10 ml of liquid containing 1.0 g of galactooligosaccharides (PH
6.0) to Aspergillus oryzae
Add 10 mg of β-galactosidase derived from Oryzae (trade name: Oryzazyme; manufactured by Yakult), and
I reacted with 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. Galactooligosaccharide is decomposed by this β-galactosidase, and after the reaction it is reduced to 1/1 of the amount before the reaction.
It became 5 or less.

[Brief explanation of drawings]

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 Comparative Example 1, Figures 4 and 5 are Comparative Example 2. This is a high-speed chromatograph showing the sugar composition of the solution before and after the reaction, respectively.

Claims (1)

[Claims] 1 Lactose and 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.) A method for producing a sweetener containing galactooligosaccharides, which comprises reducing the content of lactose in the sugar composition by allowing β-galactosidase derived from a microorganism belonging to the genus Kluyveromyces to act on the sweetener.