Method for producing composition, oligosaccharide-containing composition obtained therefrom, and uses thereof

Abstract

Provided is a method for efficiently utilizing by-products associated with the production of dairy products. In addition, the present invention addresses the problem of providing a method for producing galactosyl kojobiose having an excellent prebiotic function. The present invention addresses the problem of providing a solution and a composition containing galactosyl trilobiose in which at least 80% of galactosyl trilobiose is retained even when heated. Provided is a method for producing an oligosaccharide-containing composition, said method comprising a step in which a glucosucrase is caused to act on a composition containing lactose and sucrose to obtain a composition containing galactosyl trojobiose. It is preferable to use a lactic acid bacterium that constitutively expresses a film-bound glucan sucrase. The pH of a solution containing galactosyl trojobiose and having a pH of less than 7 and a composition containing galactosyl trojobiose is less than 7 when the solution is made into a solution having a solid content concentration of 10-80%.

Description

Technical Field

[0001] The present invention relates to a method for producing a composition containing oligosaccharides such as galactosyl kojibiose, an oligosaccharide-containing composition obtained thereby, and uses thereof. Background Art

[0002] Lactose accounts for about 99.8% of the sugar content of milk and is usually obtained as a by-product in the process of manufacturing dairy products. Regarding the use of lactose, Patent Document 1 relates to the production of oligosaccharides and records the following method: using galactose and / or lactose as sugar acceptors and starch partial hydrolysate as sugar donors, and allowing glycotransferase to act on a mixed solution of galactose and / or lactose and starch hydrolysate to produce a mixture of oligosaccharides containing galactose units in the molecule. It also records the use of the supernatant of the culture solution of Bacillus macerans (IFO 8490, IAM 1227) and the like as glycotransferase.

[0003] On the other hand, it is known that glycotransferases include lactases with lactose as a sugar donor and a sugar acceptor, enzymes with lactose as a sugar acceptor and other sugars as sugar donors, etc. Glucansucrase, one of the latter, has the activity of transferring glucose to various sugars with sucrose as a sugar donor. It is known that glucanase can be expressed in certain microorganisms, and there are some reports on its use. For example, Patent Document 2 records a reaction composition comprising at least water, sucrose, an α-glucan substrate, and a polypeptide that can form at least one α-1,2 branched chain from the α-glucan substrate. As an example of the polypeptide, glucosyltransferases derived from microorganisms such as tropical fructobacillus (Fructobacillus tropaeoli) can be cited. Patent Document 3 records a method for using glycotransferases to improve the texture of fermented milk-based products. As glycotransferases, glucosyltransferases from Streptococcus salivarius SK126 and their mutants are recorded.

[0004] In addition, lactic acid bacteria expressing glucan sucrase are known. For example, non-patent document 1 records the use of Leuconostoc mesenteroides to obtain trisaccharides from a mixed sugar culture solution of sucrose and lactose. In addition, non-patent document 2 relates to the synthesis of glucan from the genus Lactobacillus, and the document records the isolation and characteristics of glucan from 6 strains of the genus Lactobacillus, the enzyme, and the generated glucan. In addition, non-patent document 3 relates to the production of glucan using glucan sucrase derived from the red-striped liquid lactobacillus strain isolated from the fermented beverage starter culture. The document records the following technology: water-soluble dextrins composed of D-glucose units of α-(1→6) bonds and D-glucose units containing both α-(1→6) bonds and α-(1→3) bonds and water-insoluble glucan are produced by the strain from sucrose. Furthermore, non-patent document 4 relates to two kinds of glucan sucrases (GtfA and Gtf180) derived from Lactobacillus reuteri. The document records the structural characteristics of glucosylated lactose derivatives synthesized by enzymes, and records that two enzymes can be used to bond glucosyl residues (α1→2) to the reducing glucose units of lactose. In addition, patent document 4 records edible compositions of lactic acid bacteria, enzymes synthesized by the lactic acid bacteria, and extracellular polysaccharide (EPS) products containing the enzymes. As a preferred example of lactic acid bacteria, lactic acid bacteria of the genus Lactobacillus are listed, and it is recorded that EPS is obtained by fermentation of a specific species of lactic acid bacteria that can produce glucan sucrase.

[0005] On the other hand, galactosyl kojibiose with kojibiose as a constituent sugar has a structure in which a glucosyl residue is bonded to the glucose unit of lactose. Regarding kojibiose and oligosaccharides with kojibiose as a constituent sugar, Patent Document 5 records an anti-inflammatory agent containing a saccharide having a ketone hexose structure in the molecule as an effective ingredient, and as preferred examples of saccharides having a ketone hexose structure in the molecule, 3-ketotrehalose, 3-ketothiose, 3-ketoisomaltose, 3-ketolactose, 3-ketomaltose, 3-ketosucrose and 3-ketomaltitol are listed. In addition, Patent Document 6 records an in vivo lipid regulator containing one or more selected from kojibiose, kojitriose, kojibiose glycoside, kojitetraose and kojitriose glycoside as an effective ingredient.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 48-98093

[0009] Patent Document 2: WO2017 / 091533 (Japanese Patent Publication No. 2019-505171)

[0010] Patent Document 3: WO2020 / 009893

[0011] Patent Document 4: WO2004 / 013343 (Japanese Patent Publication No. 2005-534315)

[0012] Patent document 5: WO2017 / 018336 (Japanese Patent No. 6729913)

[0013] Patent Document 6: Japanese Patent Application Publication No. 2005-281188 (Japanese Patent Publication No. 4746281)

[0014] Non-patent literature

[0015] Non-patent document 1: Sugar content in sake and rice koji juice. Journal of the Japan Brewing Association 53(11), 859-854, (1958)

[0016] Non-patent document 2: Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains-Microbiology (2004), 150, 3681-3690

[0017] Non-patent document 3: The production of glucans via glucansucrases fromLactoacillus satsumensis isolated from a fermented beverage starterculture.Gregory L Cote et al., Appl.Microbiol.Biotechnol.(2013)Aug;97(16):7265-73

[0018] Non-patent document 4: Structural characterization of glucosylated lactosederivatives synthesized by the Lactobacillus reuteri GtfAand Gtf180glucansucrase enzymes. Carbohydrate Research 449(2017)59-64 Summary of the invention

[0019] Problem that the invention aims to solve

[0020] When cheese and milk protein concentrate (MPC) are produced from raw milk, whey is produced as a byproduct in the former and membrane permeate is produced as a byproduct in the latter. These mainly contain lactose and minerals and are sometimes used as livestock feed, etc., but the consumption is limited. In recent years, from the perspective of effective utilization of resources, the demand for effective utilization of these byproducts accompanying the production of cheese and milk raw materials has increased compared to the past. As one of the effective utilization of byproducts, it is considered to allow glycotransferase to act on lactose in the byproducts to produce oligosaccharides.

[0021] On the other hand, galactosyl kojibiose, one of the oligosaccharides, has a structure in which a glucosyl residue is bonded to a glucose unit of lactose, and according to the research of the present inventors, it has been found that it has excellent prebiotic function and is expected to become a new functional ingredient in food, medicine and cosmetics. However, there are almost no reports on its production method.

[0022] In addition, according to the research of the present inventors, it is known that in the production of galactosyl kojibiose, galactosyl kojibiose may be isomerized in the process of spray drying a solution containing the sugar, or in the process of heating and concentrating the sugar solution, or heat sterilizing the sugar solution. It is desirable that at least 80% of galactosyl kojibiose is retained even when the process involves heating.

[0023] Solutions for solving problems

[0024] The present inventors found that galactosyl kojibiose was obtained in high yield by mixing glucansucrase with sucrose and lactose, which is a sugar acceptor in a sugar transfer reaction, and performing an enzyme reaction under appropriate conditions.

[0025] The present inventors have also found that a solution of galactosyl kojibiose has a tendency to have high thermal stability at the acidic side and to decrease thermal stability when the pH exceeds a specific value. In addition, the present inventors have identified the chemical structure of sugars generated by isomerization of galactosyl kojibiose.

[0026] The present invention provides the following aspects in one embodiment.

[0027] [1] A method for producing an oligosaccharide-containing composition, comprising the step of allowing a composition containing lactose and sucrose to act with dextran sucrase to obtain a composition containing galactosyl kojibiose.

[0028] [2] The production method according to item 1, wherein the dextran sucrase is used in the form of lactic acid bacteria or a processed product thereof.

[0029] [3] The production method according to 2, wherein the lactic acid bacteria are lactic acid bacteria that constitutively express glucansucrase.

[0030] [4] The production method according to any one of 1 to 3, wherein the dextran sucrase is of a bacterial cell-bound type.

[0031] [5] The production method according to any one of 1 to 4, wherein the dextransucrase has dextransucrase activity.

[0032] [6] The production method according to any one of 2 to 5, wherein the lactic acid bacteria or the processed product thereof is any one selected from the group consisting of living bacteria, dead bacteria, a culture containing bacteria, a bacterial cell disrupted product, and a purified product of glucansucrase.

[0033] [7] The production method according to any one of 1 to 6, wherein the glucansucrase or the lactic acid bacteria expressing glucansucrase or the processed product thereof is derived from a lactic acid bacterium belonging to the family Lactobacillaceae.

[0034] [8] The production method according to any one of 1 to 7, wherein the glucansucrase or the lactic acid bacteria expressing glucansucrase or the processed product thereof is derived from lactic acid bacteria belonging to the genus Liquorilactobacillus.

[0035] [9] The production method according to any one of 1 to 8, wherein the glucansucrase or the lactic acid bacteria expressing the glucansucrase or the processed product thereof is derived from a lactic acid bacterium belonging to Liquorilactobacillus satsumensis.

[0036]

[10] A method for producing a composition containing oligosaccharides, comprising the step of allowing a dextran sucrase composed of the following protein (A), (B) or (C) to act on a composition containing lactose and sucrose to obtain a composition containing galactosyl kojibiose.

[0037] (A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8;

[0038] (B) a protein consisting of an amino acid sequence having a high sequence identity to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity;

[0039] (C) A protein comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted and / or added to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity.

[0040]

[11] The production method according to item 10, wherein the glucansucrase is composed of the following protein (B') or (C').

[0041] (B') a protein having a high sequence identity to the amino acid sequences described in SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1, and having glucansucrase activity;

[0042] (C') A protein having glucansucrase activity and comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted and / or added in the amino acid sequences of SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the sequence corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1.

[0043]

[12] A method for producing fermented milk containing galactosyl kojibiose, comprising:

[0044] a step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing raw material milk and sucrose to obtain a composition containing galactosylkojibiose; and

[0045] A step of adding lactic acid bacteria starter to raw milk and fermenting the raw milk to obtain fermented milk.

[0046]

[13] An enzyme agent comprising glucansucrase composed of the protein of (A), (B) or (C) below.

[0047] (A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8;

[0048] (B) a protein consisting of an amino acid sequence having a high sequence identity to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity;

[0049] (C) A protein comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted and / or added to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity.

[0050]

[14] The enzyme agent according to 13, wherein the glucansucrase is composed of the following protein (B') or (C').

[0051] (B') a protein having a high sequence identity to the amino acid sequences described in SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1, and having glucansucrase activity;

[0052] (C') A protein having glucansucrase activity and comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted and / or added in the amino acid sequences of SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the sequence corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1.

[0053]

[15] The enzyme agent according to item 13 or 14, wherein the glucansucrase is contained in the form of any one selected from the group consisting of living cells, dead cells, cultures containing cells, and cell fragments of lactic acid bacteria expressing glucansucrase.

[0054]

[16] The enzyme agent according to any one of 13 to 15, which is used in the production method according to any one of 1 to 12.

[0055]

[17] The enzyme agent according to any one of 13 to 16, which is used for producing GlucitylEpilactose.

[0056]

[18] A solution comprising galactosyl kojibiose, wherein the pH of the solution is less than 7.

[0057]

[19] A composition comprising galactosyl kojibiose, wherein the composition has a pH of less than 7 when prepared as a solution having a solid content concentration of 10 to 80%.

[0058]

[20] The solution according to item 18 or the composition according to item 19, wherein the fructose content per unit solid content is 10% or more.

[0059]

[21] The solution according to 18 or the composition according to 19 or 20, which is obtained by the production method according to any one of 1 to 12.

[0060]

[22] A composition comprising galactosyl kojibiose having the following characteristics.

[0061] (1) containing 15 to 40% galactosyl kojibiose per unit solid content;

[0062] (2) containing 15-45% fructose per unit solid content;

[0063] (3) When a solution having a solid content concentration of 12% is prepared and heated at 120°C for 20 minutes, the residual rate of galactosyl kojibiose is 80% or more.

[0064]

[23] Glucosyl epilactose or a composition comprising the same.

[0065]

[24] The production method according to any one of 1 to 12, further comprising a step of adjusting the pH of the obtained composition to less than 7.

[0066]

[25] The method for producing an oligosaccharide-containing composition according to any one of 1 to 12 and 24, wherein the oligosaccharide-containing composition comprises galactosyl kojibiose and fructose.

[0067]

[26] The method for producing an oligosaccharide-containing composition according to any one of 1 to 12, 24 and 25, wherein the oligosaccharide-containing composition contains 10% to 100% of galactosyl kojibiose per unit solid content.

[0068]

[27] The method for producing an oligosaccharide-containing composition according to any one of 1 to 12 and 24 to 26, wherein the oligosaccharide-containing composition further contains 1% or more of fructose per unit solid content.

[0069]

[28] The method for producing an oligosaccharide-containing composition according to any one of 1 to 12 and 24 to 27, wherein the mass ratio of galactosyl kojibiose to lactose (galactosyl kojibiose:lactose) in the oligosaccharide-containing composition is 15 to 40:15 to 30.

[0070]

[29] The method for producing an oligosaccharide-containing composition according to any one of 1 to 12 and 24 to 28, wherein the oligosaccharide-containing composition contains glucosyl epilactose.

[0071]

[30] The production method according to any one of 1 to 6 and 24 to 29, which is used to produce the solution according to 18 or the oligosaccharide-containing composition according to any one of 19 to 23.

[0072] The present invention provides the following aspects in one embodiment.

[0073] [1] A method for producing a composition containing oligosaccharides, comprising:

[0074] A step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to produce a composition containing galactosylkojibiose.

[0075] [2] The production method according to item 1, wherein the lactic acid bacteria are lactic acid bacteria that constitutively express glucansucrase.

[0076] [3] The production method according to 1 or 2, wherein the dextran sucrase is of a bacterial cell-bound type.

[0077] [4] The production method according to any one of 1 to 3, wherein the lactic acid bacteria expressing glucansucrase or a processed product thereof is any one selected from the group consisting of living bacteria, dead bacteria, a culture containing bacteria, a bacterial cell fragment, and a purified product of glucansucrase.

[0078] [5] The production method according to any one of 1 to 4, wherein the lactic acid bacteria expressing glucansucrase or a processed product thereof is derived from a lactic acid bacterium belonging to the family Lactobacillaceae.

[0079] [6] The production method according to any one of 1 to 5, wherein the lactic acid bacteria expressing glucansucrase or a processed product thereof is derived from lactic acid bacteria belonging to the genus Liquorilactobacillus.

[0080] [7] The production method according to any one of 1 to 6, wherein the lactic acid bacteria expressing glucansucrase or a processed product thereof is derived from lactic acid bacteria belonging to Liquorilactobacillus satsumensis.

[0081] [8] The production method according to any one of 1 to 7, which is a method for producing a composition containing galactosyl kojibiose, the production method comprising the step of purifying galactosyl kojibiose from the obtained composition containing galactosyl kojibiose.

[0082] [9] A composition containing galactosyl kojibiose, which is obtained by allowing a composition containing lactose and sucrose to act on a lactic acid bacterium expressing glucansucrase or a processed product thereof.

[0083]

[10] A method for producing fermented milk containing galactosyl kojibiose, comprising:

[0084] a step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing raw material milk and sucrose to obtain a composition containing galactosylkojibiose; and

[0085] A step of adding lactic acid bacteria starter to raw milk and fermenting the raw milk to obtain fermented milk.

[0086]

[11] An enzyme preparation comprising any one selected from the group consisting of living cells, dead cells, cultures containing cells, and cell fragments of lactic acid bacteria expressing cell-bound glucansucrase.

[0087]

[12] The enzyme agent according to item 11, wherein the lactic acid bacteria are lactic acid bacteria that constitutively express glucansucrase.

[0088]

[13] The enzyme agent according to 11 or 12, which is used for treating a composition containing lactose.

[0089]

[14] Use of a composition containing lactose and sucrose as a raw material for obtaining a composition containing galactosylkojibiose.

[0090]

[15] The use according to item 14, wherein the composition containing lactose is any one selected from the group consisting of raw milk, skim milk, a reconstituted liquid of a dried product containing lactose, skim concentrated milk, whey, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolated (WPI), a membrane permeate or membrane retentate derived from a milk raw material, a milk concentrate, and a lactic acid bacteria culture containing lactose.

[0091]

[16] The use according to item 14 or 15, which is used to obtain galactosyl kojibiose using a lactic acid bacterium expressing glucansucrase or a processed product thereof.

[0092]

[17] The use according to item 16, wherein the lactic acid bacteria are lactic acid bacteria that constitutively express glucansucrase.

[0093]

[18] The production method according to item 16 or 17, wherein the dextran sucrase is of the bacteria-bound type.

[0094] One embodiment of the present invention provides the following aspects.

[0095] [1] A solution comprising galactosyl kojibiose, wherein the pH of the solution is less than 7.

[0096] [2] The solution according to item 1, wherein the solid content concentration in the solution is 10 to 80%.

[0097] [3] The solution or dried product thereof according to 1 or 2, which is obtained by a production method comprising the step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose.

[0098] [4] A composition comprising galactosyl kojibiose and glucosyl epilicatose.

[0099] [5] The composition according to 4, further comprising any sugar selected from the group consisting of lactose, fructose and sucrose, wherein the content of galactosyl kojibiose per unit solid content is 10 to 50%.

[0100] [6] The composition according to 4 or 5, which is obtained by a production method comprising the step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose.

[0101] [7] A composition comprising galactosyl kojibiose, wherein the composition has a pH of less than 7 when prepared as a solution having a solid content concentration of 10 to 80%.

[0102] [8] The composition according to item 7, wherein the fructose content per unit solid content is 10% or more.

[0103] [9] The composition according to item 7 or 8, which is obtained by a production method comprising the step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose.

[0104]

[10] A composition comprising galactosyl kojibiose having the following characteristics.

[0105] (1) containing 15 to 40% galactosyl kojibiose per unit solid content;

[0106] (2) containing 15-45% fructose per unit solid content;

[0107] (3) When a solution having a solid content concentration of 12% is prepared and heated at 120°C for 20 minutes, the residual rate of galactosyl kojibiose is 80% or more.

[0108]

[11] A method for producing a composition containing oligosaccharides, comprising:

[0109] a step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose; and

[0110] A step of adjusting the pH of the obtained composition to less than 7.

[0111]

[12] Glucosyl epilactose or a composition comprising the same.

[0112] Effects of the Invention

[0113] According to the production method of one embodiment of the present invention in which dextran sucrase is allowed to act, oligosaccharides such as galactosyl kojibiose can be efficiently produced using a composition containing sucrose and lactose as a raw material.

[0114] According to the production method of one embodiment of the present invention, by-products produced during the production of milk protein concentrate (MPC) can be effectively utilized.

[0115] According to the embodiment of using a lactic acid bacterium constitutively expressing glucansucrase as the lactic acid bacterium expressing glucansucrase, it is not necessary to add sucrose as an inducer when preparing a crude enzyme solution, and by-production of glucan can be prevented.

[0116] According to the embodiment of using a lactic acid bacterium expressing cell-bound glucansucrase as the lactic acid bacterium expressing glucansucrase, glucansucrase is not secreted into the culture supernatant, and dilution can be prevented.

[0117] According to one embodiment of the present invention, a solution of galactosyl kojibiose having high stability, particularly high thermal stability, or a dried product thereof is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 This is a chromatogram of an enzyme reaction solution obtained by allowing dextran sucrase to act on a mixture of sucrose and lactose.

[0119] Figure 2 The chromatograms are of the enzyme reaction solution and the tri-sugar fraction and tetra-sugar fraction after fractional purification.

[0120] Figure 3 Kojibiose is produced by treating the trisaccharide fraction with lactase.

[0121] Figure 4 The chromatograms of galactosyl kojibiose solutions at various pH values ​​after heat treatment.

[0122] Figure 5 is the residual rate of galactosyl kojibiose after heat treatment.

[0123] Figure 6 Analysis of sugars generated by isomerization of galactosyl kojibiose.

[0124] Figure 7 The amino acid sequence of the dextran sucrase used in one embodiment of the present invention is shown in Figure 1. The underline indicates an important site for the target enzyme activity.

[0125] FIG. 8 is an amino acid sequence of glucansucrase used in one embodiment of the present invention. DETAILED DESCRIPTION

[0126] <Embodiment 1: Method for producing a composition containing galactosyl kojibiose and the like>

[0127] The present embodiment relates to a method for producing a composition containing oligosaccharides such as galactosyl kojibiose. The production method includes a step of allowing dextran sucrase or lactic acid bacteria expressing dextran sucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosyl kojibiose. The present invention further relates to an enzyme agent containing lactic acid bacteria expressing dextran sucrase or a processed product thereof. The present invention also relates to the use of a byproduct containing lactose obtained in the production process of a dairy product.

[0128] [Lactic acid bacteria or processed products thereof]

[0129] (Lactic acid bacteria, dextran sucrase)

[0130] The production method of this embodiment uses glucansucrase or lactic acid bacteria expressing glucansucrase or a processed product thereof. In the present invention, glucansucrase refers to an enzyme belonging to glycoside hydrolase (also known as glycosyl hydrolases) family 70 (GH70) unless otherwise specified (see (CAZy) database and Cantarel et al., Nucleic Acids Res. 37: D233-238, 2009).

[0131] The lactic acid bacteria used are not particularly limited as long as they can produce the target glucansucrase. One of the preferred examples is a lactic acid bacteria belonging to the Lactobacillus (Lactobacillaceae) family or the Streptococcus (Streptococcus) family. It should be noted that, with respect to the present invention, when describing lactic acid bacteria, except for the cases specifically recorded, according to Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB, Mattarelli P, O'Toole PW, Pot B, Vandamme P, Walter J, Watanabe K, Wuyts S, Felis GE, Ganzle MG, Lebeer S.: Ataxonomic note on the genus Lactobacillus:

[0132] Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol. 2020Apr;70(4):2782-2858. The proposed reclassification classification.

[0133] The Lactobacilaceae family includes the following 31 genera:

[0134] [1] Lactobacillus, Paralactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus acillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Pediococcus, Convivina, Leuconostoc, Fructobacillus, Oenococcus, Weissella.

[0135] In a preferred embodiment, among the lactic acid bacteria belonging to the Lactobacillus family, lactic acid bacteria belonging to the Lactobacillus family other than lactic acid bacteria belonging to Limosilactobacillus reuteri are used, and lactic acid bacteria belonging to the Lactobacillus family other than lactic acid bacteria belonging to the genus Lactobacillus mucosa are preferably used. In another preferred embodiment, lactic acid bacteria belonging to the genus Lactobacillus in the Lactobacillus family are used.

[0136] Examples of lactic acid bacteria belonging to the genus Lactobacillus include the following:

[0137] Liquid Lactobacillus aquaticus, Liquid Lactobacillus cacaonum, Liquid Lactobacillus capillatus, Liquid Lactobacillus ghanensis, Liquid Lactobacillus hordei, Liquid Lactobacillus mali, Liquid Lactobacillus nagelii, Liquid Lactobacillus oeni,

[0138] Red stripe liquid lactobacillus (Liquorilactobacillus satsumensis), apple wine liquid lactobacillus (Liquorilactobacillus sicerae), soap liquid lactobacillus (Liquorilactobacillus sucicola), grape liquid lactobacillus (Liquorilactobacillus uvarum), wine liquid lactobacillus (Liquorilactobacillus vini)

[0139] When lactic acid bacteria belonging to the family Streptococcus are used, lactic acid bacteria belonging to the genus Streptococcus are preferably used, and lactic acid bacteria belonging to Streptococcus mutans are more preferably used.

[0140] In a particularly preferred embodiment, a lactic acid bacteria as red-striped liquid lactobacillus in the lactic acid bacteria belonging to the genus liquid lactobacillus is used. An example of a particularly preferred red-striped liquid lactobacillus strain is the JCM12392 strain. It should be noted that the red-striped liquid lactobacillus belongs to red-striped lactobacillus (Lactobacillus satsumensis) according to the classification before modification. Therefore, the JCM12392 strain is recorded as Lactobacillus satsumensis in RIKEN BioResource Center, GENERAL CATALOG No.9, 2012, JAPAN COLLECTION OF MICROORGANISMS, M51. The JCM12392 strain can be obtained from the independent administrative agency RIKEN BRC Microbial Material Development Room (Japan Collection of Microorganisms) (RIKEN BRC-JCM, Japan) with JCM No. JCM12392.

[0141] From another viewpoint, the lactic acid bacteria used are preferably lactic acid bacteria that constitutively express glucan sucrase. Constitutively expressing glucan sucrase means that the gene can be expressed without induction using a substrate or the like. The constitutively expressed glucan sucrase is referred to as a constitutively expressed type or a constitutive type, and the glucan sucrase that requires expression induction is referred to as an induced expression type or an inducible type. Whether the glucan sucrase is constitutively expressed can be confirmed by evaluating the mRNA transcription level using qPCR and evaluating the protein expression amount using Western blot.

[0142] When preparing the lactic acid bacteria expressing glucan sucrase or its processed material for the manufacture method of oligosaccharide, in the case of inducible glucan sucrase, sucrose is needed for expressing glucan sucrase. At this time, sucrose is also the substrate of glucan sucrase, and therefore also participates in the reaction of adding glucose to the glucan chain, and glucan can be generated. Polymeric glucan sometimes makes the prepared liquid present high viscosity. In the case of constitutive type, sucrose is not needed and glucan is not generated when preparing the lactic acid bacteria expressing glucan sucrase or its processed material, which is preferred from this perspective.

[0143] An example of a lactic acid bacterium constitutively expressing dextransucrase is Lactobacillus rubrotii. On the other hand, one of the lactic acid bacteria expressing dextransucrase is well known to be lactic acid bacteria of the genus Leuconostoc. The dextransucrase expressed by lactic acid bacteria of the genus Leuconostoc is mainly of the inducible type.

[0144] From another viewpoint, the lactic acid bacteria used are preferably lactic acid bacteria expressing cell-bound glucansucrase. Whether the cell-bound glucansucrase is expressed can be determined by confirming the enzyme activity of the cell and the culture supernatant or by confirming whether the amino acid sequence of the enzyme or its precursor contains a secretion signal.

[0145] When preparing a lactic acid bacterium expressing glucansucrase or a processed product thereof for use in a method for producing oligosaccharides, a bacterial cell-bound glucansucrase is preferred from the viewpoint that the enzyme can be easily recovered together with the bacterial cells by centrifugation.

[0146] It should be noted that the two types of glucansucrases (GtfA and Gtf180) derived from Lactobacillus reuteri described in the aforementioned Non-Patent Document 4 are secretory.

[0147] Examples of the dextran sucrase used in the present embodiment include dextran sucrase composed of the following proteins (A), (B) or (C). The dextran sucrase composed of the following proteins (A), (B) or (C) may be in the form of lactic acid bacteria expressing the dextran sucrase or a processed product thereof, and in this case, the dextran sucrase may also be used in the production method of the present embodiment.

[0148] (A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8;

[0149] (B) a protein consisting of an amino acid sequence having a high sequence identity to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity;

[0150] (C) A protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added to the amino acid sequence of any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity.

[0151] In one embodiment, the glucansucrase consists of the following protein (B') or (C').

[0152] (B') a protein having a high sequence identity to the amino acid sequences described in SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1, and having glucansucrase activity;

[0153] (C') A protein having glucansucrase activity and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added to the amino acid sequences of SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the sequence of the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1.

[0154] Sequence number 1 shows the amino acid sequence of GTF21, which is a constitutively expressed and bacterial-bound glucansucrase derived from Lactobacillus erythrocyticus. It is known that the portions at positions 450-467, 488-499, and 559-573 in the sequence are important for the activity of glucansucrase (Non-patent document 5: J. Agric. Food Chem. 2011, 59, 4148-4155).

[0155] SEQ ID NOs: 2 to 4 show the amino acid sequences of glucansucrases (GTF68, GTF29, and GFT39, respectively) possessed by L. satsumensis and having a sequence other than SEQ ID NO: 1.

[0156] Sequence No. 5 shows the amino acid sequence of dextransucrase possessed by Leuconostoc meseteroides (Accession No. AAB40875).

[0157] Sequence No. 6 shows the amino acid sequence of dextransucrase possessed by Leuconostoc citreum (Accession No. ACY92456).

[0158] Sequence No. 7 shows the amino acid sequence of dextransucrase present in Streptococcus mutans (Accession No. AAN58705).

[0159] Sequence number 8 shows the amino acid sequence of glucansucrase possessed by Lactobacillus reuteri (Accession number AAU08001).

[0160] The sequence identities of each sequence with the amino acid sequence of SEQ ID NO: 1 are described below and in Fig. 8. Specifically, these values ​​were calculated by comparative analysis of protein amino acid sequences (BLASTP) using the BLAST algorithm (http: / / www.ncbi.nlm.nih.gov / BLAST / ) provided by NCBI (National Center for Biotechnology Information).

[0161] Serial No. 2 49%

[0162] Sequence No. 3 62%

[0163] Sequence No. 4 48%

[0164] Sequence No. 5 46%

[0165] Serial No. 6 45%

[0166] Serial No. 7 47%

[0167] Serial No. 8 56%

[0168] The portion at positions 559-573 of SEQ ID NO: 1 corresponds to the conserved sequence 4 of non-patent document 5. The dextran sucrase used in one embodiment is composed of the protein of (B) or (C), and in this case, regardless of the sequence of other parts of the dextran sucrase, it is selected from:

[0169] The amino acid corresponding to position 565 of SEQ ID NO: 1 (H),

[0170] The amino acid corresponding to position 566 of SEQ ID NO: 1 (D),

[0171] The amino acid corresponding to position 567 of SEQ ID NO: 1 (S),

[0172] The amino acid corresponding to position 571 of SEQ ID NO: 1 (D) and

[0173] One or more, preferably two or more, more preferably three or more, and even more preferably all of the amino acids (Q) corresponding to position 572 of sequence number 1 are consistent with sequence number 1. Positions 566 to 567 and 571 to 572 of sequence number 1 are important recognition sites for receptors (lactose in the present invention) (Non-patent Document 5). Positions 565 to 566 of sequence number 1 are highly conserved among glucansucrases.

[0174] The portion at positions 488-499 of sequence number 1 corresponds to the conserved sequence 3 of non-patent document 5. In one embodiment, the dextran sucrase used is composed of the protein of (B) or (C), and in this case, in the dextran sucrase, regardless of the sequence of other parts, one or more, preferably two or more, more preferably three or more, and even more preferably all of the positions 488 to 493 (HLSILE) selected from sequence number 1 are consistent with sequence number 1. In a particularly preferred embodiment, regardless of the sequence of other parts, the amino acid (H) corresponding to position 488 of sequence number 1 and the amino acid (E) corresponding to the position of sequence number 1 are consistent.

[0175] The portion at positions 450-467 of sequence number 1 corresponds to the conserved sequence 2 of non-patent document 5. The dextran sucrase used in one embodiment is composed of the protein of (B) or (C), and in this case, the dextran sucrase, regardless of the sequence of other parts, is selected from:

[0176] The amino acid corresponding to position 453 of SEQ ID NO: 1 (R),

[0177] The amino acid corresponding to position 455 of SEQ ID NO: 1 (D),

[0178] The amino acid corresponding to position 456 of SEQ ID NO: 1 (A) and

[0179] One or more, preferably two or more, more preferably three or more, and even more preferably all of the amino acids (D) corresponding to position 458 of SEQ ID NO: 1 are identical to those of SEQ ID NO: 1.

[0180]

Table 1

[0181]

[0182] The dextran sucrase used is preferably an enzyme having an activity of catalyzing a reaction of decomposing sucrose into glucose and fructose and a reaction of adding glucose to a dextran chain, and is preferably an enzyme having any one selected from the group consisting of dextran sucrase (EC 2.4.1.5) activity, alternan sucrase (EC 2.4.1.140) activity, reuteran sucrase (EC 2.4.1.-) activity, α-4,6-glucanotransferase (EC 2.4.1.-) activity, α-1,2-branched dextran sucrase (EC 2.4.1.-) activity, α-4,3-glucanotransferase (EC 2.4.1.-) activity, and mutan sucrase (EC 2.4.1.372) activity, and more preferably at least dextran sucrase (EC 2.4.1.5) activity. 2.4.1.5) Activity.

[0183] In the present invention, whether a certain protein has glucansucrase activity can be determined by whether the protein has dextransucrase (EC 2.4.1.5) activity, alternansucrase (EC 2.4.1.140) activity, reutlandsucrase (EC 2.4.1.-) activity, α-4,6-glucanotransferase (EC 2.4.1.-) activity, α-1,2-branched dextransucrase (EC 2.4.1.-) activity, and α-4,3-glucanotransferase (EC 2.4.1.-) activity. Whether a certain protein has glucansucrase activity can more preferably be determined by whether the protein has dextransucrase (EC 2.4.1.5) activity.

[0184] Regarding the amino acid sequence of glucansucrase, high sequence identity means an identity value of 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 56% or more, 62% or more, preferably 63% or more, 70% or more, more preferably 80% or more, further preferably 90% or more, further preferably 95% or more, further preferably 98% or more.

[0185] Regarding the present invention, regarding amino acid sequences, when referring to identity, except for the case of special records, it refers to the percentage of the number of consistent amino acids that both sequences have when the two sequences are arranged in the best manner. The search and analysis of the identity of the amino acid sequence can be performed by an algorithm or program well known to those skilled in the art, such as GENETIX (registered trademark) ver.14 (GENETIX Co., Ltd.), BLASTN, BLASTP, BLASTX, ClustalW. The parameters when using the program can be appropriately set by those skilled in the art, and the default parameters of each program can also be used in addition. The specific methods of these analytical methods are also well known to those skilled in the art.

[0186] With regard to the present invention, when referring to an amino acid sequence consisting of substitution, deletion, insertion and / or addition of one or more amino acids, the number of amino acids that undergo substitution, etc. is not particularly limited as long as the protein consisting of the amino acid sequence has the target activity, except for the case where there is a special record. For the dextran sucrase consisting of the sequence of sequence number 1 consisting of a full-length 1075 amino acid sequence, for example, it refers to less than 50% (specifically, less than 537), preferably less than 37% (specifically, less than 409), more preferably less than 200, further preferably less than 100, further preferably less than 50, further preferably less than 20, and further preferably 1 to 9. If it is substituted with an amino acid with similar properties, there may be a larger number of substitutions, etc. The means for preparing polynucleotides or proteins involving such amino acid sequences are well known to those skilled in the art.

[0187] In the present invention, an amino acid equivalent to position x (xth position from the N-terminus) of a reference sequence (e.g., SEQ ID NO: 1) in a certain amino acid sequence S refers to an amino acid in sequence S that corresponds to the amino acid at position x of the reference sequence when sequence S and the reference sequence are aligned. In the case where sequence S lacks one or more amino acids in the reference sequence, the position of the corresponding amino acid in sequence S is offset from the position of the reference sequence and may not be position x in sequence S. Which amino acid in sequence S the amino acid equivalent to position x of the reference sequence corresponds to can be appropriately determined by a person skilled in the art.

[0188] From other viewpoints, the glucansucrase used is preferably suitable for a large number of manufactures of oligosaccharides. Specifically, when the preferred substrate concentration is high, the active person can be fully exerted. When manufacturing oligosaccharides in a large number, from the viewpoint of production efficiency, it is preferably possible to implement efficiently under higher sucrose concentration.

[0189] From another viewpoint, the glucansucrase to be used is preferably one whose activity can be increased by calcium.

[0190] (Processing material)

[0191] The form of the lactic acid bacteria expressing glucansucrase or its processed material is not particularly limited as long as it contains glucansucrase in a state with the target activity. As examples of preferred lactic acid bacteria expressing glucansucrase or its processed material, living cells, dead cells, cultures containing cells, cell crushing products and purified products of glucansucrase can be enumerated. These can be in a state containing water or can be dried products.

[0192] Preferred examples of lactic acid bacteria expressing glucansucrase or processed materials thereof used in the embodiment using lactic acid bacteria expressing cell-bound glucansucrase as the lactic acid bacteria expressing glucansucrase include living cells, dead cells, cultures containing cells, and cell fragments.

[0193] Live bacteria can be obtained, for example, by culturing lactic acid bacteria with a commercially available culture medium, collecting bacteria from the obtained culture solution, and washing as needed. Dead bacteria can be obtained, for example, by sterilizing a collection of live bacteria under sterilization conditions that do not inactivate glucansucrase. Cultures containing bacteria can be obtained, for example, by culturing lactic acid bacteria with a commercially available culture medium. Bacterial crushed products can be obtained, for example, by physically crushing a collection of live bacteria using a homogenizer, a ball mill, a bead mill, a Dyno mill, a planetary mill, a jet mill, a French press, a cell crusher, etc., or by destroying the bacterial structure by self-digestion, enzyme treatment, a surfactant, etc., and other treatments.

[0194] The degree of purification of the purified product of glucansucrase is not particularly limited as long as the target reaction can be carried out. The purified product of glucansucrase can be purified to high purity or can be a crude purified product. The method of purification is not particularly limited and can be carried out by the prior art. In addition, the purified product of glucansucrase is not particularly limited in source as long as it can be used as an enzyme and carry out the target reaction. It can be a cell of lactic acid bacteria, a substance purified from a culture, a substance manufactured and purified in a recombinant form, or a substance synthesized and purified.

[0195] (Enzyme)

[0196] Glucansucrase or lactic acid bacteria expressing Glucansucrase or processed products thereof can be used as components of enzyme preparations. When used as components of enzyme preparations, Glucansucrase is preferably at least one of constitutive expression type and bacterial binding type, more preferably constitutive expression type and bacterial binding type. Examples of constitutive expression type and bacterial binding type Glucansucrase include Glucansucrase composed of the above-mentioned (A), (B) or (C) proteins.

[0197] The active ingredient of conventional enzyme preparations is the enzyme itself, whereas the lactic acid bacteria expressing glucansucrase or its processed product as a component of an enzyme preparation in one embodiment may not be the glucansucrase itself but may be living bacteria, dead bacteria, culture containing bacteria, or bacteria fragments, as long as the glucansucrase is contained in a state having the target activity. Such a form as a component of an enzyme preparation is particularly suitable for the case where bacteria-bound glucansucrase is used as a component.

[0198] The enzyme agent may also include excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, etc. in addition to glucansucrase or lactic acid bacteria expressing glucansucrase or their processed products. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, and glycerol. Examples of buffers include phosphates, citrates, acetates, etc. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methyl parahydroxybenzoate. Examples of preservatives include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0199] (Method for producing dextran sucrase)

[0200] Glucansucrase can be manufactured using lactic acid bacteria or its processed material, and can also be manufactured by other methods. For example, it can be manufactured by expressing Glucansucrase in a suitable expression system. That is, a polynucleotide encoding Glucansucrase consisting of the protein of above-mentioned (A), (B) or (C) can be used to transform a suitable host, and then the obtained transformant is utilized to produce Glucansucrase. As the host, bacteria such as Escherichia coli, Bacillus subtilis, lactic acid bacteria, yeast, filamentous fungi, etc. can be listed, which can be appropriately selected according to the type, operability, etc. of the recombinant vector. A vector can be used during transformation, and as such a vector, a plasmid vector, etc. can be listed, which can be appropriately selected according to the host, operability, etc.

[0201] Glucansucrase can be produced by chemical synthesis. That is, based on the amino acid sequence information of the Glucansucrase composed of the protein of (A), (B) or (C), Glucansucrase can be synthesized by chemical synthesis methods such as the Fmoc method (fluorenylmethoxycarbonyl method) and the tBoc method (tert-butyloxycarbonyl method), or can be synthesized using various commercially available peptide synthesizers.

[0202] Glucansucrase can be produced using a genome-edited organism obtained by genome editing an organism having a gene encoding Glucansucrase. Through genome editing, the expression of the gene encoding the target Glucansucrase can be upregulated. As usable genome editing techniques, ZFN, TALEN, CRISPR / Cas9 and PPR etc. can be cited, and those skilled in the art can appropriately select according to the circumstances.

[0203] The enzyme agent can be used in the fields of food processing, etc. Specific examples thereof include treatment of a composition containing lactose and production of oligosaccharides such as galactosyl kojibiose.

[0204] [Composition containing lactose and sucrose]

[0205] The production method of the present embodiment uses a composition containing lactose and sucrose.

[0206] The composition containing lactose and sucrose can be obtained by adding sucrose to the composition containing lactose. The composition containing lactose is preferably a by-product obtained in the manufacturing process of dairy products or milk raw materials. As examples thereof, raw milk, skim milk, reconstituted liquid of a dried product containing lactose, skim concentrated milk, whey, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolated (WPI), membrane permeate derived from milk (also referred to as permeate.) or membrane retained liquid (sometimes also referred to as retentate.), milk concentrate, lactic acid bacteria culture containing lactose. The reconstituted liquid of a dried product containing lactose refers to a liquid obtained by adjusting a known dried product containing lactose such as whole milk powder, skim milk powder, whey protein concentrate powder, and whey protein isolated powder to any concentration.

[0207] Examples of membranes used in the production of milk raw materials include microfiltration membranes (MF membranes), ultrafiltration membranes (UF membranes), nanofiltration membranes (NF membranes), and reverse osmosis membranes (RO membranes). The permeate obtained by passing through the MF membrane and the UF membrane among these can be used as a composition containing lactose, preferably for the production method of the present invention. This is because the permeate produced as a byproduct in the production of milk raw materials such as MPC is rich in lactose. When using NF membranes and RO membranes, the retentate contains lactose.

[0208] In a preferred embodiment, as a composition containing lactose, a by-product when a dairy product or a milk raw material is produced is used. Examples of by-products include whey when cheese is produced and permeate when WPC or MPC is produced. When a permeate is used, it is preferred that the content of an acid such as lactic acid is 1% or less, preferably 0.8% or less, and more preferably 0.7% or less, because pH adjustment for forming the optimal conditions for an enzyme reaction is not required.

[0209] [Effects of lactic acid bacteria expressing glucansucrase or processed products thereof]

[0210] (Enzyme reaction)

[0211] Generally, dextran sucrase has the activity of catalyzing the reaction of decomposing sucrose into glucose and fructose and the reaction of adding glucose to the dextran chain. According to the research of the present inventors, when dextran sucrase is allowed to act on sucrose in the reaction in which lactose, the main component of whey and protein membrane concentrated permeate, coexists as an acceptor, galactosyl kojibiose having a kojibiose skeleton can be produced. The present invention utilizes such an enzyme reaction.

[0212]

[0213] In this reaction, sugar transfer is carried out with α-1,2 bonds to generate galactosyl kojibiose (α-D-glucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-glucopyranoside). It should be noted that the structure of high molecular weight glucan produced by glucansucrase derived from a strain of Lactobacillus erythropolis was studied in the aforementioned non-patent document 3. The document describes sugar transfer with α-1,3 and 1,6 bonds, but does not describe the ability to form α-1,2 bonds by glucose transfer.

[0214] (Reaction Conditions)

[0215] When dextran sucrase or lactic acid bacteria expressing dextran sucrase or a processed product thereof is allowed to act on a composition containing lactose and sucrose, the concentration of lactose can be appropriately set as long as the target product can be obtained. The concentration of lactose can be, for example, set to 3.0% or more, preferably 6.0% or more, more preferably 12% or more, further preferably 15% or more, and further more preferably 20% or more. In addition, it can be set to 40% or less, 30% or less, or 17% or less.

[0216] When glucansucrase or lactic acid bacteria expressing glucansucrase or its processed product is allowed to act on a composition containing lactose and sucrose, the concentration of sucrose can be appropriately set as long as the target product can be obtained. The concentration of sucrose can be set to 5.0% or more, preferably 10% or more, more preferably 30% or more, and further preferably 50%. In addition, it can be set to 64% or less, preferably 60% or less, more preferably 56% or less, and further preferably 52% or less. It should be noted that, with respect to the present invention, when showing the concentration and ratio of the components, unless otherwise specified, it is shown as a value based on mass.

[0217] The ratio of sucrose to lactose can be appropriately set as long as the target product can be obtained. The ratio of sucrose to lactose (sucrose:lactose) can be set to, for example, 0.5:1 to 11:1, preferably 0.7:1 to 9:1, more preferably 0.8:1 to 6:1, and even more preferably 1:1 to 4:1.

[0218] When the lactic acid bacteria expressing glucansucrase or its processed product is allowed to act on a composition containing lactose and sucrose, the concentration of the lactic acid bacteria expressing glucansucrase or its processed product can be appropriately set as long as the target product can be obtained. When the lactic acid bacteria expressing glucansucrase are used in the form of live bacteria, the lactic acid bacteria can be cultured under appropriate conditions, and then the bacterial bodies can be collected from the obtained culture solution, and then washed as needed, and a buffer solution 0.2 to 2 times the amount of the original culture solution can be added to suspend the bacteria as the lactic acid bacteria expressing glucansucrase or its processed product referred to here. Such a suspension of lactic acid bacteria can be added to the system in a manner of reaching 1.0 to 25%, preferably 2.0 to 20%, more preferably 3.0 to 18%, and further preferably 4.0 to 15%. Alternatively, the concentrate obtained by culturing the lactic acid bacteria under appropriate conditions and concentrating the culture solution can be used as the lactic acid bacteria expressing glucansucrase or its processed product referred to here. Such a concentrate of lactic acid bacteria can be added to the system so as to be 0.01 to 10%, preferably 0.05 to 5%, 0.1 to 3%, or 0.2 to 0.6%.

[0219] The system in which a lactic acid bacterium expressing glucansucrase or a processed product thereof is allowed to act on a composition comprising lactose and sucrose may contain a component that can increase the activity of the glucansucrase, and preferably does not contain an activity-inhibiting component. When the glucansucrase is a glucansucrase composed of the above-mentioned protein (A), (B) or (C), an example of a component that can increase the activity of the glucansucrase is calcium, and an example of an activity-inhibiting component is iron ions.

[0220] When glucansucrase or a lactic acid bacterium expressing glucansucrase or a processed product thereof is allowed to act on a composition containing lactose or sucrose, conditions such as temperature can be appropriately set by those skilled in the art.

[0221] The pH can be set to pH 3.5 to 7.0, preferably pH 4.0 to 6.5, and more preferably pH 4.5 to 6.0.

[0222] The temperature can be 4 to 55°C, preferably 20 to 52°C, more preferably 30 to 51°C, and further preferably 46 to 50°C.

[0223] The time for allowing glucansucrase or lactic acid bacteria expressing glucansucrase or a processed product thereof to act on a composition containing lactose or sucrose can be set so that the target purified product can be obtained sufficiently. The time used for this is usually 4 to 96 hours, preferably 6 to 80 hours, more preferably 8 to 72 hours, and further preferably 12 to 64 hours.

[0224] [Composition containing galactosyl kojibiose, final product]

[0225] The production method of the present invention includes the step of allowing glucansucrase or a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose.

[0226] The production method of the present invention can be implemented for the purpose of producing various oligosaccharide-containing compositions. The final product, i.e., the oligosaccharide, as the target, is any one selected from the group consisting of kojibiose and oligosaccharides with kojibiose as a constituent sugar. With regard to the present invention, when referring to oligosaccharides with kojibiose as a constituent sugar, kojibiose itself is not included. It should be noted that, with regard to the present invention, when referring to "any one", it is used in the sense of "at least one" unless otherwise specified.

[0227] Examples of oligosaccharides having kojibiose as a constituent sugar include galactosyl kojibiose, which is a trisaccharide, and tetrasaccharides in which one molecule of a monosaccharide such as glucose is bonded to galactosyl kojibiose. In the present invention, galactosyl kojibiose refers to kojibiose described below in which galactose is bonded to any of positions 1 to 8 by an α or β bond, unless otherwise specified. Theoretically, galactose can be bonded to the OH at position 8 in four ways, namely, α-α, α-β, β-α, and β-β, and galactose can be bonded to the OH at positions 2 to 7 in two ways, so 18 types of galactosyl kojibiose are assumed.

[0228]

[0229] In a preferred embodiment, one of the galactosylkojibiose produced is α-D-glucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-glucopyranoside). This sugar can also be referred to as a type of glucosyl lactose.

[0230] In the present invention, glucosyl lactose refers to a substance obtained by bonding glucose to lactose except for cases where it is specifically stated. More specifically, it refers to any kind of substance selected from the following lactose in which glucose is bonded by an α or β bond at any position from 1 to 8. Theoretically, glucose can be bonded to the OH at position 1 in four ways, such as α-α, α-β, β-α, and β-β, and glucose can be bonded to the OH at positions 2 to 7 in two ways, so it is assumed that there are 18 kinds of glucosyl lactose. They are respectively called 1α-α body, 1α-β body, 1β-α body, 1β-β body, 2α body, 2β body, 3α body, 3β body, 4α body, 4β body, 5α body, 5β body, 6α body, 6β body, 7α body, 7β body, 8α body, and 8β body. α-D-glucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-glucopyranoside is a compound in which glucose is bonded to the OH group at the 8 position via an α bond (8α form).

[0231]

[0232] In one embodiment, regardless of whether the above-mentioned α-D-pyranoglucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-pyranoglucoside (8α) can be produced, glucosyl lactose other than the sugar can be produced. Specifically, any one of 17 types of glucosyl lactose (1α-α, 1α-β, 1β-α, 1β-β, 2α, 2β, 3α, 3β, 4α, 4β, 5α, 5β, 6α, 6β, 7α, 7β, 8β) other than the sugar can be produced, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 types can be produced.

[0233] (Sugars contained in the composition)

[0234] The composition obtained by the step of allowing glucansucrase or lactic acid bacteria expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose may contain unreacted lactose in addition to galactosyl kojibiose, and may also contain fructose produced by decomposition of sucrose. The fructose in the obtained composition is usually equimolar or more to galactosyl kojibiose. The composition may also contain a tetrasaccharide in which one molecule of a monosaccharide such as glucose is bonded to galactosyl kojibiose, which is a trisaccharide.

[0235] It should be noted that in the description of the present invention and the embodiments, the production method of the present invention is sometimes described by taking the case where the final product, that is, the oligosaccharide-containing composition is a composition containing galactosyl kojibiose as an example, but this description is also applicable to the case of producing a composition containing a tetrasaccharide formed by bonding one molecule of monosaccharide such as glucose to galactosyl kojibiose. Those skilled in the art can appropriately replace galactosyl kojibiose with a tetrasaccharide formed by bonding one molecule of monosaccharide such as glucose to galactosyl kojibiose.

[0236] (Form, Other)

[0237] The composition obtained by the production method can be in various forms. The form of the composition can be the original form of the obtained reaction solution, and can be made into a concentrate, a frozen product, or a dried product after the purification process described below as needed. The dried product can be made into a powder or granular form.

[0238] (use)

[0239] The obtained composition can be used as a sweetener, flavor improver, quality improver, stabilizer, anti-discoloration agent, excipient or the functional ingredients described below in the fields of food, cosmetics and medicine. Food and medicine include not only those used for humans but also those used for animals other than humans, unless otherwise specified. Food includes general food, functional food, nutritional composition, therapeutic food (food for therapeutic purposes. Food prepared based on a recipe prescribed by a doctor, a menu prepared by a nutritionist, etc.), diet therapy food, ingredient-adjusted food, nursing food, and therapeutic auxiliary food, unless otherwise specified. Food includes not only solids but also liquids, such as beverages, drinks, liquid food and soup.

[0240] Composition can also be used as functional ingredient. According to the research of the present inventors, oligosaccharides with kojibiose as constituent sugars such as kojibiose and galactosyl kojibiose can promote the proliferation of Parabacteroides bacteria in the intestinal flora, and can also promote the proliferation of Bifidobacterium bacteria in the intestinal flora. Therefore, the composition obtained by the manufacture method of the present invention can be used to promote the proliferation of Parabacteroides bacteria in the intestinal flora, and to promote the proliferation of Bifidobacterium bacteria in the intestinal flora. In addition, by promoting the proliferation of Parabacteroides bacteria, it is expected that inflammation, obesity, abnormal glucose metabolism, multiple sclerosis (MS), fatty liver, cancer and chronic obstructive pulmonary disease (COPD) will be disposed, so the composition can be used to dispose of these diseases, and can also be used for the disposal of diseases or states other than these that can be improved by promoting the proliferation of Parabacteroides bacteria in the intestinal tract. Furthermore, by promoting the growth of Bifidobacterium bacteria in the intestinal flora, it can be expected to promote the absorption of minerals and regulate the intestines. Therefore, the composition can be used to promote the absorption of minerals, regulate the intestines, and treat conditions other than these that can usually be improved by promoting the growth of Bifidobacterium bacteria in the intestines.

[0241] Additionally, the composition may be used as a prebiotic or a synbiotic.

[0242] [Specific Embodiment]

[0243] (Other processes)

[0244] The production method of the present invention may further include various steps in addition to the step of allowing glucansucrase or a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose.

[0245] The production method may further include a purification step. Examples of the target component to be purified in the purification step include galactosyl kojibiose and a tetrasaccharide in which one molecule of a monosaccharide such as glucose is bonded to galactosyl kojibiose.

[0246] The purification step may include any of means such as treatment with activated carbon, separation by chromatography, treatment with ion exchange resin, filtration, concentration, crystallization, crystal separation, drying, cooling, temperature control, humidity control, and the like.

[0247] When the purification step is performed, the degree of purification is not particularly limited. For example, the purity of galactosyl kojibiose can be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 97% or more.

[0248] The manufacturing method may further include a pH adjustment step. The preparation used to adjust the pH in the pH adjustment step is not particularly limited as long as it is suitable for the manufacture of food, medicine, and cosmetics. As examples, organic acids such as citric acid, malic acid, gluconic acid, succinic acid, ascorbic acid, tartaric acid, lactic acid, and fumaric acid, inorganic acids such as phosphoric acid, and their salts may be cited. Adjusting the resulting composition to a suitable pH is sometimes important for the stabilization of the target component contained.

[0249] The pH can be adjusted to, for example, pH 3.0 to 8.0, pH 4.0 to 7.0, or pH 4.5 to 6.0.

[0250] The manufacturing method preferably further includes a sterilization step. The stage at which the sterilization step is performed is not particularly limited, and the sterilization step can be performed multiple times as needed. For example, sterilization can be performed before allowing glucan sucrase or lactic acid bacteria expressing glucan sucrase or its processed material to act on the composition containing lactose and sucrose. In addition, after allowing lactic acid bacteria expressing glucan sucrase or its processed material to act, the obtained composition containing galactosyl kojibiose can be sterilized under conditions that do not inactivate the glucan sucrase. When the obtained composition containing galactosyl kojibiose is concentrated, the composition can be sterilized before the concentration step.

[0251] The sterilization method is not particularly limited as long as it does not affect the target component, and examples thereof include heat sterilization (UHT sterilization, retort sterilization, etc.) and ultraviolet sterilization.

[0252] (Appropriate example)

[0253] As one embodiment, a method for producing fermented milk containing galactosyl kojibiose may be implemented, comprising the following steps:

[0254] an enzyme treatment step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing raw milk and sucrose to obtain a composition containing galactosylkojibiose; and

[0255] In the fermentation step, lactic acid bacteria starter is added to raw milk and fermented to obtain fermented milk.

[0256] The order of the enzyme treatment step and the fermentation step is not particularly limited. For example, after the enzyme treatment step, a lactic acid bacteria starter may be added to perform the fermentation treatment step, or after the fermentation step, lactic acid bacteria expressing glucansucrase or a processed product thereof may be added to perform the enzyme treatment. By such a step, galactosyl kojibiose can be contained in the fermented milk without adding galactosyl kojibiose to the fermented milk.

[0257] Examples of raw material milk include raw milk, cow's milk, special milk, skim milk, partially skim milk, and a mixture of any of these.

[0258] The lactic acid bacteria used as the starter are not particularly limited as long as they are suitable for producing fermented milk. Lactobacillus delbrueckii is preferably used. Among Lactobacillus delbrueckii, Lactobacillus delbrueckii subspecies bulgaricus is preferably used. In addition, thermophilic Streptococcus can be used. Lactobacillus delbrueckii and thermophilic Streptococcus can also be used in combination.

[0259] As one embodiment, a method for producing a milk concentrate containing galactosyl kojibiose may be implemented, comprising the following steps:

[0260] A step of allowing glucansucrase or lactic acid bacteria expressing glucansucrase or a processed product thereof to act on a composition containing raw material skim concentrated milk and sucrose to obtain skim concentrated milk containing galactosyl kojibiose;

[0261] A step of drying the obtained skim concentrated milk as desired.

[0262] Examples of the milk concentrate obtained in the present embodiment include concentrated milk (concentrated raw milk, milk or special milk, with a milk solid content of 25.5% or more and a milk fat content of 7.0% or more) specified in the "Ordinance of the Ministry of Health, Labor and Welfare on the Standards of the Composition of Milk and Dairy Products", skim concentrated milk (concentrated after removing the milk fat component from raw milk, milk or special milk, with a non-fat milk solid content of 18.5% or more and a bacterial count (standard plate culture method, per 1g) of 100,000 or less), raw milk, milk or special milk concentrated with a milk solid content of less than 25.5% (concentrated milk not specified in the ordinance), raw milk, milk or special milk concentrated after removing the milk fat component and with a non-fat milk solid content of less than 18.5% (skim concentrated milk not specified in the ordinance), or milk protein concentrate (MPC).

[0263] <Embodiment 2: Composition containing galactosyl kojibiose and the like>

[0264] The present embodiment relates to a solution containing galactosyl kojibiose and having a pH of a specific value or less, and a composition containing galactosyl kojibiose and glucosyl epilicatose.

[0265] [Composition]

[0266] (Galactosyl kojibiose, pH of solution, content)

[0267] The composition of the present embodiment contains galactosyl kojibiose (defined in the present specification).

[0268] In a preferred embodiment of the composition, the composition contains α-D-glucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-glucopyranoside) as one of the galactosyl kojibiose.

[0269]

[0270] This α-D-glucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-glucopyranoside may be referred to as 4-galactosylkojibiose or 4'-galactosylkojibiose. In addition, it may be referred to as a type of glucosyl lactose (which has been defined in the present specification).

[0271] In one embodiment of the composition, regardless of whether or not the α-D-pyranoglucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-pyranoglucoside is contained, glucosyl lactose other than the sugar may be contained. Specifically, when any of 17 types of glucosyl lactose (1α-α form, 1α-β form, 1β-α form, 1β-β form, 2α form, 2β form, 3α form, 3β form, 4α form, 4β form, 5α form, 5β form, 6α form, 6β form, 7α form, 7β form, 8β form) other than the sugar is produced, the composition may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of them.

[0272] The content of galactosyl kojibiose in the composition is not particularly limited. For example, it can be set to 10% or more, 15% or more, preferably 20% or more, more preferably 25% or more, and further preferably 30% or more per unit solid content. The upper limit is not particularly limited, for example, it can be 100%, 99% or less, 80% or less, 70% or less, 50% or less, or 40% or less. In one preferred embodiment, the content of galactosyl kojibiose in the composition can be set to 10-50% per unit solid content, more preferably 15-40%, and further preferably 30-40%. It should be noted that, with respect to the present invention, when the content or concentration of a component is expressed by a numerical value, the value is based on mass unless otherwise specified.

[0273] One of the preferred forms of the composition is a solution (sugar solution) having a pH of less than 7. Alternatively, the pH is less than 7 when the solution is made into a solid content concentration of 10 to 80%. This is because if the pH is less than 7, the thermal stability of galactosyl kojibiose is high. The pH can be, for example, set to be greater than 2, can be set to be greater than 2.5, can be set to be greater than 3, preferably greater than 3 and less than 6, and more preferably greater than 4 and less than 6. This is because if it is within this range, the thermal stability of galactosyl kojibiose is higher. If appropriate, the solid content concentration of the solution can be set to 12% and the pH of the solution can be measured. It should be noted that, with respect to the present invention, the pH can be measured by a conventional measurement method in this field.

[0274] One of the preferred forms of the composition is a concentrate, frozen product, or dried product containing galactosyl kojibiose having a pH of less than 7 when made into a solution (aqueous solution) having a solid content concentration of 10 to 80%. A dried product refers to a product obtained by drying a solution to a moisture content of 7% or less, preferably 5% or less. The moisture content can be measured by a statutory method for measuring the moisture content of sugars in the food field or in the Japanese Agricultural, Forestry and Fisheries Standards (JAS). Examples of dried products include powders and granules. When a concentrate, frozen product, or dried product is made into a solution having a solid content concentration of 10 to 80%, the pH can be set to, for example, 2 or more, 2.5 or more, 3 or more, preferably 3 or more and 6 or less, and more preferably 4 or more and 6 or less. This is because, if it is within this range, the thermal stability of galactosyl kojibiose is higher. If appropriate, the solid content concentration of the solution can be set to 12% and the pH of the solution can be measured.

[0275] In the present invention, solid content concentration refers to the concentration of solid components dissolved in a solution. The solid content concentration can be measured by conventional methods in the art. In appropriate cases, the solid content concentration can be expressed as a Brix value or a sugar content. The Brix value and the sugar content can be appropriately measured using known determination methods, and can generally be measured using commercially available equipment (e.g., a digital refractometer trade name "RX-5000α" (manufactured by Atago)).

[0276] High thermal stability means that when a solution having a solid content concentration of 12% is prepared and heated at 120°C for 20 minutes, the residual rate of galactosyl kojibiose is 80% or more, preferably 90% or more, and more preferably 95% or more.

[0277] When the composition containing galactosyl kojibiose is a solution, the solid content concentration of the solution is not particularly limited, and is, for example, 10% or more, preferably 30% or more, more preferably 50% or more, and further preferably 60% or more. The upper limit is not particularly limited, and may be, for example, 80% or less, 78% or less, or 76% or less. The solid content is mainly composed of sugars, and may sometimes contain components other than sugars. Examples of components other than sugars include enzymes used in the production method described later and agents for adjusting pH. The benefits of components other than sugars are small and may sometimes be ignored.

[0278] (Other ingredients and components)

[0279] The composition may contain glucosyl epillactose in addition to galactosyl kojibiose, and more specifically glucosyl epillactose (α-D-Glcp-(1→2)-[β-D-Galp-(1→4)-]D-Manp). This sugar is sometimes referred to as 2-glucosyl epillactose. When glucosyl epillactose is mentioned in the present invention and the embodiments, it refers to a trisaccharide having the following structure unless otherwise specified.

[0280]

[0281] The composition may further contain the aforementioned α-D-glucopyranosyl-(1→2)-[β-D-galactopyranosyl-(1→4)-]D-glucopyranoside, and may contain glucosyl lactose other than the sugar regardless of whether the sugar is contained.

[0282] Glucosyl epilactose is a novel compound found by the present inventors to be generated in a solution of galactosyl kojibiose treated by heating.

[0283] Glucosyl epilactose can be produced by adjusting the pH of a composition containing galactosyl kojibiose to 7 or higher, and then heating the composition at 100° C. or higher, preferably 110° C. or higher.

[0284] When the composition contains glucosyl epillactose, the content of glucosyl epillactose is not particularly limited, and can be, for example, 5% or less, preferably 3% or less, more preferably 1% or less, and further preferably 0.1% or less per unit solid content. The lower limit is not particularly limited, and for example, it is 0.001% or more, 0.01% or more, or 0.05% or more per unit solid content.

[0285] When the composition contains glucosyl epilactose, the mass ratio of galactosyl kojibiose to glucosyl epilactose (galactosyl kojibiose:glucosyl epilactose) is not particularly limited, and is, for example, 99.99:0.01 to 80:20, 99.9:0.1 to 85:15, or 99:1 to 90:10.

[0286] The composition may further include any sugar selected from the group consisting of lactose, fructose and sucrose. It should be noted that, in the present invention, "any" is used in the sense of "at least one" unless otherwise specified.

[0287] When the composition contains fructose, the content of fructose is not particularly limited, and can contain, for example, 5% or more, preferably 15% or more, more preferably 20% or more, and further preferably 30% or more per unit solid content. The upper limit is not particularly limited, and for example, it is 45% or less, 40% or less, or 35% or less per unit solid content. When the purity of galactosyl kojibiose is preferably high, the content of fructose is preferably low, and preferably no fructose is contained.

[0288] When the composition contains fructose, the mass ratio of galactosyl kojibiose to fructose (galactosyl kojibiose:fructose) is not particularly limited, and may be, for example, 15-40:15-45, 17-37:17-37, or 20-35:25-40.

[0289] When the composition contains lactose, the content of lactose is not particularly limited, and can contain, for example, 30% or less, preferably 25% or less, more preferably 20% or less, and further preferably 15% or less per unit solid content. The lower limit is not particularly limited, and for example, it is 1% or more, 5% or more, or 10% or more per unit solid content. When the purity of galactosyl kojibiose is preferably high, the content of lactose is preferably low, and preferably no lactose is contained.

[0290] When the composition contains lactose, the mass ratio of galactosyl kojibiose to lactose (galactosyl kojibiose:lactose) is not particularly limited, and may be, for example, 15-40:15-30, 17-37:17-37, or 20-35:20-25.

[0291] When the composition contains sucrose, the content of sucrose is not particularly limited, and can contain, for example, 30% or less, preferably 25% or less, more preferably 20% or less, further preferably 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.2% or less, or 0.1% or less per unit solid content. The lower limit is not particularly limited, and for example, it can be 0.01% or more, 0.5% or more, 1% or more, 5% or more, or 10% or more per unit solid content. Depending on the conditions, all sucrose can be used for the reaction, and sometimes it is below the detection limit.

[0292] When the composition contains sucrose, the mass ratio of galactosyl kojibiose to sucrose (galactosyl kojibiose:sucrose) is not particularly limited, and may be, for example, 15-40:15-30, 17-37:17-37, or 20-35:20-25.

[0293] The composition may also contain a tetrasaccharide in which one molecule of a monosaccharide such as glucose is bonded to galactosylkojibiose as a trisaccharide.

[0294] As one of the preferred embodiments, there can be mentioned a composition containing galactosyl kojibiose having the following characteristics.

[0295] (1) The solid content contains 15 to 40% galactosyl kojibiose

[0296] (2) Contains 15-45% fructose per unit solid content

[0297] (3) When a solution having a solid content concentration of 12% is prepared and heated at 120°C for 20 minutes, the residual rate of galactosyl kojibiose is 80% or more

[0298] The above-mentioned composition may further have the following characteristics.

[0299] (4) Contains 5-30% lactose per unit solid content

[0300] The above composition may replace (3) or may further have the following characteristics.

[0301] (6) When the solid content concentration is 10-80%, the pH is less than 7

[0302] Such a composition has high stability and is suitable for processing. In addition, it can be expected to be suitable for long-term storage and circulation. Furthermore, it is also useful in terms of excellent taste and low calorie content compared to the same amount of sucrose.

[0303] (form)

[0304] The composition can take various forms. One of the preferred forms is a solution. In one embodiment, the composition can be in the form of a solution (sometimes also referred to as a reactant) obtained by the following production method, wherein the production method includes a step of allowing glucansucrase or a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosyl kojibiose.

[0305] The solution can be prepared as a concentrate, a frozen product, or a dried product. A dried product is obtained by drying the solution to a moisture content of 7% or less, preferably 5% or less. The moisture content can be determined by the statutory method for determining the moisture content of sugars in the food field or in the Japanese Agricultural, Forestry and Fisheries Standards (JAS). Examples of dried products include powders and granules.

[0306] (use)

[0307] The composition containing galactosyl kojibiose can be used as a sweetener, a taste improver, a quality improver, a stabilizer, a discoloration inhibitor, an excipient, or a functional ingredient described later in the fields of food, cosmetics, and pharmaceuticals. The description of the (Use) section of the above <Method for producing a composition containing galactosyl kojibiose> can also be directly applied to the composition of this embodiment.

[0308] Glucosyl epilactose that may be contained in the composition of one embodiment is expected to be used as a precursor of epilactose, which is reported to be a promising prebiotic (Non-Patent Document 5 described below). Therefore, the composition can be used for the purpose of obtaining a precursor of epilactose in addition to obtaining glucosyl epilactose.

[0309] [Method for producing composition]

[0310] The composition containing galactosyl kojibiose of the present embodiment can be produced by various methods. As one of the particularly preferred examples of the production method, there can be cited a production method including a step of allowing glucansucrase or lactic acid bacteria expressing glucansucrase or a processed product thereof to act on a composition containing lactose and sucrose to obtain a composition containing galactosyl kojibiose. The description of the above <Embodiment 1: Production method of a composition containing galactosyl kojibiose, etc.> can be directly applied to the production of the composition of the present embodiment.

[0311] (pH adjustment process)

[0312] The manufacture method of the composition of the present embodiment may also include a pH adjustment process. When the pH is less than 7, there is no need to adjust, but such a process for confirming the pH may also be referred to as a pH adjustment process. The agent used in the pH adjustment process for adjusting the pH is not particularly limited as long as it is suitable for food, medicine, and cosmetics manufacturers. As an example, inorganic acids such as organic acids such as citric acid, malic acid, gluconic acid, succinic acid, ascorbic acid, tartaric acid, lactic acid, and fumaric acid, and phosphoric acid and their salts may be cited. The composition obtained is adjusted to a suitable pH, which is sometimes very important for the stabilization of the contained target component.

[0313] The pH can be adjusted to, for example, less than 7. This is because the thermal stability of galactosyl kojibiose is high when the pH is less than 7. The pH can be, for example, 2 or more, 2.5 or more, or 3 or more, preferably 3 or more and 6 or less, and more preferably 4 or more and 6 or less. This is because the thermal stability of galactosyl kojibiose is higher within this range. If appropriate, the pH of the solution can be measured by setting the solid content concentration of the solution to 12%.

[0314] [Example]

[0315] [Example 1: Production of galactosyl kojibiose using lactose]

[0316] (Preparation of enzyme solution)

[0317] Using a commercially available MRS liquid medium, red-striped liquid Lactobacillus JCM12392 was cultured at 30°C overnight, and then cooled to 10°C or less to terminate the culture.

[0318] The obtained culture solution was centrifuged, the supernatant was discarded and the bacterial cells were collected, and then an equal amount of citric acid phosphate buffer (pH 5.0) was added and the bacterial cells were collected again to wash the bacterial cells. The above buffer was added to the obtained bacterial cells in an amount of half the original culture solution, and the cells were suspended to prepare an enzyme solution (amino acid sequence of enzyme protein: sequence number 1).

[0319] (Enzyme reaction)

[0320] Sucrose 32 w / w%, lactose 16 w / w%, and enzyme solution 10 w / w% were mixed and reacted at 48°C for 18 hours under static conditions. The sugar composition after the reaction was analyzed by HPLC (SUPELCO apHera TM NH2 HPLC Column, mobile phase 68% acetonitrile, column temperature 35°C, flow rate 1.0 mL / min, RI detection) and the results are shown in Figure 1 The composition contains 25-30% galactosyl kojibiose, 15-20% lactose, and 30-35% fructose per unit solid content).

[0321] [Example 2: Production of galactosyl kojibiose using whey powder]

[0322] Under the enzyme reaction conditions shown in Example 1, 20 w / w% of whey powder (Meiji Co., Ltd.) (containing 75% lactose) was added instead of lactose, and the enzyme reaction was carried out under the same conditions.

[0323] When whey powder was used, the same chromatogram as in Example 1 was obtained.

[0324] [Example 3: Purification of tri- and tetra-sugar fractions]

[0325] After the enzyme reaction solution was passed through a diatomaceous earth column, distilled water was passed through to remove non-adsorbed substances. Elution was performed in stages using aqueous ethanol solutions of varying concentrations, thereby obtaining a fraction consisting of trisaccharides containing 4'-galactosyl kojibiose as a main component and a tetrasaccharide fraction in which one molecule of glucose was also bonded to 4'-galactosyl kojibiose ( Figure 2 ).

[0326] [Example 4: Lactase treatment and structural analysis of fractionated purified products]

[0327] The purified trisaccharide fraction was freeze-dried and dissolved in a 10% concentration of phosphate citrate buffer at pH 6.5. 0.1% lactase (product name: GODO-YNL, Godo Spirits Co., Ltd.) was added thereto and the enzyme reaction was carried out at 40°C. The results of LCMS analysis using an amide column (BEH Amide, Waters) confirmed that kojibiose (two sugars of glucose linked to each other by α-1,2 bonds, elution time at 7.75 minutes) was generated after the reaction ( Figure 3 ).

[0328] From the above results, it was confirmed that the main component of the trisaccharide fraction was galactosyl kojibiose. Based on the activity of the enzyme, it is believed that lactose is also transferred to galactose residues, but surprisingly, galactosyl kojibiose is generated without almost any transfer to galactose residues (described below).

[0329]

[0330] [Example 5: Fermented milk containing galactosyl kojibiose]

[0331] (Preparation of enzyme solution)

[0332] 1% of Lactobacillus rhodopsinus JCM12392 activated and cultured in a commercially available MRS liquid medium was inoculated into the medium shown below, and the pH was kept constant at 6.4 with sodium hydroxide in a fermenter at 30°C for 27 hours, and then the culture was terminated by cooling the fermenter to below 10°C.

[0333]

Table 2

[0334]

[0335] The obtained culture solution was centrifuged, the supernatant was discarded, and the red-striped liquid Lactobacillus concentrate (the concentrate of the enzyme-producing protein having the amino acid sequence of SEQ ID NO. 1) in which the bacterial cell concentration was concentrated to 15 times was used as the enzyme solution.

[0336] (Production of fermented milk containing galactosyl kojibiose)

[0337] Raw milk is prepared by mixing 500.0 g of raw milk, 53.2 g of skimmed milk powder, 23.0 g of fresh cream, 403.6 g of tap water and 50 g of sucrose. The raw milk is heat-sterilized at a temperature of 95°C, and the heat-sterilized raw milk is cooled. After that, 0.5% red-striped liquid Lactobacillus concentrate (enzyme solution) is inoculated into the raw milk, and fermented at 48°C for 18 hours under static conditions, and then Lactobacillus bulgaricus and Streptococcus thermophilus are added as lactic acid bacteria starters. The amount of lactic acid bacteria starter added is 20 g. The raw milk to which the lactic acid bacteria starter is added is filled in a cup container (capacity: 100 ml. Made of plastic). The raw milk filled in the cup container is left to ferment in a fermentation room at a temperature of 43°C until the lactic acid acidity reaches 0.7%.

[0338] [Example 6: Evaluation of thermal stability of aqueous solution]

[0339] The purified and fractionated galactosyl kojibiose obtained in Example 3 was dissolved in a buffer solution of each pH (100 mM citric acid phosphate buffer solution at pH 3 to 6, 100 mM phosphate buffer solution at pH 6 to pH 8) to obtain 12° Bx (12 wt%). After heating at 60° C., 80° C., 100° C., and 120° C. for 20 minutes using an autoclave apparatus, the residual rate of galactosyl kojibiose was evaluated.

[0340] The residual rate was calculated by HPLC method under the following conditions.

[0341] Column: SUPELCO apHera TM NH2 HPLC Column

[0342] Mobile phase: 68% acetonitrile

[0343] Column temperature: 35°C

[0344] Flow rate: 1.0mL / min

[0345] Detection: RI

[0346] As a result of heating at 120°C for 20 minutes, the galactosyl kojibiose observed at around 15.8 minutes elution time did not show significant changes below pH 6. However, at pH 7 or above, the chromatographic peak attenuated and a new chromatographic peak believed to be derived from a compound with a structural change appeared at around 12.1 minutes elution time ( Figure 4 ).

[0347] The peak areas of the respective temperature treatment conditions are shown in Table 1, assuming that the 60°C heating condition at which no attenuation of galactosyl kojibiose was observed at any pH was 100. Figure 5 .

[0348] [Example 7: Identification of new sugars]

[0349] The molecular structure of the compound generated by the heat treatment (generated compound) was inferred by LC / MS using an amide column (ACQUITY UPLC BEH Amide Column, 130 angstrom, 1.7 μm, 2.1 mm×100 mm, Waters). Specifically, the solution of the purified and fractionated galactosyl kojibiose obtained in Example 3 was heated to generate the compound ( Figure 6 0.1% of β-galactosidase (lactase) GODO-YNL was added to the mixture, and the enzyme reaction was carried out at 40°C. As a result, in addition to kojibiose produced from galactosyl kojibiose by hydrolysis of the galactosyl group, a peak of a new disaccharide believed to be derived from the generated compound was observed ( Figure 6 The elution time of this peak is similar to that of 2-glucosylmannose ( Figure 6 above) are consistent.

[0350] Therefore, it is estimated that the compound generated by the heat treatment is glucosyl epilactose (α-D-Glcp-(1→2)-[β-D-Galp-(1→4)-]D-Manp) in which the reducing terminal glucose of galactosyl kojibiose is isomerized to mannose.

[0351]

[0352] This novel compound is expected to be used as a precursor of epilactose, which is reported to be promising as a prebiotic (Non-patent document 5: Prebiotic properties of epilactose. J Dairy Sci. 2008 Dec; 91(12): 4518-26).

[0353] [Example 8: Preparation of pH-adjusted sugar solution and dried product thereof]

[0354] The pH of the composition containing galactosyl kojibiose obtained in Example 1 or Example 2 was checked, and if the pH exceeded 7, it was adjusted to pH 7 or less using a pH adjuster permitted as a food. The obtained sugar solution having a pH of 7 or less can be dried by a conventional method to prepare a powder.

[0355] Industrial Applicability

[0356] According to the present invention, a food containing oligosaccharides such as galactosyl kojibiose and a method for producing the food can be provided. In addition, according to the present invention, it can be expected to achieve nutritional improvement for various groups of people, ensure healthy living, and enhance welfare.

[0357] [Sequences listed in the sequence listing]

[0358] SEQ ID NO: 1-8: Amino acid sequence of glycoside hydrolase

[0359] SEQ ID NO: 9-21: Amino acid sequence of glycoside hydrolase (partial)

Claims

1. A method for producing an oligosaccharide-containing composition, comprising the step of allowing a composition containing lactose and sucrose to act with glucansucrase to obtain a composition containing galactosylkojibiose.

2. The manufacturing method according to claim 1, wherein: Glucansucrase is used in the form of lactic acid bacteria or a processed product thereof.

3. The manufacturing method according to claim 2, wherein: The lactic acid bacteria are lactic acid bacteria that constitutively express glucansucrase.

4. The manufacturing method according to claim 1, wherein: Glucansucrase is a bacteria-bound type.

5. The manufacturing method according to claim 1, wherein: Glucansucrase has dextransucrase activity.

6. The manufacturing method according to claim 2, wherein: The lactic acid bacteria or processed products thereof are any selected from the group consisting of live bacteria, dead bacteria, cultures containing bacteria, disrupted bacteria, and purified products of glucansucrase.

7. The manufacturing method according to any one of claims 1 to 6, wherein: Glucansucrase, or a lactic acid bacterium expressing glucansucrase, or a processed product thereof is derived from a lactic acid bacterium belonging to the family Lactobacillaceae.

8. The manufacturing method according to any one of claims 1 to 6, wherein: The dextransucrase or the lactic acid bacteria expressing the dextransucrase or the processed product thereof is derived from the lactic acid bacteria belonging to the genus Liquorilactobacillus.

9. The manufacturing method according to any one of claims 1 to 6, wherein: Glucansucrase or lactic acid bacteria expressing glucansucrase or a processed product thereof is derived from lactic acid bacteria belonging to Liquorilactobacillus satsumensis.

10. A method for producing a composition containing oligosaccharides, comprising the step of allowing a glucansucrase composed of the following protein (A), (B) or (C) to act on a composition containing lactose and sucrose to obtain a composition containing galactosylkojibiose, (A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8; (B) a protein consisting of an amino acid sequence having a high sequence identity to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity; (C) A protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added to the amino acid sequence of any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity.

11. The manufacturing method according to claim 10, wherein: The dextran sucrase is composed of the following protein (B') or (C'), (B') a protein having a high sequence identity to the amino acid sequences described in SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1, and having glucansucrase activity; (C') A protein having glucansucrase activity and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added to the amino acid sequences of SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the sequence of the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO:

1.

12. A method for producing fermented milk containing galactosyl kojibiose, comprising: A step of allowing a lactic acid bacterium expressing glucansucrase or a processed product thereof to act on a composition containing raw material milk and sucrose to obtain a composition containing galactosylkojibiose; and A step of adding lactic acid bacteria starter to raw milk and fermenting the raw milk to obtain fermented milk.

13. An enzyme agent comprising glucansucrase composed of the following protein (A), (B) or (C), (A) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8; (B) a protein consisting of an amino acid sequence having a high sequence identity to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity; (C) A protein comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted and / or added to the amino acid sequence described in any one of SEQ ID NOs: 1 to 8 and having glucansucrase activity.

14. The enzyme agent according to claim 13, wherein The dextran sucrase is composed of the following protein (B') or (C'), (B') a protein having a high sequence identity to the amino acid sequences described in SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the portions corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO: 1, and having glucansucrase activity; (C') A protein having glucansucrase activity and comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, inserted and / or added in the amino acid sequences of SEQ ID NOs: 1 to 8, wherein the amino acid sequence is identical to the sequence corresponding to positions 450-467, 488-499 and 559-573 of SEQ ID NO:

1.

15. The enzyme agent according to claim 13 or 14, wherein The glucansucrase is contained in the form of any one selected from the group consisting of living cells, dead cells, cultures containing cells, and cell fragments of lactic acid bacteria expressing glucansucrase.

16. The enzyme agent according to claim 13 or 14, which is used in the production method according to any one of claims 1 to 6.

17. The enzyme agent according to claim 13 or 14, which is used for producing glucosylepilactose.

18. A solution comprising galactosyl kojibiose having a pH of less than 7.

19. A composition comprising galactosyl kojibiose, wherein the composition has a pH of less than 7 when the composition is made into a solution having a solid content concentration of 10 to 80%.

20. The composition according to claim 19, wherein Contains more than 10% fructose per unit solid content.

21. The solution according to claim 18 or the composition according to claim 19 or 20, which is obtained by the production method according to any one of claims 1 to 6 and 10 to 12.

22. A composition comprising galactosyl kojibiose having the following characteristics: (1) containing 15 to 40% galactosyl kojibiose per unit solid content; (2) containing 15-45% fructose per unit solid content; (3) When a solution having a solid content concentration of 12% is prepared and heated at 120°C for 20 minutes, the residual rate of galactosyl kojibiose is 80% or more.

23. Glucosyl epilactose or a composition comprising the same. 24 . The production method according to claim 1 , further comprising a step of adjusting the pH of the obtained composition to less than 7.

25. The manufacturing method according to any one of claims 1 to 6 and 10 to 12, wherein: The oligosaccharide-containing composition comprises galactosyl kojibiose and fructose.

26. The manufacturing method according to any one of claims 1 to 6 and 10 to 12, wherein: The oligosaccharide-containing composition contains 10% to 100% of galactosyl kojibiose per solid content.

27. The manufacturing method according to any one of claims 1 to 6 and 10 to 12, wherein: The oligosaccharide-containing composition contains 1% or more of fructose per solid content.

28. The manufacturing method according to any one of claims 1 to 6 and 10 to 12, wherein: In the composition containing oligosaccharides, the mass ratio of galactosyl kojibiose to lactose, i.e., galactosyl kojibiose:lactose is 15-40:15-30.

29. The manufacturing method according to any one of claims 1 to 6 and 10 to 12, wherein: The oligosaccharide-containing composition comprises glucosyl epillactose. 30 . The production method according to any one of claims 1 to 6 and 10 to 12, which is used for producing the oligosaccharide-containing composition according to any one of claims 18 to 23.

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