JP5603036B2 - Probiotic growth promoter - Google Patents

Description

  The present invention relates to a Lactobacillus casei YIT 9029 having various biologically beneficial effects, a probiotic growth promoter, a composition for promoting growth, and a medium for promoting growth.

  Lactic acid bacteria and Bifidobacterium bacteria live in the human intestine, and by producing lactic acid and acetic acid, the pH in the intestine is maintained acidic, and spoilage and pathogenic bacteria such as E. coli grow and proliferate in the intestine. By suppressing this, the intestinal environment is improved. These lactic acid bacteria and Bifidobacterium are dominant bacteria in the intestine during infancy, but harmful bacteria such as Escherichia coli become dominant as humans grow, and various harmful substances produced by harmful bacteria have an adverse effect on the human body. It is supposed to affect. Lactic acid bacteria and Bifidobacterium spp. Are not only effective in improving the intestinal environment but also beneficial physiological effects such as fecal improvement, infection protection, immunity boosting action, antiallergic effect, and cancer prevention effect. Indicates. Therefore, in order to supply these useful bacteria called probiotics into the intestines and promote health, ingestion of lactic acid bacteria beverages, fermented milk, fungal preparations and the like containing live bacteria of these fungi is performed .

  However, it is said that simply ingesting live probiotic preparations may not improve the intestinal flora, and in order to vigorously grow the ingested bacteria in the intestines, It is desirable to simultaneously take as a growth promoter a sugar source that is difficult to be digested and absorbed in the body and that can be preferentially assimilated by the bacteria, so-called prebiotics. These prebiotics are not only less assimilating to harmful bacteria, but are also desired to grow lactic acid bacteria and Bifidobacterium that have been shown to have favorable effects on drinkers. ing.

  Lactobacillus casei YIT 9029 (FERM BP-1366) is known as a lactic acid bacterium whose physiologically beneficial effect has been clarified by various animal experiments and human tests. This Lactobacillus casei YIT 9029 is able to withstand strong digestive juices such as gastric juice and bile and reaches the intestine alive, increasing the number of resident Bifidobacterium in the intestine and reducing the coliform group To improve the intestinal environment. Lactobacillus casei YIT 9029 suppresses the production of harmful substances produced by harmful bacteria in the intestines. Furthermore, the drinking effect of Lactobacillus casei YIT 9029 includes fecal improvement, infection inhibition effect of pathogenic Escherichia coli O-157, verotoxin production inhibition effect, urinary tract infection prevention effect, etc., NK cell activation It has been reported to increase immunity such as action, antiallergic effect, and cancer suppressive effect such as colon cancer and superficial bladder cancer.

  Conventionally, as a growth promoting factor of lactic acid bacteria, yeast extract, chlorella extract, certain peptides, fruit and vegetable juice, mevalonic acid, consti-prica factor, orotic acid, panthetin, adenylic acid and the like are used. And substances having hypoxanthine bases such as inosinic acid are known. However, these are only used to promote the growth of lactic acid bacteria in the production process of fermented milk and the like, and are not considered to be effective for promoting the growth of lactic acid bacteria in the human intestine. In addition, some foods are restricted from being used in food because of their influence on flavor, cost, and product stability. Furthermore, these substances non-selectively grow lactic acid bacteria, and do not selectively grow Lactobacillus casei YIT 9029, which is known for its various useful effects.

For Bifidobacterium, the following general formula is generated by β-galactosyl group transfer reaction when β-galactosidase is allowed to act on lactose.
Gal- (Gal) n-Glc
A galactooligosaccharide (where Gal represents a galactose residue, Glc represents a glucose residue, and n represents an integer of 1 to 4) is used as an effective growth promoter in the human intestine (Non-patent Document 1, 2).

  However, this galactooligosaccharide is a selective sugar source of Bifidobacterium, and among the lactic acid bacteria, many Lactobacillus bacteria are not assimilating, and are particularly useful for Lactobacillus casei species. Not.

On the other hand, an oligosaccharide containing sorbitol which is a sugar alcohol of glucose instead of glucose,
Gal-Gal-Sor
(In the formula, Gal represents a galactose residue and Sor represents a sorbitol residue)
The reduced galactooligosaccharide represented by the formula is known to promote the growth of Bifidobacterium, but is not assimilated by Lactobacillus acidophilus, a kind of lactic acid bacteria (Patent Document 1). .

Also, the following formula
Gal- (Gal-) Sor
(In the formula, Gal and Sor have the same meaning as described above.)
It is known that trisaccharide oligosaccharides mainly composed of Bifidobacterium are not utilized by Lactobacillus bacteria, although Bifidobacterium bacteria have assimilability (Non-patent Document 3).

  On the other hand, a galactosyl disaccharide obtained by acting β-galactosidase on lactose is also known as a growth promoter for lactic acid bacteria including Lactobacillus bacteria and Bifidobacterium bacteria (Patent Document 2). However, since these galactosyl disaccharides are indigestible saccharides and easily induce diarrhea due to increased osmotic pressure in the digestive tract, trisaccharides having a higher molecular weight than disaccharides are desirable, and galactooligos that specifically grow Lactobacillus bacteria There has been a long-awaited demand for polymers having three or more sugars such as sugars or derivatives thereof. In addition, although galactosyl disaccharide is an indigestible saccharide, it is known that it is slightly more digestible than galactooligosaccharides of 3 or more sugars (Non-patent Document 4). A galactooligosaccharide or a derivative thereof capable of growing Lactobacillus bacteria with high selectivity has been awaited.

JP-A-63-246391 Japanese Patent No. 2722110

Journal of Japan Society for New Materials, vol. 6, p55-66 (2003) Journal of Enterobacteriaceae, vol. 18, p25-35 (2004) J Nutr Sci Vitaminol, vol.41, p83-94, (1995) Journal of Japan Society for Food Science and Technology, vol. 51, p28-33 (2004)

  An object of the present invention is to provide Lactobacillus casei YIT 9029 having various biologically beneficial effects with high selectivity and to provide a galactooligosaccharide or a derivative thereof having a higher degree of indigestibility. is there.

  As a result of intensive studies to solve the above problems, the present inventors have selectively reduced Lactobacillus casei YIT 9029 having various biologically beneficial effects among lactic acid bacteria. And the present invention has been completed. Further, the reduced galactooligosaccharide was found to selectively grow not only Lactobacillus casei YIT 9029 but also other bacteria used in probiotics, and the present invention was completed.

That is, the present invention provides the following formula (I)
Gal-Gal-Sor (I)
(In the formula, Gal represents a galactose residue and Sor represents a sorbitol residue)
Is a Lactobacillus casei YIT 9029 (FERM BP-1366) growth promoter.

  The present invention also provides a composition for promoting growth of Lactobacillus casei YIT 9029 containing the reduced galactooligosaccharide and a solid or liquid non-toxic pharmaceutical carrier or food / drink component, and a lactobacillus containing the reduced galactooligosaccharide as a carbon source. Casei YIT 9029 growth promoting medium.

Furthermore, the present invention provides the following formula (I)
Gal-Gal-Sor (I)
(In the formula, Gal represents a galactose residue and Sor represents a sorbitol residue)
Is a probiotic growth promoter containing a reduced galactooligosaccharide represented by

  Furthermore, the present invention relates to a probiotic growth promoting composition comprising the reduced galactooligosaccharide and a solid or liquid non-toxic pharmaceutical carrier or food / drink component, and a probiotic growth containing the reduced galactooligosaccharide as a carbon source. Promotion medium.

  The reduced galactooligosaccharide represented by the formula (I), which is an active ingredient of the growth promoter of the present invention, is produced by selectively growing and proliferating Lactobacillus casei YIT 9029 in the intestines of humans and animals. -Various beneficial physiological activity derived from casei YIT 9029 can be continuously exerted in the body.

  Moreover, the said reduced galactooligosaccharide can be utilized suitably as a component of the selective culture medium of the food / beverage products which have the said effect | action, and Lactobacillus casei YIT 9029.

  Furthermore, the reduced galactooligosaccharide can be grown not only for Lactobacillus casei YIT 9029 but also for Bifidobacterium, which is a representative probiotic bacterium. The beneficial physiological activity can be continuously exerted in the body.

  Furthermore, the reduced galactooligosaccharide can be suitably used as a component of a selective medium for foods and beverages and probiotics having the above-described effects.

The Lactobacillus casei YIT 9029 (FERM BP-1366) growth promoter of the present invention (hereinafter simply referred to as “growth promoter”) is represented by the following formula (I):
Gal-Gal-Sor (I)
(In the formula, Gal represents a galactose residue and Sor represents a sorbitol residue)
The reduced galactooligosaccharide represented by the formula is used as an active ingredient.

  The binding mode in the reduced galactooligosaccharide represented by the formula (I) (hereinafter simply referred to as “reduced galactooligosaccharide”) is not particularly limited, and for example, β1-4, β1-6, β1-3, Examples include β1-2, α1-3, α1-6 bonds, and the like, and β1-4, β1-6, β1-3, β1-2 bonds are preferable in terms of easy preparation. Specific reduced galactooligosaccharides include Galβ1-4Galβ1-4Sor, Galβ1-4Galβ1-3Sor, Galβ1-6Galβ1-4Sor and the like.

Although the manufacturing method of the said reduced galactooligosaccharide is not specifically limited, For example, following formula (II) as a trisaccharide fraction
Gal-Gal-Glc (II)
(In the formula, Gal represents a galactose residue and Glc represents a glucose residue)
And a method of reducing this by hydrogenation (hereinafter referred to as “reduction method”), an enzyme having transglycosylation activity such as β-galactosidase to act on lactitol (Gal-Sor), A method of adding galactose (hereinafter referred to as “addition method 1”), galactose prepared from a substance containing galactose such as lactose and galactan by hydrolysis with an enzyme or acid, and converted into lactitol by an enzymatic method or a chemical synthesis method. It is obtained by a method of adding galactose (hereinafter referred to as “addition method 2”). These methods will be described below.

<Reduction method>
The binding mode in the galactooligosaccharide represented by the formula (II) used as a raw material in the reduction method is not particularly limited. For example, β1-6, β1-3, β1-4, β1-2, α1-3 , Α1-6 bond, and the like. The galactooligosaccharide may be obtained by any method, for example, by a method of isolating and purifying from a natural product containing galactooligosaccharide, or a method of causing lactose as a raw material to act on an enzyme or a microorganism that produces the enzyme. What was obtained is mentioned.

  Here, examples of microorganisms that produce enzymes for producing galactooligosaccharides include Streptococcus thermophilus, Lactobacillus bulgaricus, Kriveromyces fragilis, Kriveromyces lactis, Aspergillus oryzae, Penicillium multicolora, Rhizopus oryzae, and Bacillus.・ Circulance, Cryptococcus laurenti, Sporoboromyces singularis, Saccharomyces fragilis, Lipomyces lipopha, Sterigmatomyces eribiae, Rhodotorula minuta and Shirobasidium magnum, especially Sporoboromyces Singularis and Krivellomyces lactis are preferred. In order to produce enzymes from these microorganisms, culture is performed under conditions suitable for the microorganisms to be used, and according to the purpose, the enzymes can be isolated and produced from the obtained bacterial cells or their culture supernatant according to a conventional method. . For example, a method of crushing bacterial cells obtained by culturing with ultrasonic waves or a surfactant and removing insoluble matters, or a method of purification using chromatography can be mentioned. In order to produce galactooligosaccharides with these enzymes, the reaction may be carried out under conditions that do not completely deactivate the enzyme. Generally, the raw material concentration is 10 to 70% by mass (hereinafter simply referred to as “%”), pH The temperature is suitably 3 to 8, the temperature is 20 to 80 ° C., and the reaction time is suitably 2 hours to 3 days.

  In addition, the above-described microorganism producing the enzyme can be directly cultured in a medium containing the raw material. The culture conditions in this case are not particularly limited, and may be performed under conditions suitable for culturing the microorganisms to be used. In general, the raw material concentration is 10 to 40%, the pH is 3 to 8, and the temperature is A temperature of 20 to 45 ° C. and a culture time of 2 hours to 7 days are appropriate.

  Further, as the galacto-oligosaccharide, commercially available galacto-oligosaccharides such as Oligomate (registered trademark) 55N manufactured by Yakult Pharmaceutical Co., Ltd., Cup Oligo (registered trademark) manufactured by Nissin Sugar Co., Ltd., etc. can also be used. .

  The reduced galactooligosaccharide can be obtained by reducing the galactooligosaccharide obtained as described above by a high-pressure catalytic reduction method using a sponge nickel catalyst or a known reduction method using a reducing agent such as sodium borohydride.

<Additional method 1>
Examples of the lactitol used as a raw material in addition method 1 include those for foods manufactured by Mitsubishi Corporation Food Tech Co., Ltd. and Bussan Food Science Co., Ltd., in addition to the reagents of each company. Examples of the galactose source added to lactitol include not only lactitol itself but also lactose and foods containing lactose such as milk, skim milk powder, whole milk powder and the like.

  In order to add galactose by acting an enzyme having transglycosylation activity such as β-galactosidase on lactitol, a microorganism producing an enzyme for producing galactooligosaccharide used in the above reduction method is used to produce galactooligosaccharide. The reaction may be carried out under the condition that the enzyme is not completely inactivated.

<Additional method 2>
The galactose used in addition method 2 is not only commercially available galactose but also galactose as a substance containing galactose as described above. You can also.

  In order to add the obtained galactose to lactitol by an enzymatic method or a chemical synthesis method, for example, an enzymatic method uses a sugar transfer reaction such as β-galactosidase, or a chemical synthesis method usually uses Reduced galactooligosaccharide is obtained by the sugar addition reaction of the method.

  The reduced galactooligosaccharide obtained by the above method can be used for the growth promoter of the present invention even if it contains unreacted galactooligosaccharide, tetrasaccharide or more reduced galactooligosaccharide, etc. It may be used.

  The method for separating and purifying the reduced galactooligosaccharide can be performed using an activated carbon column, a gel filtration column chromatographic separation apparatus, more preferably a simulated moving bed type chromatographic separation apparatus using an ion exchange resin. The prepared reduced galactooligosaccharide can be appropriately concentrated or diluted and used as a liquid sugar having a suitable concentration. Moreover, the prepared liquid sugar can also be used in the form of a powder, after drying using a spray dryer or a freeze dryer.

  The reduced galactooligosaccharide thus prepared selectively exhibits a growth promoting action against Lactobacillus casei YIT 9029 among lactic acid bacteria. The Lactobacillus casei YIT 9029 growth-promoting action referred to in the present invention is the increase or maintenance of the number of bacteria in counting the number of bacteria using a plate medium or the like, and the number of bacteria using the bacteria-specific DNA or RNA as an index. It means an increase or maintenance, an increase in lactic acid as a metabolite, a decrease in pH or an increase in acidity of a medium such as a growth environment. Further, in the present invention, Lactobacillus casei YIT 9029 is FERM BP-1366 as of May 1, 1981, Institute of Microbial Technology, Institute of Industrial Technology, Ministry of International Trade and Industry Research Center for Biological Biology: Not only those deposited internationally in Tsukuba City, Ibaraki Prefecture, 305-8586, Japan, 1st Central, 6th), but also, for example, Lactobacillus casei YIT 9029 as a parent strain Mutants that have been mutated by a known method such as agent treatment and that retain the ability to assimilate the reduced galactooligosaccharides of the present invention are also included.

  Since the growth promoter of the present invention has the above action, for example, it is ingested or administered to humans or animals in which Lactobacillus casei YIT 9029 is present in the intestine, or Lactobacillus casei YIT 9029 is cultured. By adding to the medium or the like, the growth of Lactobacillus casei YIT 9029 can be promoted.

  When the growth promoting agent of the present invention is ingested or administered to a human or animal in which Lactobacillus casei YIT 9029 is present in the intestine, the ingested or administered amount is the age, sex, body weight, administration of the subject. It can be determined appropriately according to the method. For example, in the case of a human adult, the daily intake or dosage is in the range of 1 to 1000 mg, preferably 10 to 500 mg, as reduced galactooligosaccharide per kg of body weight. In addition, the growth promoter of the present invention may be taken in the whole daily required amount at once, or may be taken in several times.

  In order to easily ingest or administer the reduced galactooligosaccharide of the present invention to humans and animals, the reduced galactooligosaccharide of the present invention is mixed with a solid or liquid non-toxic pharmaceutical carrier to obtain a conventional pharmaceutical preparation. The composition for promoting the growth of Lactobacillus casei YIT 9029 may be prepared by mixing with a food or drink component to obtain a conventional food or drink.

  Examples of the form of the pharmaceutical preparation include solid preparations such as tablets, granules, dispersions, capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These forms of pharmaceutical preparations can be prepared by conventional means on preparations, except that the reduced galactooligosaccharide, which is an active ingredient of the growth promoter of the present invention, is contained in the required amount. Examples of non-toxic pharmaceutical carriers used in pharmaceutical preparations include, for example, starch, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin albumin, water, physiological saline Water etc. are mentioned. Furthermore, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents, excipients and the like can be appropriately added to these pharmaceutical preparations as necessary.

  Moreover, what is necessary is just to make the reduced galactooligosaccharide which is an active ingredient of the growth promoter of this invention as it is or with various nutritional components as a form of the said food / beverage products, and to make it food / beverage products. The amount of reduced galactooligosaccharide blended in the composition for promoting the growth of food and drink products only needs to be an amount that can exert its growth promoting action, the general intake of the target food and drink, the form of the food and drink, the efficacy and effects, What is necessary is just to set suitably in consideration of taste, palatability, cost, etc. For example, in the case of a solid food, the reduced galactooligosaccharide content is 0.01 to 100%, preferably 0.1% to 100%. In the case of a liquid food such as a beverage, the reduced galactooligosaccharide content is 0.001. It is prepared to be ˜50%, preferably 0.01˜20%.

  Specific examples of the food / beverage product components that can be blended with the reduced galactooligosaccharide of the present invention include the following. That is, milk beverages (raw milk, ordinary milk, concentrated milk, processed milk such as low-fat milk, skim milk, coffee milk drink, fruit milk drink), soft drink, tea drink, carbonated drink, nutrition drink, fruit drink, etc. (Including concentrated concentrates and powders for adjustment of these beverages), powdered milk such as whole milk powder, skim milk powder, prepared milk powder, yogurt (soft yogurt, hard yogurt, plain yogurt, drink type yogurt, frozen yogurt, yogurt with pulp, etc. ), Dairy products such as butter, cheese, condensed milk, ice cream, ice candy, ice sherbet, frozen confectionery such as shaved ice, buckwheat, udon, harusame, gyoza peel, Chinese noodles, instant noodles and other noodles, rice cakes, chewing gum, candy , Gummy, Gum, Caramel, Chocolate, Tablets, Snacks, Biscuit Sweets such as jelly, jam, cream, fishery products such as kamaboko, chikuwa, hamburger, croquette, ham, sausage, processed foods such as margarine, mayonnaise, shortening, whipped cream, dressing, etc., syrup, soy sauce , Ingredients such as sauces, vinegar and mirin, and food and beverage ingredients such as breads such as bread and confectionery bread, but are not limited thereto.

  The composition for promoting growth in the form of food or drink may be appropriately blended with additives usually used depending on the type. As such additives, any additives that are acceptable for food hygiene can be used. For example, sugar, fructose, isomerized liquid sugar, glucose, aspartame, sucralose, stevia, and other sweeteners, citric acid , Sour agents such as malic acid and tartaric acid, excipients such as dextrin and starch, antioxidants such as polyphenols, β-carotene and ascorbic acid derivatives, binders, diluents, flavoring agents, coloring agents, buffering agents, Examples include a sticking agent, a gelling agent, a stabilizer, a preservative, an emulsifier, a dispersant, a suspending agent, and a preservative.

  Moreover, you may mix | blend minerals suitably with the composition for proliferation promotion of the said food-drinks form. As such minerals, any additive that is acceptable for food hygiene can be used. For example, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, calcium glucuronate, calcium glycerophosphate, iron citrate, etc. As vitamins to be added, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, vitamin C, vitamin D, vitamin E, vitamin K, calcium pantothenate, nicotinamide, choline chloride Etc.

  Furthermore, if fermented milk containing Lactobacillus casei YIT 9029 is added to the composition for promoting growth in the form of the above food and drink, these foods and drinks have good proliferative properties in the intestine of Lactobacillus casei YIT 9029. The product can be highly effective.

  In addition, the said food / beverage products also contain animal feed, and can also be used as feed with the effect of promoting the growth of Lactobacillus casei YIT 9029 and feed with health effects derived therefrom.

The growth promoter of the present invention can selectively culture Lactobacillus casei YIT 9029 by adding Lactobacillus casei YIT 9029 as a carbon source to the culture medium (aerobic condition). It can be a growth promoting medium.
The amount of the growth promoter of the present invention added to the medium may be 0.1 to 5%, preferably 0.5 to 3%, as the reduced galactooligosaccharide content.

  If the above growth promoting medium is used, Lactobacillus casei YIT 9029 can be specifically selected from a medium containing various lactic acid bacteria. For example, when cultured under aerobic conditions using a plate medium containing reduced galactooligosaccharides, the viable cell count of Lactobacillus casei YIT 9029 can be determined by the difference in growth rate and the size and color of various bacterial colonies. Separation can be facilitated.

  The reduced galactooligosaccharide, which is an active ingredient of the growth promoter of Lactobacillus casei YIT 9029 of the present invention described so far, together with Lactobacillus casei YIT 9029 having various specific physiological effects, is a useful probiotic fungus. Representative Bifidobacterium genus bacteria, especially Bifidobacterium addressensetis, Bifidobacterium breve, Bifidobacterium longum, etc. are also cultured or in vitro in human or animal intestines. The separated one can be grown. Therefore, reduced galactooligosaccharides can also be used in probiotic growth promoters, probiotic growth promotion compositions containing the same, and probiotic growth promotion media, and are particularly effective as prebiotics. Is extremely high.

  The probiotic growth-promoting action referred to in the present invention means the number of bacteria in counting the number of bacteria using a plate medium of useful probiotic bacteria as in the above-described Lactobacillus casei YIT 9029 growth-promoting action. Increase or maintenance, increase or maintenance of the number of bacteria using bacteria-specific DNA or RNA as an indicator, increase of lactic acid as a metabolite, decrease in pH or increase in acidity of a medium as a growth environment.

  In addition, it can be used as a health food or food material for the purpose of increasing the probiotics in the intestine as described above, or for increasing the effectiveness of probiotics such as intestinal regulation. The food / beverage product or its container may be labeled as having the above effect.

  Hereinafter, the content of the present invention will be described in more detail with reference to examples, but the present invention is not limited by these.

Example 1
Preparation of reduced galactooligosaccharide (Gal-Gal-Sor):
(1) Preparation of β-galactosidase (a) Production of bacterial cell suspension In a 300 mL Erlenmeyer flask, 50 g / L lactose, 6 g / L yeast extract, 1 g / L 1 potassium phosphate and 0.5 g / L were added. 100 mL of a medium (pH 5.0) containing L magnesium sulfate was taken and autoclaved (121 ° C., 15 minutes). One platinum loop of Sporoboromyces singularis (FERM P-18818) was inoculated into this medium, followed by shaking culture for 6 days under conditions of 28 ° C. and 120 rpm to obtain a culture end solution.

  Next, 1.5 L of a medium having the same composition as described above was prepared in a 3 L fermenter (Maruhishi Bioengine), and autoclaved (121 ° C., 15 minutes). This was inoculated with 60 mL of the previous culture end solution, and aerated and stirred for 5 days under the conditions of 27 ° C., 425 rpm, and 1 vvm. After culturing, the culture solution was centrifuged (8000 G, 20 minutes) to recover the cells, and suspended in 50 mM disodium hydrogen phosphate-citrate buffer (pH 4.0). The activity value of this cell suspension was measured by the following activity measurement method using 2-nitrophenyl-β-galactoside as a substrate.

(B) Activity measurement method using 2-nitrophenyl-β-galactoside as a substrate 2-Nitrophenyl-β-galactoside was dissolved in 50 mM disodium hydrogen phosphate-citrate buffer (pH 4.0), and 12.5 mM. A substrate solution was prepared. To 0.8 mL of this substrate solution, 0.2 mL of a cell suspension appropriately diluted with 50 mM disodium hydrogen phosphate-citrate buffer (pH 4.0) was added and reacted at 30 ° C. for 10 minutes. The reaction was stopped by adding 4 mL of a 0.25 M sodium carbonate solution to this, and then the absorbance at 420 nm was measured with a spectrophotometer for the supernatant obtained by centrifugation (3000 g, 10 minutes) (test solution). On the other hand, sodium carbonate solution and diluted cell suspension were sequentially added to the 2-nitrophenyl-β-galactoside solution, and the absorbance at 420 nm was measured for the supernatant obtained by centrifugation (blind test). . In this method, the amount of enzyme that liberates 1 micromole of 2-nitrophenol per minute was defined as 1 unit (U).

(2) Preparation of galactooligosaccharide liquid sugar 25 kg of lactose was added to 25 kg of 50 mM disodium hydrogen phosphate-citrate buffer (pH 6), dissolved by heating, and then cooled to 60 ° C. To this, 1900 U of the cell suspension obtained in (1) (a) was added and reacted at 60 ° C. for 45 hours, and the reaction was stopped by heating. After cooling to 45 ° C., the pH is adjusted to 6.5 with 2N sodium hydroxide solution, and 550,000 U (according to the manufacturer's measurement method) β-galactosidase derived from Krivellomyces lactis (according to the manufacturer's measurement method) is added and reacted for 18 hours. The reaction was stopped by heating. Add 3 times the volume of ion-exchanged water to the reaction solution, centrifuge (8000G, 20 minutes) to remove the cells, purify with activated carbon, diatomaceous earth filtration, ion-exchange resin, and concentrate according to conventional methods. A galactooligosaccharide liquid sugar of Brix (Bx) 70 was obtained. The resulting galactooligosaccharide composition was 7% for 4 or more sugars, 35% for 3 sugars, 29% for 2 sugars, and 29% for monosaccharides.

(3a) Preparation of reduced galactooligosaccharide 2.5% sponge nickel catalyst was added to 27 kg of the galactooligosaccharide liquid sugar prepared in (2) above adjusted to 50, and the temperature was raised to 120 to 130 ° C. while stirring. Then, the hydrogen pressure was increased to 7-8 MPa to complete the hydrogenation. After removing the sponge nickel catalyst, purification by activated carbon, diatomaceous earth filtration, and ion exchange resin was performed according to a conventional method to obtain Bx30 reduced galactooligosaccharide liquid sugar (including lactitol, sorbitol, galactitol). The amount of reducing sugar in the reaction solution after the reaction (Beltrant method, galactose conversion) was reduced from 30.3% before the reaction to 0.05%, and thus the reduction reaction proceeded sufficiently. I confirmed. The sugar composition was 7% for tetrasaccharides or more, 35% for 3 sugars, 29% for 2 sugars, and 29% for monosaccharides, which was unchanged from that before reduction.

(3b) Preparation of reduced galactooligosaccharide 180 g of 10% (w / w) sodium borohydride solution was added to 180 g of the galactooligosaccharide liquid sugar prepared in (2) above adjusted to 10 and stirred gently. Then, the reaction was stopped for 3 hours, and then the reaction was stopped by adding acetic acid to adjust the pH to 7. Next, this reaction solution was passed through a column packed with 500 mL of a strongly acidic ion exchange resin (PK-218, manufactured by Mitsubishi Chemical), and sugar was recovered by flowing pure water. This was dried under reduced pressure on a rotary evaporator, redissolved in 500 mL of methanol and dried under reduced pressure four times, then recovered with pure water, and Bx34 reduced galactooligosaccharide liquid sugar (including lactitol, sorbitol, galactitol) ) In addition, in the proton NMR analysis of this liquid sugar, since the peak of the α-anomeric position of the reducing terminal glucose was not detected, it was confirmed that the reducing sugar was sufficiently reduced. The sugar composition was 7% for tetrasaccharides or more, 35% for 3 sugars, 29% for 2 sugars, and 29% for monosaccharides, which was unchanged from that before reduction.

(4) Preparation of fractions The reduced galactooligosaccharide (solid content: about 20 g) obtained in (3b) above was passed through a column packed with 430 mL of chromatographic activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) Monosaccharide and disaccharide were eluted. Subsequently, 50% ethanol was passed through to elute more than 3 sugars, and ethanol contained in the recovered solution was removed with a rotary evaporator. About 1.5 g of the solid content of the recovered liquid is injected into a column filled with 2 L of gel filtration resin (Biogel P-2, manufactured by Bio-Rad), and pure water is poured to obtain a trisaccharide and tetrasaccharide fraction. The recovered galactooligosaccharide (tetrasaccharide) was reduced and concentrated under reduced pressure to 50 (w / v)% or more.

(5) Composition of fractions Each content of the obtained reduced galactooligosaccharide and reduced galactooligosaccharide (tetrasaccharide) was 98% or more. As a result of separation and purification by high performance liquid chromatography and analysis by NMR, the main component of the reduced galactooligosaccharide is Gal-Gal-Sor, and its structure is Galβ1-4Galβ1-4Sor (about 63%), Galβ1-6Galβ1-4Sor (about 11%), Galβ1-4Galβ1-3Sor and the like. The main component of the reduced galactooligosaccharide (tetrasaccharide) was Galβ1-6Galβ1-4Galβ1-4Sor.

Example 2
Utilization test:
(Test substance)
As test substances, a reduced galactooligosaccharide fraction comprising Galβ1-4Galβ1-4Sor prepared in Example 1 as a main component, a galactooligosaccharide trisaccharide fraction, a reduced galactooligosaccharide tetrasaccharide fraction, a galactooligosaccharide tetrasaccharide fraction, lactitol, Sorbitol and galactooligosaccharide (4 ′ galactosyl lactose) were used.

(Operation)
Tests of the strains described in Tables 1 to 3 which were anaerobically cultured in each medium containing 0.5% glucose in 3 mL of medium (ILS, PY or VL medium) after autoclaving containing 0.5% of the test substance. A culture solution was prepared by adding 30 μL of the bacterial solution. After culturing this culture solution at 37 ° C. for 48 to 72 hours under anaerobic conditions, pH was measured. The determination of assimilation was performed based on the difference (ΔpH) between the pH of the culture solution containing the test substance and the pH of the control culture solution (no test substance added), and evaluation was performed according to the following criteria. Table 1 shows the results of the assimilation test for 8 strains of the genus Lactobacillus of reduced galactooligosaccharide, galactooligosaccharide (trisaccharide), reduced galactooligosaccharide (tetrasaccharide) and galactooligosaccharide (tetrasaccharide). Table 2 shows the results of the assimilation test for 12 strains of the genus Lactobacillus of reduced galactooligosaccharide, lactitol, sorbitol, and galactooligosaccharide (4 ′ galactosyl lactose). Furthermore, Table 3 shows the assimilation properties of bacteria other than the genus Lactobacillus of the reduced galactooligosaccharide.

<Usability evaluation criteria>
Evaluation: Contents ++ The pH decreased by 1.5 or more compared to the control culture + The pH decreased by 1.0 or more and less than 1.5 compared to the control culture ± The pH was 0.5 or more compared to the control culture Reduced by less than 1.0-pH decreased by less than 0.5 compared to control culture

(result)
From the results shown in Table 1, Lactobacillus casei YIT 9029 grows only when a reduced galactooligosaccharide containing Galβ1-4Galβ1-4Sor as a main component is added, and galactooligosaccharide (trisaccharide) and reduced galactooligosaccharide (tetrasaccharide). ), Galactooligosaccharide (tetrasaccharide) was found to have no growth effect. In addition, with reduced galactooligosaccharides, only Lactobacillus casei YIT 9029 out of the 8 strains tested showed significant growth.

  In addition, from the results shown in Table 2, reduced galactooligosaccharides mainly composed of Galβ1-4Galβ1-4Sor can specifically and significantly proliferate only Lactobacillus casei YIT 9029 out of 12 strains tested. all right. Lactitol and sorbitol were assimilated by Lactobacillus casei YIT 9029, but were also assimilated by several other Lactobacillus bacteria and were found to have low specificity.

  Furthermore, from the results shown in Table 3, reduced galactooligosaccharides mainly composed of Galβ1-4Galβ1-4Sor are rarely used for other intestinal bacteria except that they are assimilated by Bifidobacterium. all right. Thus, it is clear that the reduced galactooligosaccharides mainly composed of Galβ1-4Galβ1-4Sor are selectively used for probiotic strains that are considered to contribute to the maintenance of health in the intestine. became.

From the above, it was revealed that the reduced galactooligosaccharide mainly composed of Galβ1-4Galβ1-4Sor selectively proliferates Lactobacillus casei YIT 9029 (FERM BP-1366) and Bifidobacterium.

Example 3
Liquid sugar preparation:
The reduced galactooligosaccharide liquid sugar (Bx30) obtained in (3a) of Example 1 was filled in an 18 L aluminum can.

Example 4
Preparation of reduced galactooligosaccharide powder:
The reduced galactooligosaccharide liquid sugar (Bx30) obtained in (3a) of Example 1 was diluted 3 times and dried with a freeze dryer, and then reduced galactooligosaccharide powder was prepared using a pulverizer.

Example 5
Beverage preparation:
300 kg of reduced galactooligosaccharide liquid sugar (Bx30) obtained in (3a) of Example 1, 0.5 kg of citric acid and 3 kg of fragrance were dissolved in 3000 L of water, bottled in 100 mL portions, and reduced galactooligosaccharides per bottle. A beverage containing 3 g was prepared.

Example 6
Preparation of selective medium for Lactobacillus casei YIT 9029:
In 1 L, 5 g of reduced galactooligosaccharide prepared by the method of (4) of Example 1 as a solid content, 10 g of trypticase peptone (DIFCO), 5 g of yeast extract (DIFCO), 3 g of tryptose (DIFCO), hydrogen phosphate 2g 3g, potassium dihydrogen phosphate 3g, diammonium hydrogen citrate 2g, magnesium sulfate heptahydrate 0.55g, iron sulfate heptahydrate 34mg, manganese sulfate heptahydrate 0.12g, Tween 80 1g, cysteine Hydrochloride 0.2 g and agar 15 g were dissolved and autoclaved to prepare Lactobacillus casei YIT 9029 selective medium. In addition, this medium can be used as a medium for promoting the growth of Lactobacillus casei YIT 9029. Furthermore, this medium can be used as a medium for promoting probiotic growth.

  The reduced galactooligosaccharide represented by the formula (I), which is an active ingredient of the growth promoter of the present invention, has a higher degree of indigestibility and has various biologically beneficial effects among lactic acid bacteria. Casei YIT 9029 can be grown with high selectivity. The reduced galactooligosaccharide can be selectively grown not only for Lactobacillus casei YIT 9029 but also for other bacteria used in probiotics.

Therefore, the growth promoter of the present invention can be used for probiotics and the like, and is useful for promoting health.

Claims (7)

A reduced galactooligosaccharide containing Galβ1-4Galβ1-4Sor, Galβ1-6Galβ1-4Sor, Galβ1-4Galβ1-3Sor ( wherein Gal represents a galactose residue and Sor represents a sorbitol residue) , and Lactobacillus casei YIT 9029 (FERM BP-1366) and the food / beverage products characterized by the above-mentioned.   The food or drink according to claim 1, which is fermented milk. A non-toxic pharmaceutical carrier and a reduced galactooligosaccharide comprising Galβ1-4Galβ1-4Sor, Galβ1-6Galβ1-4Sor, Galβ1-4Galβ1-3Sor (wherein Gal represents a galactose residue and Sor represents a sorbitol residue) Containing Lactobacillus casei YIT 9029 (FERM BP-1366) composition for promoting growth (excluding food and drink). Lactobacillus containing a reduced galactooligosaccharide containing Galβ1-4Galβ1-4Sor, Galβ1-6Galβ1-4Sor, Galβ1-4Galβ1-3Sor (wherein Gal represents a galactose residue and Sor represents a sorbitol residue) as a carbon source Casei YIT 9029 (FERM BP-1366) growth promoting medium. Lactobacillus containing a reduced galactooligosaccharide containing Galβ1-4Galβ1-4Sor, Galβ1-6Galβ1-4Sor, Galβ1-4Galβ1-3Sor (wherein Gal represents a galactose residue and Sor represents a sorbitol residue) as a carbon source -Selection medium for casei YIT 9029 (FERM BP-1366).   A method for growing Lactobacillus casei YIT 9029, comprising culturing Lactobacillus casei YIT 9029 in a medium for promoting growth of Lactobacillus casei YIT 9029 (FERM BP-1366) according to claim 4.   A method for selecting Lactobacillus casei YIT 9029, comprising culturing Lactobacillus casei YIT 9029 in a medium for selecting Lactobacillus casei YIT 9029 (FERM BP-1366) according to claim 5.