JPH06233677A - Saccharide for proliferation of bifidobacterium - Google Patents

Abstract

PURPOSE:To obtain a saccharide sugar excellent in the resistance to acids and capable of being well assimilated by Bifidobacterium but hardly being assimilated by other indigenous or noxious bacteria by incorporating a specific glycosylxyluloside. CONSTITUTION:The objective product contains, as a saccharide for proliferrating Bifidobacterium, a glycosylxyluloside wherein a xylulosyl group binds to alpha- hydroxy group at 1-position of glucose through a beta-bond. This saccharide has a low decomposition rate before reaching ileum and large intestine when orally administered and is easily crystallized in an aqueous solution and easily pulverized to form crystalline powder.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a carbohydrate for growing Bifidobacteria, and more particularly to the use of glucosylxyluloside, which is an oligosaccharide in which a xylulosyl group is β-bonded to the α-hydroxyl group at the 1-position of glucose. is there.

[0002]

2. Description of the Related Art There are numerous reports on the physiological significance of Bifidobacterium,
It is widely known that the intestinal spoilage bacteria inhibit spoilage, the production of toxic amines is inhibited, and the growth of pathogenic bacteria is inhibited by the formation of organic acids. Therefore, for the purpose of maintaining and improving human health, studies have been conducted to keep the intestinal flora predominantly bifidobacteria, and it has been known that certain sugars become bifidobacterial growth factors.

In order to grow bifidobacteria in the intestine, it reaches the ileum and large intestine without being digested after being ingested, it is not used by bacteria other than bifidobacteria, and bifidobacteria are often used. It is necessary to meet the conditions of.

Fructooligosaccharides (trade name: May-oligo), lactulose, raffinose and the like are known as sugars for growing Bifidobacteria. However, since fructooligosaccharides are weak against acid, they may be decomposed before ingestion when used in acidic foods and drinks such as soft drinks. Furthermore, it is also used for bacteria other than Bifidobacteria. Similarly, lactulose is used for enterobacteria other than Bifidobacteria, such as Escherichia coli, and thus has a drawback that diarrhea occurs when the dose is increased a little. Regarding the raffinose, in an experiment using mice, human-derived Bifidobacterium infantis proliferated during the administration period, but at the same time, Escherichia coli was also proliferated.

Furthermore, most of the known sugars for growing Bifidobacteria cannot be pulverized by crystallization, which is a simple method. Therefore, when it is necessary to make powder for handling or food processing, it is necessary to make powder by freeze-drying, spray-drying, etc., which is inconvenient and has contributed to the cost of the product. Therefore, a saccharide that can be easily pulverized by crystallization and satisfies the above-mentioned conditions, which is a saccharide for selective growth of Bifidobacterium, has been desired.

[0006]

The inventors of the present invention have conducted extensive studies to find a further excellent carbohydrate for growing Bifidobacteria, and as a result, have found that the xylrosyl group is β-bonded to the α-hydroxyl group at the 1-position of glucose. The inventors have found that an oligosaccharide, glucosyl xyluloside, is suitable for this purpose, and completed the present invention.

That is, the present invention relates to a carbohydrate for growing Bifidobacteria which comprises the glucosyl xyluloside.

The structure of the glucosyl xyluloside of the present invention is glucose α-1-2-β xyluloside. This is a method of allowing sucrose phosphorylase to act on a mixed solution of α-D-glucose-1-phosphate and D-xylulose, a method of allowing sucrose phosphorylase to act on a mixed solution of sucrose and D-xylulose, or sucrose and D. It can be easily produced by a method of allowing a xylose isomerase and sucrose phosphorylase to act on a mixed solution of xylose. The detailed method for producing this oligosaccharide is disclosed in JP-A-3-191786.

The glucosyl xyluloside of the present invention has a concentration of 6
It can be easily pulverized by crystallization from an aqueous solution of 0 to 80 w / w%, preferably 62 to 70 w / w%, separation by a sieve and drying. In addition, this oligosaccharide shows strong resistance to acid as shown in FIG. Fig. 1 shows that various sugars (Bx12) were used at a given pH and temperature.
It shows the residual rate of sugar when rapidly cooled after holding for a minute. The strong resistance of the glucosyl xyluloside of the present invention to acid means that when it is used in a processed food, it is less decomposed by acid and much remains in the product. Further, as shown in the test example described later, this oligosaccharide is not easily decomposed by digestive enzymes, and 75.4% of the ingested amount reaches the ileum and the large intestine. This sufficiently satisfies the above conditions.

[0010]

EXAMPLES The present invention will be specifically described below with reference to examples. Test Example 1 Hydrolysis by Saliva 5.0% Glucosylxylloside (1 mM CaCl ₂
50 mM Bis-Tris buffer, pH 6.0) 90
100 μl of human saliva (36.0 U / ml) was added to 0 μl and reacted at 37 ° C. for 30 minutes. After inactivation by heating, when analyzed by high performance liquid chromatography (HPLC),
The hydrolysis rate of glucosyl xyluloside was 2.7%. One unit of the enzyme activity of human saliva was the amount of enzyme that produced 1.0 μmol of glucose per minute from 1% soluble starch at pH 6.0 and 37 ° C.

Test Example 2 Hydrolysis by gastric acid: 200 μm of 250 μl of 3.0% glucosyl xyluloside
M hydrochloric acid-potassium chloride buffer (pH 2.0, artificial gastric juice)
750 μl was added, and the mixture was reacted at 37 ° C. for 100 minutes. Then, after neutralizing with 0.5M sodium hydroxide, HP
When analyzed by LC, the hydrolysis rate of glucosyl xyluloside was 5.9%.

Test Example 3 Hydrolysis by pancreatic juice 0.5% glucosyl xyluloside (1 mM CaCl ₂
50 mM Bis-Tris buffer, pH 6.0) 90
Porcine pancreatic α-amylase (10.0 U / ml,
Add Boehringer Mannheim Yamanouchi Co., Ltd. to add 3
The reaction was carried out at 7 ° C for 6 hours. Then, after deactivation by heating, HPL
When analyzed by C, the hydrolysis rate of glucosyl xyluloside was 0.0%, and it was not decomposed at all. The enzymatic activity of porcine pancreatic α-amylase was defined as in the case of salivary amylase.

Test Example 4 Hydrolysis by Small Intestinal Mucosal Enzyme 900 μl of 0.5% glucosyl xyluloside (50 mM maleic acid buffer, pH 6.6) was added with enzyme solution (18.8 U).
(/ Ml) 100 μl was added, and the mixture was reacted at 37 ° C. for 3 hours. Next, after inactivation by heating, HPLC analysis revealed that the hydrolysis rate of glucosyl xyluloside was 17.6%. One unit of the enzyme activity of the small intestinal mucosa was defined as the amount of enzyme that produces 2.0 μmol glucose per minute from 1% maltose at 37 ° C and pH 6.0.

Test Example 5 Crystallization Test An aqueous glucosylxyluloside solution (concentration 64 w / w%) having a purity of 96% (analysis by HPLC) was shaken at 25 ° C. for 48 hours, and 1.0 to 4.0 mm large Crystals were precipitated, separated by sieving and dried to obtain white crystalline powder. The crystal recovery rate was about 20%.

The HPLC analysis conditions in Test Examples 1 to 5 are shown below. Column TOSOH TSK-gel Amide-80 (4.6 x 250 mm, manufactured by Tosoh Corporation) Solvent 71% Acetonitrile Flow rate 1.0 ml / min Temperature 35 ° C Detector Differential refractometer

The following examples show that the glucosyl xyluloside of the present invention satisfies the above-mentioned conditions as a carbohydrate for growing Bifidobacteria to the same extent as fructooligosaccharides and the above conditions are more than fructooligosaccharides. explain.

Example Using 108 representative strains of human-derived intestinal bacteria, the assimilation of glucosyl xyluloside was tested. For comparison, glucose and fructooligosaccharide (trade name: Mei-oligo P, manufactured by Meiji Seika Co., Ltd.) were also tested in the same manner. The test was conducted as follows. First, the test strain was purely cultured on BL agar medium. That is, a pure culture strain was obtained by streaking a frozen strain on a BL agar plate and performing an operation to obtain an isolated colony twice. The BL agar plate used was a BL agar medium manufactured by Nissui Pharmaceutical Co., Ltd. to which 5% horse defibrinated blood manufactured by Kojin Co., Ltd. was added. Culture is performed in an anaerobic incubator (SANYO / FORMA).
(Manufactured by K.K.) and anaerobically performed at 37 ° C. for 48 hours.

The pure culture strain thus obtained was used as Fildes
solution GAM broth was anaerobically cultivated at 37 ° C for 24 hours and subcultured, and 0.03 ml of this culture solution was added to anaerobic medium [Pepton-Yeast-Fildes (PYF) semi-fluid agar medium having the following composition and various sugars. It was added to a final concentration of 0.5% and sterilized by autoclaving at 115 ° C. for 20 minutes] and inoculated into 1.5 ml [Automatic multi-species inoculator, MD-1
20 (manufactured by LSFETEC) was used]. 4 at 37 ° C
After anaerobic culture for a day, the pH of the culture solution was measured. As in the case of the above-mentioned pure culture, an anaerobic incubator (SANYO / FORMA) was used for the culture, and the atmosphere was CO.
₂ 10%, H ₂ 10%, N ₂ balance mixed gas was used. In addition, pH measurement and data processing are BI
S-120 (manufactured by LEFETEC) was used.

Composition of PYF semi-liquid agar medium (pH 7.2) Trypticase (BBL) 10.0 g Yeast extract (Difco) 10.0 g Fildes solution 40.0 ml Salts solution ^* 40.0 ml L-cysteine hydrochloride monohydrate 0.5g agar 1.5g purified water 920.0ml

^* Composition of Salts solution CaCl ₂ anhydrous 0.2 g MgSO ₄ 0.2 g K ₂ HPO ₄ 1.0 g KH ₂ PO ₄ 1.0 g NaHCO ₃ 10.0 g NaCl 2.0 g Purified water 1000.0 ml

The presence or absence or strength of assimilation by the test strain was judged according to the following criteria. That is, the degree of pH decrease was used as a criterion, and the greater the pH decrease, the greater the assimilation ability. Assessment of assimilation pH 6.0 ≤ actually measured pH-pH 5.5 ≤ actually measured pH <6.0 ± pH 5.0 ≤ actually measured pH <5.5 + pH 4.5 ≤ actually measured pH <5.0 + + pH 4.0 ≤ actually measured pH <4.5 +++

The results are shown in Table 1. Column A in the table shows the genus name of the strain used and the number of different strains belonging to that genus. Bifidobacterium is a so-called Bifidobacterium, which is a useful bacterium. All other strains are plain harmless bacteria (plain bacteria are harmless, but in some cases harmful) or harmful bacteria. Therefore, as shown in the column B, in the case of Bifidobacterium, the growth is strong (+++, +
+, +), Weak growth in the case of other strains (-, ±)
Is desirable. Column C shows the percentage of the bacterial strains that gave the desired results shown in Column B when cultured with each sugar in the medium. The larger the value shown in this column C is, the more preferable as the sugar for the selective growth of Bifidobacteria in the case of Bifidobacterium and other strains. In column D, for comparison with fructooligosaccharide, the value of glucosyl xyluloside in column C is 10 more than that of fructooligosaccharide.
If the difference is 10% or less, the difference is 10%.
Those that are smaller than 0% are shown as −.

[0023]

[Table 1]

Thus, glucosyl xyluloside is
It is assimilated by bifidobacteria to the same extent as fructooligosaccharides, but it can be said that other plain harmless bacteria or harmful bacteria are not assimilated as fructooligosaccharides. That is, the glucosyl xyluloside of the present invention satisfies the conditions as a carbohydrate for growing Bifidobacterium to the same extent as fructooligosaccharides and the conditions more than fructooligosaccharides.

[0025]

EFFECT OF THE INVENTION The glucosyl xyluloside of the present invention is well assimilated by bifidobacteria, but is hardly assimilated by other plain harmless or harmful bacteria. Also, when ingested,
Since the rate of digestive degradation until reaching the ileum or large intestine is low, it satisfies all the conditions required for a carbohydrate for growing Bifidobacteria. Moreover, since it has strong resistance to acid, it is not easily decomposed at the processing stage, and it can be easily crystallized in an aqueous solution and can be easily crystallized into powder, if necessary. It can be applied to various foods.

[Brief description of drawings]

FIG. 1 is a graph showing the effects of pH and temperature on the decomposition of various sugars.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuhiro Kuwahara 13-46 Daikokucho, Tsurumi-ku, Yokohama-shi, Kanagawa Shimizu Port Sugar Co., Ltd.

Claims (1)

[Claims] 1. A carbohydrate for growing Bifidobacterium, which comprises an oligosaccharide in which a xylulosyl group is β-bonded to the α-hydroxyl group at the 1-position of glucose.