JP2823640B2 - Intestinal flora improving substance - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an intestinal flora improving substance having an effect of promoting the growth of bifidobacteria and lactic acid bacteria and an effect of inhibiting the growth of C. perfringens.

[Background Art] In recent years, it has been known that the flora (bacteria flora) in the intestine is related to human health, and interest in intestinal bacteria is increasing. Bifidobacterium, for example, is one of the major bacterial species that make up the human intestinal flora, and has various beneficial physiological roles for humans and animals, such as inhibiting the growth of putrefactive bacteria and pathogens in the intestine. It is known to work. Since this bifidobacterium decreases or disappears with various diseases and aging, various attempts have been made to increase the bifidobacteria in the intestine.

Foods or pharmaceuticals for such purposes include, for example, ruffled bifidobacteria, bifidobacterium powder,
Bifidobacterium-proliferating oligosaccharides and the like are known. Among these, bifidobacterium-proliferating oligosaccharides have recently been in the spotlight, and fructooligosaccharides, soybean oligosaccharides, konjac oligosaccharides, isomaltoligosaccharides, galacto-oligosaccharides, and the like have been reported as having a bifidobacterial growth effect. . Some of them are already commercially available as health food materials.

On the other hand, lactic acid bacteria have long been known as beneficial bacteria that affect the intestinal flora. Lactic acid bacteria, like Bifidobacterium, are thought to play a role in the growth suppression of putrefactive bacteria in the intestine, and live lactic acid bacteria are put into intestinal medicines, and drinks containing lactic acid bacteria are marketed. I have.

"Problems to be Solved by the Invention" The effect of oligosaccharides on promoting the growth of bifidobacteria is that bifidobacteria can utilize the oligosaccharides and use them as nutrients, but many other bacteria use the oligosaccharides. As a result, bifidobacteria selectively grow. However, these oligosaccharides are not necessarily selectively used only for bifidobacteria, and there are bacteria other than bifidobacteria that grow using these oligosaccharides. The kind of bacteria that each oligosaccharide is used by and the kind of bacteria that cannot be used depend on the type of oligosaccharide, and at present, it is impossible to know without actually conducting experiments.

Moreover, the intestinal flora is constituted by the coexistence of many bacteria, and even if the bifidobacteria temporarily become influential in the intestine, the power easily changes. Therefore, in order to stably obtain the effect of improving intestinal flora,
It is preferable to promote the growth of not only bifidobacteria but also other beneficial bacteria such as lactic acid bacteria. That is, by increasing the power of lactic acid bacteria and the like together with the bifidobacteria, the growth inhibitory effect of putrefactive bacteria such as C. perfringens can be more stably obtained.

However, previous studies on the improvement of intestinal flora by oligosaccharides have focused solely on the growth-promoting effects of bifidobacteria, and their effects on other beneficial bacteria, such as lactic acid bacteria, have been poorly studied. It is not at present.

Regarding lactic acid bacteria, although live lactic acid bacteria are used in intestinal medicines and beverages, they do not improve the growth environment of lactic acid bacteria in the intestine. Even if the concentration of bacterium is increased, the presence of other bacteria that are more adapted to the environment will cause the power to fluctuate easily, so that a stable intestinal flora improving effect cannot be expected.

Accordingly, an object of the present invention is to provide an intestinal flora-improving substance that has not only bifidobacteria but also a growth-promoting effect of lactic acid bacteria, a growth-suppressing effect of C. perfringens, and a more stable effect. To provide.

“Means for Solving the Problems” The present inventors have developed β-
Research on industrial production and utilization of gluco-oligosaccharides has been repeated.In the process, β-gluco-oligosaccharide was found to have an effect of promoting the growth of not only bifidobacteria but also lactic acid bacteria. Was completed.

That is, the intestinal flora improving substance of the present invention contains a gluco-oligosaccharide comprising a β-glucoside bond and / or a reduced product thereof as an active ingredient. In a preferred embodiment, cellobiose, sophorose, laminaribiose, and gentiobiose are used. , 4- O -β- D - Gen thio oligosilsesquioxane - D - glucose and 6- O -β- D - Gen thio oligosilsesquioxane - D - 1 kind or 2 selected from glucose
More than one species is the active ingredient.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

The β-gluco-oligosaccharide used in the present invention is obtained by converting β-glucosidase from various microorganisms into glucose and / or β-glucosidase.
By acting on gluco-oligosaccharides and maximizing the limiting function of the condensation / transfer action of β-glucosidase, it can be easily produced in high yield. This method is disclosed in Japanese Patent Application No. 63-46 proposed by the present inventors.
No. 313 and Japanese Patent Application No. 1-41289.

To explain the outline of this production method, it is possible to use β-glucosidases derived from various microorganisms, for example, Trichoderma viridi ( Tr.
ichoderma viride , trichoderma lesai ( Trich)
oderma reesei ), Trichoderma konsei ( Tricho)
derma koningii , Aspergillus niger ( Aspergi)
llus niger ), Penicillium friqutans ( Pen
icillium frequentans ), wood rot fungi such as Polypolus tulipiferae and Chrysosporium lignoru.
m), Schizophyllum commune (Shizophyllum commu
ne ), and other bacteria such as Pseudomonas fluorescens var. cellulosa , Cellulomonas uda , and Clostridium thermocellu.
m ), Ruminococcus albu
Enzymes of microbial origin such as s ) are preferably used. All of these microorganisms are known and can be easily obtained and the enzyme can be prepared.

As a substrate, D-glucose and / or β-
Gluco-oligosaccharides are used. Here, β-
Glucooligosaccharides are cellobiose, gentiobiose,
Alternatively, it means a gentio-oligosaccharide having a higher degree of polymerization. When β-gluco-oligosaccharide is used as a substrate, β-gluco-oligosaccharide having a higher polymerization degree can be obtained by the present enzyme reaction. Particularly preferably, at least one selected from glucose, cellobiose and gentiobiose is used as the substrate.

When β-glucosidase is allowed to act on glucose and / or β-gluco-oligosaccharide in this manner, cellobiose, sophorose, laminaribiose,
Gentiobiose, 4- O -β- D - Gen thio oligosilsesquioxane - D - glucose, 6- O -β- D - Gen thio oligosilsesquioxane - D - Various β- glucooligosaccharide such as glucose can be obtained. Here, 4- O -β- D - Gen thio oligosilsesquioxane - D - and glucose, 4- O -β- D - Genchiobioshiru - D - glucose, 4- O -β- D - Gen thio Trio sill - D A glucose or higher degree of polymerization is meant. Also, 6- O-.beta.-D - Gen thio oligosilsesquioxane - D - and glucose, 6- O-.beta.-D - Genchiobioshiru - D - glucose (gentiotriose),
6- O -β- D -gentiotriosyl- D -glucose (gentiotetraose) or a gentio-oligosaccharide having a higher degree of polymerization.

These reaction products vary depending on the enzyme used, but when glucose or cellobiose is used as a substrate, there is a tendency that several kinds of the above-mentioned various β-gluco-oligosaccharides are easily mixed and produced. When gentiobiose is used as a substrate,
6- O -β- D -gentiobiosyl- D -glucose,
There is a tendency that only gentio-oligosaccharides such as 6- O -β- D -gentiotriosyl- D -glucose are easily produced.

In addition, when explaining the enzyme reaction conditions, the substrate concentration is not particularly limited, but is usually preferably 1 to 90% (solid content / volume), and more preferably 5 to 80% (solid content / volume). The higher the enzyme concentration with respect to the substrate, the better. However, it is usually preferable to use 100 mg or more per 1 g of the substrate. The reaction temperature and the reaction pH may be performed under the optimum reaction conditions of the enzyme used. Usually, the reaction temperature is preferably from 30 to 80 ° C, more preferably from 50 to 70 ° C. The reaction pH is preferably about 3 to 8. The reaction time may be a time during which the desired β-gluco-oligosaccharide is sufficiently generated and accumulated.
About 2 minutes to 72 hours is appropriate. The reaction may be carried out by adding the enzyme to the substrate, or may be carried out in a continuous reaction system using the immobilized enzyme by adsorbing the enzyme to an appropriate immobilizing agent to form the immobilized enzyme. The reaction product thus obtained can be further fractionated by various methods to separate and purify various β-oligosaccharides.

The intestinal flora improving substance of the present invention contains, for example, the β-glucooligosaccharide obtained as described above and / or its reduced product as an active ingredient. The reduced product can be obtained by subjecting these oligosaccharides to catalytic reduction (hydrogenation). Such a treatment is a treatment method conventionally employed in the production of sugar alcohols and the like.

The intestinal flora improving substance of the present invention can be used as it is as a pharmaceutical or health food, but can also be used as an additive to various foods and drinks. When added to food and drink, besides the effect of improving intestinal flora, β
Gluco-oligosaccharides are low-calorie saccharides, so they can be used as dietary saccharides, and because they have a bitter taste that has never been seen before, they can also be used as saccharides for improving the taste of various foods. Furthermore, since gentio-oligosaccharides are rich in moisturizing properties, they can also be used as moisturizers for foods, as well as crystallization inhibitors, shining agents, and body-imparting agents.

The intestinal flora-improving substance of the present invention includes, for example, soy sauce, miso, mayonnaise, dressing, vinegar, Chinese ingredients, tentsuyu, sauce, ketchup, grilled meat sauce, curry pork,
It can be used for various seasonings such as stew base, soup base, dash base, composite seasoning, and mirin. In addition, various Japanese sweets such as rice crackers, hail, mochi, manju, uiro, bean jam, yokan, jelly, castella, candy, bread,
Biscuits, crackers, cookies, pies, puddings, butter cream, cream puffs, sponge cakes, donuts, chocolate, chewing gum, caramel, hard candy, and other Western confections, ice cream,
Ice confectionery such as sherbet, pickled fruit syrup, syrups such as ice honey, pastes such as flower paste, peanut paste, fruit paste, processed fruit products such as jam, marmalade, syrup pickled, sugarcane fruit, Fukujin pickled, senmai pickled, rakkyo Pickles such as pickles, meat products such as ham and sausage, fish meat products such as kamaboko, bamboo rings, various delicacies, boiled foods, alcoholic beverages such as beer, liqueur, sake, coffee, cocoa, juice, etc.
It can also be used for various drinks and drinks, such as soft drinks such as carbonated drinks, stamina drinks, lactic acid drinks, and lactic acid bacteria drinks, and instant drinks and drinks such as instant juice and instant coffee.

As described above, the intestinal flora improving substance of the present invention can be used by adding it to foods, foods, foods, feeds, feeds, pharmaceuticals, and all other orally used substances. Can be improved. Further, as the intestinal flora improving substance of the present invention, β-gluco-oligosaccharide obtained by subjecting a β-gluco-oligosaccharide to a reduction treatment to convert a glucose residue at the reducing end to a sugar alcohol can be similarly used.
Gentiooligosaccharides are also known to be contained in small amounts in honey and acidified syrup, and can be safely used from the viewpoint of safety because they are considered natural products. When the intestinal flora improving substance of the present invention is added to various foods and drinks, the compounding amount is preferably 0.5 to 50% by weight, more preferably 1.0 to 30% by weight.

"Action and Effect" The intestinal flora improving substance of the present invention includes cellobiose, sophorose, laminaribiose, gentiobiose,
- O-.beta.-D - Gen thio oligosilsesquioxane - D - glucose and 6- O -β- D - Gen thio oligosilsesquioxane - D - but as an active ingredient and consisting of β- glucooligosaccharide glucose, these As is clear from the test results described later, it was found that the oligosaccharide is often used by lactic acid bacteria as well as by bifidobacteria. On the other hand, it was found that harmful bacteria such as Clostridium perfringens and other putrefactive bacteria cannot utilize these oligosaccharides and hardly grow. Therefore, by ingesting the intestinal flora improving substance of the present invention, beneficial bacteria such as bifidobacteria and lactic acid bacteria are selectively grown in the intestine, and the growth of harmful bacteria and putrefactive bacteria is suppressed. It can be seen that the flora can be improved.

On the other hand, fructooligosaccharides, isomaltooligosaccharides and the like that have been conventionally proposed as bifidobacterial proliferating oligosaccharides are those that are digested in the body and used as an energy source, and even if they have a bifidobacterial proliferating effect, If these are taken in large amounts, adult diseases such as obesity may be caused. In addition, since these oligosaccharides have a sweetness, if not as much as sugar, they may not be suitable for modern tastes that tend to lose their sweetness. On the other hand, β-glucooligosaccharide is a low-calorie saccharide that is difficult to digest in the body, and thus is useful for preventing adult diseases such as obesity. Furthermore, β-gluco-oligosaccharides, especially genti-oligosaccharides, have a bitter taste in addition to sweetness, and thus can impart a new taste that matches modern tastes that tend to lose sweetness.

"Example" Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 (Production of Genthio-oligosaccharide) (1) Purified and prepared from 300 g of D -glucose from crude cellulase preparation "Meiselase" (trade name, manufactured by Meiji Seika Co., Ltd.) derived from Trichoderma viride . β-glucosidase was converted to 5.8 × 10 ⁵ units (500 m
l) Add (approximately 60% glucose, w / v), pH 5.0, temperature 60
Reaction was performed at 48 ° C. for 48 hours. After completion of the reaction, the reaction was stopped by heating at 100 ° C. for 5 minutes.
After deionization purification, the solution was concentrated under reduced pressure to a solid content of 72% (W / W).

The obtained reaction product sugar solution is applied to a jacketed (60 ° C.) column having an inner diameter of 2 cm and a length of 120 cm (Dowex 9).
9 "(Na ⁺ type, manufactured by Dow Chemical Company) were filled with the sugar solution so that the solid content of the resin amount per 5~7% (w / v) was loaded, the space velocity (SV.hr ^{- 1} ) Fractionation at 0.35 yielded a gentio-oligosaccharide fraction (OS-1). Table 1 shows the results obtained by analyzing the sugar composition of this fraction by high performance liquid chromatography. The analysis conditions of the high performance liquid chromatography are as follows.

Column; Shimadzu SCR-101 detector; Differential refractometer Column temperature; 55 ° C Column flow rate; 0.8 ml / min The above operation was repeated 10 times to obtain about 40 g of gentiooligosaccharide (OS-1). This was freeze-dried and powdered.

(2) The above gentiooligosaccharide fraction (OS-1) is further fractionated and prepared by the same method as described above, and the β-glucodisaccharide fraction (F-2) and the β-glucotrisaccharide fraction are prepared. (F-3) and β
-A fraction (F-4) of glucotetrasaccharide or more was obtained. Table 1 shows the results obtained by analyzing the sugar composition of these fractions by high performance liquid chromatography.

In the above description, β-glucodisaccharide means a disaccharide having a β-glucoside bond such as cellobiose, sophorose, laminaribiose, and gentiobiose. Further, the β- glucoside trisaccharide, 4-O-.beta.-D - Genchiobioshiru - D - glucose, 6- O-.beta.-D - Genchiobioshiru - D - glucose (gentiotriose)
And so on. Furthermore, β-glucotetrasaccharide or more means 6-
O-.beta.-D - Gen thio Trio sill - D - those of glucose or more polymerization degree, 6- O -β- D - Gen thio Trio sill - D - glucose (gentiotetraose) or more polymerizable Degree means.

Example 2 (Assimilation test by intestinal bacteria) An assimilation test of various oligosaccharides by intestinal bacteria was performed by the following method.

(1) Test strains 17 Bacteroides , 20 Bifidobacterium , 26 Clostridium , Eubacteri
um ) 6 strains, 5 Fusobacterium strains,
Peptstreptococcus 6
Strains, 9 strains of Lactobacillus , 5 strains of Enterococcus, 5 strains of Escherichia coli , and 21 other strains were used.

(2) Test group 1. Control (no carbohydrate added) 2. Glucose 3. Mayoligo P (commercially available fructooligosaccharide, trade name, manufactured by Meiji Seika Co., Ltd.) 4. OS-1 (β-glucooligosaccharide fraction) 5.F-2 (β-glucodisaccharide fraction) 6.F-3 (β-glucotrisaccharide fraction) 7.F-4 (fraction of β-glucotetrasaccharide or higher) Mayoligo P has the following sugar composition.

Sucrose (GF) ... 4 wt% 1-ketose (GF ₂₎ ... 35 wt% nystose (GF ₃₎ ... 50% by weight of 1-fructosyl oligofructose (GF ₄₎ ... 11% by weight (3) using medium Pepton- Various sugars of the test group were added to a yeast-Fildes solution (PYF) semi-fluid agar medium so as to have a final concentration of 0.5%, and used after autoclaving at 115 ° C for 20 minutes.

In the above, the composition of the PYF semi-fluid agar medium is as follows.

Trypticase (BBL) 10.0 g Yeast Extract (Difco) 10 g Fildes Solution 40.0 ml Salts Solution 40.0 ml L-cysteine hydrochloride 1 hydrate 0.5 g Agar 1.5 g Purified water 920.0 ml (adjusted to pH 7.2) In the above, Salts Solution is , Consisting of the following composition:

CaCl ₂ Anhydorous 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 (4) Test method Frozen strains of test strains are streaked on BL agar medium The operation of culturing and obtaining an isolated colony was repeated twice to obtain a pure culture strain.
The BL agar plate was obtained by adding 5% of horse defibrinated blood manufactured by Kojin Co. to a BL agar medium manufactured by Nissui Pharmaceutical. Culture was performed using an anaerobic incubator manufactured by SANYO / FORMA.

The test strain thus pure-cultured was filtered with Fildes Solution G
AM bouillon (Nissui Pharmaceutical GAM bouillon with Fildes Solution
The culture medium was inoculated into a medium (supplemented with 0.4%), anaerobically cultured at 37 ° C. for 24 hours using an anaerobic incubator manufactured by SANYO / FORMA, and subcultured.

This culture solution was transferred to LIFETEC's automatic multi-strain inoculator “MD
Using -120 ", 0.03 ml of the above test medium was inoculated into 1.5 ml, and anaerobically cultured at 37 ° C for 4 days (96 hours), and then the pH was measured. After inoculation, contamination and poor growth of the inoculated bacterial solution were examined, and those corresponding to those were deleted from the data.
The anaerobic culture was performed using an anaerobic incubator manufactured by SANYO / FORMA, and the atmosphere was a mixed gas of CO ₂ 100%, H ₂ 10%, and N ₂ balance. For pH measurement and data processing,
"BIS-120" (manufactured by LIFETEC) was used.

(5) Judgment of assimilation property The presence or absence or strength of assimilation property was judged based on how much the pH of the culture solution was lowered. The criteria are as follows.

pH 6.0 or more ...-pH 5.5 or more and less than 6.0 ... ± pH 5.0 or more and less than 5.5 ... + pH 4.5 or more and less than 5.0 ... ++ Less than pH 4.5 ... +++ (6) Test results The test results are shown in Table 2. The following can be seen from this table.

Regarding Bacteroides , each test group shows almost the same assimilation.

For the Bifidobacterium (Bifidobacterium), showing an overall stronger assimilative also each test group. However,
Bifidobacterium
bifidum ) has no assimilability with Mayoligo P, F-3, or F-4, but contains OS-1, F containing β-glucodisaccharide.
In the case of -2, assimilation is recognized.

Regarding Clostridium , most of glucose has assimilation,
Most of Meioligo P, OS-1, F-2, F-3 and F-4 show no assimilation.

Oy Agrobacterium (Eubacterium), Fusobacterium (Fusobacterium), Peptostreptococcus (Pep
tstreptococcus ), Escheric
hia coli ), mayoligo P, OS-1,
Many of F-2, F-3, and F-4 do not show assimilation.

In Lactobacillus , Mayoligo P is often not assimilated, but OS-
As for 1, F-2, F-3 and F-4, assimilation is recognized in a considerably large number. In particular, Lactobacillus casei ( La
ctobacillus casei ) shows that OS-1, F-2, F-3, F-3
At -4, strong assimilation can be recognized.

From the above results, OS- consisting of β-glucooligosaccharide
1, F-2, F-3, and F-4 are often used not only for bifidobacteria but also for lactic acid bacteria, indicating that they are hardly used for other pathogenic bacteria or spoilage bacteria. Therefore, by ingesting these β-glucooligosaccharides, Bifidobacteria and lactic acid bacteria are selectively grown,
It can be seen that the growth of pathogens or putrefactive bacteria can be suppressed and the intestinal flora can be improved.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 1/38 C12N 1/38 (56) References JP-A-59-39287 (JP, A) JP-A-1-3111090 (JP) , A) (58) Fields surveyed (Int. Cl. ⁶ , DB name) A23L 1/30 A61K 31/70

Claims (2)

(57) [Claims] 1. An intestinal flora improving substance comprising a gluco-oligosaccharide comprising a β-glucoside bond and / or a reduced product thereof as an active ingredient. 2. A cell selected from cellobiose, sophorose, laminaribiose, gentiobiose, 4-O-β-D-gentio-oligosyl-D-glucose and 6-O-β-D-gentio-oligosyl-D-glucose. The intestinal flora-improving substance according to claim 1, wherein one or more of the selected substances are an active ingredient.