JP3459837B2 - Live bacteria powder - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides an excellent viable bacterium containing tannins, which has an increased ability to grow useful intestinal bacteria in humans or animals, suppresses the growth of harmful bacteria, and has increased storage stability. Regarding powder.

[0002]

BACKGROUND OF THE INVENTION Useful enteric bacteria such as bifidobacteria and lactic acid bacteria are resident in the intestines of infants and adults, and are used to prevent spoilage by enterobacteriaceae, prevent the production of toxic amines, and treat organic acids such as lactic acid and acetic acid. Growth inhibition of pathogenic bacteria by production, vitamin B ₁ ,
It is known that there are physiological actions such as the synthesis of B ₂ , B ₆ and B _12, etc., and they have a beneficial effect on the health of the host. In addition, it has been reported that feeding certain kinds of Bacillus subtilis or Miyairi bacteria to livestock improves growth, improves feed efficiency, and prevents the occurrence of diarrhea.

From the above, a large number of intestinal useful bacteria-containing preparations / foods for the prevention and treatment of diseases of the intestinal system, particularly diarrhea, intestinal catarrh, dyspepsia, constipation or bacterial symptom after administration of antibiotics. Livestock feed is commercially available.

The majority of useful enteric bacteria are obligately anaerobic bacteria, which makes it difficult to store them for a long period of time in the presence of air and difficult to store them even in the presence of water. For improving, a method of mixing lactulose with a dried product of Bifidobacterium (JP-A-52-15178), a method of mixing fats and oils (JP-A-57-32221), and a method of mixing vitamin E (JP-B-63-3585) , A method of mixing raw starch (Japanese Patent Publication No. 63-12594), a method of mixing inulooligosaccharide (Japanese Patent Application Laid-Open No. 3-49670), etc., and useful enteric useful bacterial agents which can withstand long-term storage are also disclosed. It is known.

However, when the above-mentioned known probiotic agent is ingested, it can pass through the upper gastrointestinal tract barrier such as gastric acid and bile acid in the host body and grow in the lower gastrointestinal tract as a growth site. The ability of live bacteria to colonize and proliferate such as sex is a serious problem. Furthermore, it has been proposed that various sugar sources such as lactulose, palatinose, fructooligosaccharides, raffinose, stachyose, and xylooligosaccharides are effective for the growth of bifidobacteria, which are useful bacteria. , It is assimilated against harmful bacteria such as Clostridium and is also involved in their growth. From the above, it is the present situation that it is selectively assimilated by Bifidobacterium and only the number of Bifidobacterium does not specifically increase in the intestine of the host.

[0006] On the other hand, catechins in green tea are in vitro intestinal harmful bacteria Clostridium perfringens ( Clostridium perfringeng)
s ), Clostridium difficile (C. diff
It has been disclosed that the growth of Clostridium bacteria such as icile ) is selectively suppressed (JP-A-2-29).
5923). In addition, actually ingesting catechins in green tea in vivo lowers the pH in the intestine to facilitate the growth of useful bacteria, but only selectively inhibits the growth of harmful bacteria Clostridium. It is extremely difficult to specifically increase the number of bifidobacteria in Japan (Abstracts of the 1991 Annual Meeting of the Japanese Society of Agricultural Chemistry, p.251).

[0007]

[Problems to be Solved by the Invention] How to suppress the growth of harmful intestinal bacteria such as Escherichia coli and Clostridium bacteria that have already colonized in the intestine, and how to improve the colonization and growth ability of ingested useful intestinal bacteria, Furthermore, it is important to improve the storage stability of the useful bacterial preparation. The object of the present invention is to provide such a live bacterial powder.

[0008]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that the above-mentioned dried product of useful intestinal useful bacteria and the tea oolong tea that we usually drink.・ Black tea ingredients, namely (+)-catechin, (+)-
Gallocatechin, (+)-Gallocatechin gallate, (-)
-Epicatechin, (-)-epicatechin gallate,
Tannins such as (−)-epigallocatechin, (−)-epigallocatechin gallate, free theaflavin, theaflavin monogallate A, theaflavin monogallate B, theaflavin digallate, etc. can solve the above problems. The inventors have found that a bacterial powder can be obtained, and that the ingestion of this live bacterial powder remarkably promotes the colonization and growth ability of useful bacteria in the intestine to improve the intestinal disease, thus completing the present invention. Further, it was also found that the storage stability of this live bacterial powder was significantly improved.

The present invention will be described in detail below. The tannins used in the present invention can be obtained from an ultrafiltration membrane and reverse osmosis membrane treatment of water or alcohol extract of green tea, oolong tea or black tea, or an ethyl acetate-soluble fraction, but other raw materials such as persimmon shibu It may be of origin or a chemically synthesized product.

A typical method for preparing the tannins used in the present invention is disclosed in detail in the previously filed patent (JP-A-2-6499).

The useful bacteria in the present invention are microorganisms which bring about beneficial effects on human or animal health, and are representative of bifidobacteria and lactic acid bacteria. As the Bifidobacteria, any strain may be used as long as it is a known strain of Bifidobacterium, but in the case of human, it is known that Bifidobacterium is resident in the intestine regardless of age. Umm Adressentis ( Bifidobacte
rium adolescentis ), Bifidobacterium longum ( B. longum ), Bifidobacterium bifidum ( B. bifidum ), and in the case of animals, Bifidobacterium pseudolongum ( B. B. thermophilum
It is desirable to use m 1 ) or the like alone or in combination. Lactic acid bacteria, Lactobacillus, Enterococcus, may be used any strain as long as it is a known strain of Streptococcus bacteria, for example Lactobacillus casei (Lactobacillusc asei), Lactobacillus Ashidofirisu (L. Acidophilu
s ), Lactobacillus plantarum ( L. plant)
arum ), Enterococcus faecalis ( Ent
erococcus faecalis ), Enterococcus faecium ( E. faecium ), Enterococcus hirae ( E. hirae ), Streptococcus bovis ( Streptococcus bovi)
s ), Streptococcus thermophilus ( Stre
Ptococcus thermophilus ) or the like is preferably used alone or in combination. Also,
Any bacterium other than the bacteria listed above can be used in the present invention without any particular limitation as long as it is a microorganism beneficial to the health of the host. For example, Bacillus Toyoi (Bacillus toyo is a bacterium belonging to the genus Bacillus
i ), Bacillus coagulans ( B. coagula
ns ), Bacillus subtilis varietas natto ( B. subtilis var . natto ), Bacillus licheniformis ( B. licheniformi)
s ), Bacillus subtilis ( B. subtilis )
Or Clostridium butyricum belonging to the genus Clostridium bacteria (Clostridium buty
ricum ) and the like.

The live bacterial powder described above is subjected to large-scale culture by a known medium and a culturing method, and then a known dispersion medium is added to the microbial cell suspension, followed by freeze-drying, or JP-A-4- It can be prepared by a method of coating an enteric substance as disclosed in No. 41434 and is not particularly limited.

The dried product of the useful bacterium obtained above, the powdering agent and the tannins described above are mixed in a low humidity room using a V-type mixer or the like and immediately sealed and packed, and if necessary, nitrogen gas, The product of the present invention is obtained by gas displacement packaging with carbon dioxide gas or the like.

In the present invention, the mixing ratio of the dried product of useful bacteria and the tannins is preferably 99: 1 to 1:99 by weight.

In use, the thus obtained live bacterial powder may be used as it is as a live bacterial preparation, or may be used by adding it to foods, feeds and the like, and the use form is not particularly limited.

The intake of the product of the present invention is preferably 0.0001 g to 0.03 g / kg body weight per day as tannins, and 10 ⁴ to 10 ¹⁰ cells / body weight k as viable bacterial cells.
g is preferred. Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

[0017]

EXAMPLES Example 1. Preparation of dried Bifidobacterium glucose 5.0 g, Bact River leachate 1 liter, proteose peptone 10 g, trypticase
5.0 g, yeast extract 3.0 g, Tween 801.0
g, L-Cysteine hydrochloride 2.0 g of a medium was added to Bifidobacterium adressentitis ( Bifidoba).
Cerium adolescentis ) was inoculated and anaerobically cultured at 37 ° C. for 3 days. The obtained culture solution is centrifuged to collect the cells, the cells are washed with physiological saline, and after centrifugation, 4% by weight of skim milk, 1% by weight of sodium glutamate, 0.4% by weight of gelatin, 5% of sucrose. The suspension was suspended in a dispersion medium containing wt% and freeze-dried. The dried bifidobacteria product had a water content of 2.0% by weight, and the viable bifidobacteria count was 2.3 × 10 ¹¹ per gram.

Example 2. Preparation of Tannins 200 g of sencha was extracted with 4 liters of hot water at 85 ° C. while stirring for 30 minutes, and tea leaves were removed by filtration to obtain 3.4 liters of extract. This liquid was passed through an ultrafiltration device (manufactured by DDS, membrane type GR-81PP, molecular weight cutoff 6000) to obtain 3 liters of a passing liquid. To the concentrated residual liquid, 1 liter of water was added and the same operation was performed to obtain 1.2 liters of the passing liquid. Both liquids were combined and concentrated with a reverse osmosis membrane (manufactured by DDS, membrane type HC-50) to give 200 ml and freeze-dried to obtain 48.6 g of tannins having a purity of 35%. The component composition of the obtained tannins was (+)-catechin 3.5% by weight,
(+)-Gallocatechin 14.8 wt%, (+)-Gallocatechin gallate 11.6 wt%, (-)-Epicatechin 7 wt%, (-)-Epicatechin gallate 4.6 wt%, (-) 15.0% by weight of epigallocatechin and 18.0% by weight of (−)-epigallocatechin gallate.

Example 3. Preparation of bifidobacteria powder 50 g of the dried bifidobacteria obtained in Example 1, 45 g of lactulose powder as a dispersant and 5 g of tannins obtained in Example 2 were mixed in a low humidity room using a V-type mixer, 100 g of Bifidobacteria powder of the present invention was obtained.

Example 4. Preparation of Lactic Acid Bacteria Powder A dried live bacterium of Lactobacillus acidophilus was obtained in the same manner as in Example 1, and this dried dry bacterium and the tannins obtained in Example 2 were mixed in the same manner as in Example 3.
800 g of this mixture was added to vegetable oil 20 having a melting point of 30 to 32 ° C.
After mixing with 0 g and kneading thoroughly, the mixture is mixed with 0.5 g.
Granulation was carried out by a small screen type extrusion granulator having extrusion holes with a diameter of mm to obtain 940 g of a granulated product. This granulated product was passed through a 16-mesh shifter, and then 2.5 g each was packed in a moisture-proof stick package to obtain 300 packaged products.

Test Example 1. Clinical test Feces were collected from 5 healthy adults during a control period in which they normally eat, and then the bifidobacteria powder obtained in Example 3 was ingested at 5.0 g / day for 10 days. Feces were collected twice on the 5th and 10th days. As a control, instead of the bifidobacteria powder obtained in Example 3, 1.0 g of the tannin powder was ingested daily, the feces were cultured on the BS medium and the CW medium at the total of three times of the feces collection. However, in the BS medium, the number of Bifidobacterium was
In the medium, the number of Clostridium perfringens was measured. As a control, instead of the bifidobacteria powder obtained in Example 3, a group (control group-1) in which a commercially available bifidobacteria preparation was ingested and a group in which 1.0 g of tannin powder was ingested per day ( The same test was carried out for 5 healthy adults by dividing the control group-2). The results are shown in Tables 1 and 2. In addition, the numerical value has shown the average of 5 persons.

[0022]

[Table 1]

[0023]

[Table 2]

[0024]

[Table 3]

From Tables 1 and 2, in Control Group-1, the number of Bifidobacterium in the intestine slightly increased during the ingestion period, but the number of Clostridium perfringens in the intestine hardly decreased. , It was confirmed that the intestinal flora dominated by bifidobacteria could not be formed, and in control group-2, the intestinal Clostridium perfringens bacterial count fell below the detection limit, The number of Bifidobacterium in the intestine hardly increased. On the other hand, when the Bifidobacteria powder of the present invention is ingested, the number of Bifidobacterium in the intestine is extremely remarkably increased, and the number of Clostridium perfringens in the intestine is lowered to below the detection limit, and Bifidobacterium. The formation of intestinal flora in which the bacteria were dominant was observed. Further, as a result of comparing the storage stability with a commercially available Bifidobacterium preparation, it was confirmed from the results in Table 3 that the storage stability was significantly improved.

Test Example 2. Diarrhea Prevention Test for Piglets The preventive effect of the powder of the present invention on the occurrence of diarrhea in piglets was tested. The test method used 10 piglets in the control group and the test group, the control group was bred only with basic feed, and the test group was fed with the lactic acid bacteria powder obtained in Example 4 per day per day.
Each animal was fed with 0 g of the standard feed for 7 days. The results are shown in Table 4.

[0027]

[Table 4]

As is clear from Table 4, feeding the lactic acid bacteria powder of the present invention was very effective in preventing the development of diarrhea in piglets.

[0029]

By incorporating tannins in the viable bacterial powder of the present invention, the growth of harmful bacteria that have originally settled due to the action of tannins when ingested is selectively suppressed, and the intestinal useful bacteria It is considered that the intestinal beneficial bacteria ingested proliferated due to the environment in which colonization and growth easily occurred.

Furthermore, the product of the present invention can be supplied with a live bacterial powder having a significantly improved storage stability as compared with the conventionally known live bacterial powder. Moreover, since the tannins contained in this product are tea ingredients that have been used for drinking since ancient times, their safety is extremely high, and they can be supplied in large quantities at low cost. The present invention greatly contributes to the promotion of human health and the prevention of diseases of livestock.

Continuation of the front page (56) Reference JP-A-1-191680 (JP, A) JP-A-2-211863 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1 / 20 BIOSIS / WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A live bacterium powder containing tannins selected from the group of green tea, oolong tea and black tea extracts, and one or more selected from bifidobacteria and lactic acid bacteria. 2. The tannins are (+)-catechin, (+)
-Gallocatechin, (+)-Gallocatechin gallate,
From (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, (−)-epigallocatechin gallate, free theaflavin, theaflavin monogallate A, theaflavin monogallate B, theaflavin digallate The live bacterial powder according to claim 1, which is one or more compounds selected from the group consisting of: