JPH0349670A - Bacterial composition of survival bifidobacterium - Google Patents

Abstract

PURPOSE:To enable to remarkably improve the survivability of Bifidobacterium bacteria by mixing the powder of the Bifidobacterium bacteria with inulooligosaccharide. CONSTITUTION:The subject composition contains inulooligosaccharide in an amount of 20-60wt.% based on the solid content of the composition and Bifidobacterium bacteria in a viable bacterial count of at least 10<8> per g of the whole composition. The inulooligosaccharide can be obtained by hydrolyzing inulin with a dilute hydrochloric acid solution or with an enzyme, but in order to efficiently prepare the highly pure inulooligosaccharide by hydrolyzing the inulin with e.g. the endo type inulinase of a Penicillium genus fungus.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a mixture of bifidus and inulo-oligosaccharides.
The present invention relates to a viable bifidobacteria composition capable of maintaining the number of viable bifidobacteria at a high level for a long period of time.

[Conventional technology]

Traditionally, Bifidobacterium has been used as a useful intestinal laxative, to inhibit putrefaction caused by intestinal putrefaction, and to improve various intestinal diseases such as diarrhea, in the form of various Bifidobacteria preparations.
It is commercially available including for livestock. However, there is a general problem that Bifidobacteria easily die during storage.
In order to improve this problem, formulations have been formulated using skim milk powder, starch, etc. as protective agents, but the survivability is still low and the effects cannot be said to be sufficient.

Therefore, lactulose is mixed with bifidobacteria powder as in JP-A No. 52-151787, vitamin E is added as in JP-B No. 63-3585, and as in JP-A-63-12594, There have also been disclosures of attempts to improve the survival of bifidus by mixing raw starch.

On the other hand, examples of extensions of Bifidus genus with reproductive activity include balatinose (Japanese Unexamined Patent Publication No. 57-91193), Frac 1 oligolli (Japanese Patent Publication No. 53834),
Branched, t- IJ gosaccharide (special 15Fl lr716 1-
227777) and xylooligo v! ! (Japanese Unexamined Patent Publication No. 112979/1983) and the like are disclosed.

[Fill problem to be solved by the invention] As mentioned above, various ears are said to be useful for increasing the activity of bifidobacteria, but lactulose is useful not only for bifidobacteria but also for coliforms, cross 1, lysium, etc. It also has the disadvantage of being non-selectively utilized by intestinal cells.

Also, Palatinose, Frac 1, Oligo Ear, 1 minute! It is said that ζ-oligosaccharides and xylo-oligosaccharides are selectively assimilated by Bifidobacteria and serve as selective growth factors for Bifidobacterium in the intestines, but both are Survivability is not clear, and no immunoenhancing activity has been shown.

An object of the present invention is to provide a Bifidobacterium composition that increases the survival of Bifidobacteria during storage and in vivo, and furthermore, is a selective growth factor for Bifidobacteria, and furthermore, is an immune-enhancing composition due to macrophage activating activity. It is an object of the present invention to provide a preparation for retaining bifidobacteria activity, which contains a protective agent that also has bifidobacterial activity.

[Means for solving the problem] In order to solve the above problem, the wood-pounders have conducted intensive research and found that if Imelooligo-tang is mixed with the powder of mucus and bifidobacteria, the survival of bifidobacteria will be significantly reduced. In order to complete the present invention, we have discovered that ingestion of this composition not only promotes the proliferation of Bifidobacteria in the intestines and improves intestinal diseases, but also extremely enhances immune-enhancing activity. It has arrived.

That is, the present invention is a viable bifidobacteria composition which contains canine oligosaccharide in an amount of 20 to 60 fflE1% on a solid basis, and which contains at least 108 viable bifidobacteria per gram of the entire composition.

The present invention will be explained in detail below.

The inulooligosaccharide used as a protective agent in the present invention is an oligosaccharide in which 2 to 5 molecules of fructose are bonded to fructose with β-2.1 viscosity. Aus (F5)
, canine hexaose (F6), and contains about 10% fructooligosaccharide with a glucose residue at the end.

Inulooligosaccharides can be obtained by dissecting inulin with dilute acids or enzymes, but in order to obtain highly pure inulooligosaccharides with high efficiency, there is a method, for example, of hydrolyzing inulin using endo-type inulinase from Penicillium fungi. There is.

In addition, if you do not need highly purified canine oligosaccharide for livestock, etc., inulin-containing plants such as Jerusalem artichoke and chicory can be diluted directly with endo-type inulinase. It can be obtained efficiently by the following method. These methods make it possible to obtain inulooligosaccharide-containing products in which the content of inulooligosaccharides in the solid content is at least 30%.

Next, the Bifidobacterium used in the present invention may be any known strain belonging to Bifidobacterium, but it tends to colonize in the intestine of the host to which the composition with canine oligosaccharide is administered. In the case of humans, for example, Bifidobacterium adresentis (Bll'ld
obacLcrlumadolescentis),
Bifidobacterium ◆ Bifidum (B. bifldo
s), Bifidobacterium longum ([1.
Iongum), and in the case of livestock, Bifidobacterium pseudolongum (n. psoudol).
onguI1). ．． It is preferable to use Bifidobacterium Thermophilum (11. thorl01) hlrul).

After culturing bifidobacteria using a commonly used medium and culture method, the cells are centrifuged to collect the bacteria and washed to obtain 6i bacteria containing about 10 to 10 liters of bifidobacteria per gram. Sterile water containing approximately 20% dispersion medium 1
About 100 g of this wet bacterial body is added to and mixed with g, freeze-dried, and pulverized to obtain bifidobacteria powder. As the dispersion medium used here, for example, a general one mainly containing 5% sucrose and 4% skim milk can be used. Therefore, there are no limitations on the dispersion medium and its content.

The moisture content of the Bifidobacterium powder thus obtained is 5%.
and contains at least 109 viable Bifidobacteria per gram.

When this Bifidobacterium powder is added and mixed with the above-mentioned canine oligosaccharide-containing composition and freeze-dried, the canis oligosaccharide content is 20 to 60% by weight, and at least 108 Bifidobacterium bacteria per 1 g of these compositions are obtained. A composition containing Bifidobacterium can be obtained.

The composition of the present invention obtained in this manner may be immediately sealed and used as a bifidobacteria preparation, or when administered to livestock, it may be mixed with feed by adding 0.1 to 5% by weight. Just use it.

In addition, in the present invention, in the Rizan containing 20 to 60% by weight of canine oligosaccharide, if it is less than 20% by weight, it is not possible to sufficiently achieve the survival effect of bifidus and the multiplication effect in the intestine. If it exceeds 60% by weight, the effect will be saturated and no further effect will be observed, making it uneconomical.

In addition, the number of viable bacteria per gram of composite material was set to 108 or more.
This is to enhance the survival of bifidobacteria and to sufficiently improve intestinal diseases in the intestines.

Since the bifidobacterium composition containing canine oligosaccharide of the present invention is protected by the canine oligosaccharide, the survival rate of bifidobacteria is extremely high, and the survival rate after being kept at room temperature for 6 months is lower than that of commercially available bifidobacteria. In the case of the composition of the present invention, it is 60-85%, whereas it is 20% or less in the bacterial preparation.
%.

Furthermore, after the composition is administered and enters the body, the inulooligosaccharide is selectively assimilated by Bifidobacterium, and as a result, the intestinal flora becomes dominated by Bifidobacteria, leading to the growth of putrefactive bacteria such as Escherichia coli and Clostridium. is suppressed, and intestinal diseases such as diarrhea are improved.

In addition, by applying this composition, the macrophage activating activity of canine oligosaccharide and the strong immunity effect derived from the bifidobacterial cell wall are activated, and the infection defense ability of the living body can be enhanced.

〔Example〕

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but it goes without saying that the present invention is not limited to these Examples.

(Manufacture of products containing inulo-oligo) Inulin (separated and purified from chicory root) 06
5% by weight maltose
0.5% by weight NH4H2PO4
0.27% by weight K2HPO4
0.1% by weight MgSO4●7H,,O
O. 05 heavy melon% tween 80
0.02% by weight These were further prepared with tap water to a pH of 7.0, then 500% by weight.
Transfer 100 ml to a ml Sakaguchi flask, add FERM P-8705 (Ponlclllus) to the sterilized
purpurogonum war rubrl-sc
lerotleus) was inoculated with one platinum loop and incubated at 28°C for 48 hours.
Pre-incubate for an hour. Separately, put the same medium of 3.51 in a mini jar, sterilize it, inoculate it with the pre-culture solution, and heat it at 28℃ for 96 hours.
The cells were cultured under aeration during the day. Culture in which bacterial cells are separated by filtering the culture solution; Toto solution (3.'l inulinase activity 6 u/ml
) was added with ammonium sulfate, a 40-80% saturated precipitate fraction was collected by centrifugation, and a solution of M/100 acetic acid buffer (pH
5.0).

The activity of 100 ml of the obtained crude enzyme solution is 210
u/ml. This crude enzyme solution was added to a 20% inulin solution at pH 5, 0,
Enzymatic digestion was performed at 60°C for 48 hours. This reaction product is a canine oligosaccharide-containing product.

(Manufacture of Bifidobacterium powder) Glyose 5. Og bactoliver infusion IJ7 proteose peptone
101l tripty case 5.0g
Yeast extract 3.0g Tween 80
1.0. L-cystine hydrochloride 2. Of these media DSM20213
(formerly Rldo-bacterlus breve) was inoculated and statically cultured at 37°C for 3 days.

The obtained culture solution was centrifuged to collect the bacteria, and the empty body was washed with physiological saline (δ) and after centrifugation again, 12% by weight of casein,
It was suspended in a dispersion medium containing 20% by weight of lactose and 2% by weight of sodium glutamate and freeze-dried.

In this way, a bifidobacteria powder having a water content of 2.1% by weight and a viable count of bifidobacteria of 2×10'' per gram was obtained.

(Example 1) 100 g of the above bifidobacteria powder was suspended in 130 g of a canine oligosaccharide-containing material with a solid content of 75% by weight, and then freeze-dried by a conventional method to obtain about 190 g of the viable bifidobacteria composition according to the present invention. Obtained. The water content of this powder is 1. -8% by weight, contained about 45% by weight of inulooligosaccharide in the solid content, and the number of Bifidobacterium was 1010/g.

The composition of the present invention thus obtained and the commercially available 2
Bifidobacteria preparations A and B were tested for bacterial survival.

30 g of each sample was sealed and stored at 25°C for 6 months, the number of viable bacteria was measured over time, and the survival rate was determined from the ratio of test start nr7 to the number of viable bacteria. The results are shown in Table 1.

As is clear from Table 1, the survivability of the composition according to the present invention is extremely high compared to commercially available products.

(Example 2) The composition of the present invention produced in Example 1 was given to a river, and an eating test was conducted on the elderly. 5 per person per day for 6 elderly people
Table 2 shows the number of bifidobacteria in feces measured before and during sampling after ingestion of this composition.

In order to compare with the composition of the present invention, a similar test was conducted using a commercially available bifidobacteria preparation. The determination of the number of Bifidobacteria was carried out using a commonly used selective medium (BS medium).

Table 2 Ingestion test in the elderly As is clear from Table 2, even when commercially available products are injected, the number of bifidobacteria only increases by 6.1 times.
When the composition of the present invention was ingested, the amount increased to 32 times after 8 days. Since the total number of bacteria in the feces hardly increased, it was clearly seen that Bifidobacterium became predominant in the intestine by collecting the composition of the present invention.

(Example 3) The underground part of chicory, which is an inulin-containing plant, is ground into a slurry. On the other hand, in I'l? produced in Example 1?
30,000 units of the base solution was added to 30 kg of this slurry, and the slurry was heated at 55° C. for 24 liters of enzyme.

After completion of the decomposition, 3 kg of the bifidobacterium powder of the present invention obtained in Example 1 was added and mixed to the slurry after completion of the decomposition, and about 8.5 kg of the bifidobacterial composition containing canine oligosaccharides of the present invention was obtained by freeze-drying. The water content of this composition is 2.0% by weight
It contains about 35% by weight of canine oligosaccharide based on the solid content, and the number of Bifidobacterium is 6. OxlO9/g. Using livestock feed to which 3% by weight each of this composition and a commercially available bifidobacteria preparation were added, piglets 3 weeks old were fed, and secreted immunoglobulin IgA in feces was measured.

Secretory immunoglobulin IgA is Microplate-1・EL
ISA method (Kuchimoto Pediatric Society Journal, 88 305.198
4). Table 3 shows the results of rearing 6 animals each for 2 weeks.

Table 3 Secretory IgA value in piglet feces (value before the start of the test was 1.0
As shown in Table 3, by adding the composition of the present invention, secretory 1gA increases significantly compared to adding a commercially available product. As a result, immune-enhancing activity is clearly recognized.

〔Effect of the invention〕

As is clear from the examples above, the ↑■ composition of the present invention has extremely high survivability compared to general commercial products, and promotes the growth of bifidobacteria in the intestines. It has a very high intestinal disease improving effect and a high level of activity against demon plague I.

Claims (1)

[Claims] 20-60% by weight of canine oligosaccharide based on solid content
A viable bifidobacteria composition comprising at least 10 degrees of viable bifidobacteria per gram of the total composition.