JPH01221319A - Composition for promoting enteral colonization -of useful bacteria and method therefor - Google Patents

Abstract

PURPOSE:To promote enteral colonization of useful bacteria, by obtaining the title composition, consisting of live microbial cells of useful bacteria consisting of bacteria belonging to the genus Bifidobacterium and/or lactic acid bacteria and lactoferrin and useful for suppressing and preventing diarrhea and administering an effective amount thereof. CONSTITUTION:The subject composition consisting of live microbial cells consisting of bacteria belonging to the genus Bifidobacterium and/or lactic acid bacteria and lactoferrin. Live microbial cell powder is prepared by adding a dispersion to a solution of microbial cells obtained by culturing in a large amount and freeze-drying the resultant mixture. The lactoferrin is a protein, binding to iron and present in cow's or human milk and can be powdered by separating and purifying the lactoferrin from the cow's or human milk with an adsorbent resin and freeze-drying the resultant substance. The subject composition can be administered in the form of powder, tablet and solution or added to a food or feed and administered. The composition is preferably used in a dose so as to provide >=10<6> live microbial cells/day and >=1mg/day lactoferrin based on 1kg body weight.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to promoting and improving intestinal colonization of useful bacteria in humans or animals, and more particularly, to promoting intestinal colonization of useful bacteria in humans or animals. The present invention relates to compositions and methods for promoting intestinal colonization of beneficial bacteria in humans or animals.

The composition and method for promoting intestinal colonization of useful bacteria of the present invention can be used to maintain the health of humans or animals, and can also be used to suppress and prevent the occurrence of phthisis in humans or animals. Can be done.

[Technical background and explanation of conventional technology]

It has been widely known that bifidobacteria and lactic acid bacteria inhabit the intestines of humans and animals and are useful bacteria for carrying out the normal functions of the human or animal intestinal tract. It has already been confirmed that when lactic acid bacteria colonize and live in the intestines of humans or animals, the intestinal tract of the host human or animal functions normally and is effective in maintaining the health of that human or animal. has been done. The mechanism is that bifidobacteria or lactic acid bacteria that colonize and inhabit the intestinal tracts of humans and animals produce organic acids (particularly acetic acid in the case of bifidobacteria) in the intestines of humans and animals. It is thought to suppress the growth of undesirable bacteria such as Clostridium perfringens and pathogenic Escherichia coli in the intestines of humans and animals, and prevent abnormal fermentation and spoilage of food in the intestinal tract.

From this point of view, in order to replenish the intestinal tracts of humans and animals with useful bacteria such as bifidobacteria and lactic acid bacteria, many foods and feeds containing live cells of these useful bacteria have been developed. . However, the reality is that even if food or feed containing live cells of these useful bacteria is ingested, it is often difficult to produce satisfactory physiological effects. This is because even if humans and animals ingest foods and feed containing these viable bacteria, many bacterial groups that can coexist with humans and animals live in the intestines of humans and animals. It is difficult for live useful bacteria ingested with foods and feed to colonize the intestinal tracts of humans and animals, and these viable bacteria often simply pass through the intestines of humans and animals, or are inactivated. It is considered.

Microorganisms have the characteristic of breeding and colonizing species that are adapted to the given environmental conditions, so it is possible to create conditions in the intestines of humans and animals that allow these beneficial bacteria to grow and colonize. Although conceivable, it is now virtually impossible to alter the conditions within the human and animal intestines without jeopardizing human and animal health. This is because humans and animals also live with optimal conditions in their intestines.

On the one hand, contacting skim milk with an H-type ion exchanger,
The iron-binding proteins contained in skim milk are adsorbed on an H-type ion exchanger, the iron-binding proteins adsorbed on the ion exchanger are eluted with saline, and the iron-binding protein lactoferrin is separated and collected from the eluate. It has been proposed that (
Patent Application No. 122548 of 1988).

The present inventors continued their research with the intention of colonizing the intestines of humans and animals with useful bacteria, and found that lactoferrin colonized the intestines of humans and animals with useful bacteria such as bifidobacteria and lactic acid bacteria. The present invention was developed based on this finding.

[Object of the invention and summary of the invention]

An object of the present invention is to provide effective means and compositions for colonizing the intestines of humans or animals with useful bacteria that are administered to humans or animals.

The present invention is a composition for promoting intestinal colonization of useful bacteria, characterized by comprising live bacterial cells of useful bacteria such as bifidobacteria and/or lactic acid bacteria, and lactoferrin.

The composition for promoting intestinal colonization of useful bacteria of the present invention can be administered to humans and animals in an amount of at least 106 viable bacteria per kg of body weight and at least 1 μg of lactoferrin per day. , whereby Bifidobacterium and/or Lactobacillus bacteria can colonize the intestines of humans and animals.

In the composition of the present invention that promotes intestinal colonization of useful bacteria, lactoferrin is blended with lactulose, fructooligophores, lactoperoxidase, or lysozyme, thereby improving the colonization rate of bifidobacteria and/or lactic acid bacteria in the intestines. be able to.

[Detailed description of the invention]

The "useful bacteria" in the present invention are bacteria that coexist in the gastrointestinal tract of humans or animals and have a beneficial effect on the survival of humans or animals, and representative examples thereof include bifidobacteria and lactic acid bacteria. Bifidobacterium belongs to the genus Bifidobacterium and is a bacterium that widely inhabits the intestinal tract of humans and animals, but when used on humans, it is preferable to use human-derived bifidobacteria. When used against animals, it is preferable to use bifidobacteria derived from that animal. Lactic acid bacteria are mainly bacteria belonging to the genus Lactobacillus or Streptofuccus.

Bacteria other than bifidobacteria and lactic acid bacteria can be used as "useful bacteria J" in the present invention if they coexist in the digestive tract of humans or animals and have a beneficial effect on the survival of humans or animals. For example, 7f41LsllI (R#IpH1nxS 1m@
tXRil! +? % book-f can also be used or used in combination with bifidobacteria or lactic acid bacteria.

The viable bacterial powder of useful bacteria in the present invention can be prepared by culturing a preculture of the above bacterial species in large quantities by a conventional method, adding a dispersion medium to the bacterial cell liquid, and freeze-drying.

"Lactoferrin" in the present invention is a harmless, natural iron-binding protein present in cow's milk or human milk, and has mild bacteriostatic activity. Lactoferrin powder can be produced by separating and purifying cow's milk or human milk using adsorbed fat, followed by freeze-drying and powdering. It is possible to use products manufactured by the same method.

In the method of promoting intestinal colonization of useful bacteria of the present invention, per kg of human or animal body weight,! By administering 0.6 or more live cells of useful bacteria per day and lactoferrin at 1.0 or more per day, it is possible to improve the colonization rate of the administered useful bacteria in the intestines.

When a known bifidobacteria growth promoting substance (bifidus factor) such as lactulose or fructo-oligos is co-administered with viable cells of useful bacteria and lactoferrin, the colonization rate of bifidobacteria in the intestines can be further improved. Furthermore, lactoperoxidase or lysozyme, which have a bacteriostatic effect, can be used in combination to improve colonization of the intestines by useful bacteria.

In the present invention, live useful bacteria and lactoferrin can be used in the form of powder, tablet, or liquid.
The composition for promoting intestinal colonization of useful bacteria of the present invention can be administered to humans and animals, but can also be administered by adding it to food or feed. The composition may be in any form, and it may also be a composition in which live useful bacteria and lactoferrin are added to food, feed, drink, or nutrient. In the composition for promoting intestinal colonization of useful bacteria of the present invention, when live useful bacteria and lactoferrin are made into a powder mixture and a tablet composition, 10 or more live useful bacteria per Ig of the composition. The amount of lactoferrin is preferably 10 or more.

In the following, the present invention will be explained in more detail by means of test examples and examples.

Test example! A test was conducted on the effect of lactoferrin on the intestinal colonization rate of Bifidobacterium longum M-8201 in adult males.

(1) Sample preparation Row 1) Sample 1 (U-P) Product of Example 1.

(1-2) Sample 2 (S-P) Lactulose (υ
) instead of Milk III (S).

(1-3) Sample 3 (U-5) Lactoferrin (P
) instead of milk W (S).

(1-4) Sample 4 (S-5) A product in which milk Ml (S) was used in place of lactulose and lactoferrin in the blending ratio of the product in Example 1.

(2) Test method: Intestinal bacteria are tested, and Bifidobacterium and Bifidobacterium
Adult males (average weight: 63 kg) who do not have Longum M-8201 as resident bacteria were selected as test panelists.

The 16 adult male panelists were divided into 4 groups of 4 people each, and one panelist in each group was given the sample shown in Table 1! Daily allowance 2.
5g was administered for 2 weeks. 7 days after the start of administration, 10
After 14 days, 21 days, and 21 days, each panelist's feces (
Bifidobacterium longum M-8201 was detected, and the percentage (%) of the test samples in which Bifidobacterium longum M-8201 was detected relative to the total test samples.
was calculated as the detection rate.

(3) Test results It was as shown in Table 1.

(Leaving space below) Table 1 Human intestinal colonization rate (4) Discussion According to Table 1, the detection rate of groups 1 and 2 is much higher than the detection rate of groups 3 and 4. It can be seen that the administration is effective in improving the intestinal colonization rate of Bifidobacterium longum M-8201 (Bifidobacterium). Furthermore, from the difference in detection rate between Group 1 and Group 2, it can be seen that administering lactoferrin together with lactulose is more effective in improving the intestinal colonization rate of bifidobacteria.

(5) Test supplements and test results! So, the bacterial body of Bifidobacterium is one of the panelists.
12 x 108 doses were administered per day, and 225 fng of lactoferrin was administered per panelist per day.

A test was conducted in which the number of Bifidobacterium cells and the dose of lactoferrin were independently varied, and the results showed that the detection rate of Bifidobacterium in the faeces of the panelists was 50-60% or more. It was found that it is necessary to administer at least 106 bifidobacterial cells/day and lactoferrin at least 1/day per kilogram of body weight.

Test Example 2 A test was conducted on the effect of lactoferrin on preventing diarrhea caused by Lactobacillus acidophilus in piglets.

(1) Preparation of sample Lactobacillus acidophilus bacterial powder was prepared in Example 2.
(Number of viable bacteria: 1so x lo8/Jil)
and its 0. IJil (Number of viable bacteria: 15x 1o
8) /1 day was added to the feed.

Lactoferrin used was that obtained in Example 1.
0971 days were added to the feed.

(2) Test method Landrace pigs 4 to 5 days old (average weight = 460
.
The animals were divided into 3 groups (11 animals) under the following conditions and administered for 7 days, and observation of the occurrence of diarrhea was continued for several months.

Control X: Breeding was carried out only with standard feed, and Lactobacillus acidophilus bacterial powder and lactoferrin were not administered at all.

Test 1 (M) section: Lactobacillus obtained in Example 2
0.1g of acidophilus bacterial powder per head/day (
Several mouthfuls of live bacteria (5XI08) were fed with standard feed for 7 days.

Test 2 (M-P) group: Lactobacillus obtained in Example 2
acidophilus bacterial powder per tB/day. IJl
(Number of viable bacteria: 15 x 108) and lactoferrin obtained in Example 1 were fed together with standard feed for 10119.7 days per head per day.

(3) Test results It was as shown in Table 2.

(4) Discussion According to Table 2, administering Lactobacillus acidophilus bacterial powder is effective in suppressing the occurrence of diarrhea in piglets, but administering Lactobacillus acidophilus bacterial powder together with lactoferrin This increases its effectiveness and proves to be effective in preventing the occurrence of diarrhea in piglets.

The difference in the number of animals with diarrhea in the Test + (M) section and the Test 2 (M-P) section in Table 2 is thought to be due to the improvement in the intestinal colonization rate of Lactobacillus acidophilus due to the administration of lactoferrin.

(5) Supplementary Tests and Results In Test Example 2, Lactobacillus acidophilus cells were administered at 15 x 108 cells per head per day, and lactoferrin was administered at 1O2 per head per day.

A test was conducted in which the dose of Lactobacillus acidophilus (bacterial cell count) and the dose of lactoferrin were independently varied, and the results showed that it was necessary to suppress the occurrence of diarrhea in piglets. The number of Lactobacillus acidophilus cells per pig per day is 106 or more, and the dose of lactoferrin required to prevent the occurrence of diarrhea in piglets is 1μ or more per pig per day. I found out something.

Example 1 (Preparation of Bifidobacterium cell powder) Bifidobacterium Longum (01fLdobacterium Longum)
After subculturing M-8201 (Feikoken Bacteria I 6548) for 10 generations in Brix River Pross,
Synthetic medium a50/! consisting of glucose, yeast extract, peptone and phosphate! and incubated at 37°C for 14
Mass culture was carried out for hours. The culture was centrifuged, the bacterial cells were collected, and the resulting bacterial liquid 17! A dispersion medium 500- containing 100 g of glutamic acid and Sho W50.9 was added to the mixture, and this was freeze-dried. Milk to 275g of the obtained bacterial powder! 12Kg
Add 9 to 2.5 and dried cornstarch, mix,
Triturate the bacterial powder of Bifidobacterium longum (
Several copies of live bacteria (00 x 108/g) 4.7 K9 were obtained.

(Preparation of lactoferrin powder) CM Sepharose FF (manufactured by Pharmacia) 25/! has a carboxymethyl group as an ion exchange group, has a hemoglobin adsorption capacity of 6.1/l/100d, and a volume change of 1.0. was packed into a column and 0. IN)
After passing through IC+ 501, the solution was washed with water to prepare an H-type ion exchanger.

This H-type ion exchanger is removed from the column and its H-type ion exchanger is removed from the column.
type ion exchanger was added to raw skimmed cow milk 25001 at pH: 6.7, stirred at 4°C for 6 hours,
After bringing the H-type ion exchanger into good contact with cow skim milk,
The ion exchanger was separated and removed by filtration. The pH of skim milk from cows from which the ion exchanger was removed was 6.7, which was unchanged, and there was no change in appearance or flavor at all.

The ion exchanger taken out from skimmed cow milk is packed into a column again, and water is passed through this column to remove the skim milk from cows that has adhered to the ion exchanger.
57 salt solutions! The components adsorbed on the ion exchanger were desorbed by passing liquid at a rate of 251/hour to obtain a recovered solution of +251.

This recovered liquid 1221 was transferred to a DD with a molecular weight cutoff of 20,000.
In a laboratory module equipped with an ultrafiltration membrane manufactured by Company S,
WIMi flow rate is 87! /min and average pressure 30 to 9/min
After ultrafiltration under the conditions of J, water was added, diafiltration was performed to remove salt, and the IIIra solution from which the salt had been removed was freeze-dried to obtain 102 g of a freeze-dried product. Ta. The lactoferrin content of this freeze-dried product was 95% (by weight).

(Composition for promoting intestinal colonization of useful bacteria) Using the Bifidobacterium longum bacterial powder and lactoferrin powder obtained above, commercially available lactulose, dried corn starch powder, lysozyme and skim milk powder were mixed in the following blending ratio. The ingredients were mixed uniformly in a V-type mixer to obtain a composition for promoting intestinal colonization of useful bacteria.

(Blending ratio) Bifidobacterium longum bacterial powder (several copies of live bacteria 00 x 108/, 9) 501 Lactoferrin powder (purity: 95%) 100.9 Lactulose powder (purity: 90%) 5oog dry Q Cornstarch 150g Lysozyme 5g Skimmed milk powder
1959 total
1000, lit This composition for promoting intestinal colonization of useful bacteria was packaged into moisture-proof sachets to obtain 360 packaged products containing 2.59 pieces.0 Per 11 g of this packaged product, Bifidobacterium longum M- 8201 live bacteria 4.
It contained 8 x 108 and 90 lactoferrin.

(Product evaluation) This sachet product (2.5g) was given to an adult male (weight: 5g).
0 and 9) were administered 1 packet per day for 2 weeks. The number of administered bacteria detected in the feces is as follows. According to the above results, it can be seen that the product of Example 1 is effective in improving the intestinal colonization rate of useful bacteria.

Example 2 Using Lactobacillus acidophilus obtained from unpasteurized fresh milk, in the same manner as in Example 1, 9 was added to 3.7 of lactic acid bacteria powder with a viable count of 150 x 108/9. Obtained.

This lactic acid bacteria (Lactobacillus acidophilus) powder, lactoferrin powder prepared in the same manner as in Example 1, and the following materials were mixed in the same manner as in Example 1.

(Blending ratio) Lactoferrin powder (purity: 95%) 50g dry cornstarch +00 /i dextrin 100 /l skim milk powder
620g Sodium ascorbate (commercially available) 301 go tj 10
00 g 800 g of the above mixture was mixed with vegetable oil 2 with a melting point of 30-32°
00/i and sufficiently kneaded, the mixture was granulated using a small screen type volatilization granulator having extrusion holes with a diameter of 0.5s+s+ to obtain granulated product 940i.

The granules were passed through a 16-mesh sifter and then divided into moisture-proof stick packages (1,59 pieces) to obtain 500 moisture-proof stick packages (1,577 people).

This moisture-proof stick packaged product contained 14 x 108 live Lactobacillus acidophilus bacteria and 4719 lactoferrin per gram.

This moisture-proof stick packaged product was administered once a day to an adult male (weight: 60-9) who had been exhibiting diarrhea symptoms for 2 days. (corresponding to the administration of 1.17 tng of lactoferrin) and the progress was observed.

Three days later, the diarrhea symptoms disappeared and the stools returned to normal.

Example 3 Bifidobacterium breve (Blfldobacterium breve)
Bifidobacterium breve cell powder having a viable cell count of 400 x 108/p was obtained in the same manner as in Example 1 using a microorganism strainer (ATCC 15700).

Apart from this, Streptococcus faecalis (St
reptococcus faealis) S
Using PA-05 and in the same manner as in Example 1, a powder of Streptococcus faecalis cells having a viable cell count of 200 x 108/I was obtained.

The Bifidobacterium breve bacterial powder, the Streptococcus faecalis bacterial powder, the lactoferrin powder prepared in the same manner as in Example 1, and the following materials were uniformly mixed in a V-type mixer to produce a useful product. A composition for promoting intestinal colonization of bacteria was obtained.

(Margin below C) (Blending ratio) Bifidobacterium breve bacterial powder (Number of viable bacteria:
400 × 108/, 9) 2009 Streptococcus fueca 1 nos bacterial powder (number of viable bacteria:
200 x 10”/l) 100 g
Lactoferrin powder (purity: 95%) 20 (1
Lactoperoxidase (content: O, 12%) 50g Dry Q Corn Starch 50g Skimmed Milk Powder + 501 Dextrin 250g Total
1000 g of this composition for promoting intestinal colonization of useful bacteria was packaged into capsules to obtain 3200 B-do capsules each containing 280 ml. Each gram of this capsule contains 6 x 10 live bacteria of Bifidno (Cuticum breve), 1.8 x 10 live bacteria of Streptococcus fueca, and 176 lactoferrin. .

A 4-year-old girl (weight: 16 kg) with chronic diarrhea symptoms + 3 capsules per day (note: 3.9 x + 08 Bifidobacteria per kg of body weight per day) When administered (equivalent to the amount of bacterial cells of U. breve and 9.2 m9 of lactoferrin), the stool quality changed from soft to normal, and after one month, the stool became completely normal.

Example 4 Bifidobacterium pseu isolated from cow feces
Bifidobacterium pseudolongum bacterial powder with a viable cell count of 100XIO/g was obtained in the same manner as in Example 1 using Bifidobacterium pseudolongum (2.6 to 9).

This Bifidobacterium pseudolongum bacterial powder, lactoferrin powder prepared in the same manner as in Example 1, and calf milk replacer were uniformly mixed in the following proportions,
A composition for promoting intestinal colonization of useful bacteria was obtained.

(Left below) (Blending ratio) Bifidobacterium pseudolongum bacterial powder (
Several volumes of live bacteria oo x to8/g) 1.5Kg lactoferrin powder (purity: 95%) 0.4 K9
Calf milk replacer (powder) 998.1
9 total 1000 K9 This composition promoting intestinal colonization of useful bacteria is 1.5 per phrase.
Contains X 101θ live bacteria of Bifidobacterium pseudolongum and 400μ of lactoferrin.

This composition promoting intestinal colonization of useful bacteria was administered to 6 calves (average weight: 58 to 9) aged 2 to 3 weeks at a dose of 520 I/day.
When the mice were given 1 dose at a time until they were 6 to 7 weeks old, the incidence of diarrhea was 16.9%.

On the other hand, calves of the same age that received only calf milk replacer had a diarrhea incidence of 34.3%.

This shows that it is also effective in preventing diarrhea in calves.

〔Effect of the invention〕

The composition of the present invention can effectively colonize the intestines of humans or animals with useful bacteria, and since its active ingredient, lactoferrin, is extracted from milk, a natural food, it is harmless and It's safe.

Furthermore, colonization of the intestines with useful bacteria is effective in suppressing and preventing the occurrence of diarrhea caused by disturbances in intestinal flora.

Claims (2)

[Claims] (1) A composition that promotes the colonization of useful bacteria in the intestine, characterized by comprising viable cells of useful bacteria consisting of bifidobacteria and/or lactic acid bacteria, and lactoferrin. (2) For humans or animals, bifidobacteria and/or
Alternatively, in a method of administering viable cells of useful bacteria such as lactic acid bacteria and colonizing the intestines with the useful bacteria, the above-mentioned live bacteria of the useful bacteria should be administered at least 10^6/day and lactoferrin should be administered at 1 mg per 1 kg of body weight. 1. A method for promoting intestinal colonization of useful bacteria, characterized by administering the drug for 1 day or more.