JPH0838064A - Feed capable of preventing infection of harmful bacterium - Google Patents

Abstract

PURPOSE:To obtain a feed capable of preventing livestock or poultry from being infected with harmful bacteria through inhibiting adherence of such bacteria to come via the enteric canal to the intestine, thus effective for maintaining or improving the health of livestock/poultry, by formulating the original feed with specific harmful bacteria infection-preventive ingredient(s). CONSTITUTION:This feed is obtained by formulating the original feed with, as harmful bacteria infection-preventive ingredient(s), pref. 0.1-1wt.% of at least one kind selected from mannoses (pref. mannose or methyl-alpha-mannoside), sialic acids (pref. N-acetylneuraminic acid or ganglioside) and lactol-N- fucopentaose. It is preferable that this feed begin to administer at the time of fledging or juvenile livestock stage having small number of harmful enterobacteria.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a feed for preventing the infection of harmful bacteria, and more particularly to a feed for the purpose of preventing the infection of harmful bacteria that infect via the intestinal tract.

[0002]

2. Description of the Related Art Up to now, antibiotics have been widely used in animal feeds, especially livestock and poultry feeds, for the purpose of increasing breeding efficiency, promoting growth such as weight gain, and preventing bacterial diarrhea. Is also recognized. However, the problem is that the antibiotics are transferred to meat and eggs and remain. Furthermore, chronic use of antibiotics is feared to cause emergence of resistant bacteria, disturb the intestinal flora of livestock, and reduce the resistance of livestock to infection. Therefore, in May 1976, the "Act on Ensuring Feed Safety and Improving Quality" came into force, and the addition of antibiotics to feed has become stricter.

Under such circumstances, attention has been paid to substances that achieve the above-mentioned objects in place of antibiotics and the like, and viable bacterial preparations (probiotics) have been developed as such substances. In other words, attempts have been made to improve the intestinal flora of animals by formulating useful bacteria that live in the intestines of animals and feeding them to animals. Lactobacillus (Lactobacillus bacterium) and lactococcus (eg Streptococcus faecalis) have been most often used as such viable bacterial preparations. Butyricum) and other active ingredients are commercially available.

However, useful bacteria used as a viable bacterial preparation have high sensitivity to antibiotics, and many of them cannot grow and settle in the intestine when used in combination with antibiotics. In addition, many useful intestinal bacteria cannot settle in the intestines of other animal species, and even bacteria specific to the animal species tend to be eliminated when they are fed from outside the body. Further, there remains a problem of formulation stabilization such as reduction or fluctuation of efficacy due to low survival rate of useful bacteria in the formulation.

Therefore, in recent years, search has been carried out for highly safe natural sugars and the like capable of controlling infection of microorganisms. As such a substance, a mannoglucan derivative for preventing infection of Escherichia coli etc. 61-40219), glycopeptide composition (JP-A 61-103829) and sialic acids (JP-A 62-208261). However, the mannoglucan derivative has the main effect of increasing the number of leukocytes or hyperactivity and indirectly suppresses the onset of infectious diseases, so that it is inevitable to use it together with an antibiotic.

The glycopeptide composition is a pathogenic bacterium having type I pili (Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, etc.)
Only the target. Sialic acids are targeted at pathogenic E. coli causing human diarrhea. Such an infection preventive agent is limited in the combination of the target harmful bacteria and the host. For harmful bacteria that are infected via the digestive tract of animals, fructooligosaccharides (Symbol 3-501971), and D-mannose (BAOyofo et al; Poultry Science 68: 135) that prevents Salmonella colonization on broilers.
7-1360, 1989) and the like are only reported, and substances having various harmful bacterial infection preventive effects are desired.

On the other hand, the attachment phenomenon of bacteria and viruses to host cells has been attracting attention as the beginning of infectious diseases. That is, in order for infection to occur, bacteria and viruses must first attach to the infected site of the host. Those that do not have the ability to adhere are eliminated from the infected site by the body's defense ability and self-cleaning action. It is becoming clear that carbohydrates are involved in such adhesion phenomenon (CSEden; Current topics in infectious disease
No. 3, 1989). That is, some bacteria have a lectin (adhesin) on their surface, and it is considered that the bacteria use this adhesin to bind to complementary sugar receptors existing on the surface of target cells.

The specificity of the epithelial cell receptor depends on the sugar composition of the cell surface and is present in the form of glycoprotein or glycolipid by binding to proteins and lipids. The sugar composition of the cell surface varies greatly not only between species and individuals but also between tissues of the same individual. This difference in sugar composition is due to the difference in bacterial binding to each target tissue, that is,
It is considered to define the host and site of infection by bacteria. As described above, the phenomenon of bacterial attachment to the host is understood as a model of sugar-mediated cell recognition system. Research is progressing on bacterial lectins and sugar chains on the surface of host cells, and sugars that specifically inhibit bacterial adhesion are
Research is also being carried out for use in the prevention of bacterial infections. The development of sugars that control the adhesion phenomenon of such bacteria and the development of methods for controlling the infection of harmful bacteria using such sugars are expected.

[0009]

An object of the present invention is to provide a feed for preventing harmful bacterial infection by using a substance that inhibits the attachment of bacteria to host cells as an active ingredient.

[0010]

According to the present invention, a substance which inhibits harmful bacteria from colonizing the intestinal tract of livestock and poultry by inhibiting the adhesion of harmful bacteria to intestinal epithelial cells is contained as an active ingredient. Feed is provided. Hereinafter, the present invention will be described in more detail. In the present invention, the effectiveness of the sugar compounded as an infection-preventing ingredient is determined by using human intestinal epithelial cell Intestine 407 using bacteria such as Escherichia coli derived from healthy people, Escherichia coli pathogenic to chicken, Campylobacter jejuni, etc.
It was evaluated by conducting an in vitro adhesion inhibition test on broilers and an infection test on broilers.

As a result, mannose; methyl-α-mannoside; mannooligosaccharide; or mannose, which is a hydrolyzate of mannan obtained from guar gum, locust bean gum or yeast with an enzyme and / or an acid, and N-acetylneuramine. Acid, N-acetylneuraminic acid binding oligosaccharide, N-acetylneuraminic acid binding peptide,
It was discovered that N-acetylneuraminic acid binding protein, ganglioside sialic acids, and lacto-N-fucopentaose are effective, and the present invention has been completed.

The mannose used in the present invention may be a mixture prepared from a raw material containing mannose by a method including hydrolysis with an enzyme and / or an acid, or a fractionated product or a commercially available product thereof can also be used. . To give a specific example, for example, mannose is a low-viscosity galactomannan (trade name "Guafiber", manufactured by Meiji Seika Co., Ltd.) made from guar gum, milk yeast (trade name "PROTIVEL", Bell Indus). The polysaccharide fraction obtained from Dries can be obtained by acid hydrolysis with sulfuric acid or the like or enzymatic hydrolysis with mannanase or the like.

Further, N-acetylneuraminic acid-binding protein, N-acetylneuraminic acid-binding peptide, N-acetylneuraminic acid-binding oligosaccharide such as sialyllactose,
Sialic acids such as N-acetylneuraminic acid and ganglioside can also be prepared from milk or chicken egg by a known method in addition to commercially available products. Further, lacto-N-fucopentaose can be prepared from human milk or the like by a known method in addition to a commercially available product.

The mannose or sialic acid or lacto-N-fucopentaose thus obtained is added to itself or its concentrate or additives such as an excipient to obtain 0.05% to 5. 0%, preferably 0.1
It is suitable to add 1.0%. Further, these mannoses, sialic acids, or lacto-N-fucopentaose may be added to drinking water. The excipient is not particularly limited, and lactose, calcium carbonate, skim milk powder, corn grits and the like can be used.

The livestock and poultry that feed the feed of the present invention are not particularly limited. For example, cow, pig, chicken, horse, sheep, turkey,
The invention is not limited to quails and the like, and can be applied to pets such as dogs and cats, ornamental animals such as zoos, and experimental animals such as mice, rats, rabbits, and guinea pigs. The powder can be administered by mixing it with feed when it is made into powder and by mixing it with drinking water when it is liquid. The time of administration is not particularly limited, but since it is different from general antibacterial agents, it is preferable to start the administration at a time when the number of harmful intestinal bacteria is small, such as chicks or larvae.

[0016] The feed of the present invention, for example, in the case of chickens, inhibits adhesion of Salmonella bacteria, pathogenic Escherichia coli, Campylobacter bacteria, Clostridium perfringens, etc., which may cause food poisoning, to the intestinal tract. , Can prevent infection.

[0017]

EXAMPLES Examples of the present invention will be described below. Example 1 (Escherichia coli adhesion inhibition test) Human intestinal epithelial cells Intestine 407 were cultured in a minimal Eagle medium (hereinafter referred to as MEM medium) supplemented with 10% fetal bovine serum at 37 ° C. for 24 hours in a CO ₂ incubator. To form a monolayer and washed with MEM medium to remove serum components. Escherichia coli derived from a healthy person or chicken pathogenic Escherichia coli cultivated in Luria-Bertani broth at 37 ° C. for 18 hours was collected and washed with PBS (-) (phosphate buffered saline, Ca and Mg-free), It was suspended in a MEM medium in which the test sugar was dissolved so that the concentration was 10 ⁸ / ml. After 10 minutes, this E. coli suspension was added to the above Intestine 407. 90 at 37 ° C
After incubating in CO ₂ incubator for min, MEM
After washing the medium three times to remove non-adhering bacteria, the number of bacteria adhering to Intestine 407 was counted for 30 cells using a scanning electron microscope, and the average number of adhering bacteria was calculated. The adhesion rate A was calculated as follows, where B0 is the average number of adherent bacteria in the control section and B is the average number of adherent cells in the test section.

A = B / B0 × 100 (%) Table 1 and Table 2 show the results of evaluating effective carbohydrates by conducting an Escherichia coli adhesion inhibition test on various carbohydrates. As is clear from Table 1, with respect to Escherichia coli derived from a healthy person, the sialic acids and lacto-N-fucopentaose were confirmed to have an Escherichia coli adhesion inhibitory effect. Further, as is clear from Table 2, mannoses, sialic acids, and lacto-N-fucopentaose were found to have an Escherichia coli adhesion-inhibiting effect on chicken pathogenic Escherichia coli.

[0019]

TABLE 1 Adhesion inhibition test test samples of healthy subjects from E. coli (amount) adhesion rate (%) Control (no addition) 100 lacto -N- fucopentaose ^{I 1) (0.05%) 55} * G M1 - penta Saccharide ²⁾ (0.1%) 58 * N-acetylneuraminic acid (0.1%) 78 * G _M1 ³⁾ (0.1%) 13 * G _M2 ⁴⁾ (0.1%) 11 * G _M3 ⁵⁾ (0.1%) 34 * Fetuin ⁶⁾ (0.1%) 61 * * There is a significant difference in the t test (p <0.01) 1) Lact N-fucopentaose I; Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glc 2) G _M1 -pentasaccharide; Galβ1 → 3GalNAcβ1 → 4 (NeuAcα2 → 3) Galβ1 → 4Glc G _M1 sugar chain (oligosaccharide portion) 3) G _M1 ; Galβ1 → 3GalNAcβ1 → 4 (NeuAcα2 → 3) Galβ1 → 4Glcβ1 _{→ 1'Cer 4) G M2; GalNAcβ1} → 4 (NeuAcα2 → 3) Galβ1 → 4Glcβ1 → 1'Cer 5) G M3; NeuAcα2 → 3 Galβ1 → 4Glcβ1 → 1'Cer 6) N- acetylneuraminic acid If protein

[0020]

[Table 2] Test sample for adhesion inhibition test of chicken pathogenic Escherichia coli (amount added) Adhesion rate (%) Control (no addition) 100 Lacto-N-fucopentaose I ¹⁾ (0.5%) 47 * Man ²⁾ ( 0.1%) 49 * αMan → OMe ³⁾ (0.05%) 71 * αMan → OMe (0.1%) 37 * αMan1 → 2αMan-1 → OMe (0.1%) 64 * αMan1 → 3αMan- 1 → OMe (0.1%) 75 * αMan1 → 4 αMan-1 → OMe (0.1%) 57 * Ganglioside mixture (0.5%) 14 * * Significantly different by t test (p <0.01) 1) Lacto-N-fucopentaose I; Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glc 2) Man; mannose 3) αMan → OMe; Methyl-α-mannoside

Example 2 (Campylobacter jejuni adhesion inhibition test) Human intestinal epithelial cells Intestine was prepared in the same manner as in Example 1.
407 was monolayer-cultured and washed with MEM medium to remove serum components. Campylobacter jejuni cultivated in Brucella broth at 37 ° C for 18 hours was collected, washed with PBS (-), and the test sugar was dissolved to 10 ⁸ / ml ME.
Suspended in M medium. After 10 minutes, the Campylobacter jejuni suspension was added to the above Intestine 407.
After incubation in a CO2 incubator at 37 ° C for 90 minutes, the adhesion rate was calculated by the same method as in Example 1.
As a result of conducting a Campylobacter jejuni adhesion inhibition test on various sugars and searching for effective sugars, the effective sugars are shown in Table 3.

[0022]

[Table 3] Adhesion inhibition test of Campylobacter jejuni Test sample (addition amount) Adhesion rate (%) Control (no addition) 100 Man (50 mM) 56 * αMan → OMe (50 mM) 75 * Guafiber ** ( 1.0%) 14 * * There is a significant difference in t test (p <0.01) ** Low viscosity galactomannan (Meiji Seika Co., Ltd.)

Example 3 (Escherichia coli infection test) 12 kg of guar fiber was dissolved in 77.8 liters of tap water, and 2.2 liters of concentrated sulfuric acid was added. 9 this solution
The mixture was heated to 5 degrees and reacted for 5 hours. The reaction solution was neutralized to pH 7.0 with sodium hydroxide, and then desalted by a continuous desalting system (CDI) manufactured by Millipore to obtain a Bx34.5 guafi fiber decomposition product.

On the other hand, 20 kg of PROTIVEL was suspended in 85 liters of tap water, and 15 liters of 40% sodium hydroxide was added. The suspension was heated to 93 ° C., reacted for 8 hours, neutralized to pH 7.5 with concentrated hydrochloric acid, and solid-liquid separated. The obtained reaction solution (60 liters) was subjected to ultrafiltration to collect a fraction having a molecular weight of 10,000 or more, desalted by Millipore's continuous desalination system (CDI), and freeze-dried. In this way, about 1 kg of crude yeast polysaccharide was recovered. The yeast polysaccharide obtained was a brown powder and was not completely soluble in water. Sugar content is about 70%, protein content is about 12%, sugar composition is about 1.4: galactose: glucose: mannose.
1: 4. The yeast polysaccharide thus obtained,
B is obtained by acid decomposition in the same manner as guafiber.
x20 yeast polysaccharide degradation product was obtained.

Five 4-day-old broiler groups fed with a feed prepared by adding 0.3% of mannose to a test feed for broilers (for chicks, manufactured by Japan Blended Feed Co., Ltd.) and 4 guafi fiber degradation products were fed. 5x4 day-old broilers bred in drinking water supplemented with 3% (v / v) or 5.0% (v / v) of yeast polysaccharide degradation product, and 6 x 107 of chicken pathogenic E. coli
/ Ml Sterile saline suspension was forcibly orally administered to the sacs in an amount of 1 ml / feather. In addition, rectal contents were collected 7 days after the administration of Escherichia coli and the number of Escherichia coli was measured. The results are shown in Table 4.

[0026]

[Table 4] Chicken pathogenic E. coli infection test (rectal contents) test group log (bacteria / g) Control group 7.2 Mannose administration group 5.6 * Guafiber degradation product administration group 6.6 * Yeast polysaccharide degradation product administration group 5.8 * * t-test Is significant (p <0.05)

From the feed, a test feed for broilers (for chicks, manufactured by Nippon Formulated Feed Co., Ltd.) was added with 5.0% methyl-α-mannoside (αMM) or 0.1% ganglioside (Gg). 5 chickens in each group were fed with 6 × 10 ⁷ / ml of chicken pathogenic Escherichia coli at 1 or 2 days of age.
Sterile physiological saline suspension was orally administered by intraperitoneal injection in an amount of 1 ml / feather. In addition, fresh feces were collected at the age of 6 days and the number of E. coli was measured. Gg is Type IIgang manufactured by Sigma.
A mixture of liosides and Type III gangliosides 3: 2 was used. The results are shown in Table 5.

[0028]

[Table 5] Chicken pathogenic Escherichia coli infection test (feces) test group log (bacteria / g) control group 8.1 αMM administration group 6.8 * Gg administration group 7.2 * * Significant difference by t test (p <0.05) Table 4 and Table As is clear from Fig. 5, mannose, guar fiber degradation product, yeast polysaccharide degradation product, αMM, and Gg suppress colonization and growth of Escherichia coli in the chicken intestine.

Example 4 (Campylobacter jejuni infection test) Test feed for broilers (for chicks, Nippon Formulated Feed Co., Ltd.)
Manufactured), mannose, guafiber, or yeast polysaccharide 0.3% was added to the diet to ingest it, and four-day-old broilers each group contained 5 birds, and guafiber degradation product was 4.3%.
(V / v) or yeast polysaccharide degradation product 5.0% (v / v)
To each group of 5 4-day-old broilers bred with the added drinking water,
Campylobacter jejuni 1.7 × 10 6 / m
l Sterile physiological saline suspension was orally administered by intraperitoneal injection in an amount of 1 ml / feather. Immediately before the administration of Campylobacter jejuni and one day after the administration, feces were collected from the total excretory cavity with a swab to detect Campylobacter jejuni.
The results are shown in Table 6. As is clear from Table 6, when mannoses are ingested in advance, colonization and growth in the chicken intestine are suppressed immediately after administration of Campylobacter jejuni.

[0030]

[Table 6] Campylobacter jejuni infection testDays after infection Test group 0 1  Control group 0/5 5/5 Mannose administration group 0/5 3/5 Guafiber degradation product administration group 0/5 0/5 Guafiber administration group 0/5 2/5 Yeast polysaccharide degradation product administration group 0/5 1/5 Yeast polysaccharide administration group 0/5 1/5 Number of detected samples / number of tested samples, detection limit 10²/ Swab

[0031]

INDUSTRIAL APPLICABILITY By using the feed of the present invention, infection can be prevented by inhibiting adhesion of harmful bacteria of livestock and poultry to the intestinal tract, and it is possible to maintain and improve the health of livestock and poultry and improve productivity. It is valid. Furthermore, food poisoning that may occur when eating the livestock product can be avoided. In addition, mannoses, sialic acids, which are active ingredients,
Lacto-N-fucopentaose is a naturally occurring substance, and is therefore safe and harmless.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Tadaaki Okada 760 Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Confectionery Co., Ltd., Pharmaceutical Research Laboratory (72) Masao Hirayama 5-3-1, Chiyoda, Sakado, Saitama Prefecture No. Meiji Confectionery Co., Ltd. Biological Science Research Institute

Claims (3)

[Claims] 1. Mannose, sialic acid and lacto
-A feed comprising one or more selected from N-fucopentaose as a component for preventing harmful bacteria infection. 2. The mannoses are mannose; methyl-α-
The feed according to claim 1, which is a hydrolyzate of mannoside; manno-oligosaccharide; or a hydrolyzate of mannan obtained from guar gum, locust bean gum or yeast with an enzyme and / or an acid. 3. The sialic acid is N-acetylneuraminic acid,
The feed according to claim 1, which is an N-acetylneuraminic acid-binding oligosaccharide, an N-acetylneuraminic acid-binding peptide, an N-acetylneuraminic acid-binding protein or a ganglioside.