JPH0211519A - Weight-increasing and immunoenhancing substance, its production and feed containing the same substance - Google Patents

Abstract

PURPOSE:To obtain the subject substance exhibiting remarkable weight- increasing and immunoenhancing effect especially to young animal and poultry with a simple process at a low cost by removing capsule from microbial cell and using the cell as an active component. CONSTITUTION:Cells of bacterium belonging to genus Bifidobacterium, Bacillus, Corynebacterium, Lactobacillus, etc., is treated with 0.05-5vol.%, especially 0.1-1.5vol.% of a solution of a surfactant, preferably nonionic surfactant at room temperature or 30-60 deg.C for 1-24hr to obtain bacterial cells free from capsule. It is preferable to perform heat-treatment (e.g., at 56-75 deg.C for 15-60hr) of the bacterial cell before the surfactant treatment to inactivate the autolyzing enzyme existing in the cell and protect the cell wall. The cell free from capsule is administered at a rate of usually 10mug-1mg daily par 1kg of the body weight.

Description

[Detailed description of the invention] [Industrial application field] This invention increases the body hair of livestock and poultry.

A weight gain and immunity enhancer having the effect of enhancing immune function, its manufacturing capacity 27: Relating to a feed containing the same.

[Conventional branch] When poultry is young, the immune organs and immune cells are generally weak, and they are in a state of so-called congenital immunodeficiency, resulting in digestive problems such as diarrhea and white diarrhea. There are also many cases of respiratory infections, which is a major problem for the livestock industry. For example, the incidence of dysentery and dysentery in suckling pigs, or dysentery during the weaning period is 40% to 100%, and the economic loss caused by the migration of piglets to the north or the spread of filtration is extremely large, and the pig farming industry is This has become a big problem.

Currently, various antibiotics and antibacterial agents such as sulfa drugs are used to prevent and treat these diseases, but their effectiveness is not necessarily sufficient. Due to the emergence of drug-resistant bacteria and drug-resistant bacteria, etc.

Therefore, the livestock industry is desperate for the development of non-antibacterial drugs such as antibiotics that can prevent infectious diseases in young livestock and poultry, provide R1 therapy, and also have the effect of increasing weight. has been done.

In recent years, B CG (Racillus Calmett
e-Guerin) or Nocardia (Nocardia).
a) It was discovered that the cell wall component isolated from the microorganism i had an immune-enhancing effect, and that the component was peptidoglycan ("Journal or National
Cancer In5titude” No. 52a, +5
71 pages, 1974; Gann, No. 59! i
5.669 pages, 1976; “Cancer
Immunology and Immunothera
py, Vol. 4, p. 95, 1978), and since then, much attention has been focused on Durham-positive bacteria, which are widely distributed in nature and do not have to worry about the presence of endotoxins. It has been discovered that peptidoglycan has an immune-enhancing effect on piglets, and also has an effect on increasing body weight.Japanese Journal
of Veterinary 5science”, No. 4
9, p. 235, 1987).

The cell wall of Durham-positive bacteria is composed of peptidoglycan, polysaccharide, or ticoic acid, etc., as its main components, and is a covalently bonded bag-like structure that circumscribes the cytoplasmic membrane.
It plays the role of maintaining the shape of the bacterial cell and protecting the cytoplasm and its functions from various external stimuli. In particular, the peptidoglycan moiety is not only important as one of the immune-enhancing effects, but also plays an important role in maintaining the shape of the entire MB bacterial cell. In other words, peptidoglycan is a three-dimensional structure formed by bonding peptide chains with clecan buttons consisting of N-acetylglucosamine and N-acetylmuramic acid in a network, and as a whole is water-insoluble and physically extremely rigid. forming a polymer.

Conventionally, in order to separate and extract the cell wall from bacteria, the cells inside the bacterial body were first extracted under specific conditions. After the components are separated and removed, the cell wall remaining as a water-insoluble residue is recovered, and this is fermented with protease and nuclease.

However, the above-mentioned treatment is complicated in terms of production, yields are low, and the production cost is too high to be used as a $1 additive for livestock and poultry feed, making it extremely impractical. There isn't.

[Problems to be Solved by the Invention] Therefore, the object of the present invention is to provide a method for increasing body Φ and immunity that has excellent body Φ, increasing and immune-enhancing effects, and that can be produced at low cost using a simple method. The objective is to provide feedstuffs and feed containing the same.

Furthermore, it is an object of the present invention to provide a method for increasing the number of L letters and producing an immune enhancer.

[Efforts to solve the problem] As a result of intensive research, the inventors of the present application have determined that a product that simply removes the yellow membrane from bacterial cells, or a product that is particularly effective against young livestock and poultry. The present invention has been completed based on the discovery that it has the effect of increasing body weight and enhancing immunity, and that one pod can be removed from the bacterial cells by simply treating the bacterial cells with a surfactant.

That is, the present invention provides a weight gain and immunity enhancer containing bacterial cells from which the yellow membrane has been removed as an effective ingredient.

The present invention also provides a feed containing the weight gain and immunity enhancer of the present invention.

Furthermore, the present invention provides a method for increasing E resistance and using an immune enhancer, which comprises treating bacterial cells with a surfactant.

[Effects of the Invention] The present invention provides a novel weight gain and immunity enhancer that exhibits excellent weight gain and immunity enhancement effects. The weight gain and immunity enhancer of this invention is particularly suitable for young livestock,
It exhibits excellent effects on poultry and farmed fish (hereinafter referred to as livestock).

Furthermore, the weight gain and immunity enhancer of the present invention can be obtained by treating bacterial cells with a surfactant, and does not require any untreated M as in the prior art. Therefore, the feed containing the weight gain and immunity enhancer of the present invention can be produced much more cheaply than conventional products and is economically viable even when used as a feed ingredient for livestock, etc. By giving this to livestock, especially young livestock, death due to diarrhea and delayed growth of livestock can be avoided, which is extremely advantageous for the industry.

[Detailed Description of the Invention] As described in F, the weight gain and immunity enhancer of the present invention,
The active ingredient is bacterial cells from which the yellow membrane has been removed. The bacterial body may be any bacteria or yeast, or Bifidobacterium thermophilum (Ri
fidobacLeriumther■ophi lu
g) Bifidobacterium bacteria such as Bacillus
Rac i l 1 ussub Lili
s), Corynebacterium bacteria such as Corynebacterium ovis, Achnanomyces 0
Actinomyccs pyogenes
Bacteria of the genus Actyromyces, such as Lactobacillus ac
Lactobacillus bacteria, such as Lactobacillus idophilus,
Streptococcus 0.
Streptococcus bacteria such as Streptococcus faecdlis) and Staphylococcus conii (5T
Bacteria belonging to the genus Staphylococcus, such as Aphylococcus cohnii, as well as Kluyveromyces lactis,
s), Satucharomyces cerevisiae (5acchar
Bacteria of the genus Bifidobacterium are particularly preferred.

The bacterial cells may be killed.

The body TrE increasing and immune enhancing agent of this invention can be produced by treating bacterial cells with a surfactant, and does not require any other enzyme treatment. This can also be done by suspending the bacteria in an active agent solution, or by adding the bacterial cells to water or a buffer solution. ! This can also be done by adding a surfactant to this.

Any type of surfactant can be used as the surfactant, but nonionic surfactants are preferred since they can be removed relatively easily in subsequent steps. Reprints of such nonionic surfactants can be used, such as Tween 20 (trade name) and TriLon X100 (trade name).

When treating bacterial cells with a surfactant solution, the concentration of the surfactant used is preferably 0.05$ to 5z (V/V), more preferably 0.1% to 1.5X (
V/V), and the amount of surfactant solution used is the amount of wet bacterial cells (3000-8000 rp+s, l
centrifuged for 60 minutes) per 50 to 100 g of l
~10 times the volume (10 to 100 times the volume for dry bacterial cells) is preferable. When suspending in water or buffer in advance, add the surfactant at 1 volume after suspension. I can do it.

Surfactant treatment can be carried out at room temperature, and
It can also be carried out under heating at 60°C to 60°C. The treatment time is preferably 1 hour to 24 hours, and the suspension is preferably stirred during the treatment.

After treatment, in order to remove the surfactant adhering to the cJ body, it is desirable to wash it with wood or a buffer solution or by filtration.

Bacteria have their own autolytic enzymes, which are known to lyse the cell walls of bacteria (Masaru Chizutsu and Daisuke Nishiki, [Bacteriolytic Enzyme J p. 14, J-dan 21, 1977] ).

For this reason, it is preferable to inactivate the autolytic enzyme and protect the cell wall of the bacterial cells before performing the surfactant treatment. This can be done by heat-treating the bacterial cells. The heat treatment conditions were 56°C to 75°C for 15 minutes to 6°C in order to completely inactivate the autolytic enzyme and prevent heat denaturation of proteins in the bacterial cells.
Short-term treatment such as after 0 minutes 111 meters or 10 minutes 1 meter after spring water width is recommended.

Note that it is not always necessary to use a surfactant to remove the yellow membrane from the bacterial cells; depending on the type of bacterial cells, the yellow membrane can be removed by simply stirring or shaking the bacterial cell suspension. There is also. In such cases, stirring or shaking the bacterial cell suspension without surfactant treatment can eliminate this reaction.
A weight gain and immune enhancer for JI can be produced.

The bacterial cells from which one pod has been removed by treatment with a surfactant can be used as is, or can be further used after being freeze-dried.

The weight gain and immunity enhancer of the present invention is particularly suitable for domestic animals such as pigs, cows, horses, mountain animals, dogs, cats, and snails, poultry such as chickens, and freshwater fish such as carp; Rainbow trout, sweetfish, eel, and tilapia.

It has the effect of increasing the weight of farmed fish and shellfish such as conger eel, salmon, and trout, as well as saltwater fish such as sea bream, yellowtail, flounder, pufferfish, and shrimp, strengthening immunity, and preventing diarrhea. In addition, the early childhood period refers to 2 months to 3 months after birth.
It means the period of strength.

The weight gain and immunity enhancer of this invention may be added as is to the feed of livestock, etc., or it may be suspended in wood and orally administered to livestock, etc., or it may be mixed with other excipients and dried. It may also be administered by mixing it with feed.

The amount thrown for livestock, etc. is usually 1 kg per 1 animal.

10. per day. It is about g to 1 mg.

[Example] Next, Examples of the present invention will be shown, and the effects of the present invention will be specifically explained. Note that this invention is not limited to the following examples.

Example 1 Production of weight gain and immunity enhancer Bifidobacterium thermophilum (IlifidobacLc)
rium Ll+ermophilu meeting PNA 1-2
4. Cultivate 100 g of the obtained wet bacterial cells in a 0.05 M phosphate buffer (p.
H7,0) suspended in 1 sentence, heated at 75°C for 30 minutes,
The autolytic enzyme possessed by the bacteria itself was inactivated. after that,
Surfactant (Tween
After adding 20) and stirring at room temperature for 12 to 15 hours.

The cells were centrifuged at 8,000 rpm for 20 minutes, and the surfactant-treated cells were collected, followed by centrifugal washing three times to obtain cells. The weight gain and immunity enhancer of the present invention obtained by freeze-drying the bacterial cells was used as a feed material in the following examples. This feed material weighed approximately 12 g as a freeze-dried product.

Example 2 Analysis of weight gain and immune enhancer Hexosamine (main components: N-acetylglucosamine and N-acetylmuramic acid), which is an immune enhancer, and Bifidobacterium thermophilum cells obtained in Example 1 We also analyzed reducing sugars to see if they were contained in the lyophilized product.

Hexosamine was analyzed by the Morgan-Elson method and the Dische-Bordenfreund method (indole-hydrochloric acid method), and the results were 2.74K and 3.84$, respectively. Furthermore, as a result of measuring reducing sugar using the phenol method, it was found to contain 134 dollars. As is clear from the following results, it is clear that the weight gain and immunity enhancer of the present invention obtained in Example 1 contains hexosamine, which is a constituent sugar of the immune enhancer. was made into

Example 3 Effect of preventing diarrhea and increasing body weight in piglets Feed ingredients obtained in Example 1 (hereinafter referred to as
We investigated whether or not the addition of feed ingredients (referred to as feed ingredients) to piglets and oral administration to piglets would reduce the incidence of diarrhea in piglets, and whether or not this would result in an increase in the number of piglets per pig.

In the administration group (8 pigs), feed ingredients were added to the feed for the early lactation period of the pigs.
The supplement containing p■ was orally administered to the mice from 3 weeks of age to 5 weeks of age. The non-working group (6 pigs) was orally fed only feed for pigs in the early lactation period without the addition of feed K (N1).
In both cases, the pigs were fed a feed for the late lactation period without the addition of feed ingredients.The pigs were fed continuously for a period of a year and were observed until they were 9 weeks old, and the weight gain was measured and the diarrhea score was calculated. Here, the diarrhea score is calculated using a linear equation with 0 points for normal stool, 1 point for soft stool, 2 points for muddy stool, and 3 points for watery stool. .

Diarrhea score (score 7 days/head) = The results are shown in Table 1. Compared to the non-role J group, the incidence of diarrhea was smoothly reduced, and the increase in body hair was also significantly lower. it was high.

Example 4 11! Enhancement effect of immune function in vascular tissue Regarding the injection lj group and non-injection group in the farm of Example 3,
Pig+4[i feed for early lactation'1-H7 (5 weeks old) and pig 14ft At the end of feeding for late lactation (9@years of age) r pigs were slaughtered and dissected, and piglets Il! The tubes were immunohistologically examined for the presence or absence of enhanced immune function.

The test pigs were 2 pigs from YakuK group and 2 pigs from J(
There was one horse in the throwing group. Plasma cells, which are immunoglobulin-producing cells, are stained using vironin methyl green staining.
l! The 1g^ cell that is important for the immunity of the lumbar duct is PAP.
It was dyed by method. The count of plasma cells and 1gA-bearing cells is 4.
Coefficients were calculated in 00x x 20x field of view and expressed as the total number.

Pig 1 (1 milk + At the end of feeding for the 3rd period (5il age)
As shown in Table 2, the number of positive and negative plasma cells in the Yakuga group was clearly higher than that in the Non-Yakuga group. In addition, the number of Ig cells was also higher in the denum.

What is the shape of the toni denum, jejunum, and ileum of the throwing group at the end of feeding the feed for the later stages of lactation (9 weeks of age)? The number of jm cells is higher than that of the non-academic group, and the number of IgA-bearing cells is also higher in the heptadactyl group.
l! , Sky 11! Similarly, the number of patients was also high (Table 3).

As a result of Section F, the number of antibody-producing cells in the intestinal tissues of piglets administered with the feed supplemented with the weight gain and immunity enhancer of the present invention was clearly higher than that of non-administered piglets. It was shown that immune function was enhanced.

Table 2. /-Number of cells produced per gram in the right layer of the mucosa in the 1st grade pig of the pig (during stirring during the early lactation period of the older pig) Throwing group + 1 Throwing group + 2 Table 3 1g in the lamina propria of the 1st grade r pig Number of cells produced (at the end of feed feeding for pigs 1-6 in the late lactation period) Throwing group 1, II-throwing Ij, group 1mu AJ Immune enhancement effect in mice Example 1 The freeze-dried bacterial cells obtained in mouse After intraperitoneal administration, E. coli (septic type) or Salmonella typhimurium (Salaone
lla Lyphimurium, ATCII:1:
111 strain), and its effectiveness was determined using the survival rate as an index.

Five mice (ICR strain) were used for each administration group and non-nuclear-1. The lyophilized bacterial cells were administered intraperitoneally 4 days before the E. coli challenge, before IH, and intraperitoneally 4 days after the Salmonella typhimurium challenge. Also, E. coli is 1.8 x 10'
CFU/mouse, Salmonella typhimurium is 3.4 x 10'C
FU/animal was challenged intraperitoneally.

The results are shown in Table 4. As shown in Table 4, all treatment groups were extremely effective against E. coli. In addition, the 40 μg dose group was ineffective against Salmonella Typhimurium, but the 200 μg dose group was ineffective.
It was significantly more effective in the groups administered ug or more. In addition, in the non-administered control group, all died between 1 and 5 days after challenge with Escherichia coli and Salmonella typhimurium. In addition, in Table 1, the immune-enhancing effect is (#'! Number of surviving animals in the strong drug administration group: 4, number of surviving animals in the Chisho group: 4)
Calculated by ÷1A mouse quadruply x 11111 (z),
*(275% extremely effective), 0 (50-75% effective), /\(25-50% moderately effective), ×(≦25%,
! (effective).

From the above, it can be seen that the immunostimulant and immunostimulant of this invention has protective activity against highly pathogenic microorganisms such as Salmonella typhimurium and Salmonella typhimurium even in mice, and has a high immunity-enhancing effect. It was shown to increase the activity by 41%.

Table 4. Immune-enhancing activity in mice: 1.1 Immune-enhancing effect [duration g/mouse/time 1 E, coli challenge S, Lypl+iou+riu+*challenge (intraperitoneal administration,) (intraperitoneal injection 'i-) ◎ ◎

Claims (4)

[Claims] (1) A weight gain and immunity enhancer containing bacterial cells from which the yellow membrane has been removed as an active ingredient. (2) The weight gain and immunity enhancer according to claim 1, wherein the bacterial cells are bacterial cells belonging to the genus Bifidobacterium. (3) A feed containing the weight gain and immunity enhancer according to claim 1 or 2. (4) The method for producing a weight gain and immunity enhancer according to claim 1 or 2, which comprises treating the bacterial cells with a surfactant.