JPS5988042A - Purified blood plasma and artificial milk for animal containing the same - Google Patents

Abstract

PURPOSE:A purified blood derived from pig blood, obtained by treating a blood plasma separated from a pig blood with an ultrafiltration membrane for a given molecular weight range, and removing a salt, low-molecular weight protein, etc., capable of exhibiting an antimicrobial activity and an antibody value against pathogenic coliform bacilli, effective for preventing diseases caused by coliform bacilli of newborn animals. CONSTITUTION:A fresh blood collected from a pig body by the direct blood collecting method while avoiding the contact with air is rapidly cooled to a temperature at which the multiplication of microorganisms will not occur, e.g. about 4-5 deg.C. The resultant cooled blood is then centrifuged at <=5,000r.p.m. rotational frequency to give a supernatant fraction, which is then ultrafiltered through a filtration membrane for 6,000-13,000 fractionated molecular weight while being kept at a low temperature to purify the blood plasma component. Preferably, the removal ratio of foreign materials in the purification treatment is about 70-90%. The resultant purified concentrated liquid is then dried into powder by the freeze-dryig method, etc. and give the aimed purified blood plasma containing albumin and total globulin in (40-50):(50-60) ratio between the albumin and the total globulin in the whole protein. An antibiotic substance ''Colistin '' is added to the purified blood plasma and used together to give an artificial milk for animals.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to purified blood plasma, which has antibacterial activity and antibodies against pathogenic Escherichia coli and is effective in preventing coliosis in neonatal animals. To provide purified plasma derived from blood. Furthermore, it is possible to provide artificial milk processed with the addition of the same.

In general, in newborn animals, for example, piglets are
It is known that around 3 weeks of age, children are susceptible to infection by pathogenic bacteria in a normal environment, resulting in frequent episodes of diarrhea. Pathogenic Escherichia coli is known to be the causative agent of diarrhea, and sulfa drugs and antibiotics have traditionally been used to treat diarrhea (late-onset colibacillosis and early-onset colibacillosis). However, for early-onset colibacillosis, there is no treatment available after symptoms appear, and for late-onset colibacillosis, the disease progresses to anaphylactic diarrhea after symptoms appear.
Treatment with drugs may not be sufficient.

To solve these problems, attempts have been made to artificially give antibodies to piglets. However, such antibody products are produced by inoculating large amounts of various pathogens into healthy domestic animals such as horses, cows, pigs, goats, and sheep, and then collecting blood to obtain sufficient antibody titers. It is obtained by extracting immunoglobulin, which requires a large amount of money,
There were restrictions on its use in large quantities. In addition, as an antibody product, dried cow colostrum powder is known to be effective in treating diarrhea in live piglets, but there was a problem in that the raw material, colostrum, was not available in large quantities.

The present inventor researched an efficient method different from the conventional method of obtaining special antibodies or γ-globulin from the blood of livestock animals and colostrum of cows, and as a result, the inventor discovered that the bacteria that causes diarrhea in newborn piglets has been found. We have discovered purified plasma that is effective for this purpose and a method for its production, and have hereby completed the present invention.

In other words, fresh pig blood is used as a raw material, the plasma fraction is collected under low temperature conditions while avoiding air contact, and low molecular components are removed using dialysis to obtain a concentrated solution under mild conditions. The product was dried to obtain powdered plasma, and it was found that this product showed excellent antibacterial activity against E. coli and also showed antibody titers against pathogenic E. coli derived from pigs or cows. Also,
This product has also been found to exhibit a strong E. coli growth inhibitory effect in coexistence with colistin, which is known as an antibiotic that is usually effective against Durham-negative bacteria. Therefore, the combination of this product and colistin should not be used alone. We have found the advantage that it can be used at a lower concentration than the effective formulation when using.

To explain the method of the present invention in more detail, first, the blood that is the raw material for obtaining purified plasma is not limited to the type of livestock, but blood that is generally available in large quantities in fried chicken, such as blood derived from pigs, can be used. It will be done. However, conventional blood
Air contact is unavoidable, and contamination with bacteria and other ingredients may occur.
It was very difficult to collect fresh blood. Therefore, in the present invention, a direct blood collection method that avoids air contact from within the animal body is adopted, and fresh blood obtained by this method is used.

Next, the blood is heated to a temperature below which microbial growth does not occur (
For example, it is necessary to quickly cool it to about 4 to 5°C. Conventionally, blood is not cooled, which can lead to putrefaction due to proliferation of contaminating bacteria or denaturation of proteins, etc., making it unusable, so cooling blood is important.

Next, the cooled blood is centrifuged at a rotation speed of 5,000 rpm or less to obtain both supernatants, but if necessary, an appropriate amount of citrate may be added to prevent blood coagulation.
Alternatively, a clear Hiko solution is obtained by appropriately removing precipitated insoluble substances such as fibrin.

It is necessary to purify the plasma components of the supernatant obtained in this way by ultrafiltration using a filtration membrane with a molecular weight cutoff of 6,000 to 13,000 while keeping it at a low temperature. . The foreign substances removed in this process include inorganic salts, low molecular weight proteins, and sugars;
It not only reduces the purity of the final product, but also has a negative effect on the drying process, resulting in a non-homogeneous or dry product.
Removal of foreign substances is important as this results in a product with low antibacterial activity.

The filtration membrane used to remove foreign substances may be of the tube type or the flat membrane type, but the molecular weight cut-off is 6. If Od is less than 0, the removal of foreign matter will not be sufficient; if the molecular weight cut-off exceeds 15,000, the filterability will decrease and the operation will be difficult and therefore unsuitable. The foreign matter removal rate is preferably about 70 to 90 degrees, using the decrease in salt concentration as an index.

Through the ultrafiltration process described above, it is possible to simultaneously concentrate albumin and various globulins (α, β, γ).
- A concentrated solution of high-molecular protein whose main component is globulin, etc.) can be obtained. The solid content concentration of such a concentrated solution is about 15-2
Reach 0chi.

Next, the obtained concentrate is dried and powdered using a freeze-drying method or a spray-drying method that avoids harsh conditions. Purified plasma is obtained which contains albumin at a ratio of 40 to 50 and total globulin at a ratio of 50 to 600 in the total protein of the product.

Hereinafter, the present invention will be explained by giving examples.

Example 1 Approximately 150 tons of fresh blood collected from 50 conventionally bred adult pigs
While cooling to about 4℃, add 0.2 of sodium citrate.
~0.5 t was added. Next, the rotation speed s, o o o・r
The supernatant was collected by centrifugation under pm conditions.

6- After removing inorganic salts, saccharides, and low molecular weight proteins by dialysis using an ultrafiltration membrane with a molecular weight cutoff of 6,000, the obtained supernatant liquid was concentrated to a liquid volume of about 30 t1. The solid content in the concentrate was about 19%. The concentrated liquid was blown at a temperature of 130°C.
The purified plasma of the present invention was spray-dried at an outlet temperature of 70 to 75° C. to obtain 5.15 kp.

For the obtained product, Escherichia coli (E. coli 0111
Stoke) was investigated for its antibacterial effect. At the same time, as a comparison, the antibacterial activity against Escherichia coli was also measured for commercially available dried serum powder derived from pig blood.

The results are shown in Table 1.

Measurement conditions: A predetermined amount of a sterile solution prepared by dissolving each dry powder in water was added to a meat juice liquid medium, and after inoculating Escherichia coli, the mixture was cultured at 37°C overnight.

7- Table 1 Escherichia coli (E, coliol 5toke)
) growth rate As described above, it is recognized that the product of the present invention is a purified plasma with stronger antibacterial activity than commercially available products.

Next, various pathogenic E. coli antibodies were assayed for the product of the present invention and a commercially available dried serum product. The results shown in Table 2 show that the purified plasma is effective against two or more types of E. coli antigens from pigs and cows.

Measurement method: A solution was prepared by dissolving each powder to a concentration of 5% in physiological saline, and the agglutination reaction with each killed E. coli was performed, and positive results were indicated as + and negative values were indicated as -.

= 8- Table 2 Dry Powder Antibody Assay Furthermore, it is recognized from Table 3 below that when the purified plasma of the present invention is used in combination with the antibiotic colistin, the antibacterial activity against Escherichia coli increases.

Measurement conditions: A predetermined amount of a solution in which each additive was dissolved in water and sterilized was added to the broth liquid medium, and after inoculation with Escherichia coli, the mixture was cultured at 37°C overnight.

9- From this, when N plasma and colistin coexist, it is possible to suppress the growth of E. coli at a much lower concentration than the effective concentration when each is added alone.

Example 2 5 parts of purified plasma obtained in Example 1, 95 parts of artificial milk for piglets (main components: 22.0 parts of crude protein, 3.0 parts of crude fat, 2.0 parts of crude fiber, 5 parts of crude ash) .5 parts, calcium 0.7
3 parts of purified plasma obtained in Example 1 and 97 parts of artificial milk for piglets (main component: same as above), as well as the antibiotic colistin at 20p
-Purified plasma 3 chycolistin 2
Op-voice-added formula milk was prepared.

Next, these purified plasma-added artificial milks were fed to animals infected with pathogenic E. coli to examine their effects on the infected bacteria.

The test method used 6-day-old LWH pigs, which were in good health on the day of the test start and weighed 1.35-2.
Select piglets weighing 40 kg and divide them into groups of 4 pigs so that the weight distribution in each group is equal. Group 2 is a test group with purified plasma added and a test group with a combination of purified plasma and colistin, and the other groups are purified pigs. This experiment was carried out as a control test group in which no plasma was added.

On the other hand, infection of pathogenic Escherichia coli to laboratory animals occurs only after the start of feeding.
1.5 x 10 copies of Es per head to each district in 4 hours
cherichia coli (0-101, K-9
9) was orally administered. During the breeding period, the characteristics of each piglet's feces were observed every day, and the weight was measured at the start of the test and after 7 days. Furthermore, after oral infection with pathogenic Escherichia coli, Escherichia coli (
The effectiveness of the purified plasma against pathogenic Escherichia coli was determined by testing the numbers (0-101, -99).

The test results are shown in Table 4.

The explanations for the items in the table are as follows.

× 100 (3) Detection rate of infectious bacteria (2) Infectious bacteria E, coli (Q-101, Ni-9
Measurements were made by performing a cell agglutination reaction between standard antiserum of Escherichia coli groups ○1 and K1 and the detected bacteria at the ratio of 9).

Table 4 As is clear from the above results, the purified plasma according to the present invention is effective against pathogenic E. coli, and is also effective when used in combination with colistin.

-13-

Claims (4)

[Claims] (1) Plasma separated from pig blood with fractional molecular weight is,
Purified plasma containing high molecular weight protein as a main component obtained by treating with an ultrafiltration membrane of 000 to 15,000 to remove salts, low molecular weight proteins, sugars, etc. (2) Purified plasma has a concentration of 4,000 p% against E. coli.
The plasma according to claim 1, which exhibits an antibacterial effect at a concentration above. (3) The plasma according to claim 1, wherein the produced plasma exhibits an antibody titer against two or more types of E. coli among pathogenic E. coli derived from pigs or cows. . (4) Plasma separated from pig blood with a molecular weight cutoff of 6,000
~15,000 ultrafiltration membranes to remove salts, low molecular weight proteins, sugars, etc. Purified plasma, which is mainly composed of high molecular weight proteins, is used alone or in combination with the antibiotic colistin. Artificial milk for animals made with additives.