JPH0466050A - Preparation of sterilized milk containing immunoglobulin - Google Patents

Abstract

PURPOSE:To obtain a sterilized milk without lowering immunoglobulin activity by thermally sterilizing a milk containing immunoglobulin under specific condition. CONSTITUTION:A milk containing immunoglobulin is thermally sterilized at a sterilization ratio of >=99.9% in such a manner as to leave >=70% of the immunoglobulin activity based on the level before sterilization. The thermal sterilization is carried out at 62 deg.C for 30min, at 75 deg.C for 15sec or under a condition attaining the equivalent effect. The milk containing immunoglobulin is e.g. preferably milk obtained from cow immunized with plural kinds of bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing sterilized milk without reducing the immunoglobulin activity of milk containing immunoglobulins.

l Raitagi distorted milk contains 0, including secreted immunoglobulin.
．． It contains about 1 μ/d of immunoglobulin. This immunoglobulin is known to be effective in preventing bacterial colonization and infection in the intestinal tract.

However, in conventional milk production methods, most of the milk is inactivated by sterilization and cannot be used effectively. Recently, attention has been focused on antibodies in milk, and research is being conducted on colostrum, which has a particularly high antibody content.

In order to obtain such sterilized milk with high antibody content, an alkali is added to the milk so that the antibodies are not inactivated by sterilization, and
Methods for preventing thermal denaturation such as coagulation have been studied. For example, Japanese Patent Application Laid-Open No. 160664/1984 discloses a method of adding a monovalent alkaline agent to colostrum and sterilizing it by heat.

Although several methods have been studied for colostrum with a high antibody content, no such studies have been conducted for normal pores because the antibody content is low.

Recently, in addition to the issue of the amount of antibodies in milk, the existence of specific antibodies has attracted attention, and research into the use of these antibodies has been progressing. JP-A-54-113425, JP-A-57-188
Publication No. 523 discloses a method in which 24 types of bacterial antibodies are injected as a booster and the milk extracted from the cow is used as an anti-inflammatory agent.

It is also known that such milk has the effect of improving immune function. This effect is thought to be due to the antibodies contained in milk, but the amount of immunoglobulin in this milk is slightly higher or about the same as that of regular milk. It is completely unknown whether milk containing such specific antibodies is stable after pasteurization. Because immunoglobulins are expected to be easily deactivated during the normal milk sterilization process, milk is consumed without sterilization or low-temperature drying methods are used. Therefore, the number of bacteria contained in milk is
l1li! The number of doses per patient was more than 10,000, which is not preferable from a safety standpoint, and in particular, administration to patients with immunodeficiency was counterproductive.

As a result of conducting research on the use of milk containing specific antibodies, the present inventors discovered a method for producing sterilized milk without reducing immunoglobulin activity. .

Therefore, an object of the present invention is to provide a method for producing sterilized milk without deactivating antibodies.

Normally, sterilization of milk such as milk is done by pasteurization, which involves heat treatment at 60"C for 30 minutes, and H, which involves heat treatment at 72℃ to 75℃ for 15 seconds.
TST sterilization and UHT sterilization in which processing is performed at 120°C to 150°C for 0.5 to 4 seconds are known. Through these sterilization treatments, the number of general bacteria specified by the Ministerial Ordinance on Milk, etc. of the Ministry of Health and Welfare is 0 to 1.
00 pieces/ld. However, whether or not sterilization under such conditions affects the activity of the immunoglobulin contained therein has not been rigorously investigated.

The present invention is based on the viewpoint that in order to effectively utilize the immunoglobulin contained in milk, it is necessary to prevent deactivation due to heating to the maximum extent possible, and that strict temperature control is necessary for this purpose. As a result of these studies, the invention was completed.

That is, the present invention provides a method for sterilizing immunoglobulin-containing milk by heat-sterilizing the immunoglobulin-containing milk at a sterilization rate of 99.9% or more and in such a way that at least 70% of the immunoglobulin activity remains before sterilization. This is the manufacturing method.

Such sterilization conditions include 62°C for 30 minutes, 75°C
Any condition for 15 seconds can be selected.

The present invention further includes heat sterilization conditions that have the same effects as these conditions.

The immunoglobulin-containing milk used as a raw material in the present invention includes:
Skim milk, cow's milk, etc. to which immunoglobulin extracted from the serum of mammals such as humans and cows has been added, and milk that has passed 72 hours or more after calving and has passed the so-called colostrum period, but has not exceeded the 10th day. Milk expressed from a mother, JP-A-54-1
Examples include milk milked from a mother cow immunized with a mixture of various killed bacteria disclosed in Japanese Patent Application Laid-Open No. 57-188523, and milk that has not been heat-treated after milking.

These milks are prepared using conventional processing and, in unpasteurized conditions, have a bacterial count of 40. Over 6 Oo.

In the present invention, these bacteria are sterilized without deactivating the antibodies contained in these milks.

It is necessary to strictly set temperature conditions for sterilization. The amount of antibodies remaining in milk and the bactericidal effect can be confirmed by the following experiment.

Experiment 1 (method for sterilizing cow milk) The milk used for sterilization contained 0.5 g/d of IgG and had a general bacterial count of 75,000 cells/ML1.

Sterilization conditions were 62°C for 30 minutes, 70°C for 30 minutes, and 70°C for 30 minutes.
Sterilization was carried out at 5°C for 15 seconds, 80'C for 15 seconds, 110°C for 1 second, and 150'C for 1 second. The sterilization rate and the residual rate of antibody titer when sterilized under these conditions were determined. The content was measured by immunodiffusion method.

The results are shown in Table 1.

Table 1: Under the conditions of 70°C for 30 minutes, 80°C for 15 seconds, 110°C for 1 second, and 150°C for 1 second, the remaining bacteria were below the detection limit (10 cells/d) and were sufficiently sterilized. , IgG
The remaining amount was 30% or less in all cases.

On the other hand, under the conditions of 62° C. for 30 minutes and 75° C. for 15 seconds, residual bacteria were detected, but the sterilization rates were 99.93% and 99.97%, respectively, indicating sufficient sterilization.

Moreover, the residual IgG showed high values of 95% and 83%.

Experiment 2 (method for sterilization of a mixture of IgG and IgA in skim milk) The sample was prepared by adding 1 portion each of IgG and IgA to skim milk.
It was added at a rate of 0.0cm/d and 0.5cm/M1. In addition, to examine the bactericidal effect, 100,000 E. coli/I1
It was prepared by adding it so that it became 1.

This solution was heated at 62°C for 30 minutes and at 70°C for 30 minutes at 75°C.
°C for 15 seconds, 80 °C for 15 seconds, 110 °C for 1 second, 1
Sterilization was performed at 50°C for 1 second.

The sterilization rate and residual rate of antibody titer when sterilized under these conditions were determined. The content of IgG was measured by immunodiffusion method. The results are shown in Table 2.

Table 2: Under the conditions of 70°C for 30 minutes, 80°C for 15 seconds, 110°C for 1 second, and 150°C for seconds, the number of remaining bacteria was below the detection limit (10 cells/ll11) and was sufficiently sterilized. However, the residual IgG was 30% or less in all cases.

On the other hand, under the conditions of 62° C. for 30 minutes and 75° C. for 15 seconds, residual bacteria were detected, but the sterilization rates were 99.9% and 99.98%, respectively, indicating sufficient sterilization.

Moreover, the residual IgG showed high values of 96% and 88%.

Experiment 3 (sterilization method of immunized milk) l) Method of immunizing cows a) JP-A-54-113425, JP-A-57-1885
The method described in Publication No. 23 was used.

b) After fertilization of cows, a mixture of killed bacteria shown in Table 1 of the above-mentioned publication was continuously fed to the feed until calving.

Milk obtained by immunization by method a) or b) was heated at 4℃ for 1
The sample was stored for 1 day and used as a sample.

This solution was heated at 62°C for 30 minutes, at 70°C for 30 minutes, at 75°C.
-15 seconds, 80℃-15 seconds, 110℃-1 second, 1
Sterilization was performed at 50°C for 1 second.

The sterilization rate and residual rate of antibody titer when sterilized under these conditions were determined. The content of IgG was measured by immunodiffusion method.

Further, the specific antibody titer was determined by immunoassay method using antigen-antibody reaction.

The results are shown in Table 3.

Table 3 Under the conditions of 70°C for 30 minutes, 80°C for 15 seconds, 110°C for 1 second, and 150°C for 1 second, the number of remaining bacteria was below the detection limit (10 cells/M1), and the bacteria were sufficiently sterilized. ,Ig
The residual amount of G was 30% or less in all cases.

On the other hand, under the conditions of 62° C. for 30 minutes and 75° C. for 15 seconds, residual bacteria were detected, but the sterilization rates were 99.9% and 99.95%, respectively, indicating sufficient sterilization.

Moreover, the residual amount of IgG was as high as 98% and 90%. Furthermore, when immunized with 25 types of killed bacterial cells, the specific antibody titer also showed a high survival rate of 97% and 92%.

From the above experiments, in the present invention, antibody activity can be reduced by 70%.
In order to make the milk remain as long as 99.9% or more and to obtain a sterilization effect of 99.9% or more, if the conditions are either 62℃ for 30 minutes or 75℃ for 15 seconds, denaturation such as browning and coagulation will occur in the milk. It is possible to provide sterilized milk in which antibody activity is maintained without causing any sterilization. In the present invention, we have discovered a sterilization method that allows 70% or more of antibody activity to remain and a sterilization effect of 99.9% or more by adjusting the heat sterilization temperature and time. The temperature-time conditions that occur are included in the present invention. Sterilization in the present invention is
A busterizer may be used, or a known device such as an HTST sterilizer may be used. The antibody-containing sterilized milk thus obtained can be stored in the refrigerator as it is, or, if necessary, can be dried at low temperatures such as freeze drying or continuous vacuum drying. The resulting milk can then be consumed as is. Furthermore, it can be administered to patients with immunodeficiency for medical purposes to enhance their antibodies, and can also be used for the prevention or treatment of various infectious diseases. EXAMPLES Below, Examples and Experimental Examples will be shown to further explain the present invention in detail.

Example 1 Production of immunized milk using bacterial mixed antigen
The amount of antibody was obtained according to the method for producing immunized milk disclosed in Publication No. 88523.

Five cows were immunized against a polyvalent vaccine consisting of the bacterial strains listed in Table 4. As the primary immunization, a vaccine containing 4×10 cells/d of each heat-sterilized bacterial cell was administered 5 d each once a week, 4 times in a row.

After confirming the antibody titer of each cow by an agglutination method, the cows were milked every day while administering the same dose of killed bacteria at 14-day intervals.

The expressed milk is collected, defatted by centrifugation, and then
The milk was sterilized using a Basterizer at ℃ for 30 minutes, and the sterilized milk was filled into a 200 d paper container. The bactericidal effect and antibody residual rate of this milk were compared with that of the same unpasteurized milk.

As a result, the number of bacteria after treatment was 30/d compared to the number of bacteria ao and ooo/111 before treatment. Furthermore, the antibody amount was 98 when the IgG value before treatment was taken as 100, and the antibody titer against mixed bacteria was 97 when the value before treatment was taken as 100, and sterilized milk with maintained antibodies was obtained.

Table 4 (Bacterial antigens) Example 2 Immediately after fertilization of 5 cows, 50 d of heat-sterilized vaccine (4 x 10" cells/11) of bacterial cells shown in Table 4 was mixed with the feed, and this was administered daily. This administration was continued until the time of delivery, and milk was collected from the 4th day after delivery to the 100th day.
It was sterilized at 70°C for 15 seconds using an ST sterilizer. Sterilized milk was filled into a 200 volume paper container.

The bactericidal effect and antibody residual rate were measured in the same manner as in Example 1. The number of bacteria before treatment was 100,000/I11, and the number of bacteria after treatment was 10/-. In addition, the amount of IgG before treatment,
When the specific antibody titer was set as 100, these values after treatment were each 72.75. Therefore, sterilized milk with maintained antibody activity was obtained.

Experimental Example This experimental example shows the therapeutic effect of sterilized skim milk obtained by freeze-drying the skim milk obtained in Example 1 on opportunistic infections.

One group of 7-week-old female ICR mice was 10 mice, each with 700 mice.
A model animal was created in which opportunistic infections are caused by irradiation with R X-rays, thereby reducing gastrointestinal immune function. The therapeutic agents to be administered include the skim milk obtained in Example 1 (FZ-09) and the skim milk powder (GZ-08) obtained by spray-drying skim milk obtained from ordinary sterilized milk at high temperature in sterilized water. An absorption bottle was filled with the solution dissolved at a concentration of 12.5% and allowed to seed freely instead of water.

Administration began 8 days before X-ray irradiation and 7 days after irradiation. Each individual was bred and observed, and for animals that died, observation was continued for 30 days while recording the number of days they died.

The survival days of each sample administration group were as shown in Table 5. Furthermore, changes in mortality rates for each group are shown in Figure 1.

For each deceased animal, a necropsy was performed and a liver homogenate was prepared. A large amount of E. coli was detected in this homogenate, confirming that an opportunistic infection of intestinal bacteria had taken place. Furthermore, it was confirmed from the autopsy findings that each animal's cause of death was due to an infectious disease.

The mortality rate was significantly lower in the group administered with the sample prepared by the method of the present invention, and the number of days of survival was significantly higher in the group administered with the sample prepared by the method of the present invention. It was confirmed that the drug has therapeutic and preventive effects.

Table 5 According to the method of the present invention, it is possible to provide a large amount of sterilized milk in which antibodies are not deactivated by a simple method.

Such sterilized milk can be used as ordinary commercial milk for drinking as it is, or can be administered for medical purposes to patients with immunodeficiency to enhance their antibodies, or can be used to treat various infectious diseases. It can also be used for prevention.

[Brief explanation of the drawing]

FIG. 1 shows the survival rate curve of animals to which a therapeutic agent for opportunistic infections prepared from sterilized milk obtained according to the present invention was orally administered. ↑ indicates that the therapeutic agent for opportunistic infections of the present invention was administered. -〇- (water) used water as a control, and -l- (GZ-08) received orally a sterile aqueous solution of skim milk powder obtained by spray-drying skim milk obtained from regular sterilized milk at high temperature. The survival rate curve when administered is shown. Again...
-(FZ-09) shows a survival rate curve when an aqueous solution of skim milk powder processed and prepared according to Example 1 was orally administered.

Claims (3)

[Claims] (1) Sterilization rate of milk containing immunoglobulin is 99.9%
A method for producing sterilized immunoglobulin-containing milk, which is characterized in that it is heat sterilized so that 70% or more of the immunoglobulin activity remains before sterilization. (2) The sterilized immunoglobulin-containing milk according to claim (1), wherein the heat sterilization is carried out under heat sterilization conditions selected from 62°C for 30 minutes, 75°C for 15 seconds, or heat sterilization conditions that produce similar effects. manufacturing method. (3) The method for producing sterilized immunoglobulin-containing milk according to claim (1) or (2), wherein the immunoglobulin-containing milk is milk expressed from a cow immunized with various types of bacteria.