JPH0276822A - Immune activator - Google Patents

Abstract

PURPOSE:To obtain an immune activator containing a proteolized extract containing a peptide obtained by decomposing a protein by a proteolytic enzyme and having a specific molecular weight as an active ingredient, free from side effects and capable of activating immune function of an animal. CONSTITUTION:The aimed immune activator containing a proteolized extract containing a peptide obtained by decomposing a protein (e.g., fishes and shellfishes or edible animals) and having 1000-15000, especially 3500-10000 molecular weight as an active ingredient. The above-mentioned proteolized extract is obtained by heating a raw material protein such as fishes and shellfishes or edible animal so as to completely inactivate an autolysin, decomposing a protein contained in the raw material up to proteose class by Bacillus subtilis-producing proteolytic enzyme and then decomposing the protein to the peptide having the above-mentioned molecular weight by Aspergillus producing proteolytic enzyme. Then dose of the above-mentioned activator is preferably >=10mg/kg, especially >=25mg/kg in the case of oral administration and preferably >=5mg/kg, especially 15mg/kg in the case of vein injection.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to an immunostimulant that activates immune function.

<Prior art> In general, animals have a wide variety of defense functions that try to eliminate foreign substances such as foreign microorganisms and foreign self-substances, that is, immune functions. It is believed that it can fight against diseases. Therefore, recently, the development of substances to activate the above-mentioned immune function has been progressing. For example, research has been carried out on BRM, polysaccharides, lignin, lectins, and low-molecular substances as substances with immunomodulatory functions in food ingredients. has been achieved. However, at present, it is not known at all whether proteolytic extracts containing peptides obtained by decomposing proteins have immunostimulatory effects.

<Problems to be Solved by the Invention> An object of the present invention is to provide an immunostimulant that has no side effects and activates the immune function of animals.

<Means for Solving the Problems> According to the present invention, there is provided an immunostimulant characterized by containing as an active ingredient a proteolytic extract containing peptides with a molecular weight of 1,000 to 15,000 obtained by decomposing proteins with a proteolytic enzyme. agent is provided.

The present invention will be explained in more detail below.

The immunostimulant of the present invention contains a proteolytic extract containing a specific peptide as an active ingredient.

In the present invention, the molecular weight of the peptide contained as an active ingredient is 1000 to 15000, preferably 35
The range is from 00 to 10000.

If the molecular weight is less than 1,000 or more than 15,000, a sufficient immunostimulatory effect cannot be obtained.

As the proteolytic extract containing the peptide used in the present invention, fish and shellfish extracts and/or food animal extracts obtained by decomposing fish and shellfish and/or food animals with proteolytic enzymes can be used.

The fish and shellfish extract can be produced, for example, as follows. Specifically, raw fish and shellfish such as horse mackerel, mackerel, sardines, saury, bonito, atka mackerel, cod, squid, octopus, shrimp, oysters, freshwater clams, clams, mussels, snails, red clams, clams, etc. are shredded into slurry, etc. Pour into the reactor as a whole without any pretreatment, and immediately after charging, 75%
℃ or more.

Preferably, the temperature is raised to 80°C or higher to completely inactivate the autolytic enzymes contained in fish and shellfish, and at the same time remove odors such as the dullness and foul odor characteristic of fish and shellfish that are generated by the action of autolytic enzymes. 1 Then, 40°C to 70°C, pH 5.0
~7. O1 Preferably at pH 5.5 to 6.5, Bacillus subtilis-produced protease is added to decompose proteins contained in fish and shellfish to proteose grade. Next, the temperature is raised to at least 65°C or higher, preferably 75°C or higher, usually for 3
Bacillus subtilis-produced proteases are inactivated for 1 to 1.5 hours, preferably 10 minutes to 1 hour, and then the pH is adjusted again.
It is decomposed by adding a protease produced by Aspergillus at 30-60℃ and pH 5-7 without adjusting it, and the molecular weight is substantially 10.
Decomposed into 00-15000 peptides. Decomposition time is 1
It is carried out for about 0 minutes to 3 hours, preferably about 30 minutes to 2 hours.

If the decomposition time is less than 30 minutes, proteose remains and a peptide constituting a specific amino acid composition cannot be obtained, whereas if it exceeds 3 hours, the immunostimulatory effect decreases, which is not preferable.

The decomposed liquid thus obtained can be classified into undecomposed substances such as fish and shellfish extract layer, oil layer and bone fragments using a centrifuge or the like in a conventional manner, and then subjected to ultrafiltration or heated at 60°C or below. It can be manufactured by using a method such as concentration under reduced pressure.

The components of the fish and shellfish extract include various pavtide amino acids and free amino acids such as glutamic acid, aspartic acid, lysine, arginine, glycine, alanine, leucine, proline, histidine, phenylalanine, and serine, and have a molecular weight of substantially 1000. The main component is ~15,000 peptides. Edible animal extracts can also be produced as follows. That is, for example, the meat, bones, skin, etc. of food animals such as seaweed, cows, and pigs as raw materials are cut into appropriate sizes, placed in a reaction vessel, and heated to 75°C or higher, preferably 80°C or higher. to prevent denaturation of the raw materials and inactivate autolytic enzymes, then heat at 40°C to 70°C, pH 5.0.
~7.0, preferably at pH 5.5 to 6.5, a Bacillus subtilis-produced protease is added, and the stirring reaction is carried out to decompose food animal proteins to proteose grade. Next, the temperature is raised to at least 65°C or higher, preferably 75°C or higher, and the Bacillus subtilis-produced protease is inactivated, usually for 3 minutes to 1.5 hours, preferably 10 minutes to 1 hour. Then, without adjusting the pH again, at 30-60℃, p l-l5~
In step 7, a protease produced by Aspergillus oryzae is added to decompose the mixture into peptides having a molecular weight of 1,000 to 15,000. Finally, these can be separated into oil, fat, aqueous solution, and bone fragments using a centrifugal three-layer separator or the like, and can be produced by ultrafiltration or vacuum concentration at 60° C. or lower.

The method for producing the fish and shellfish extract and/or edible animal extract as the proteolytic extract is one example and is not limited thereto. For example, in both of the above two methods, the proteolytic enzyme is allowed to act in two steps, but it is also possible to use a proteolytic extract obtained by a method in which the proteolytic enzyme is allowed to act in only one step and various conditions are changed. Furthermore, as the protease, any enzyme that can decompose proteins can be used alone or in combination.

The above-mentioned active ingredient, the proteolytic extract, is non-toxic because it is made entirely of natural products.

It is an extremely safe substance.

The immunostimulant of the present invention can be administered, for example, by oral administration, intravenous injection, etc. In the case of oral administration, the effective amount is preferably 10 μ/kg or more, particularly 25 μ/kg.
g or more is desirable. In addition, in the case of intravenous injection, 5■/k
The amount is preferably 15 mg/kg or more, particularly 15 mg/kg or more. In addition to the proteolytic extract containing the peptides mentioned above, it is also possible to contain, for example, quinone, α-tocopherol, etc., which are commonly used in pharmaceuticals.

In the present invention, when administering the immunostimulant orally,
It can be ingested by adding it to food or feed, and excellent immune activity can be obtained by ingesting it every day.

<Effects of the Invention> By using the immunostimulant of the present invention, immune activity can be improved. Furthermore, since the immunostimulant of the present invention has a peptide obtained by decomposing natural proteins as its main component, it is non-toxic and there is no fear of side effects even if it is taken daily.

<Examples> The present invention will be explained in more detail below using Examples and Comparative Examples, but the present invention is not limited thereto.

5 tons of bar was poured into a reaction vessel from a rice cooker, and the temperature was raised to about 90°C to completely inactivate the autolytic enzyme and remove the odor at the same time.

Next, a Bacillus subtilis-produced protease was added at 60°C and pH 6.0, and the mixture was allowed to react for 1 hour. Next, the temperature is 75℃
The temperature was raised to 30 minutes to inactivate the B. subtilis-produced protease, and at 50° C. and pH 6, the Aspergillus aspergillus-produced protease was added and decomposed for 30 minutes. The resulting decomposition solution was processed in a centrifuge for 20 minutes to obtain a proteolytic mackerel extract, which was then subjected to ultrafiltration to prepare an immunostimulant. The molecular weight of the peptide in the obtained immunostimulant was measured by gel permeation chromatography (GPC), and the molecular weight was 7,000.

S] 11 breasts Adult male rabbits weighing around 2 kg were fed regular feed for 4 weeks.

After fasting for 12 hours, peripheral blood was collected from the ear vein, and the lymphocyte rejuvenation response to ConA stimulation was measured. Next, 1 g of mackerel extract, which is an immunostimulant prepared in Example 1, was added to 100 g of feed and administered for 30 days. Thereafter, after fasting again for 12 hours, peripheral blood was collected from the ear vein and lymphocyte development was measured. Figure 1 shows the measurement results of the lymphocyte blastogenesis reaction. Further, the method for measuring lymphocyte development is shown below.

Lymphocytes 6 The peripheral blood collected on the other side was subjected to specific gravity centrifugation to separate lymphocytes, washed, and suspended in 11 PMI-1640 supplemented with 10% FC5.

ConA stimulation was performed using ConA for 0.5 mQ of cell suspension.
0.5 m of 10% FC3-added RPMI-1640 containing A
Prepared by adding Q. As a control, a system without the addition of ConA was prepared, and a 24-well plastic plate was prepared for 72 hours.
Culture was carried out for hours (37°C, 5% CO2.95% air).

Similarly, for PHA stimulation, circle I was applied to 0.5 mQ of cell suspension.
0.5 mQ of FC5-added 11 PMI-1640 containing A was added and cultured. Collect the cultured lymphocyte suspension into a test tube.

After washing the lymphocytes, the supernatant was removed. To the remaining cell pellet, add 2+nQ of 0.1% Triton
After adding 0.15 mQ of Q and EB solution (200 μg/mQ) and reacting at 4°C for 20 minutes, the spectrum m (
It was measured by Spe-ctrumll [ ) (manufactured by Oats Co., Ltd.).

Lymphocyte blastogenesis was measured in the same manner as in Example 2, except that the immunostimulant prepared in Example 1 was not used. 1st
The figure shows the measurement results of the lymphocyte blastogenesis reaction.

As shown in Fig. 1, the results of Example 2 and Comparative Example 1 show that when no immunostimulant is administered, there is a correlation between the immature response of rabbit ear vein peripheral blood lymphocytes to Con A on the day of the start of the experiment and after 30 days. There was no sex. However, when an immunostimulant was administered, the rejuvenation response to ConA was 3.3% before administration.
5±3.35 (M±SD), whereas 30
After daily administration, it was 9.26±2.99 (MfSD).

Therefore, it has been found that administration of the immunostimulant of the present invention clearly increases the blastogenesis response of lymphocytes.

The immunostimulant that had been adapted to y4 in Example 1 was orally administered to 11 healthy subjects (age 30±3.3, weight 61±5.3 kg, male) at a total of 6 g/day, 3 g in the morning and evening, for 30 days. did. The intake of alcohol, tobacco, and drugs was prohibited during treatment, but there was no restriction on one meal.

Next, about 24 hours after the final administration of the immunostimulant, blood was collected on an empty stomach before breakfast, and PIIA and C were collected in the same manner as in Example 2.
Peripheral blood lymphocyte blastogenesis response to onA stimulation was measured. Figures 28 and b show the measurement results of peripheral blood lymphocyte blastogenesis responses to PIIA and ConA stimulation, respectively.

The results of Example 3 are as shown in Figures 2a and b.

It was found that administration of the immunostimulant of the present invention increased the peripheral blood lymphocyte blastogenesis response to ConA and PIA. In particular, the ConA-stimulated lymphocyte blastogenesis response was 1.769±0.304 (M±SD) before administration.
) to 2.342±0.614 (M+SD) after administration.

Male C57B was used to examine the ability of lung cells to produce antibodies.
L/6 mice were kept in a constant environment for 6 days after introduction.

Next, 1 g of the immunostimulant prepared in Example 1 was added to 100 g of normal powdered feed, and the mixture was orally administered to the mice for 10 days. On day 5 of administration, 5 x sheep red blood cells (SRBG)
0.2 mQ of tO'/mQ was injected into the tail vein. Five days after the end of administration, the lungs were removed from the mice and made into a single cell suspension, and then washed three times with IE-MEM (1500 rpm 51
1 inch) L/, a suspension prepared at 1,25XIO/mQ, 50% 5RBC, and complement [guinea pig serum (Guin
-er Pig Serum) dry powder as E-HEM.
solution] were mixed in a ratio of 8:1:1 and placed in a Cunningham chamber to a thickness of 50 μΩ. Next, the tube was sealed with white petrolatum and reacted for 90 minutes in a 37° C. incubator. After the reaction, the plaque R (number of antibody-producing cells/number of lung cells per mouse) was examined using a microscope and found to be 401.
/106±54/10'. Figure 3 shows the results of measuring the number of plaques on mouse lung cells.

While feeding the powdered feed used in Example 4 ad libitum, the immunostimulant prepared in Example 1 was administered at 25 μ/kg or 50 μ/kg.
The number of plaques on mouse lung cells was measured in the same manner as in Example 4, except that 25 mg/kg was orally administered.
g when administered orally, 401/10'±19/10'', and when administered orally at 50■/kg, 523/10'
It was ±10/10'. Figure 3 shows the results of measuring the number of plaques on mouse lung cells.

The number of plaques on mouse lung cells was measured in the same manner as in Example 4, except that the immunostimulant prepared in Example 1 was not used, and it was found to be 246/10'±31/10''.

Figure 3 shows the results of measuring the number of plaques on mouse tile m cells.

The number of plaques on mouse lung cells was measured in the same manner as in Example 5 or 6, except that the immunostimulant prepared in Example 1 was not used, and the number was 222/10'±8/10'. Ta. Figure 3 shows the results of measuring the number of plaques on mouse lung cells.

The results of Examples 4 to 6 and Comparative Examples 2 and 3 revealed that administration of the immunostimulant of the present invention increased the ability of mice to produce antibodies in lung cells.

[Brief explanation of the drawing]

Figure 1 is a graph showing the measurement results of the lymphocyte rejuvenation response of rabbits to ConA stimulation, Figure 2a is a graph showing the measurement results of the lymphocyte rejuvenation response of healthy subjects to PHA stimulation, and Figure 2b is the graph showing the measurement results of the lymphocyte rejuvenation response of a rabbit to ConA stimulation. FIG. 3 is a graph showing the measurement results of the lymphocyte rejuvenation response of healthy individuals to stimulation, and FIG. 3 is a graph showing the measurement results of the number of plaques on mouse lung cells.

Claims (1)

[Claims] Molecular weight 100 obtained by decomposing proteins with proteolytic enzymes
An immunostimulant characterized by containing a proteolytic extract containing 0 to 15,000 peptides as an active ingredient.