JPH0573731B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to an immunostimulant that activates immune function.

<Prior art> Animals generally have a wide variety of defense functions, or immune functions, that try to eliminate foreign substances such as foreign microorganisms and foreign self-substances. It is believed that it can fight against various 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 fish and shellfish 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, an immune system containing as an active ingredient a proteolytic extract containing peptides with a molecular weight of 3,500 to 15,000 obtained by decomposing fish and shellfish with a proteolytic enzyme. An activator 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 in the range of 3,500 to 15,000. If the molecular weight is less than 3,500 or more than 15,000, a sufficient immunostimulatory effect cannot be obtained.

The proteolytic extract containing the peptide used in the present invention is a fish extract obtained by decomposing fish with a proteolytic enzyme. The fish extract can be produced, for example, as follows. That is,
Raw material fish, specifically, horse mackerel, mackerel, sardines, saury, bonito, sea bream, cod, etc., are preferably put into a reactor in their whole form without any pretreatment such as cutting into pieces and making into a slurry. Preferably, the temperature is raised to 80°C or higher to completely inactivate the autolytic enzymes contained in the fish, and at the same time remove the fish-specific odors such as dullness and foul odor generated by the action of the autolytic enzymes, and then , 40℃~70℃, PH
At a pH of 5.0 to 7.0, preferably 5.5 to 6.5, Bacillus subtilis-produced protease is added to decompose proteins contained in fish to proteose grade. Next, the temperature is raised to at least 65°C or higher, preferably 75°C or higher to inactivate the Bacillus subtilis-produced protease, usually for 3 minutes to 1.5 hours, preferably 10 minutes to 1 hour. 30-60℃, PH5 without adjusting the PH again
In steps 7 to 7, a protease produced by Aspergillus oryzae is added to decompose the mixture into peptides having a molecular weight of 3,500 to 15,000. Decomposition time is 10 minutes to 3 hours, preferably
Do this for about 30 minutes to 2 hours. If the decomposition time is less than 30 minutes, proteose remains and a peptide having a specific amino acid composition cannot be obtained, whereas if it exceeds 3 hours, the immunostimulatory effect will be reduced, which is not preferable.

The decomposed liquid thus obtained can be separated into undecomposed substances such as fish extract layer, oil layer and bone fragments using a centrifuge or the like in a conventional manner, and then subjected to ultrafiltration or
It can be produced by using a method such as vacuum concentration at 60°C or lower.

The components of the fish extract include various peptide amino acid groups and free amino acids such as glutamic acid, aspartic acid, lysine, arginine, glycine, alanine, leucine, proline, histidine, phenylalanine, and serine, and have a substantially low molecular weight.
The main component is 3500-15000 peptides.

The above-mentioned method for producing fish as a proteolytic extract is one example, and the present invention 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 made entirely of natural products, so it is non-toxic and extremely safe.

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 mg/Kg or more, and particularly preferably 25 mg/Kg or more. In addition, in the case of intravenous injection, the amount is preferably 5 mg/Kg or more, especially 15 mg/Kg or more.
mg/Kg or more is desirable. 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 the immunostimulant is orally administered, it can be added to food or feed and ingested, 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 fish 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.

Example 1 5 tons of mackerel were placed in a reaction vessel, and the temperature was raised to about 90°C to completely inactivate the autolytic enzymes 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 was raised to 75°C to inactivate the protease produced by Bacillus subtilis over a period of 30 minutes, and the protease produced by Aspergillus oryzae was added at 50°C and pH 6 to decompose 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 found to be 7,000.

Example 2 Adult male rabbits weighing approximately 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.

Lymphocyte blastogenesis measurement method Lymphocytes were separated from the collected peripheral blood by specific gravity centrifugation, washed, and suspended in RPMI-1640 supplemented with 10% FCS. ConA stimulation is for 0.5ml of cell suspension.
It was prepared by adding 0.5 ml of RPMI-1640 containing ConA and 10% FCS. As a control, a system without the addition of ConA was prepared, and a 24-well plastic plate was prepared for 72 hours.
The cells were cultured for hours (37° C., 5% CO ₂ , 95% air). Similarly, for PHA stimulation, PHA was added to 0.5ml of cell suspension.
0.5 ml of FCS-added RPMI-1640 was added and cultured. The cultured lymphocyte suspension was collected in a test tube, the lymphocytes were washed, and the supernatant was removed. To the remaining cell pellet, add 2 ml of 0.1% Triton (manufactured by).

Comparative Example 1 Lymphocyte blastogenesis measurement was carried out in the same manner as in Example 2, except that the immunostimulant prepared in Example 1 was not used. Figure 1 shows the measurement results of the lymphocyte blastogenesis reaction.

As a result of Example 2 and Comparative Example 1, as shown in Figure 1, when no immunostimulant is administered, the experiment start date and
There was no correlation between the rejuvenation response of rabbit ear vein peripheral blood lymphocytes to ConA after 30 days. However, when an immunostimulant was administered, the rejuvenation response to ConA was 3.35 ± 3.35 (M ± SD) before administration, but 9.26 ± 2.99 (M ± SD) after 30 days of administration.
±SD). Therefore, it was found that administration of the immunostimulant of the present invention clearly increases the blastogenesis response of lymphocytes.

Example 3 The immunostimulant prepared in Example 1 was administered to 11 healthy people (age 30 ± 3.3, weight 61 ± 5.3 kg, male) three times in the morning and evening.
A total of 6 g/day was administered orally for 30 days. Alcohol, tobacco, and drug intake were prohibited during treatment, but there were no restrictions on diet. Next, about 24 hours after the final administration of the immunostimulant, blood was collected on an empty stomach before breakfast, and PHA and ConA were collected in the same manner as in Example 2.
The peripheral blood lymphocyte blastogenesis response to stimulation was measured. Figures 2a and 2b show the measurement results of peripheral blood lymphocyte rejuvenation responses to PHA and ConA stimulation, respectively.

As shown in Figures 2a and 2b, as a result of Example 3, by administering the immunostimulant of the present invention,
It was found that the peripheral blood lymphocyte blastogenesis response to ConA and PHA was increased. In particular, the ConA-stimulated lymphocyte blastogenesis response was 1.769±0.304 (M±SD) before administration.
- Increased to 2.342±0.614 (M±SD) after administration.

Example 4 To examine the antibody production ability of mouse splenocytes, male
C57BL/6 mice were kept in a constant environment for 6 days after delivery. 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 the fifth day of administration, 0.2 ml of sheep red blood cells (SRBC) at 5×10 ⁸ /ml was injected into the tail vein. Five days after the end of administration, the spleen was removed from the mouse and made into a single-cell suspension, washed three times with E-MEM (1500 rpm 5 min), and mixed with the suspension adjusted to 1.25 x 10/ml, 50% SRBC, and complement [guinea pig serum]. (Guin-er Pig Serum) dry powder
A solution dissolved in MEM] was mixed in a ratio of 8:1:1, and 50μ of the mixture was placed in a Cunningham chamber. After sealing with white vaseline, the mixture was reacted for 90 minutes in an incubator at 37°C. After the reaction, the number of plaques (number of antibody-producing cells/number of spleen cells per mouse) was examined using a microscope and found to be 401/10 ⁶ ±54/10 ⁶
It was hot. FIG. 3 shows the results of measuring the number of plaques on mouse spleen cells.

Examples 5 and 6 Mice were treated in the same manner as in Example 4, except that 25 mg/Kg or 50 mg/Kg of the immunostimulant prepared in Example 1 was orally administered while feeding the powdered feed used in Example 4 ad libitum. When the number of plaques on spleen cells was measured, when 25 mg/Kg was orally administered, it was 401/10 ⁶ ±
19/10 ⁶ , and when administered orally at 50 mg/Kg,
It was 523/10 ⁶ ±10/10 ⁶ . FIG. 3 shows the results of measuring the number of plaques on mouse spleen cells.

Comparative Example 2 The number of plaques on mouse spleen cells was measured in the same manner as in Example 4, except that the immunostimulant prepared in Example 1 was not used, and the result was 246/10 ⁶ ±31/10 ⁶
It was hot. FIG. 3 shows the results of measuring the number of plaques on mouse spleen cells.

Comparative Example 3 The number of plaques on mouse spleen 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 result was 222/10 ⁶ ±
8/10 It was ⁶ . FIG. 3 shows the results of measuring the number of plaques on mouse spleen 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 antibody production ability of splenocytes in mice.

[Brief explanation of the drawing]

Figure 1 is a graph showing the measurement results of rabbit lymphocyte blastogenesis response to ConA stimulation, Figure 2a is
Figure 2b is a graph showing the measurement results of the lymphocyte rejuvenation response of healthy individuals to PHA stimulation. It is a graph showing the measurement results of the number of plaques.

Claims (1)

[Claims] 1. An immunostimulant characterized by containing as an active ingredient a proteolytic extract containing peptides with a molecular weight of 3,500 to 15,000 obtained by decomposing fish with a proteolytic enzyme.