JPS62135433A - Immunoactivator comprising low molecular peptide as active ingredient and preparation thereof - Google Patents

Abstract

PURPOSE:A low molecular peptide useful as an immunoactivator, having molecular weight in a specific range, obtained by enzymatically decomposing a solution prepared by dispersing milk protein into water with pepsin alone or pepsin and papain. CONSTITUTION:Milk protein is dispersed into water to give a solution. Pepsin alone or pepsin and papain are simultaneously or successively added to the solution and enzymatic decomposition is carried out at 25-60 deg.C to give low molecular peptide having 4-10 chain length and 500-3,000 average molecular weight. The peptide is recognized to have improvement in biophylaxis function from the fact that administration group has lower incidence of infectious diseases after operation than reference group and immunoactivating action of the peptide is recognized from the fact that rise in complement titer is observed in the administration group by significant difference. The peptide is directly applicable as an immunoactivator as it is and can be pharmaceutically manufactured into various forms (drink agent, tablet or capsule).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunostimulant whose active ingredient is a specific low-molecular-weight peptide obtained by enzymatically decomposing milk protein, and a DI manufacturing method thereof.

Immune function plays a major role in the prognosis of diseases such as post-surgical infections and infections in the elderly and hospitalized patients, as well as in the treatment of cancer.

Conventionally, polysaccharide preparations such as Krestin (PSK), lentinan, and glucan, and preparations using bacteria or bacterial components such as BCG and picibanil have been known for immunostimulation.

Undernutrition, immunity and other resistance weaken, increasing an individual's susceptibility to infection. In the process, complications occur frequently and symptoms worsen, leading to increased mortality. On the other hand, it is known that as infection, cancer, etc. progress, nutritional requirements increase, promoting nutritional deficiency and further worsening the symptoms, creating a vicious cycle.

By the way, it is well known that many of the above-mentioned immunostimulants have little effect under the aforementioned malnutrition conditions.

Under the above-mentioned circumstances, there is a strong desire to provide an immunostimulant that can be used as a nutritional source in addition to its immunostimulatory effect.

The present invention has been made to meet the above-mentioned needs, and provides an immunostimulant that has an excellent immunostimulatory effect and is composed of ingredients that can also be used as a nutritional source in itself. The object of the present invention is to provide a method for producing the same at low cost.

That is, an object of the present invention is to provide an immunostimulant that is particularly suitable for use in the prevention or treatment of post-surgical infections, mainly in the digestive system, where oral intake of food is difficult, and for patients with digestive cancer. do.

The present invention will be explained in detail below.

The immunostimulant according to the present invention is characterized by having an average molecular weight of N500 to 3000, consisting of peptides with a chain length of 4 to 10, obtained by enzymatically decomposing milk protein with pepsin alone or in combination with pepsin and papain. The purpose of this invention is to use a low-molecular-weight peptide having the following as an active ingredient. Further, the feature of the preparation method of the above-mentioned immunostimulant of the present invention is that pepsin alone or pepsin and papain are added simultaneously or sequentially to a solution in which milk protein is dispersed in water, and the mixture is heated at 25°C to 60°C. Enzymatic decomposition at high temperature results in a chain length of 4 to 10, with an average molecular weight of 500 to 3.
The aim is to produce 1,000 low-molecular-weight peptides and formulate formulations using the obtained low-molecular-weight peptides as active ingredients.

4. One way to solve the horoscope The low molecular weight peptide which is the active ingredient of the immunostimulant according to the present invention is prepared by the following procedure.

Milk protein, such as whey protein concentrate, whey powder, lactalbumin, casein, skim milk powder, whole milk powder, etc., is preferably dispersed in water to a concentration of 2 to 20 W/V%. After adjusting to 1 to 4 with hydrochloric acid,
Pepsin to substrate milk protein 0.05-10-/-
%, preferably 0.5 to 5-/-% at 25°C to 6
Enzymatically decompose at a temperature of 0°C for 3 to 24 hours, or add 1 and papain whose pH is adjusted to around 5.3 to the obtained enzymatically decomposed solution, or enzymatically decompose the above dispersion. to 5.1 to 5.5, preferably 5.
．． After adjusting the papain to 0.3 to the substrate milk protein.
05 to 10 W/-%, preferably 0.5 to 5-%, and enzymatically decomposed at a temperature of 25 to 60°C for 3 to 24 hours,
Next, the pi of the obtained enzymatically decomposed solution is readjusted to around 3.0, and then pepsin is added to the enzymatically decomposed solution for further enzymatic decomposition.

Each of the above enzymatic degradation conditions is a condition that allows each enzymatic activity to occur, and the enzymatic degradation produces a peptide mixture having a chain length of 4 to 10 and an average molecular weight of 500 to 3,000.

After heating the enzymatically decomposed product obtained by the above-mentioned enzymatic decomposition to 100°C to inactivate the enzyme, neutralize as necessary, and then separate and remove unmeltable substances, and salt the resulting clear liquid. , concentrate and lyophilize to obtain the above-mentioned peptide in powder form.

Although the peptide thus obtained can be used as an immunostimulant as it is, it is of course also possible to formulate it into various forms such as drinks, tablets, and capsules according to conventional methods.

Furthermore, the peptide can be blended into various nutritional supplements and health foods and given to patients after surgery. For example, it can be used in addition to enteral nutrition, bread, kutsky, etc.

Next, the immunostimulatory effect of the peptide prepared by the method of the present invention will be explained.

The following test was conducted to investigate the above-mentioned immunostimulatory effect.

Test method: (1) Preparation of test samples Using each of the peptides obtained in Examples 1 and 2 described later as an active ingredient, a diet having the composition shown in Table 1 below was prepared and subjected to the test. As a comparative example, a sample using an amino acid mixture as an active ingredient instead of the above peptide was prepared and subjected to the same test.

The chain length is 4.7, and the average molecular weight is 1 and 200), respectively.

Furthermore, the amino acid compositions of each of the above peptides and amino acid mixtures are as shown in Table 2.

Table 2 (T%) ■Immunostimulation test specimen ffi Each group of 15 SD male rats weighing 120 to 150 g was acclimatized, then fasted for 18 hours, and then subjected to total gastrectomy. After 18:00 hours after surgery, the necropsy results after 4 weeks of feeding ad libitum to each diet with the composition shown in Table 1 showed the incidence of macroscopically observed infections such as pneumonia and some suppurative foci. Serum 1gG and C were measured for individuals with no abnormalities. The results are shown in Tables 3 and 4.

Table 3 As shown in Table 3, the incidence of postoperative infection was lower in the group fed the diet containing the peptide according to the present invention than in the comparative example, indicating an improvement in the biological defense function. In addition, the above results showed a statistically significant difference at a risk rate of 1% or less.

Table 4 As shown in Table 4, there is no significant difference in IgG, but according to the present invention, Cyo increases to nearly twice that of the comparative example, and this value also has a probability of 1% based on the estimation.
Significant differences were observed in the following.

From the above test results, it can be said that the effect of the immunostimulatory action at the biological level using the low molecular weight peptide according to the present invention as an active ingredient is recognized.

Incidentally, originally, complement (comple+++ent) is 1
When foreign substances such as microorganisms invade the body, they are thought to be the main non-specific humoral factors that react first, but include cell membrane destruction, negative action, and immune adhesion to blood vessels. Urakami plays an important role in biological defense mechanisms such as changes in permeability and desalting effects.

In particular, among these actions, C3, which plays an important role in the activation of the classical pathway and the secondary pathway, amplifies each of the above-mentioned immune phenomena during the activation process, and conversely, There is an increased susceptibility to pyogenic bacterial infections and autoimmune diseases seen in patients with deficiencies in inflammatory and other complement system components;
Furthermore, its role as a complement for defense after bacterial infection is extremely important.

Therefore, the complement value (C3
) can be said to be extremely significant.

Furthermore, an increase in complement levels is known to promote the activation of macrophages, and it has also been reported that they recover before other immune systems, making it an appropriate indicator of immunostimulatory effects. It is something that becomes. And this means that infection prevention at the biological level shown in Table 3 above is W! ! This is in good agreement with the functional enhancement results, and it can be confirmed that the immunostimulant according to the present invention has an immunostimulatory effect on complement and other substances, and enhances the biological defense function.

Hereinafter, the method for preparing the low molecular weight peptide which is the active ingredient of the immunostimulant of the present invention will be specifically explained with reference to Examples.

Example 1 Whey protein Biopro (lactose-free whey protein manufactured by Bioisolate) log was dispersed in water to a concentration of 10 W/V%, heat sterilized at 90°C for 15 minutes, and the dispersion After adjusting the PL of the solution to 3.0 with hydrochloric acid, 0.3 g of pepsin (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was added and reacted at 55°C for 8 hours. After the reaction, the enzymatic decomposition product obtained was After heating at ℃ for 10 minutes to inactivate the enzyme, neutralize with sodium hydroxide, remove insoluble matter by centrifugation and membrane separation, and further desalt and concentrate the resulting clear liquid. , lyophilized.

The yield of the obtained dry product was 7.2 g based on the raw material protein, the average molecular weight was 2.000, and the average peptide chain length was 6.7.

The average molecular weight is Sephadex G-10 (1,5
Gel filtration chromatography using a column (150 cm x 150 cm) (eluent: 1% acetic acid).

Moreover, the measurement of average peptide chain length (APL) was calculated as follows.

Number of moles of amino groups Example 2 Whey protein, BioPro (Bio Isolate Co., Ltd.)
Disperse 10 g in water to a concentration of 15 W/V%, heat sterilize at 90°C for 15 minutes, adjust the pll of this dispersion to 5.3, and add papain (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) to 0.3.
g was added and reacted at 55°C for 8 hours, then the pH of the reaction product was readjusted to 3.0, and 0.3g of pepsin (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was added and reacted at 55°C for 8 hours. Ta. After the reaction, the reaction product was heated at 90°C for 10 minutes to inactivate the enzyme, then neutralized with sodium hydroxide, and insoluble matter was removed by centrifugation and membrane fractionation, and the resulting clear liquid was It was further desalted, concentrated and freeze-dried.

The yield of the obtained dry product was 8.4 g, with an average molecular weight of 1i1200 and an average peptide chain length of 4.7.

Claims (4)

[Claims] (1) Consisting of peptides with chain lengths of 4 to 10, obtained by enzymatically decomposing milk proteins with pepsin alone or in combination with pepsin and papain, with an average molecular weight of 500 to 3,000.
An immunostimulant containing a low-molecular-weight peptide having the following as an active ingredient. (2) Add pepsin alone or pepsin and papain simultaneously or sequentially to a solution of milk protein dispersed in water, and perform enzymatic decomposition at a temperature of 25°C to 60°C to reduce the chain length from 4 to 60°C. 10, average molecular weight 500 to 3000
1. A method for preparing an immunostimulant, which comprises producing a low-molecular-weight peptide and formulating the obtained low-molecular-weight peptide as an active ingredient. (3) The preparation method according to claim (2), wherein milk protein is dispersed in water to a concentration of 2 to 20 W/V%. (4) The preparation method according to claim (2), wherein enzymatic decomposition is carried out for 4 to 24 hours.