JPH06199686A - Immunostimulating agent suitable for oral administration - Google Patents

Abstract

PURPOSE:To obtain an immunostimulating agent suitable for oral administration by culturing a microbial strain belonging to the genus Aspergillus and capable of producing a mitogen-active substance in a medium containing a milk component, removing the microbial cells from the cultured product and using the obtained supernatant as an active component or fractionating the supernatant and using the obtained active fraction as an active component. CONSTITUTION:An immunostimulating agent composed mainly of a peptide obtained from a microbial strain belonging to the genus Aspergillus is mixed with a milk component to obtain an immunostimulating agent developing improved effect by oral administration. Preferably, a powdery immunostimulating agent is mixed with milk powder such as whole milk powder and skim milk. It has been difficult to develop the effect of an immunostimulating agent composed mainly of a peptide obtained from a microbial strain belonging to the genus Aspergillus by oral administration, however, the effect can be developed even by oral administration by the use of the subject immunostimulating agent.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention is effective on a culture supernatant obtained by culturing a microorganism having the ability to produce mitogenic active substances belonging to the genus Aspergillus, or an active fraction obtained by fractionating the culture supernatant. The present invention relates to an immunostimulant suitable for oral administration, which is a mixture of an immunostimulant as a component and milk.

[0002]

2. Description of the Related Art In recent years, there has been increasing interest in foods from the viewpoint of their biological or functional functions in addition to their nutritional or taste functions, such as cholesterol elevation suppressing action, blood pressure lowering action and immunostimulating action. The development of food ingredients with excellent latent functions in various fields has been started. And, naturally, it is required for these food materials having a biological function controlling action to exhibit the activity even when orally administered. Further, in the field of medicine as well, in consideration of the possibility of use for home treatment and patient's pain, the present situation is that there are great expectations for the manifestation of effects by oral administration. However, a cultured cell system or a component that has been found to exhibit physiological activity by intraperitoneal injection does not necessarily exhibit physiological activity even after oral administration. In particular, when a peptide or protein drug is orally administered, there is a problem that the action and effect of the active substance is reduced due to the action of proteolytic enzyme in the digestive tract. Further, since peptides and proteins have relatively large molecular weights, there is a problem that they are poorly absorbed from the intestinal tract.

Various attempts have heretofore been made in order to express various activities possessed by peptide and protein substances [Verhoef et al. , Eur. J. Dru
g Metab. Pharmacokinet. , First
5:83, 1990]. For example, in order to reduce the effects of substances on digestion, protease
A method of administering with an inhibitor, encapsulation, and the like have been proposed, and a method of administering with a surfactant, a chelating agent, a fatty acid, or the like in order to enhance the absorbability of a substance, liposome formation, and the like have been proposed. Moreover,
There is also a report that the activity of an antihypertensive peptide derived from sardines was orally expressed using an egg yolk solution (Suetsuna et al., Journal of Japan Society of Nutrition and Food Science, Vol. 42, p. 47, 1989).
It is believed that this phenomenon is due to the increased absorption of phospholipids in the intestinal tract.

However, using these methods to enhance the activity of peptide- or protein-based substances is effective in high value-added products such as pharmaceuticals, but these physiologically active substances are used in the food industry. When it is produced and used in a relatively large amount, it is often impractical. For example, an encapsulating agent requires a special device for encapsulation, has a relatively limited dose during administration, and has a texture that is an important factor when ingested orally. It is difficult to use as a food material because it significantly reduces In addition, when a physiologically active substance is continuously administered together with a protease inhibitor or a surfactant, nutritional and physiological effects such as the effect of the protease inhibitor or surfactant on the digestive function and the effect on intestinal mucosal cells are observed. It is necessary to consider it from the viewpoint. Although the use of egg yolk to enhance the absorption of physiologically active substances can be sufficiently utilized in the food industry, the amount of lipid contained in egg yolk is high, so that it is recommended for those who are restricted in calorie intake. There is also the problem that it cannot be applied.

[0005] Therefore, it is desired to put into practical use a formulation using a relatively inexpensive material which does not have a fear of affecting the digestive tract and digestive function even when ingested in a relatively large amount and which can be used in the food industry.

On the other hand, for the purpose of application as a food material for imparting immunostimulatory ability, the filamentous fungus Aspergillus sp. The mitogen activity in a system using cultured cells and the antitumor activity by intraperitoneal injection in mice have been revealed for the fraction fraction of the culture supernatant obtained by culturing the EF8 strain in skim milk medium [Fujiwara et al. a
l. J. Agric. Food Chem. , 40th
Vol. 1280, 1992]. However, Aspergillus sp. The fraction having immunostimulatory activity produced in the skim milk culture of the EF8 strain is considered to be a peptide having a molecular weight of about 3,000, but when the fraction is orally administered, a good effect is not always obtained. There was a problem that I could not get it.

[0007]

The present invention has been made in view of the above situation, has a good immunostimulatory activity, can be used in the food industry, and can be used for nutrition. It is an object of the present invention to provide an immunostimulant which is excellent in physical or texture and suitable for oral administration.

[0008]

Means for Solving the Problems The present inventors have previously cultivated a filamentous fungus derived from Aspergillus oryzae belonging to the genus Aspergillus in a medium containing a milk component and removed the cells to obtain a culture supernatant or An immunostimulant containing the fraction as an active ingredient has been proposed [JP-A-3-11021]. Then, as a result of studying the expression of the effect of the oral administration of this immunostimulant, it was found that the expression of the immunostimulatory activity is enhanced by mixing and administering it with milk, and the present invention has been completed.

The immunostimulant which is the active substance of the present invention is obtained by culturing a microorganism having the ability to produce a mitogen active substance belonging to the genus Aspergillus, which is a filamentous fungus derived from koji, in a medium containing milk components, A culture supernatant obtained by removal or a fraction obtained by fractionating the culture supernatant [JP-A-3-
11021].

The mycological properties of strains belonging to the genus Aspergillus that have the ability to produce mitogenic active substances are shown below. (1) Growth In the malt extract agar medium, the growth at 25 ° C is fast, and the diameter of the colony reaches 3 to 5 cm within 5 days. The basal hyphal layer is white, trichome-like, dense, and forms a long conidia stalk. The surface of the colony is green-yellow, the back surface is white, and the back surface is smooth and without folds. The Tuabeck agar medium shows almost the same growth properties as the malt extract agar medium, but the growth at 25 ° C is slower than that of the malt extract agar medium, and the formation of conidia is delayed.

(2) Morphology The conidia peduncle is pale brown and transparent, and the tip forms a subspherical apex, and a phialide is further formed from the tip, but a metre is not formed. Conidia handle length is 400
At ~ 800μ, the surface is rough. Conidia are divided into several or more than ten lines from the tip of the phialide and are arranged radially to form conidia heads. The conidia head has a pale blue-green color and the size is 100 to 200 μ. Conidia are 5-6 in diameter
It exhibits a spherical shape of μ and the surface is rough.

Based on the above-mentioned mycological properties, Shunichi Udagawa,
When classified by referring to "Fungus Pictorial Book" by Keisuke Tsubaki (Kodansha, 1982), this strain was judged to belong to the genus Aspergillus. In addition, this strain is Aspergillus ( Aspergillus ) sp. Micromachine Research Deposited No. 1065 to Institute of Microbial Technology as EF8 strain
It has been deposited under the number 9 (FERM P-10659).

Next, a microorganism having the ability to produce a mitogen active substance belonging to the genus Aspergillus, which is the active substance of the immunostimulant used in the present invention, is obtained by culturing in a medium containing milk components and removing the cells. The method for preparing the obtained culture supernatant or the active fraction obtained by fractionating the culture supernatant will be described.

A medium containing milk components, for example, non-fat milk solid 8
%, Yeast extract 0.3%, glucose 1%, pH
The liquid medium adjusted to 7.0 was inoculated with a microorganism capable of producing a mitogen active substance belonging to the genus Aspergillus, shake-cultured at 27 ° C. for about 72 hours, and the bacterial cells were separated from the culture solution by filtration. Get Qing. This culture supernatant can be used as the active ingredient of the immunostimulant, and the pH of the culture supernatant thus obtained is 4.6.
Alternatively, the coexisting protein is removed by isoelectric point precipitation, and then the culture supernatant is fractionated by a treatment such as gel filtration chromatography or adsorption chromatography to obtain an active fraction. The active fraction obtained by fractionating this culture supernatant had a molecular weight of about 3 by urea SDS-polyacrylamide gel electrophoresis using 12% concentration gel or by gel filtration using Sephadex G-50. , 0
It is estimated to be 00 and is soluble in water and 50% methanol, but is insoluble in ether, chloroform and acetone, and is colored by silver nitrate staining, Coomassie brilliant blue staining and ninhydrin staining, but not by PAS staining. It has the property of The active fraction obtained by fractionating this culture supernatant exhibits physiological activities such as mitogenic activity and antitumor activity by intraperitoneal injection in mice.

In the present invention, this immunostimulant is mixed with a milk component to give an immunostimulant suitable for oral administration. In this case, the liquid culture supernatant or the active fraction obtained by fractionating the culture supernatant may be appropriately concentrated and mixed with milk, but is preferably the culture supernatant or the culture supernatant thereof. The lyophilized powder and powdered milk are mixed with the fraction thus fractionated to give an immunostimulant suitable for oral administration. This powdery immunostimulant also exhibits physiological activities such as mitogenic activity and antitumor activity. Examples of the powdered milk mixed with the powdery immunostimulant include powdered milk protein such as casein and whey protein, whole milk powder, skim milk powder, and the like. Next, the present invention will be specifically described with reference to examples.

[0016]

Example 1 100 ml of liquid medium (1% glucose, 0.5% yeast extract, 0.3% peptone) was dispensed into a 300 ml Erlenmeyer flask, and a small amount of conidia was inoculated.
After shaking culture at 28 ° C. for 3 days, Aspergillus sp.
A pellet-shaped preculture (3-5 mm in diameter) of the EF8 strain (Deposit No. 10659, Mikken Institute) was obtained. 150 ml of this pellet-shaped preculture was dispensed into a 500 ml Erlenmeyer flask.
Of the reduced skim milk medium (containing 10% skim milk powder, 1% glucose and 0.3% yeast extract) at 28 ° C.
The culture was carried out with shaking at 150 rpm for 3 days. After culturing,
36% hydrochloric acid was added to the culture solution from which the bacterial cells had been removed with a cloth to adjust the pH to 4.6, and the precipitate generated by centrifugation (7,500 rpm) was removed to obtain a culture supernatant. This culture supernatant 400
ml was passed through a C18-octadedecylsilane reverse phase column (about 70 ml), and 200 ml of water and 50 ml were passed.
After washing the column with% methanol, 250 ml of 9
The active fraction was eluted with 9.8% methanol. Then, the eluate was subjected to methanol removal and concentration with an evaporator, and then freeze-dried to obtain 320 mg of a powdered immunostimulant. The powdered immunostimulant contained 89.9% peptide and 1.8% sugar.

[0017]

[Example 2] Powdered immunostimulant obtained in Example 1 and whole milk powder (sugar 39.3%, protein 25.5%, fat 2)
6.2%, ash content 6.0%, and water content 3.0%) were mixed in equal amounts to prepare an immunostimulant suitable for oral administration.

[0018]

Example 3 Powdered immunostimulant obtained in Example 1 and skim milk powder (53.3% sugar, 34.0% protein, fat 1.
0%, ash 7.9%, water 3.8%) were mixed in equal amounts to prepare an immunostimulant suitable for oral administration.

[0019]

Comparative Example 1 Powdered immunostimulant obtained in Example 1 and milk-rich phospholipid-rich powder (sugar 18.6%, protein 15.7%, fat 61.4%, phospholipid 13. 1%, ash content 3.8%, water content 0.5%) were mixed in equal amounts to prepare an immunostimulant.

[0020]

[Test Example 1] 10 mg of the immunostimulant prepared in Example 1 was dissolved in 0.1 ml of water and orally administered to a 6-8 week-old ICR female mouse (Japan Charles River KK) with a catheter for 7 days. did. The day after the oral administration for 7 days was completed, the tumor cell Sarcoma 180 was added to the cells at a cell count of 1 × 10 ^{4 to} 3 ×.
10 ⁴ cells were intraperitoneally transplanted to the mouse, and the same amount of the immunostimulant was orally administered every other day from the day after the transplantation of the tumor cells, 5 times in total, and the survival rate of the mouse was observed. As a control, the survival rate of the mice was also observed in the same group in which only water was administered. The result is shown in Figure 1.
Shown in. In the group to which the immunostimulant was administered, the survival rate was not improved as compared with the control group to which only water was administered, and the effect as the immunostimulant was not recognized.

[0021]

[Test Example 2] Regarding the immunostimulant prepared in Example 2,
The survival rate of the mice was observed in the same manner as in Test Example 1. As a control, the survival rate of the mice was also observed in the group to which only the whole milk powder was administered. The result is shown in Figure 2.
Shown in. In the control group to which only whole milk powder was administered, some mice died on the 10th day after tumor implantation, and all the mice died on the 21st day. On the other hand, in the group to which the immunostimulant was administered, the frequency of death of mice was low,
Even when all the mice in the control group died, the survival rate was 70%.

[0022]

[Test Example 3] Regarding the immunostimulant prepared in Example 3,
The survival rate of the mice was observed in the same manner as in Test Example 1. As a control, the survival rate of the mice was also observed in the group to which only skim milk powder was administered by the same method. The result is shown in Figure 3.
Shown in. In the control group to which only skim milk powder was administered, some mice died on the 12th day after tumor implantation, and all the mice died on the 23rd day. On the other hand, in the group to which the immunostimulant was administered, the frequency of death of mice was low,
Even when all the mice in the control group died, the survival rate was 40%.

[0023]

[Test Example 4] Regarding the immunostimulant prepared in Comparative Example 1,
The survival rate of the mice was observed in the same manner as in Test Example 1. As a control, the survival rate of the mice was also observed in the group to which only the phospholipid-rich powder was administered. The result is shown in FIG. In the group to which the immunostimulant was administered, the survival rate was not improved as compared with the control group to which only the phospholipid-rich powder was administered, and the effect as the immunostimulant was not recognized.

[0024]

[Effects of the Invention] When administered orally, by coexisting milk protein such as casein or whey protein, or milk powder such as whole milk powder or skim milk powder, with an immunostimulant, which is difficult to exert its effect by the active substance alone, It is possible to provide an immunostimulant suitable for oral administration that exhibits effects even when administered.

[Brief description of drawings]

FIG. 1 shows the number of surviving mice in Test Example 1.

FIG. 2 shows the number of surviving mice in Test Example 2.

FIG. 3 shows the number of surviving mice in Test Example 3.

FIG. 4 shows the number of surviving mice in Test Example 4.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 21/00 A 8214-4B (C12P 21/00 C12R 1:66)

Claims (4)

[Claims] 1. A culture supernatant obtained by culturing a microorganism belonging to the genus Aspergillus and capable of producing a mitogen active substance in a medium containing a milk component, and then removing the cells or fractionating the culture supernatant. An immunostimulant suitable for oral administration, which comprises a mixture of an immunostimulant containing the active fraction obtained as an active ingredient and a milk component. 2. The oral administration according to claim 1, wherein the active fraction obtained by fractionating the culture supernatant is a fraction obtained by adsorption chromatography after removing the protein from the culture supernatant. Immunostimulant. 3. A microorganism belonging to the genus Aspergillus and capable of producing a mitogenic active substance is Aspergillus sp. The immunostimulant suitable for oral administration according to claim 1 or 2, which is strain EF8 (Deposit No. 10659, Micro Engineering Lab.). 4. The immunostimulant suitable for oral administration according to claim 1, wherein the powdered immunostimulant and milk powder are mixed.