JPH08280332A - Additive for feed - Google Patents

Abstract

PURPOSE: To obtain the subject additive containing yeast cells treated with a yeast cell with lysing enzyme, capable of manifesting immunocompetence enhancing effects by orally administering a minute amount to human, live stocks and fishes. CONSTITUTION: This additive for feed contains yeast cells such as Saccharomyces, Endomycopsis, Saccharomycodes, Nematospora, Candida, Torulopsis, Brethanomyces, Rodotorula, etc., treated with a yeast cell wall lysing enzyme.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to feed additives and feeds. More specifically, the present invention relates to a feed additive containing yeast cells treated with a yeast cell wall lysing enzyme and a feed containing the same.

[0002]

2. Description of the Related Art Generally, in the livestock industry and the aquaculture industry, in order to reduce the production cost, they are bred at a high density, and pathogens such as bacteria and viruses are more likely to spread.
In the past, in order to prevent the spread of these pathogens, antibiotics and other fungicides have been mixed in the feed, but due to the heavy use of these antibiotics, opportunistic infections or the emergence of resistant bacteria due to the bacterial replacement phenomenon have occurred. , There are problems such as drug residues in livestock products and fish meat. As a method for solving the above-mentioned problems, a so-called immunopotentiating substance, which is a substance that stimulates and activates the immune system of a living body and enhances immunity without using antibiotics, is added to feed. ing. Peptidoglycan (Japanese Patent Publication No. 6-250
No. 67), and β-glucan, which is a cell wall constituent of bacteria, yeasts, mushrooms, and the like are known. The yeast cells contain a high concentration of highly nutritious proteins, vitamins, minerals, etc., and their cell walls contain glucan, mannan, etc. However, humans, livestock, fish and the like do not have an enzyme that digests the yeast cell wall, and the yeast cell wall is extremely strong, resulting in poor digestibility and low utilization rate of cell components. Various methods for destroying the yeast cell wall have been investigated in order to increase the utilization rate of the yeast cells themselves. For example, Japanese Patent Application Laid-Open No. 6-256199 discloses a feed immunopotentiating agent obtained by finely pulverizing yeast cells and a method for producing the same. Japanese Patent Publication No. Sho 47-1571
No. 2 discloses a water-soluble polymer polysaccharide obtained by subjecting yeast to autolysis, enzyme treatment and alkali treatment. Further, in JP-A-4-253703, yeast is alkali-extracted to obtain a first insoluble yeast residue, which is subjected to hot alkali extraction to obtain a second insoluble yeast residue, which is obtained at a predetermined pH. It is washed to obtain a third insoluble yeast residue, which is acid hydrolyzed to obtain a fourth insoluble yeast residue, which is boiled in water to obtain a fifth insoluble yeast residue, which is treated with ethanol. Boiled in water to give a sixth insoluble residue, which was washed with water and consisted mainly of glucopyranose units linked by β-1,3-glycosidic bonds from which β-1,
Disclosed is a method of making a yeast glucan having at least one branch of glucopyranose units linked by a 6-glycosidic bond.

[0003]

SUMMARY OF THE INVENTION In order to promote the digestion of the cell wall of yeast, the conventional techniques are obliged to perform complicated steps and are unsuitable for industrial scale. The present invention eliminates such drawbacks, develops a method suitable for industrialization by a relatively simple process, and utilizes yeast treated with a yeast cell wall lysing enzyme for the purpose of enhancing immunity.

[0004]

Means for Solving the Problems As a result of repeated studies in consideration of the above points, the inventors of the present invention have digested the yeast cell wall with a yeast cell wall lysing enzyme having a low protease activity to obtain the enzyme-treated yeast obtained. When orally administered as an additive for feed, an excellent immune enhancing effect was observed, which led to the completion of the present invention. The yeast used in the present invention may be any as long as it can be lysed by a yeast cell wall lysing enzyme, for example, bacteria belonging to Saccharomyces, Saccharomyces, Nematospora, Candida, Torulopsis, Pretanomyces, Rhodotorula, or brewer's yeast, baker's yeast. , Sake yeast and the like. The yeast cell wall lysing enzyme is preferably an enzyme having a low protease activity, and examples thereof include enzymes produced by bacteria belonging to Arthrobacter and Oerscovia. Preferable specific examples of such yeast cell wall lysing enzyme include yeast cell wall lysing enzyme produced by Arthrobacter luteus (see JP-B-47-32674 and JP-B-48-2790). The yeast cell wall lysing enzyme treatment may be performed as an aqueous suspension containing about 1 to 20% by weight of bacterial cells. Then, the yeast cell wall lysing enzyme is added in an amount of 1 to 200 units, preferably 5 to 50 units, per 1 g of dry yeast cells, and the pH is about 5 to 8 and 20 to 50 °.
The reaction is carried out by gently stirring at C, preferably 30 to 45 ° C for 1 to 20 hours. The activity unit of the enzyme referred to here is obtained as follows. Cell concentration 5m
3 ml g / ml yeast suspension, 1 / 15M at pH 7.5
Yeast cell wall lysing enzyme solution (1 ml) and water are added to 5 ml of phosphate buffer solution to make a total volume of 10 ml, and this is reacted at 25 ° C. for 2 hours, and the optical density (OD) at 800 nm after the reaction is measured. As a control, water is added instead of the enzyme solution and the same operation is performed, and the reduction rate of the optical density at 800 nm is calculated by the following formula. Reduction rate = (control OD-enzyme reaction solution OD) /
The control O. D. × 100 A unit showing a reduction rate of 30% is defined as 1 unit of enzyme activity. There is a proportionality between the decrease rate and the enzyme concentration up to 60%, and when measuring the unit accurately, the enzyme solution should be 60%.
It must be diluted appropriately within the range showing a reduction rate of not more than%. Prior to this enzymatic treatment, the yeast cells were
It is preferable to heat at -100 ° C, preferably 90-100 ° C, because the amount of enzyme can be reduced.
Specifically, for example, 90 ° C by blowing of steam
The heating may be performed at the temperature above and below the boiling point for about 30 to 120 minutes. It is preferable that the yeast after the enzyme treatment is dried in order to enhance the preservability. The drying method may be any of the commonly known methods such as warm air drying.

[0005]

[Effect] When this enzyme-treated yeast was orally administered as an immunopotentiating substance, a slight amount of the immunopotentiating effect was observed. The dose of the present invention naturally varies depending on the subjects such as human beings and domestic animals. For example, when administered to livestock,
Approximately 0.5 mg / kg body weight is preferable in terms of dry yeast.

[0006]

[Reference Example 1] Preparation of enzyme-treated yeast Live beer yeast cells collected from a brewery are suspended in water at a concentration of 5%, and heat-treated at a temperature of 90 ° C or higher for 1 hour and cooled. Yeast cell wall lysing enzyme derived from Arthrobacter luteus was added to 5000 per 1 kg dry weight of yeast.
A unit is added and the reaction is carried out at 45 ° C. for 16 hours with gentle stirring. Then, if necessary, it was dried to obtain an enzyme-treated yeast preparation. This enzyme-treated yeast was suspended in physiological saline and shaken with a shaker for 15 minutes to prepare a feed immunopotentiating substance.

[0007]

Reference Example 2 Immunopotentiation Test Method (1) The enzyme-treated yeast suspension obtained in Reference Example 1 is orally administered to BALB / c mice (7-week-old, male). Two days after the administration, peritoneal macrophages, spleen lymphocytes and spleen neutrophils were collected. These cells were subjected to measurement of phagocytic ability, NK activity and cytokine using a chemiluminescence reader. In addition, the same enzyme-treated yeast was orally administered to C3H / HeN mice, and 3 days later, spleen-derived mononuclear cells were collected and the juvenile reaction due to glucose consumption was measured. (2) Measurement of phagocytic ability by chemiluminescence reader "The phagocytic cells to be measured are collected and collected in a small plastic test tube, and the photosensitizer luminol is added and set in the chemiluminescence reader. Opsonized zymocell is added as a stimulating factor. The chemiluminescence reaction associated with phagocytosis is measured with a reader, and the peak value of each administration group when the peak value of the chemiluminescence intensity of the control group is 1.
Index). (3) Measurement of NK activity K-562 is used as the target cell (T) and labeled with a fluorescent dye before measurement. The collected spleen lymphocytes were suspended in RPMI-1640 supplemented with 10% FCS to obtain effector cells (E). From the preliminary test, the optimum ratio (E: T) of effector cells to target cells was set to 40: 1. After mixing in a 96-well plate under the condition of E: T = 40: 1 and reacting at 37 ° C. for 4 hours in the presence of 5% CO _{2, the} amount of fluorescent dye remaining in the cells was measured by a fluorescent dye measuring device. The cytotoxic activity was calculated by the following formula. (4) Juvenile test of spleen-derived mononuclear cells 2 × 10 ⁶ / ml of spleen-derived mononuclear cells were concanavalin A.
(Con A) -added group, pokeweed mitogen (PWM) -added group and untreated group were divided, and mixed and cultured. After 96 hours, measure the amount of glucose in the culture supernatant,
From its consumption, S. I. The value (the amount of glucose consumed / the amount of glucose in the medium × 100) was determined and evaluated. (5) Measurement of interleukin 2 (IL-2) and interleukin 5 (IL-5) production amount Spleen mononuclear cells were stimulated with Con A and PWM, cultured for 4 days, and then subjected to ELISA (Enzyme-linked immunosorbent as
IL-2 and I in the culture supernatant by a conventional method using
The L-5 amount was measured.

[0008]

Example 1 An enzyme-treated yeast was orally administered to BALB / c mice, and the chemiluminescence amount, which is an index of the phagocytic ability of peritoneal macrophages, was measured by a chemiluminescence reader. When the peak value of the control group is 1, the peak value of each administration group is S.M. I. Expressed as a value. The result is as shown in FIG. The results in FIG. 1 indicate that oral administration of enzyme-treated yeast enhances the phagocytic ability of peritoneal macrophages.

[0009]

Example 2 NK was found in the group to which the enzyme-treated yeast was orally administered.
The activity was significantly increased and there was no difference in yeast dose. The result is as shown in FIG.

[0010]

[Example 3] The effect of spleen-derived mononuclear cells of orally administered yeasts treated with enzyme-treated yeasts on the weakening was investigated. As a result, the yeast-administered group showed S. I. The value was high. The results are as shown in FIG. It is shown that oral administration of treated yeast enhances spleen-derived mononuclear cell blastogenesis.

[0011]

Example 4 The amount of IL-2 and IL-5 produced in the spleen cell culture supernatant of orally administered yeast treated with enzyme was examined. As a result, the yeast-administered group had IL-2 and I compared to the non-administered group.
The amount of L-5 produced was large and the reactivity to mitogen was high. The results are as shown in Figures 4 and 5.

[0012]

Example 5 Mononuclear cell weakening reaction of enzyme-treated yeast and untreated yeast was examined. Samples of 100 ug were orally administered to 5 males of each C6H / HeN 6 week old group, and spleen-derived mononuclear cells were collected 3 days later. Stimulate the collected cells with PWM, and
Cultured for 6 hours. Glucose consumption in the culture solution was measured, and S. I. The value evaluated the weakening reaction (FIG. 6). The weakening reaction of the enzyme-treated yeast administration group was stronger than that of the untreated yeast administration group, and the untreated yeast administration group was not different from the negative control. The enzyme-treated yeast-administered group showed high reactivity to PWM stimulation. From this, it can be seen that yeast enzymatic treatment is effective for enhancing the immune reaction.

[0013]

[Example 6] The phagocytic ability of spleen-derived mononuclear cells of mice orally administered with yeast was compared using the amount of chemiluminescence as an index. C3H / He
A 100 μg sample was orally administered to 5 males of each N 6 week old group, and 3 days later, spleen-derived mononuclear cells were collected. Luminol was added to the collected cells, which was then reacted with zymosan and measured by a chemiluminescence reader (Fig. 7). As a result, the enzyme-treated yeast-administered group had higher phagocytic ability than the untreated yeast-administered group.

[0014]

Example 7 An enzyme-treated yeast and an untreated yeast were compared for IL-2 production ability. C3H / HeN 6-week-old males of 6 weeks each were orally administered with a sample of 100 μg, and 3 days later, spleen-derived mononuclear cells were collected. The collected cells were stimulated with PWM and cultured for 96 hours. IL-2 in the culture supernatant
Was measured by the ELISA method (FIG. 8). The IL-2 producing ability was higher in the enzyme-treated yeast administration group than in the untreated yeast administration group. The enzyme-treated yeast-administered group showed high reactivity to PWM stimulation.

[0015]

[Example 8] The productivity of IL-5 was compared by the method of Example 7 (Fig. 9). The IL-5 producing ability was higher in the enzyme-treated yeast administration group than in the untreated yeast administration group. The enzyme-treated yeast-administered group showed high reactivity to PWM stimulation.

[0016]

[Example 9] The immunopotentiating effect of enzyme-treated yeast and acid / alkali-treated yeast was compared. For the alkaline treatment, the yeast cells were stirred in a 3% NaOH solution at 37 ° C. for 24 hours, and for the acid treatment, the yeast cells were similarly treated in a 0.5N acetic acid solution. Then, the cells were washed 3 times by centrifugation to collect the yeast cells. These yeasts were orally administered to mice, and 3 days later, the chemiluminescent ability of spleen-derived mononuclear cells was measured. The phagocytic ability with chemiluminescence as an index was almost the same as that of the negative control even after the acid / alkali treatment, and the enzyme-treated yeast-administered group had the highest phagocytic ability. Throughout FIGS. 5 to 10, it is considered that enzyme treatment is effective for enhancing immunity.

[Brief description of drawings]

FIG. 1 shows the phagocytic ability of peritoneal macrophages in mice orally administered with enzyme-treated yeast.

FIG. 2 shows the NK activity of mice orally administered with enzyme-treated yeast.

FIG. 3 shows the debilitating reaction of spleen-derived mononuclear cells of a mouse orally administered with enzyme-treated yeast.

FIG. 4 shows the amount of IL-2 in the spleen cell culture supernatant of a mouse that was orally administered with enzyme-treated yeast.

FIG. 5 shows the amount of IL-5 in the spleen cell culture supernatant of a mouse that was orally administered with enzyme-treated yeast.

FIG. 6 shows the debilitating reaction of spleen-derived mononuclear cells in the enzyme-treated yeast administration group and the untreated yeast administration group.

FIG. 7 shows the phagocytic ability of spleen-derived mononuclear cells in the enzyme-treated yeast administration group and the untreated yeast administration group.

FIG. 8: IL of enzyme-treated yeast administration group and untreated yeast administration group
2 shows productivity.

FIG. 9: IL of enzyme-treated yeast administration group and untreated yeast administration group
-5 shows productivity.

FIG. 10 shows effects of various treated yeasts on phagocytosis of spleen-derived mononuclear cells.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Katsunori Inoue 6-26-1, Jingumae Shibuya-ku, Tokyo Kirin Beer Co., Ltd. (72) Inventor Hajime Matsumoto 6-26-1, Jingumae Shibuya-ku, Tokyo Kirin Beer Within the corporation

Claims (4)

[Claims] 1. A feed additive containing yeast cells treated with a yeast cell wall lysing enzyme. 2. The feed additive according to claim 1, which is in a dry state. 3. The feed additive according to claim 1 or 2, wherein the yeast is one selected from the group consisting of Saccharomyces, Endomycopsis, Saccharomyces, Nematospora, Candida, Torulopsis, Pretanomyces and Rhodotorula. 4. A feed containing the feed additive according to any one of claims 1 to 3.