JPH0379986B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Bacillus licheniformis MN-001. More specifically, the present invention relates to Bacillus licheniformis MN-001, which produces large amounts of protease and cellulase outside the cells and is suitable for addition to feed. At present, compound feeds are shifting from mainly homemade feeds to compound feeds produced in modern factories, and the proportion of compound feeds in total feed has reached an extremely high value. In addition to the main ingredients of grains, oil cakes, fish, and fats and oils, these compound feeds also contain amino acids, minerals, and vitamins for purposes such as nutritional supplementation, improved digestion and absorption, growth promotion, and disease prevention. , antibiotics, enzymes, probiotics, etc. are used as auxiliary raw materials. However, among these antibiotics, there is a risk that residual migration may cause allergies or changes in intestinal bacteria in humans, so restrictions on their use are being tightened, and the range of use is being limited or reduced. Enzymes include lipase agents, amylase agents, cellulase agents, protease agents, mixed enzyme agents, etc., and viable bacteria agents include Bacillus natto, Bacillus natto,
Lactic acid bacteria, acid bacteria, bifidobacteria, and the like have been studied, but their effects vary and a constant effect cannot be expected at present. The present inventors isolated many Bacillus bacteria from nature based on the assumption that mutant strains with better enzyme production could be found among Bacillus bacteria that are extremely safe for living organisms. However, as a result of further mutational manipulation, we were able to isolate and obtain Bacillus licheniformis MN-001, which has a remarkable ability to produce protease and cellulase outside the cell. Bacillus licheniformis isolated here
Although MN-001 has the same mycological properties as Bacillus licheniformis in that it grows under anaerobic conditions and other properties, it is completely different in that it has remarkable protease and cellulase productivity. It was recognized as a mutant strain of Bacillus licheniformis. Bacillus licheniformis MN-001 has been deposited with the Microtech Institute as FERM BP-266 under the designation MN-001. Next, Bacillus licheniformis MN−001
The mycological properties of A Morphology (1) A culm fungus with a width of 0.5 to 0.8 μm and a length of 2 to 3 μm. (2) Gram staining: positive. (3) Forms oval endospores. (4) Motility: Yes. B. Growth (1) Ordinary agar medium: Grows very well. No pigment formation. (2) Medium based on molasses and ammonium sulfate: Grows very well. No pigment formation. (3) 5% saline liquid medium: Grows. 7%: Grows. 10%: Grow. C Physiological properties (1) Catalase production + (2) Growth under anaerobic conditions + (3) VP reaction + (4) Growth status at high temperature 50℃ + 55℃ - (5) Presence or absence of acid production from sugars Glucose + L-arabinose + D-xylose + Mannite + D-tagatose + (6) Arginine dihydrolase + (7) Lecithinase - (8) Hydrolyzability of starch + (9) Hydrolyzability of gelatin + (10) DNA Hydrolyzability + (11) Citrate availability + (12) Propionate availability + (13) Nitrate reduction + (14) Indole formation − (15) Deamination of phenylalanine − ( 16) Specific properties: Protease (Bacillus licheniformis
Cellulase (Bacillus licheniformis)
Compared to IFO 12199, it has a significantly higher extracellular production capacity (approximately 15 times more than IFO 12199). Bacillus licheniformis MN− of the present invention
001 can be cultured by liquid culture or solid culture according to the usual fermentation method. As the medium, any of natural medium, semi-synthetic medium, synthetic medium, etc. can be used. The composition of the medium is as follows: () Carbon sources include glucose, sutucarose, dextrin, glycerin, starch,
Molasses etc. ()Nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean meal, urea,
Thiourea, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds are used. () As inorganic salts, phosphates, nitrates, carbonates, chlorides, etc. of magnesium, manganese, potassium, calcium, iron, etc. are used. Also, amino acids, vitamins,
Nucleic acids and related compounds may also be added. The culture temperature is the same as for normal fermentation methods, but for example, 20
It can be cultured appropriately at temperatures between ℃ and 50℃. The culture period is also similar to that of a normal fermentation method, but for example, 12 hours to 5 days is suitable for culturing. The culture thus obtained can be prepared as it is or by repeated washing to obtain only bacterial cells, which can be freeze-dried, spray-dried, etc. to form a preparation. When used as animal feed or feed additives, for example, the culture solution may be used as is, or wheat flour, starch, dextrin, etc., grains as raw materials for feed, etc.
It can be formulated using a suitable diluent such as saccharide such as defatted rice bran or oil cake. Alternatively, only the bacterial cells may be washed and separated from the culture solution, formulated in the same manner, and used. The Bacillus licheniformis MN-001 preparation thus obtained can be used as animal feed or feed additive for beef cattle, dairy cattle, calves, pigs, piglets, etc.
It is effective when given to livestock such as sheep, goats, horses, rabbits, dogs, and cats, and poultry such as meat chickens, egg-laying hens, chicks, breeding hens, ducks, geese, turkeys, quail, and baby birds. be. The dosage cannot be determined unconditionally as it may vary depending on the target animal, the age of the target animal, and other ingredients of the feed, but usually Bacillus licheniformis MN-001 cells are equivalent to 1 kg of feed.
It is preferable to add 10 ² to 10 ¹⁵ pieces, preferably 10 ⁴ to 10 ¹² pieces, and most preferably 10 ⁵ to 10 ¹⁰ pieces. Next, test examples, examples, and reference examples of the present invention will be shown. Test Example 1 Bacillus lichenylformis IFO12199 2 Bacillus lichenylformis MN-001,
FERM BP-266 Each of the strains 1 and 2 above was placed in a 500 ml Sakaguchi flask with 2 g of molasses, 1 g of ammonium sulfate, and K ₂ HPO ₁ 0.1
g, add 0.05g of yeast extract and add 100ml of tap water.
After adjusting the pH to 8 with 0.1N HCl and 0.1N NaOH, the cells were inoculated into a medium that had been sterilized at 121°C for 15 minutes.
After culturing with shaking at 37°C for 24 hours, each extracellular enzyme (protease, amylase, cellulase) was measured. The measurement method is to use the culture broth as a crude enzyme solution, to measure amylase activity, use the dinitrosalicylic acid method using starch as a substrate, to measure cellulase activity, use the dinitrosalicylic acid method using carboxymethylcellulose as a substrate, and to measure protease activity, use the dinitrosalicylic acid method using carboxymethyl cellulose as a substrate. , copper using milk casein as substrate
The Folin method was used to compare the extracellular enzyme production ability.
The numbers in the table are relative values when the IFO 12199 value is set to 1. The results are shown in Table 1 below.

[Table] Example: Culture medium 1 with the same composition as the test example was placed in a jar fermenter, and after sterilization, pre-culture was carried out in advance.
Bacillus licheniformis MN−001, FERM BP
-226 was inoculated and cultured with shaking for 24 hours. After separating the bacterial cells from the culture source by centrifugation, 1 kg of starch
Bacillus licheniformis MN−
001 spore preparation was prepared. The number of viable bacteria in this preparation is 10 ⁸
pieces/g. Reference example: Using 120 broiler chickens (40 male and female each), 40 in each group.
Divided into two groups (20 males and 20 females), the control group (starch added group) was added to the basic feed shown in Table 2, and the MN-001 formulation obtained in the example was added (10 ⁸ per 1 g of starch).
An 8-week feeding test was conducted. The results are shown in Table 3.

【table】

[Table] As shown above, the MN-001 formulation exhibits excellent effects on animals. In addition, the MN-001 preparation, which has excellent extracellular enzyme production ability, is effective not only as animal feed or feed additive, but also for use in fish feed, food, pharmaceuticals, cosmetics, enzyme production, etc. It is.

Claims (1)

[Claims] 1. Bacillus licheniformis MN-001 having the following mycological properties. A Morphology (1) A culm fungus with a width of 0.5 to 0.8 μm and a length of 2 to 3 μm. (2) Gram staining: positive. (3) Forms oval endospores. (4) Motility: Yes. B Growth (1) Ordinary agar medium: Grows very well. No pigment formation. (2) Medium based on molasses and ammonium sulfate: Grows very well. No pigment formation. (3) 5% saline liquid medium: Grows. 7%: Grows. 10%: Grow. C Physiological properties (1) Catalase production + (2) Growth under anaerobic conditions + (3) VP reaction + (4) Growth status at high temperature 50℃ + 55℃ - (5) Presence or absence of acid production from sugars Glucose + L-arabinose + D-xylose + Mannite + D-tagatose + (6) Arginine dihydrolase + (7) Lecithinase - (8) Hydrolyzability of starch + (9) Hydrolyzability of gelatin + (10) DNA Hydrolyzability + (11) Citrate availability + (12) Propionate availability + (13) Nitrate reduction + (14) Indole formation − (15) Deamination of phenylalanine − ( 16) Specific properties: Extremely high ability to produce proteases and cellulases outside the cells.