CN1908155A - Microbial viable bacteria combination and preparation method thereof - Google Patents

Abstract

the invention discloses microbe viable bacteria combined agent and preparing method, which consists of Bacillus subtilis and Rhodotorula glutinis, wherein the Bacillus subtilis is seeded firstly and then Rhodotorula glutinis, which need 15-100 h as combined culturing time.

Description

A kind of living microorganism combination and preparation method thereof

Technical field:

The invention relates to a combination preparation of living microorganisms and a preparation method thereof, belonging to the technical field of microbial fermentation engineering.

Background technique:

Since the discovery in the middle of the last century that adding antibiotics to feed has obvious antibacterial and growth-promoting effects, antibiotics and other pharmaceutical feed additives have played a huge role in preventing animal diseases, improving animal production performance and economic benefits, and promoted the livestock and poultry breeding industry, especially It is the development of intensive farming. However, it was later discovered that the long-term use and abuse of antibiotics in feed has caused other worrying problems; the resistance of pathogenic bacteria has been significantly enhanced, the animal's own immunity has declined, drug residues have caused human health hazards, and excrement has caused environmental pollution, etc., which have seriously affected human health and safety. Countries have taken measures to strictly limit the types, doses and methods of use of antibiotics in feed. The European Union has decided to completely ban the use of antibiotics in feed in 2006.

Microecological preparation refers to the preparation of known beneficial microorganisms into biological preparations applied to animals through modern bioengineering technology. It can effectively supplement the beneficial microorganisms in livestock and poultry, improve the balance of flora in animals, improve the body's disease resistance and feed Absorptive capacity. Because probiotics have no residue, no resistance, and can prevent diseases and promote animal growth, they can effectively overcome some adverse effects caused by long-term use of antibiotics. They are considered to be potential, natural, and harmful to the environment and the environment. Healthy and harmless alternative. Microecological preparations can be divided into Bacillus preparations, Lactobacillus preparations, yeast preparations and compound preparations according to the types of microorganisms. Their application in livestock and aquaculture has developed rapidly. In 1989, the total sales of global feeding microecological preparations were 75 million U.S. dollars, 122 million U.S. dollars in 1993, 186 million U.S. dollars in 2000, and an estimated 350 million U.S. dollars in 2006.

Invention content:

The purpose of the present invention is to provide a novel combination preparation of living microorganisms.

The technical scheme adopted to achieve the above-mentioned purpose: adopt the compound microecological preparation, the live bacteria components of which are Bacillus subtilis (Bacillus subtilis) and Rhodotorula glutinis (Rhodotorula glutinis), which are produced by liquid submerged fermentation mixed culture technology.

The preparation can significantly improve the body's disease resistance and feed absorption and utilization capacity, and has low production cost.

Concrete content of the present invention is as follows:

1. Identification of active strains:

(1) Bacillus subtilis ZHBal 78:

Bacteria rod-shaped, seldom in chains, usually 0.7-0.8×2.0-3.0 microns, Gram-positive, cell protoplasm stained evenly, motile, perinatal flagella. The spores are oval or columnar, 0.9×0.6 micron, mesozoic or partially mesozoic, and the colonies are wrinkled, dry or granular. The growth temperature is 15-55°C, the catalase and V-P reactions are positive, and the lysozyme is positive. The pH in the V-P culture medium is 5.8, and it grows in 7% sodium chloride and grows at pH 5.7. Utilize glucose, sucrose, arabinose, xylose and mannitol to produce acid, hydrolyze starch, liquefy gelatin, use citrate, reduce nitrate to nitrite, decompose casein, do not decompose tyrosine, produce litmus milk Alkaline peptonization. Aerobic, anaerobic growth and yolk reaction negative, does not form soluble melanin on dextrose and tyrosine agars, does not hydrolyze hippurate, does not utilize propionate.

(2) Rhodotorula viscosus ZHRh028:

Cells are ovoid to spherical, 2.3-5.0×4.0-7.8 microns, single or several cells are connected in clusters, and buds are multiterminal. On the slant of malt juice agar, the bacterial lawn is coral red to orange red, the surface of the colony is smooth, shiny, sticky and sometimes hard, the cross-section is flat to wide bulge, the edge is irregular to neat, and does not form hyphae , no pseudohyphae. Does not ferment any sugar, assimilates potassium nitrate, decomposes myricetin, and does not produce starch-like. Can assimilate glucose, maltose, galactose, sucrose, trehalose, cellobiose, L-sorbose, ethanol, D-fructose, melezitose, glycerin, etc., but cannot assimilate lactose, melibiose, D-mannose, murine Plum sugar, D-xylose, inulin, raffinose, inositol, soluble starch, D-arabinose, L-arabinose, D-sorbitol and erythritol. Asexual reproduction, growth temperature 25-36 ℃, pH 2.5-10.0 range can grow.

2. Preparation process:

(1) Production strains:

The microbial strain used in the present invention is Bacillus subtilis ZHBa178 (Bacillus subtilis ZHBa178), preserved in CGMCC, preservation number CGMCC NO.1744, preservation date is June 27, 2006; Rhodotorula glutinis ZHRh028 (Rhodotorula glutinis ZHRh028), preservation In CGMCC, the deposit number is CGMCC NO.1745, and the deposit date is June 27, 2006.

(2) Medium:

①Seed slant culture medium:

Bacillus subtilis: beef extract 0.5% peptone 1.0% sodium chloride 0.5%

Dextrose 0.2% Agar 2.0% pH 7.0-7.2

Rhodotorula viscosus: glucose 2% peptone 1.0% yeast extract 0.2%

Agar 2.0% pH 6.0

② Fermentation medium:

The nutrient source in the culture medium adopts commonly used carbon source and nitrogen source. Among them, the carbon source can be starch, dextrin, sucrose, glucose, maltose, etc.; the nitrogen source can be peptone, soybean cake powder, yeast extract, corn steep liquor, urea, ammonium sulfate, etc. On this basis, add some other nutrients, such as salt, phosphate, potassium salt, calcium salt, magnesium salt, etc.

(3) Training method:

The combination preparation of living microorganisms described in the present invention is inoculated and mixed cultured step by step through liquid submerged fermentation technology. First inoculate Bacillus subtilis at a culture temperature of 34-39°C; after culturing for 10-30 hours, inoculate Rhodotorula viscosus, lower the culture temperature to 28-33°C, and put it into the tank after 15-100 hours of joint culture.

(4) Product post-processing:

The culture solution is adjusted to a certain amount of bacteria, added with an appropriate amount of preservatives and repacked, that is, a water product; or added with an appropriate amount of adsorbent for adsorption, dried and crushed, and then repacked, that is, a powder product.

See the accompanying drawings for the specific process flow.

Significant advantage of the present invention:

1. Multiple effects:

The product is rich in two beneficial active bacteria, Bacillus subtilis and Rhodotorula viscosus, as well as physiologically active substances such as β-carotene, vitamins, and various enzymes produced by the metabolism of the two bacteria, so it has multiple functions. It has the functions of improving the balance of flora in livestock and poultry, improving animal immunity, increasing feed palatability, promoting animal digestion and absorption of feed, and purifying water quality to improve the quality of aquaculture environment.

2. Security:

This product is a live bacterial preparation composed of beneficial bacteria. It has no side effects, no residue problems, and no drug resistance after animal feeding. It is a relatively very safe active feed additive.

3. Low cost:

The production adopts the step-by-step inoculation of Bacillus subtilis and Rhodotorula viscosus followed by the mixed culture process. Compared with the mixed process after separate culture, it can greatly reduce the cost of raw materials and energy and power consumption, so the cost is significantly reduced.

Description of drawings:

The accompanying drawing is a flow chart of the preparation process of the present invention.

Detailed ways:

The present invention has following embodiment:

Embodiment one: the total number of viable bacteria is greater than the preparation of 5 billion/ml water preparation products

In a 70L fermenter, a 50L culture medium was formulated with 3% glucose, 0.4% ammonium sulfate, 0.2% yeast extract, 0.02% dipotassium hydrogen phosphate, and 0.02% magnesium sulfate. Sterilize according to conventional methods and cool to 37°C, inoculate an eggplant bottle with Bacillus subtilis production strains at a culture temperature of 37°C and a rotation speed of 250 rpm; after culturing for 18 hours, inoculate an eggplant bottle with Rhodotorula viscosus production strains species, the culture temperature was lowered to 28°C, and the joint culture was put into the tank after 20 hours. Add water to dilute to a suspension with a total viable count of more than 5 billion/ml, add 0.1% sodium benzoate as a preservative, and pack to obtain the product. The total number of live bacteria in this product is more than 5 billion/ml, of which Bacillus subtilis is more than 3.5 billion/ml, and Rhodotorula viscosus is more than 1.5 billion/ml.

Embodiment two: total viable count is greater than the preparation of 10 billion/gram powder product

In a 70L fermenter, a 45L culture medium was formulated with 4.0% sucrose, 0.5% corn steep liquor, 0.3% urea, 0.02% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, and 0.2% sodium chloride. Sterilize according to conventional methods and cool to 35°C, inoculate 1 eggplant bottle with Bacillus subtilis production strains at a culture temperature of 36°C with a rotation speed of 220 rpm; after 24 hours of cultivation, inoculate 2 eggplant bottles with Rhodotorula viscosus production strains species, the culture temperature was lowered to 30°C, and the joint culture was put into the tank after 76 hours. Add an appropriate amount of corncob powder to absorb and adjust the total number of viable bacteria to more than 10 billion/g, and then pack it after boiling and drying to obtain the product. The total number of viable bacteria in this product is more than 10 billion/g, of which Bacillus subtilis is more than 6 billion/g, and Rhodotorula viscosus is more than 4 billion/g.

Claims (2)

1. A combination preparation of live microorganisms, characterized in that the live bacteria components of the preparation are Bacillus subtilis and Rhodotorula glutinis. 2. A production method dedicated to the combined preparation of living microorganisms as claimed in claim 1, which is produced by the culture technology of microbial liquid submerged fermentation mixed bacteria, and the characteristics of its preparation process are as follows: (1) Production strains: Bacillus subtilis ZHBa178 and Rhodotorula glutinis ZHRh028 were used, and the strains were preserved in CGMCC, and the preservation numbers were CGMCC NO.1744 and CGMCC NO.1745; (2) Fermentation medium: The nutrient source in the culture medium is carbon source and nitrogen source, wherein the carbon source is starch, dextrin, sucrose, glucose or maltose; the nitrogen source is peptone, soybean cake powder, yeast extract, corn steep liquor, urea or ammonium sulfate, and added table salt, phosphate, potassium, calcium or magnesium salts; (3) Training method: Step-by-step inoculation of mixed culture through liquid submerged fermentation technology, first inoculate Bacillus subtilis, culture temperature is 34-39 ℃, after 10-30 hours of culture, inoculate Rhodotorula viscosus, culture temperature drops to 28-33 ℃, joint culture 15-100 Put the tank after hours; (4) Product post-processing: After the culture solution is prepared, preservatives are added, and then packaged into water products; or adsorbents are added for adsorption, dried and pulverized, and then packaged into powder products.