CN1908155B - Microbial viable bacteria combination and preparation method thereof - Google Patents

Abstract

The invention relates to a combination preparation of living microorganisms and a preparation method thereof. The live bacteria components of the preparation are Bacillus subtilis (Bacillus subtilis) and Rhodotorula glutinis (Rhodotorula glutinis); its preparation is produced by microbial liquid submerged fermentation mixed culture technology, first inoculated with Bacillus subtilis, then inoculated with Rhodotorula glutinis, and jointly cultivated Time 15-100 hours. The preparation can significantly improve the disease resistance of livestock and poultry bodies and the ability to absorb and utilize feed, and the production cost is low.

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. the

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. the

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. the

Invention content:

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

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. the

The specific content of the present invention is as follows:

1. Identification of active strains:

(1) Bacillus subtilis ZHBa178:

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 reaction are positive, and the lysozyme is positive, the pH in the V-P culture medium is 5.8, and the growth is in 7% sodium chloride, and the pH is 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. the

(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 phenomenon, growth temperature 25-36 ℃, pH 2.5-10.0 range can grow. the

2. Preparation process:

(1) Production strains:

The microbial bacterial classification used in the present invention is Bacillus subtilis ZHBa178 (Bacillus subtilis ZHBa178), preserved in CGMCC, guarantee number CGMCC NO.1744, preservation date is on June 27th, 2006; Rhodotorula glutinis ZHRh028 (Rhodotorula glutinis ZHRh028), Preserved in CGMCC, General Microorganism Center of China Microbiological Culture Collection Management Committee, preservation number CGMCC NO.1745, address: No. 13, North Zhongguancun, Haidian District, Beijing, and the preservation date is June 27, 2006. the

(2) Medium:

①Seed slant medium:

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

                                                                                                                           

Rhodotorula viscosum: 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. the

(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. the

(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. the

See the accompanying drawings for the specific process flow. the

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. the

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. the

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. the

Description of drawings:

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

Detailed ways:

The present invention has the following embodiments:

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

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. the

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. the

Claims (2)

1. microorganism live bacteria combination preparation, the viable bacteria composition that it is characterized in that preparation is: subtilis Bacillus subtilis ZHBa178, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on June 27th, 2006, preserving number: CGMCC NO.1744; With rhodotorula glutinis Rhodotorula glutinis ZHRh028, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on June 27th, 2006, preserving number: CGMCCNO.1745.

2. a production method that is exclusively used in the described microorganism live bacteria combination preparation of claim 1 adopts the microbial liquid submerged fermentation to mix the bacterium culture technique and produces, and its preparation technology's feature is as follows:

(1) produce bacterial classification:

Adopt subtilis Bacillus subtilis ZHBa178 and rhodotorula glutinis Rhodotorulaglutinis ZHRh028, culture presevation is in CGMCC, and preserving number is respectively CGMCC NO.1744 and CGMCC NO.1745;

(2) fermention medium:

Nutrition source in the substratum adopts carbon source and nitrogenous source, and wherein carbon source is starch, dextrin, sucrose, glucose or maltose; Nitrogenous source is peptone, soybean cake powder, yeast extract paste, corn steep liquor, urea or ammonium sulfate, and adds salt, phosphoric acid salt, sylvite, calcium salt or magnesium salts;

(3) cultural method:

Cultivate by liquid submerged fermentation technology step inoculation is mixed, at first inoculate subtilis, culture temperature is 34-39 ℃, cultivates and inoculates rhodotorula glutinis after 10-30 hour, and culture temperature is reduced to 28-33 ℃, unites to cultivate after 15-100 hour and puts jar;

(4) product aftertreatment:

After the nutrient solution modulation, add sanitas again, packing is aqua product; Or nutrient solution puts jar back absorption and modulation, and packing is powder product behind the fluidized drying.