CN1908155B - 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, the method adopts microbe liquid deep fermentation mixed fungus cultivation technology, wherein the Bacillus subtilis is seeded firstly and then Rhodotorula glutinis is seeded, the combined culturing time is 15-100 hours. The agent can improve the anti-disease capability of the livestock, and the assimilating and utilizing ability, furthermore, the cost is relatively low.

Description

A kind of microorganism live bacteria combination and preparation method thereof

Technical field:

The present invention relates to a kind of microorganism live bacteria combination preparation and preparation method thereof, belong to the microbial fermentation engineering technical field.

Background technology:

Since the middle of last century is found to add in the feed microbiotic and has obviously antibacterial, promotes growth effect, antibiotic etc forage additive has been brought into play huge effect to prevention Animal diseases, raising breeding performonce fo animals and economic benefit aspect, has promoted the especially development of intensive culture industry of livestock and poultry breeding industry.But it is found that afterwards,, produced the problem of people's worry in addition in medium-term and long-term use of feed and abuse of antibiotics; The pathogenic bacteria resistance significantly strengthens, and animal autoimmunity descends, and drug residue causes human health risk, and movement causes environmental pollution etc., has had a strong impact on human public health and safety.Various countries take measures aspects such as the kind of feeding antibiotic, dosage and using method are strictly limited one after another, and European Union has determined to begin in 2006 to completely forbid and use microbiotic in feed.

Probiotics is meant known beneficial microorganism is prepared into the biotechnological formulation that is applied to animal by modern biotechnology, it can effectively replenish the intravital beneficial microorganism of livestock and poultry, improve colony balance in the animal body, improve body disease-resistant power and feed and absorb ability.Owing to the probiotics noresidue, do not produce resistance, and effect with preventing disease and promotion growth of animal, can effectively overcome some detrimentally affects that the life-time service microbiotic causes, be considered to the feeding antibiotic potential, natural, to environment and healthy harmless surrogate.Probiotics can be divided into bacillus preparation, lactobacillus preparation, yeast preparation and compound formulation etc. by microbe species, their application developments aspect livestock and poultry and aquaculture are very fast, global feeding micro-ecological preparation total sales volume was 7,500 ten thousand dollars in 1989,1993 is 1.22 hundred million dollars, 2000 is 1.86 hundred million dollars, and estimation in 2006 can reach 3.5 hundred million dollars.

Summary of the invention:

The present invention seeks to provide a kind of novel microorganism viable bacteria combination preparation.

The technical scheme of taking for achieving the above object: adopt compound micro-ecological preparation, its viable bacteria composition is subtilis (Bacillus subtilis) and rhodotorula glutinis (Rhodotorula glutinis), adopts liquid submerged fermentation to mix the bacterium culture technique and produces.

Said preparation can significantly improve body disease-resistant power and feed absorbs ability, and low production cost.

Particular content of the present invention is as follows:

1. active bacterial strain is identified:

(1) subtilis ZHBa178:

Thalline is shaft-like, and seldom chaining is generally 0.7-0.8 * 2.0-3.0 micron, Gram-positive, cellular plasm even dyeing, motion, peritrichous.Gemma ellipse or column, 0.9 * 0.6 micron, middle life or middle partially giving birth to, bacterium colony is scabriculous shape, drying or particulate state.Growth temperature 15-55 ℃, catalase and V-P reacting positive, the N,O-Diacetylmuramidase positive, the pH5.8 in the V-P nutrient solution grows in 7% sodium-chlor, the pH5.7 growth.Utilize glucose, sucrose, pectinose, wood sugar and N.F,USP MANNITOL to produce acid, hydrolyzed starch, liquefy gelatin can utilize Citrate trianion, and reduction nitrate becomes nitrite, decomposes casein, does not decompose tyrosine, and litmus milk produces alkali and peptonizes.Aerophil, anaerobic growth and egg yolk reaction feminine gender do not form solubility melanochrome on agar glucose and tyrosine agar, and not hydrolysis hippurate does not utilize propionic salt.

(2) rhodotorula glutinis ZHRh028:

Cell is avette to spherical, 2.3-5.0 * 4.0-7.8 micron, and single or several cells link bunchiness, and multiterminal sprout.Lawn presents coral red to orange red on the wort agar inclined-plane, and bacterium colony smooth surface, glossy, thickness harden sometimes, and its square section is flat to the projection that broad is arranged, and the edge is irregular to neatly, does not form mycelia, no pseudohypha.Any sugar of nonfermented, assimilation saltpetre decomposes Myricitroside, does not produce kind of starch.Glucose, maltose, semi-lactosi, sucrose, trehalose, cellobiose, L-sorbose, ethanol, D-fructose, melizitose, glycerine etc. can be assimilated, lactose, melibiose, D-seminose, rhamnosyl, D-wood sugar, synanthrin, raffinose, inositol, Zulkovsky starch, D-pectinose, L-arabinose, D-sorbyl alcohol and red tinea alcohol can not be assimilated.No syngenesis phenomenon, growth temperature 25-36 ℃, the pH2.5-10.0 scope all can be grown.

2. preparation technology:

(1) produce bacterial classification:

The used microbial strains of the present invention is subtilis ZHBa178 (Bacillus subtilis ZHBa178), is preserved in CGMCC, protects minus sign CGMCC NO.1744, and preservation date is on 06 27th, 2006; Rhodotorula glutinis ZHRh028 (Rhodotorula glutinis ZHRh028), be preserved in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, preserving number CGMCC NO.1745, the address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, preservation date are on 06 27th, 2006.

(2) substratum:

1. seed slant medium:

Subtilis: extractum carnis 0.5% peptone 1.0% sodium-chlor 0.5%

Glucose 0.2% agar 2.0% pH 7.0-7.2

Rhodotorula glutinis: glucose 2% peptone 1.0% yeast extract paste 0.2%

Agar 2.0% pH 6.0

2. fermention medium:

Nutrition source in the substratum adopts carbon source and nitrogenous source commonly used.Wherein carbon source can be starch, dextrin, sucrose, glucose, maltose etc.; Nitrogenous source can be peptone, soybean cake powder, yeast extract paste, corn steep liquor, urea, ammonium sulfate etc.Add some other nutritions on this basis again, as salt, phosphoric acid salt, sylvite, calcium salt, magnesium salts etc.

(3) cultural method:

Microorganism live bacteria combination preparation of the present invention is cultivated by liquid submerged fermentation technology step inoculation is mixed.At first inoculate subtilis, culture temperature is 34-39 ℃; Cultivate and inoculate rhodotorula glutinis after 10-30 hour, culture temperature is reduced to 28-33 ℃, unites to cultivate after 15-100 hour to put jar.

(4) product aftertreatment:

Nutrient solution is modulated into certain bacteria containing amount, adds packing after an amount of sanitas, is aqua product; Or adding an amount of adsorbents adsorb, packing after the drying and crushing is powder product.

Figure of description is seen in concrete technical process.

Remarkable advantage of the present invention:

1. multiple-effect:

Product is rich in subtilis and two kinds of useful active bacterium of rhodotorula glutinis, and the β-Hu Luobusu that metabolism produces when being cultivated by two bacterium, VITAMIN, and physiologically active substance such as plurality of enzymes, thereby possesses multi-efficiency.Have colony balance in the livestock and poultry of the improvement body, improve animal immunizing power, increase the feed palatability, the promotion animal digests and assimilates feed, and the functions such as improving the culture environment of aquatic products quality that purifies water.

2. safety:

The active bacteria formulation that this product system is made up of probiotics, the feeding back of animal is without any side effects, and the noresidue problem does not cause resistance, is activated feed additive as safe as a house relatively.

3. low-cost:

Mix culture process after producing employing subtilis, rhodotorula glutinis step inoculation, compare, can reduce material cost and energy source and power consumption greatly, thereby cost reduces obviously with cultivating the mixed technology in back respectively.

Description of drawings:

Accompanying drawing is preparation technology's schema of the present invention.

Embodiment:

The present invention has following examples:

Embodiment one: total viable count is greater than the preparation of 5,000,000,000/ml aqua product

In the 70L fermentor tank, be made into the 50L substratum by glucose 3%, ammonium sulfate 0.4%, yeast extract paste 0.2%, dipotassium hydrogen phosphate 0.02%, sal epsom 0.02%.Sterilize according to a conventional method postcooling to 37 ℃, the subtilis of 1 eggplant bottle of inoculation produces bacterial classification, and culture temperature is 37 ℃, 250 rev/mins of rotating speeds; Cultivate 1 eggplant bottle rhodotorula glutinis production of inoculation bacterial classification after 18 hours, culture temperature is reduced to 28 ℃, unites to cultivate after 20 hours to put jar.Thin up becomes the above suspension of total viable count 5,000,000,000/ml, adds 0.1% Sodium Benzoate again and makes sanitas, and packing promptly gets product.This product total viable count is greater than 5,000,000,000/ml, and wherein subtilis is greater than 3,500,000,000/ml, and rhodotorula glutinis is greater than 1,500,000,000/ml.

Embodiment two: total viable count is greater than the preparation of 10,000,000,000/gram powder product

In the 70L fermentor tank, be made into the 45L substratum by sucrose 4.0%, corn steep liquor 0.5%, urea 0.3%, dipotassium hydrogen phosphate 0.02%, sal epsom 0.02%, sodium-chlor 0.2%.Sterilize according to a conventional method postcooling to 35 ℃, the subtilis of 1 eggplant bottle of inoculation produces bacterial classification, and culture temperature is 36 ℃, 220 rev/mins of rotating speeds; Cultivate 2 eggplant bottle rhodotorula glutinis production of inoculation bacterial classification after 24 hours, culture temperature is reduced to 30 ℃, unites to cultivate after 76 hours to put jar.Add the absorption of an amount of corn cob meal and be modulated to that total viable count 10,000,000,000/more than the gram, packing is product behind the fluidized drying.The total viable count of this product is greater than 10,000,000,000/gram, and wherein subtilis is greater than 6,000,000,000/gram, and rhodotorula glutinis is greater than 4,000,000,000/gram.

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.