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A pig-derived Bacillus subtilis and its feed probiotics prepared by solid-state fermentation

A Bacillus subtilis, solid-state fermentation technology, applied in animal feed, animal feed, bacteria, etc., can solve problems such as poor solubility, inability to directly feed animals, and animals unable to obtain satisfactory results

Inactive Publication Date: 2017-10-27
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rice husks and straws mainly contain cellulose and hemicellulose, which are not palatable and cannot be directly fed to animals. Most of them are directly discarded or incinerated, which pollutes the environment and wastes resources.
Soybean meal is a by-product of soybean oil extraction. Although the crude protein content is high and the amino acid composition is balanced, most of the soybean meal is a by-product of high-temperature processing at present. The protein denaturation is serious, the solubility is poor, and there are some anti-nutritional factors. Direct feeding to animals cannot be satisfactory. effect, leading to waste of nutrient resources

Method used

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  • A pig-derived Bacillus subtilis and its feed probiotics prepared by solid-state fermentation
  • A pig-derived Bacillus subtilis and its feed probiotics prepared by solid-state fermentation
  • A pig-derived Bacillus subtilis and its feed probiotics prepared by solid-state fermentation

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Experimental program
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Effect test

specific Embodiment approach 1

[0019] Specific embodiment one: Screening of YSJB-30 bacterial strain

[0020] The method of isolating and screening a strain of Bacillus subtilis YSJB-30 from the fresh feces of healthy sows is as follows: take fresh sow feces and put them in a sterile container, seal them, store them in an ice box, and quickly send them to the laboratory for routine strain isolation . The separation plate medium was CM medium supplemented with skim milk. Under sterile conditions, after gradient dilution of the sample, incubate the coated plate upside down at 30°C for 1 to 2 days, pick out a single colony with a transparent circle around the colony and put it on the CM slant medium, a total of 50, and culture at 30°C for 1 to 2 days. 2 days, 4 ℃ standby.

[0021] Under sterile conditions, respectively insert a bacterial loop of each of the above 50 slant surfaces into the sterilized liquid CM medium, cultivate on a shaker at 37°C and 180rpm for 16 hours, centrifuge the bacterial liquid to t...

specific Embodiment approach 2

[0022] Specific embodiment two: PCR amplification of bacterial genome DNA and 16SrDNA extraction:

[0023] Pick a bacterial ring slant and insert it into 2ml of CM liquid medium, incubate at 37°C for 16h-18h, take 1.5ml of activated bacterial liquid, centrifuge at 12 000r / min for 1 min, discard the supernatant, collect the bacterial cells, and follow the bacterial genome Extract bacterial genomic DNA according to the method in the instruction manual of the DNA extraction kit, and store at -20°C for future use. 0.8% agarose gel was used to detect the extracted genomic DNA, and the gel imaging system was used to observe and take pictures, as shown in the accompanying drawings.

[0024] PCR amplification of 16SrDNA sequence:

[0025] Primers: 799F 5'-AACAGGATTAGATACCCTG-3', 1492R 5'-GGTTACCTTGTTACGACTT-3'.

[0026] Using the extracted bacterial genome DNA as a template, PCR amplification of 16SrDNA was carried out. PCR amplification reaction system 25 μL:

[0027] 10×PCR buff...

specific Embodiment approach 3

[0030] Specific embodiment three: Bacillus subtilis YSJB-30 bacterial strain antibacterial test:

[0031] Take the sterilized liquid CM medium 10 mL / 100 mL Erlenmeyer flask, put into each bottle the activated Bacillus subtilis YSJB-30 slant cell one ring, incubate at 35-36 °C for 24 h in aerobic conditions, centrifuge at 8000 rpm for 1 min, Take the supernatant, pick one loop each of the activated Listeria monocytogenes and Salmonella bacteria, put them into the CM liquid culture medium at 37 ℃ for overnight culture, serially dilute the appropriate multiple, smear the plate, and let it stand for 20 After 1 min, punch holes with a puncher, add 100 ul of the above supernatant to each hole aseptically, let it stand for 30 min, add supernatant to fill the hole, and incubate overnight at 37 °C, observe and measure the diameter of the inhibition zone;

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Abstract

The present invention isolates and screens a strain of Bacillus subtilis YSJB-30 from the fresh feces of healthy sows, and combines the results of morphology and 16SrDNA sequence comparison to preliminarily identify it as Bacillus subtilis. The strain secretes extracellular protease, and can inhibit enteropathogenic bacteria Salmonella and Listeria tenzing in vitro. The bacterium was applied to the solid-state fermentation of probiotics with straw, rice husk and soybean meal as raw materials, and the optimal ratio of raw materials was determined by combining the cost of raw materials and the number of viable bacteria; Plackett-Burman design, steepest climbing method and The process parameters were optimized by response surface analysis. Under the optimized culture conditions, the fermentation time was 36 h, and the number of viable Bacillus subtilis reached 5.35 x109 CFU / g. The invention provides basic data for the preparation of feed probiotics for replacing antibiotics by solid-state fermentation, and at the same time has great significance for the comprehensive utilization of grain and oil by-products and resource development in northern my country.

Description

technical field [0001] The invention relates to a pig-derived bacillus subtilis and its solid-state fermentation application. It involves the comprehensive utilization of grain and oil by-products, the screening of strains that inhibit pathogenic bacteria, and the optimization of solid-state fermentation technology. Background technique [0002] The addition of antibiotics to livestock and poultry feed has become a common phenomenon at present, and the residues of antibiotics in livestock, poultry, meat, eggs and dairy products have seriously threatened human health. Finding safe alternatives to antibiotics has become a research hotspot in the academic field. Bacillus subtilis, as one of the feed bacteria allowed to be added by the Ministry of Agriculture of my country, has a strong ability to secrete extracellular enzymes such as protease, lipase and amylase, which can improve the digestion and absorption rate and feed conversion rate of animals, and can secrete Antibacteri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A23K10/18C12R1/125A23K20/195
CPCC12N1/205C12R2001/125
Inventor 徐速江连洲于殿宇孙立斌孙慧赵清霞李相昕
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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