CN104651270A - Swine bacillus subtilis and feed probiotic prepared by solid fermentation by using swine bacillus subtilis - Google Patents
Swine bacillus subtilis and feed probiotic prepared by solid fermentation by using swine bacillus subtilis Download PDFInfo
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- CN104651270A CN104651270A CN201510017923.2A CN201510017923A CN104651270A CN 104651270 A CN104651270 A CN 104651270A CN 201510017923 A CN201510017923 A CN 201510017923A CN 104651270 A CN104651270 A CN 104651270A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Abstract
The invention discloses a bacillus subtilis YSJB-30 which is separated and screened from fresh excrement of a healthy sow. The bacillus subtilis YSJB-30 is primarily identified as bacillus subtilis by combining morphology with a16SrDNA sequence alignment result. The bacillus subtilis is capable of secreting extracellaluar proteases and an in vitro test proves that the bacillus subtilis is capable of inhibiting pathogenic entero becteria such as salmonella and listeria monocytogenes; the bacillus subtilis is applied to solid fermentation of raw material such as straw, rice hull and bean pulp to prepare a probiotic; the optimal raw material proportion is determined by combining the raw material cost and viable count; technological parameters are optimized by adopting a Plackett-Burman design, a steepest ascent and a response surface analysis method; when the fermentation lasts for 36 hours under the optimized culture condition, the viable count of the bacillus subtilis reaches 5.35*10<9>CFU/g. According to the invention, basic data are provided for the feed probiotic which is prepared by solid fermentation to replace an antibiotic; and meanwhile, the bacillus subtilis is of great significance for comprehensive utilization and resource development of grain and oil by-products in the northern area in China.
Description
Technical field
The present invention relates to the application of a strain pig source subtilis and its solid state fermentation.Relate to the research contents such as the comprehensive utilization of grain and oil by product, the screening suppressing pathogenic bacterium bacterial strain and solid-state fermentation process optimization.
Background technology
Current antibiotic adds and becomes universal phenomenon in animal and fowl fodder, and the residual serious threat human health of microbiotic in livestock and poultry and meat, eggs and milk goods, the Substitutes For Antibiotic finding safety has become sphere of learning study hotspot.Subtilis allows one of feed spawn added as the Ministry of Agriculture of China, there is the secretion capacity of the extracellular enzymes such as very strong proteolytic enzyme, lipase and amylase, digestibility and the feed conversion rate of animal can be improved, antibacterial substance can be secreted and do not cause antibiotic remains problem again, animal diseases prevention and growth promoting effects are played an important role, be the Substitutes For Antibiotic of safety, green, environmental protection, be developed into increasing microbial preparation.
Rice hulls, stalk, dregs of beans are the northern area of China grain and oil processed side products, and along with China's grain yield is increased production year by year, these by product output also constantly increase.Containing Mierocrystalline cellulose, hemicellulose components, palatability is not good because of main for rice hulls, stalk, can not Direct-fed animal, is mostly directly discarded or burns, not only contaminate environment but also waste resource.Dregs of beans is the byproduct after soybean extracts grease, although crude protein content is high, amino acid Compositional balance, but dregs of beans is mostly the byproduct through high temperature process at present, protein denaturation is serious, poorly soluble, there are some antinutritional factor, Direct-fed animal can not obtain satisfied effect, causes the waste of nutritional resource.How effectively to utilize these grain and oil by products, improve its economic worth and become the task of top priority.
Summary of the invention
Subtilis is applied to stalk, rice hulls and dregs of beans by the present invention to be raw material solid state fermentation prepares probiotic agent, bacterial strain uses therefor can obviously suppress pathogenic bacterium Salmonella and Listeria monocytogenes, made bacillus subtilis microbial agent makes an addition in pig feed in advance, for substituting microbiotic in feed further, solving antibiotic remains problem in feed and pork, provide a kind of green feed additive.
The present invention includes following steps:
1, relate to a boar source subtilis in the present invention, strain name YSJB-30, Classification And Nomenclature is subtilis
bacillus subtilis, preserve and be numbered CGMCC No.9660, preservation date: on September 15th, 2014, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
2, the pig source subtilis YSJB-30 bacterial strain vitro inhibition pathogen enterobacteria pellet that the present invention relates to increases listeria bacteria, Salmonella is positive.
3, the pig source bacillus subtilis strain YSJB-30 that the present invention relates to produces extracellular protease.
4, utilize subtilis YSJB-30 solid state fermentation to prepare pig source probiotic agent, with grain and oil by product stalk, rice hulls, dregs of beans for raw material, mass ratio is stalk: rice hulls: dregs of beans=3 ~ 5:1:4 ~ 6.
5, utilize subtilis YSJB-30 solid state fermentation to prepare pig source probiotic agent, solid-state fermentation process is initial pH 5 ~ 8, leavening temperature 30 ~ 38 DEG C, amount of water 50 ~ 80%, K
2hPO
40.8 ~ 1.6%, MgSO
40.04 ~ 0.08%, corn steep liquor 0.5 ~ 1%, glucose 1 ~ 2%, (NH
4)
2sO
40.1 ~ 0.2%.
6, utilize subtilis YSJB-30 solid state fermentation to prepare pig source probiotic agent, fermentation time 36h, subtilis number of viable reaches 1 x10
9cFU/more than g.
The present invention, in conjunction with raw materials cost and quality product, is inspection target with viable count, determines the optimum material proportion meeting thalli growth, adopts Plackett-Burman design, steepest hill climbing and Responds Surface Methodology optimization for fermentation technology parameter.The present invention is that feed probiotic agent that solid state fermentation prepares substitute antibiotics provides the foundation data, simultaneously to the comprehensive utilization of the northern area of China grain and oil by product and development of resources significant.
Accompanying drawing explanation
Fig. 1 production technological process of the present invention
The antibacterial flat board of Fig. 2 subtilis YSJB-30 vitro inhibition pathogenic bacterium
Fig. 3 YSBJ-30 bacterial strain STb gene agarose gel electrophoresis detects figure
Fig. 4 YSBJ-30 bacterial strain 16S rRNA gene PCR product detected through gel electrophoresis figure
Fig. 5 YSBJ-30 bacterial strain 16S rRNA gene order
Fig. 6 YSBJ-30 bacterial strain 16S rRNA gene order with
bacillus subtilisstrain B-A 16S ribosomal RNA gene comparison result.
Embodiment
The screening of embodiment one: YSJB-30 bacterial strain
From healthy sow fresh excreta, to obtain the method for a bacillus subtilis YSJB-30 as follows for separation screening: get fresh sow ight soil and put into sterile chamber, sealing, ice chest is preserved, and sends into laboratory fast and carries out conventional strain separating.Separating plate substratum is the CM substratum that with the addition of skimming milk.Under sample aseptic condition, after gradient dilution, spread plate is inverted cultivation 1 ~ 2 day in 30 DEG C, occurs that single bacterium colony of transparent circle is in CM slant medium respectively, totally 50 around picking colony, cultivate 1 ~ 2 day for 30 DEG C, 4 DEG C for subsequent use.
Under aseptic condition, respectively by the liquid CM substratum after each for above-mentioned 50 inclined-planes collarium access sterilizing, 16h cultivated by 37 DEG C of 180rpm shaking tables, bacterium liquid centrifuging and taking supernatant liquor, carry out the bacteriostatic test of Salmonella, Listeria monocytogenes, obtain bacterial strain 6 strain that inhibition zone is large, suppress above-mentioned two kinds of pathogenic bacterium.Again respectively by each for 6 strain inclined-planes collarium access liquid CM substratum, 37 DEG C of 180rpm that spend the night cultivate, and measure proteinase activity in supernatant liquor, get most live high-enzyme strain, are experimental strain, name YSJB-30.
Embodiment two: the pcr amplification extracting phage gene group DNA and 16SrDNA:
Picking one collarium inclined-plane thalline access 2ml CM liquid nutrient medium, cultivate 16h ~ 18h for 37 DEG C, get 1.5ml and activate bacterium liquid, centrifugal 1 min of 12 000r/min, abandon supernatant liquor, collect thalline, the method extracted on test kit specification sheets according to bacterial genomes DNA extracts bacterial genomes DNA, and-20 DEG C save backup.The genomic dna of the sepharose Detection and Extraction of 0.8%, gel imaging system is observed and is taken pictures, and sees Figure of description.
Pcr amplification 16SrDNA sequence:
Primer: 799F 5 '-AACAGGATTAGATACCCTG-3 ', 1492R 5 '-GGTTACCTTGTTACGACTT-3 '.
With the phage gene group DNA extracted for template, carry out the pcr amplification of 16SrDNA.Pcr amplification reaction system 25 μ L:
10 × PCR buffer 2.5 uL, dNTP (2.5mM) (TaKaRa) 2 uL, 799F (10pmol/uL) (invitrogen) 0.1 uL, 1492R (10pmol/uL) (invitrogen) 0.1 uL, Taq polysaccharase (5U/uL) (TaKaRa) 0.125 uL, the DNA that Templet(extracts) 0.5 uL, ddH2O to 25 uL.
Pcr amplification program: 94 DEG C of denaturation 5min.94 DEG C of sex change 1min, 52 DEG C of annealing 1min, 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min.4 DEG C for subsequent use.Above-mentioned PCR product through 1% agarose gel electrophoresis, voltage 100 V, electrophoresis terminates, gel imaging system observe take pictures, see Figure of description.
Reclaim 16SrDNA pcr amplification product, send to order-checking, sequencing result is shown in Figure of description.Sequencing result is carried out Blast comparison at NCBI, and combining form feature, is initially identified as subtilis.
Embodiment three: subtilis YSJB-30 bacterial strain bacteriostatic test:
Get the liquid CM substratum 10 mL/100mL triangular flask of sterilizing, every bottle graft enters subtilis YSJB-30 inclined-plane thalline one collarium activated, cultivate 24 h for aerobic 35 ~ 36 DEG C, the centrifugal 1min of 8000 rpm, get supernatant liquor, Listeria monocytogenes after picking activation, the each collarium of Salmonella thalline, access 37 DEG C of overnight incubation in CM liquid nutrient medium respectively, gradient dilution suitable multiple, be coated with dull and stereotyped, after leaving standstill 20 min, punch with punch tool, the aseptic technique of every hole adds above-mentioned supernatant liquor 100 ul, leave standstill 30 min, add supernatant liquor to expiring hole, 37 DEG C of overnight incubation, observe and measure antibacterial circle diameter,
Embodiment four: subtilis YSJB-30 extracellular protein enzymatic determination:
Liquid CM substratum 10 mL/100mL triangular flask, accesses subtilis YSJB-30 inclined-plane thalline one collarium activated, and cultivates the centrifugal 1min of 24 h, 8000 rpm, gets supernatant liquor for aerobic 35 ~ 36 DEG C, measure extracellular protein enzyme activity 39U/ml.
Embodiment five: the determination of fermentation raw material proportioning:
Respectively in stalk: rice hulls: dregs of beans ratio 3:1:6,4:1:5,5:1:4,6:1:3,7:1:2,8:1:1 carry out the preparation of solid-state fermentation culture medium, and moisture addition is 50%, under the condition of 36 DEG C, cultivate 36 h, sampling and measuring subtilis viable count, result is as following table 1:
Table 1 fermentation raw material proportioning
| Stalk: rice hulls: dregs of beans | 3:1:6 | 4:1:5 | 5:1:4 | 6:1:3 | 7:1:2 | 8:1:1 |
| Viable count (CFU/g) | 1.2×10 9 | 8.5×10 8 | 4.8×10 8 | 5.6×10 7 | 2.5×10 7 | 3.6×10 6 |
In conjunction with northern area plantation processing food crop kind (corn, paddy rice, soybean etc.), to reduce the cost of fermentation raw material and to improve raw material cost performance for foundation, optimum material proportion selects stalk: rice hulls: dregs of beans ratio is 4:1:5, microbial inoculum viable count is 8.5 × 10
8cFU/g.
The significant factors of embodiment six: PB test design screening impact test.
Test according to Plackett-Burman test design, often organize 3 parallel tests, get its mean value, the test design scheme of PB and the results are shown in following table 2, each factorial effect and significance analysis the results are shown in Table 3.
Table 2 Plackett-Burman test design and result
| Sequence number | A | B | C | (D) | E | F | (G) | H | I | (J) | K | Thalline quantity (× 10 9CFU/g） |
| 1 | 8 | 0.16 | 0.04 | -1 | 2 | 38 | 1 | 1 | 0.2 | -1 | 50 | 3.18 |
| 2 | 8 | 0.08 | 0.08 | 1 | 1 | 30 | -1 | 1 | 0.2 | 1 | 80 | 2.67 |
| 3 | 8 | 0.08 | 0.04 | -1 | 1 | 38 | -1 | 2 | 0.2 | 1 | 50 | 3.12 |
| 4 | 5 | 0.16 | 0.08 | 1 | 1 | 38 | -1 | 1 | 0.1 | 1 | 50 | 3.94 |
| 5 | 8 | 0.16 | 0.08 | 1 | 1 | 38 | 1 | 2 | 0.1 | -1 | 80 | 4.49 |
| 6 | 8 | 0.16 | 0.04 | -1 | 2 | 30 | -1 | 1 | 0.1 | -1 | 80 | 3.5 |
| 7 | 5 | 0.08 | 0.04 | 1 | 1 | 30 | 1 | 1 | 0.1 | -1 | 50 | 2.69 |
| 8 | 8 | 0.08 | 0.08 | 1 | 2 | 30 | 1 | 2 | 0.1 | 1 | 50 | 3.45 |
| 9 | 5 | 0.16 | 0.08 | 1 | 2 | 30 | -1 | 2 | 0.2 | -1 | 50 | 3.82 |
| 10 | 5 | 0.08 | 0.08 | -1 | 2 | 38 | 1 | 1 | 0.2 | 1 | 80 | 4.86 |
| 11 | 5 | 0.08 | 0.04 | -1 | 2 | 38 | 1 | 2 | 0.1 | 1 | 80 | 4.65 |
| 12 | 5 | 0.16 | 0.04 | -1 | 1 | 30 | -1 | 2 | 0.2 | -1 | 80 | 3.86 |
Table 3 Plackett-Burman tests each factorial effect value and significance analysis
Find out by table 3, K
2hPO
4concentration, MgSO
4concentration, corn steep liquor content, leavening temperature, glucose content and amount of water numerical value be positive number, illustrate that above-mentioned factor is on viable count impact display positive-effect; Initial pH value, ammonium sulphate content effect value all shows negative, illustrates that these two factors are on viable count impact display negative effect.In table 3, confidence level is greater than the factor of 90% is initial pH value, corn steep liquor content, temperature, glucose content and amount of water, and wherein the confidence level of initial pH value, temperature and amount of water is greater than 95%, is the remarkable factor affecting viable count.Therefore to improve solid state fermentation thalline quantity, suitably should reduce initial pH value, raising leavening temperature and amount of water.
Embodiment seven: steepest hill climbing test.
Affect 3 remarkable factors vary step-lengths of solid state fermentation viable count, the test design in direction and the results are shown in Table 4.
Table 4 steepest hill climbing test
| Sequence number | Initial pH value | Leavening temperature (DEG C) | Amount of water (%) | Thalline quantity (× 10 9CFU/g） |
| 1 | 7.8 | 32 | 60 | 3.2 |
| 2 | 7.4 | 34 | 65 | 3.9 |
| 3 | 7.0 | 36 | 70 | 4.6 |
| 4 | 6.6 | 38 | 75 | 4.3 |
| 5 | 6.2 | 40 | 80 | 3.7 |
As can be seen from Table 4, the rangeability that steepest hill climbing test chooses initial pH value, leavening temperature and amount of water concentration is followed successively by 0.2,2,5, wherein the 3rd group of thalline quantity is maximum, illustrate that optimal conditions of fermentation is near this, therefore using the central point that the level of the 3rd group is tested as Box-benhnken, carry out following test.
Embodiment eight: Box-benhnken test design determination optimised process.
The factor determined is tested according to PB, and the central point that steepest hill climbing test obtains, adopt Box-benhnken center combination test design, with initial pH value (A), leavening temperature (B) and amount of water (C) for independent variable(s), thalline quantity (R
1) be response value design experiment.Independent variable(s) level code in table 5, test design scheme and the results are shown in Table 6.
Table 5 Box-benhnken test design level of factor coding schedule
Table 6 Box-benhnken testing program and result
Utilize Design Expert 7.0 software to carry out variance analysis to test-results, the results are shown in Table 7(P value <0.05 is remarkable item).
By carrying out multiple regression matching to testing data, obtain viable count (R
1) to the regression equation of independent variable(s) initial pH value (A), leavening temperature (B) and amount of water (C) be: thalline quantity=+ 5.77+0.095*A+0.12*B+0.27*C+0.24* A * B-0.43* A * C-0.41* B * C-1.54* A2-1.26* B2-0.83* C2.
Table 7 Box-benhnken tests the results of analysis of variance
Utilize Design Expert 7.0 response surface optimization analytical procedure to analyze regression model, find optimal response and the results are shown in Table 8.
Table 8 response surface optimizing result
| Factor | Actual value transforms | Arrangement value | Thalline quantity (× 10 9CFU/g） |
| Initial pH value | 7.0 | 7.0 | |
| Leavening temperature (DEG C) | 36.1 | 36.0 | 5.79 |
| Amount of water (%) | 70.1 | 70.0 |
For the reliability of inspection Responds Surface Methodology acquired results, under optimal conditions of fermentation, carry out three revision tests, the actual thalline number average of gained is 5.35 × 10
9cFU/g, the good fit property between predictor and trial value confirms the validity of model.Therefore, best fermentation parameter: initial pH 7.0, leavening temperature 36 DEG C, amount of water 70%, K
2hPO
4content 1.2%, MgSO
4content 0.06%, corn steep liquor content 0.75%, glucose content 1.5%, ammonium sulphate content 0.15%, fermentation time 36 h, solid state fermentation thalline quantity 5.35 × 10
9cFU/g.
Claims (7)
1. a boar source subtilis, is characterized in that: strain name YSJB-30, and Classification And Nomenclature is subtilis
bacillus subtilis, preserve and be numbered CGMCC No.9660, preservation date: on September 15th, 2014, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
2. the pig source bacillus subtilis strain according to right 1, is characterized in that: described bacterial strain vitro inhibition pathogen enterobacteria pellet increases listeria bacteria, Salmonella is positive.
3. the pig source bacillus subtilis strain according to right 1, is characterized in that: bacterial strain YSJB-30 produces extracellular protease.
4. utilize bacterial strain solid state fermentation described in right 1 to prepare pig source probiotic agent, it is characterized in that: with grain and oil by product stalk, rice hulls, dregs of beans for raw material, mass ratio is stalk: rice hulls: dregs of beans=3 ~ 5:1:4 ~ 6.
5. utilize bacterial strain solid state fermentation described in right 1 to prepare pig source probiotic agent, it is characterized in that: solid-state fermentation process is initial pH 5 ~ 8, leavening temperature 30 ~ 38 DEG C, amount of water 50 ~ 80%, K
2hPO
40.8 ~ 1.6%, MgSO
40.04 ~ 0.08%, corn steep liquor 0.5 ~ 1%, glucose 1 ~ 2%, (NH
4)
2sO
40.1 ~ 0.2%.
6. utilize bacterial strain solid state fermentation described in right 1 to prepare pig source probiotic agent, it is characterized in that: fermentation time 36h, subtilis number of viable reaches 1 x10
9cFU/more than g.
7. the subtilis probiotic agent that method described arbitrarily in claim 1-6 obtains.
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| CN116391792A (en) * | 2022-11-17 | 2023-07-07 | 东北农业大学 | Protein fermented feed prepared under non-sterilization condition |
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| CN116391792A (en) * | 2022-11-17 | 2023-07-07 | 东北农业大学 | Protein fermented feed prepared under non-sterilization condition |
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