CN111713607B - Antibacterial peptide-containing feed prepared based on bacillus solid state fermentation, preparation process thereof and fermentation strain - Google Patents
Antibacterial peptide-containing feed prepared based on bacillus solid state fermentation, preparation process thereof and fermentation strain Download PDFInfo
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Abstract
The application provides a preparation method of feed additive containing antibacterial peptide based on bacillus solid state fermentation, which selects bacillus (CGMCC 19541) as main matrix, and generates antibacterial peptide and protease by solid state fermentation; the technological conditions for producing the antibacterial peptide by solid state fermentation are as follows: sodium glutamate addition amount is 1-5%, potassium chloride addition amount is 0.1-0.5%, magnesium sulfate addition amount is 0.1-0.6%, dipotassium hydrogen phosphate addition amount is 0.1-0.3%, and glucose addition amount is 1-4%; the feed liquid ratio is 1:0.9, the inoculum size is 3-8%, the fermentation temperature is 37-45 ℃, and the fermentation time is 24-72h; feS (FeS)The addition amount is 0.5-3 mg/kg, cuS1.0‑3.0 mg/kg，MnS 0.2‑0.8 mg/kg，ZnSThe addition amount is 0.2-0.8mg/kg. The protease and the novel antibacterial lipopeptid protease produced by fermentation can effectively remove the anti-nutritional factors such as antigen protein and the like in the bean pulp, and prevent and treat diarrhea caused by dyspepsia of piglets due to the anti-nutritional factors; the novel antibacterial lipopeptid can show good inhibition effect on intestinal pathogenic bacteria, so that intestinal microbiota of piglets is improved.
Description
Technical Field
The application relates to the technical field of feeds, in particular to a feed containing antibacterial peptide prepared based on bacillus solid state fermentation, a preparation process thereof and a fermentation strain.
Background
One of the biggest problems faced by the breeding industry is that animals have multiple diseases in a growing period, and the key of animal health is to enhance the disease resistance of the animals. Antibiotics are often used in livestock and poultry farming as growth promoting factors in large quantities, which also makes a great contribution to the farming industry. However, the long-term and large-scale use of antibiotics breaks the microecological balance of animal organisms, causes the generation of drug-resistant bacteria and the variation of bacteria, is easy to cause endogenous infection or secondary infection of animals, and meanwhile, the drug residue in meat products occurs at the same time, thereby directly threatening the health of human beings.
The antibacterial peptide is also called as antimicrobial peptide or host defensive peptide, is a small peptide substance widely existing in natural organisms, is an important component of the innate immune system of the organisms, and is an ideal antibiotic substitute product because the antibacterial peptide has wide inhibition effect on bacteria, fungi, parasites, viruses, tumor cells and the like, and is not easy to generate drug resistance.
Disclosure of Invention
The application provides a method for preparing a feed additive containing antibacterial peptide based on bacillus solid state fermentation, which uses bacillus fermentation technology to create a special feed additive specially aiming at animals with imperfect intestinal development such as piglets; the soybean meal is used as a raw material for fermentation, so that protease and novel antibacterial lipopeptid can be synthesized.
The protease can effectively remove anti-nutritional factors such as antigen proteins and the like in the soybean meal, and prevent and treat diarrhea caused by dyspepsia of piglets due to the anti-nutritional factors; the novel antibacterial lipopeptide can show good inhibition effect on intestinal pathogenic bacteria, so that the intestinal microbiota of piglets is improved, and an important foundation is laid for the establishment of early intestinal microecological environment of the piglets.
The application provides a preparation process of feed containing antibacterial peptide based on bacillus solid state fermentation, which selects bacillus (CGMCC 19541) as a main matrix, and generates antibacterial peptide and protease by solid state fermentation; the technological conditions for producing the antibacterial peptide by solid state fermentation are as follows: sodium glutamate addition amount is 1-5%, potassium chloride addition amount is 0.1-0.5%, magnesium sulfate addition amount is 0.1-0.6%, dipotassium hydrogen phosphate addition amount is 0.1-0.3%, and glucose addition amount is 1-4%; the feed liquid ratio is 1:0.9, the inoculum size is 3-8%, the fermentation temperature is 37-45 ℃, and the fermentation time is 24-72h; feS (FeS)The addition amount is 0.5-3 mg/kg, cuS>1.0-3.0 mg/kg，MnS 0.2-0.8 mg/kg，ZnS/>The addition amount is 0.2-0.8mg/kg.
Preferably, the fermentation seed liquid medium formulation: peptone 10.0 g, K2HP0.05 g, glucose 0.85 g,KCl 0.17 g,MgS->0.1 g, L-sodium glutamate 0.85 g, feS/>0.13 mg，MnS/> 0.16 mg，CuS/>0.13 mg，ZnS/>0.13 mg, distilled water 35.0 mL, pH 7.0.
The application relates to a bacillus used for fermentation in a preparation process of a feed containing antibacterial peptide, which is prepared based on bacillus solid state fermentation, wherein the strain is named as bacillus subtilis (Latin school name Bacillus subtilis N-2), the preservation number is CGMCC No.19541, the preservation date is 30 days in 3 months in 2020, the preservation unit is the China general microbiological culture Collection center, and the preservation place is North Chen and West Lu No.1 in the Korean region of Beijing city, and the preservation place is No. 3.
The application relates to a feed containing antibacterial peptide, which is prepared based on bacillus solid state fermentation, and the antibacterial peptide is novel antibacterial lipopeptid (AP-I, 1070.2Da and AP-II, 1084.0 Da) with a primary structure of beta-NH-fatty acid-Asn-Tyr-Asn-Ser-Asn-Gln-Pro.
The content of the antibacterial peptide produced by the optimized fermentation medium can reach 450mg/kg-510mg/kg, and the experimental measurement value is 487.92mg/kg.
After fermentation, the protein in the soybean meal raw material is efficiently degraded, and the small-molecular acid-soluble protein in the product accounts for more than 50% of the total protein (molecular weight is less than 10000 Da), so that the product has high digestibility and is beneficial to intestinal health and nutrient absorption of animals; and at the same time, more than 90% of digestion inhibition factors such as antigen proteins, oligosaccharides and the like which are rich in the soybean meal raw materials are removed. The bacillus CGMCC19541 is used for producing protease through solid fermentation, so that the protein in the soybean meal can be effectively degraded, the content of anti-nutritional factors such as antigen protein and the like is reduced, and animal nutrition is promoted; meanwhile, the proper substrate is provided for the synthesis of the antibacterial peptide through degradation of the produced small molecular protein, and the most effective antibacterial peptide yield is achieved through the combination of the small molecular protein and the antibacterial peptide.
After the antibacterial peptide feed additive is used for replacing soybean meal in the common ration of piglets, the original anti-nutritional factors in the soybean meal raw materials are effectively removed due to the fact that protease is produced by bacillus fermentation, and the main causes of diarrhea of the piglets are eliminated; and the antibacterial peptide can effectively inhibit harmful microorganisms related to intestinal inflammation of piglets, and improve intestinal microecological environment. Therefore, compared with a control group, the daily gain of the piglets of the experimental group is improved by 25 g/day, and the feed weight ratio is also improved, which proves that the feed digestion and absorption level of the weaned piglets can be effectively improved by the feed additive. More importantly, the weaning diarrhea rate of the piglets in the experimental group is only 6.8%, and the diarrhea rate of the piglets in the control group is as high as 32.1%, so that the follow-up significant influence on the survival rate of the piglets is generated.
Research shows that the L-sodium glutamate is a good organic nitrogen source generated by the antibacterial peptide; the effective addition of potassium chloride can promote the secretion of the antibacterial peptide; magnesium sulfate is an important stimulus in the synthesis of antimicrobial peptides. On the basis, the content of the antibacterial peptide produced by using the optimized fermentation medium reaches 487.92mg/kg, and the culture condition not only can obviously improve the yield of the antibacterial peptide, but also has low cost, and is suitable for large-scale industrial fermentation production, thereby having important application significance.
Drawings
FIG. 1 is a graph showing the effect of the addition amount of sodium L-glutamate on lipopeptide yield and antibacterial rate;
FIG. 2 is a graph showing the effect of potassium chloride addition on lipopeptide yield and bacteriostatic rate;
FIG. 3 is a graph showing the effect of magnesium sulfate addition on lipopeptide yield and bacteriostatic rate;
FIG. 4 is a graph showing changes in ultraviolet absorbance at 260nm and 280nm of the culture supernatant of the indicator bacteria;
FIG. 5 is a LC-ESI-MS/MS analysis and molecular structure diagram of antibacterial peptide m/z 1070.2.
Detailed Description
The present application will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The application provides a preparation process of feed containing antibacterial peptide based on bacillus solid state fermentation, which selects bacillus (CGMCC 19541) as a fermentation strain, and generates antibacterial peptide and protease by solid state fermentation; the optimal technological conditions for producing the antibacterial peptide by solid state fermentation by taking the antibacterial rate and the antibacterial peptide yield as evaluation indexes are as follows: sodium glutamate addition amount is 1-5%, potassium chloride addition amount is 0.1-0.3%, magnesium sulfate addition amount is 0.1-0.4%, dipotassium hydrogen phosphate addition amount is 0.1%, glucose addition amount is 1%; the feed liquid ratio is 1:0.9, the inoculation amount is 8%, the fermentation temperature is 37 ℃, and the fermentation time is 48 hours. Carrying out single-factor optimization experiments on metal elements influencing the fermentation process to finally determine FeSThe addition amount is 1 mg/kg, cuS->1.8 mg/kg，MnS/> 0.6 mg/kg，ZnS/>The addition amount is 0.5mg/kg. The content of the antibacterial peptide produced by the optimized fermentation medium reaches 487.92mg/kg.
The formula of the fermentation seed liquid culture medium comprises the following steps: peptone 10.0 g, K2HP0.05 g, glucose 0.85 g,KCl 0.17 g,MgS->0.1 g, L-sodium glutamate 0.85 g, feS +.> 0.13 mg，MnS/> 0.16 mg，CuS/>0.13 mg，ZnS/>0.13 mg, distilled water 35.0 mL, pH 7.0.
The application relates to a bacillus used for fermentation in a preparation process of a feed containing antibacterial peptide, which is prepared based on bacillus solid state fermentation, wherein the strain is named as bacillus subtilis (Latin school name Bacillus subtilis N-2), the preservation number is CGMCC No.19541, the preservation date is 30 days in 3 months in 2020, the preservation unit is the China general microbiological culture Collection center, and the preservation place is North Chen way No.1 and No. 3 in the North Chen area of the Korean of Beijing city.
The content of the antibacterial peptide produced by the optimized fermentation medium reaches 487.92mg/kg.
Extraction of antibacterial peptide: the antibacterial peptide is extracted by water-mentioned acid precipitation method. Mixing the fermented substrate with distilled water at a ratio of 1:10 (w/v) at room temperature, stirring for 1h, leaching, centrifuging at a speed of 4800 r/min for 10 min to remove insoluble substances, obtaining antibacterial peptide crude extract, taking the antibacterial peptide crude extract as detection liquid for detecting antibacterial effect of the fermented product, and calculating antibacterial rate; 6 mol/L HCl is added to the crude extract to a final pH of 2.0, and the crude extract is stored at 4 ℃ overnight to precipitate crude lipopeptide, the precipitate is taken out after centrifugation at 4800 r/min for 20 min at 4 ℃, the precipitate is dried in an oven at 60 ℃ for 24h, and the dry weight of the crude extract of the antimicrobial lipopeptide is calculated by weighing, and the minimum inhibitory concentration MIC and the minimum inhibitory concentration MBC of the antimicrobial peptide are determined.
And (3) carrying out further structural analysis and identification on the antibacterial peptide component obtained by extracting the solid fermentation product with alcohol by adopting a liquid chromatography-mass spectrometry technology. Two fractions with m/z 1070.2, 1084.0 were obtained and subjected to secondary mass spectrometry.
A series of y+ fragment ion peaks with N-terminal fragmentation are visible according to CID collision principle:
m/z 426 (Ser4-Asn3-Gln2-Pro1)，
541 (Asn5-Ser4-Asn3-Gln2-Pro1)，
703 (Tyr6-Asn5-Ser4-Asn3-Gln2-Pro1)，
817 (Asn7-Tyr6-Asn5-Ser4-Asn3-Gln2-Pro1)。
b+ ion fragment peak from C-terminal cleavage:
b+ fragment ion 252 ( β-NH fatty acid )，
366 ( β-NH fatty acid-Asn7)，
643 ( β-NH fatty acid-Asn7-Tyr6-Asn5)，
730 (β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4)，
844 (β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4-Asn3)，
972(β-NHfattyacid-Asn7-Tyr6-Asn5-Ser4-Asn3-Gln2)。
the antibacterial peptide with m/z of 1070.2 is proved to be a lipopeptide structure, and the primary structure is beta-NH-fatty acid-Asn-Tyr-Asn-Ser-Asn-Gln-Pro. m/z 1084.0 is a lipopeptide having a molecular weight that differs from m/z 1070.2 by 14 Da (CH 2), which are homologs of the same amino acid sequence but different carbon chain lengths.
Evaluation of the Effect on disruption of bacterial cell membranes:
determination of the MIC of the antibacterial peptide by a double dilution method: the initial concentration of 1.28. 1.28 mg/mL of the antibacterial peptide extract was prepared to a concentration of 640, 320, 160, 80, 40, 0. Mu.g/mL using a beef extract peptone liquid medium, 200. Mu.L of the above culture solution was added to each tube using a sterile test tube, and 1 mL indicator bacteria liquid (concentration of about 10 4 Per mL), culturing at 37 ℃ for 20-24 h, and observing the growth of the strain to indicate that the minimum concentration of the strain which does not grow is the Minimum Inhibitory Concentration (MIC). In the beef extract peptone mixed culture solution, the growth of bacteria was observed visually in the tube at a concentration of 40. Mu.g/mL, and the growth of the indicator bacteria was not observed in the remaining tubes, so that the MIC thereof was 80. Mu.g/mL.
The activated indicator bacteria were inoculated at 1% (initial bacterial density: about 10) 3 cfu/mL) is inoculated in beef extract peptone liquid culture mediumCulturing at 37deg.C at 160r/min, and adding antibacterial peptide extractive solutions at 0h, 2h, 3h, 5h, and 6h respectively to obtain final concentration of antibacterial peptide of 1×MIC; the control group was incubated with sterile water at 0 h. Samples were taken at different times, centrifuged at 4800 r/min for 5min, the supernatant was retained, absorbance values were measured at 260nm and 280nm, and absorbance change curves were plotted.
Antibacterial peptides lead to leakage of bacterial macromolecular substances such as nucleic acids by causing rupture of cell membranes, resulting in an increase in ultraviolet absorbance values at 260nm and 280 nm. As shown in fig. 4, the ultraviolet absorption of the supernatant of the control group decreased with the increase of the culture time, and the absorbance value of the experimental group increased with the increase of the culture time after the addition of the antibacterial peptide.
Animal experiment design:
50 weaned piglets of 28 days are selected and randomly divided into two treatment groups, and the total experiment is 32 days. Wherein the control group is fed with common daily ration (the content of the soybean meal is 28%), the experimental group replaces the soybean meal in the common daily ration of piglets with the product of the application, and the rest components are unchanged.
The experimental results are shown in the following table:
after the antibacterial peptide feed additive is used for replacing soybean meal in the common ration of piglets, the original anti-nutritional factors in the soybean meal raw materials are effectively removed due to the fact that protease is produced by bacillus fermentation, and the main causes of diarrhea of the piglets are eliminated; and the antibacterial peptide can effectively inhibit harmful microorganisms related to intestinal inflammation of piglets, and improve intestinal microecological environment. Therefore, compared with a control group, the daily gain of the piglets of the experimental group is improved by 25 g/day, and the feed weight ratio is also improved, which proves that the feed digestion and absorption level of the weaned piglets can be effectively improved by the feed additive. More importantly, the weaning diarrhea rate of the piglets in the experimental group is only 6.8%, and the diarrhea rate of the piglets in the control group is as high as 32.1%, so that the follow-up significant influence on the survival rate of the piglets is generated.
The above-described embodiments should not be construed as limiting the scope of the application, but it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the inventive concept. Accordingly, the scope of the application should be assessed as that of the appended claims.
Claims (4)
1. A preparation process of feed containing antibacterial peptide based on bacillus solid state fermentation is characterized in that bacillus subtilis is selected as a fermentation strain, and antibacterial peptide and protease are produced through solid state fermentation; the antibacterial peptide component is antibacterial lipopeptide, and the primary structure is beta-NH-fatty acid-Asn-Tyr-Asn-Ser-Asn-Gln-Pro; the technological conditions for producing the antibacterial peptide by solid state fermentation are as follows: sodium glutamate addition amount is 1-5%, potassium chloride addition amount is 0.1-0.5%, magnesium sulfate addition amount is 0.1-0.6%, dipotassium hydrogen phosphate addition amount is 0.1-0.3%, and glucose addition amount is 1-4%; the feed liquid ratio is 1:0.9, the inoculum size is 3-8%, the fermentation temperature is 37-45 ℃, and the fermentation time is 24-72h; feSO 4 The addition amount is 0.5-3 mg/kg, cuSO 4 1.0-3.0 mg/kg，MnSO 4 0.2-0.8 mg/kg，ZnSO 4 The addition amount is 0.2-0.8mg/kg; the formula of the fermentation seed liquid culture medium is as follows: peptone 10.0 g, K 2 HPO 4 0.05 g, glucose 0.85 g,KCl 0.17 g,MgSO 4 0.1 g, L-sodium glutamate 0.85 g, feSO 4 0.13 mg，MnSO 4 0.16 mg，CuSO 4 0.13 mg，ZnSO 4 0.13 mg, distilled water 35.0 mL, pH 7.0; the bacillus subtilis is named as bacillus subtilis (Latin brand Bacillus subtilis) N-2, the preservation number is CGMCC No.19541, the preservation date is 30 days of 3 months in 2020, the preservation unit is China general microbiological culture Collection center, and the preservation address is North Chen Xili No.1 and 3 in the Korean region of Beijing city.
2. The preparation process of the antibacterial peptide-containing feed prepared based on the solid state fermentation of bacillus according to claim 1, wherein the addition amount of dipotassium hydrogen phosphate is 0.1% and the addition amount of glucose is 1%; the feed liquid ratio is 1:0.9, the inoculation amount is 8%, the fermentation temperature is 37 ℃ and the fermentation time is 48 hours; feSO 4 Additive amount of 1 mg/kg, cuSO 4 1.8 mg/kg，MnSO 4 0.6 mg/kg，ZnSO 4 The addition amount is 0.5mg/kg.
3. An antibacterial peptide-containing feed prepared by solid state fermentation based on bacillus, which is prepared by adopting the preparation process according to one of claims 1-2, and is characterized in that the antibacterial peptide component is antibacterial lipopeptide, and the primary structure is beta-NH-fatty acid-Asn-Tyr-Asn-Ser-Asn-Gln-Pro; the content of the antibacterial peptide is 450mg/kg-510mg/kg.
4. The antibacterial peptide-containing feed prepared by solid state fermentation based on bacillus according to claim 3, wherein the antibacterial peptide content is 487.92mg/kg.
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