CN108504601B - Pediococcus acidilactici HEW-AP27 and application thereof - Google Patents
Pediococcus acidilactici HEW-AP27 and application thereof Download PDFInfo
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Abstract
The invention discloses a newly separated Pediococcus acidilactici (Pediococcus acidilactici) HEW-AP27 which is preserved in the China general microbiological center of the Committee for culture Collection of microorganisms (CGMCC for short, with the address of the microbiological research institute of China academy of sciences No. 3, West Lu No.1 Hotel, North Cheng, south China, the name of Beijing), the preservation number is CGMCC NO.15419, and the preservation date is 3 months and 7 days in 2018. The invention also provides a preparation method of the pediococcus acidilactici viable bacteria preparation, which specifically comprises the following steps: taking pediococcus acidilactici as a fermentation strain, adopting an optimized post-fermentation process and a fermentation culture medium, putting the fermentation medium into a tank after primary, secondary and tertiary culture, centrifuging to obtain active bacterial sludge, adding a stabilizing protective agent, granulating by a granulator, and adding a coating solution into a coating machine for coating to finally obtain the pediococcus acidilactici viable bacteria preparation. The pediococcus acidilactici HEW-AP27 has the advantages of acid resistance, cholate resistance, high temperature resistance and strong acid production performance, and can also inhibit the growth and the propagation of pathogenic bacteria.
Description
Technical Field
The invention relates to probiotics, in particular to Pediococcus acidilactici (Pediococcus acidilactici) HEW-AP27 and application thereof.
Background
Pediococcus acidilactici (Pediococcus acidilactici) is one of the important members of the group of probiotics, most of which are one of the probiotics on the surface and internal mucosal surfaces of animals. In addition, pediococcus acidilactici also produces acid, can regulate gastrointestinal flora, and maintain intestinal microecological balance. The product has antagonistic action on pathogenic microorganism in animal body, and can competitively inhibit pathogenic microorganism, enhance animal organism immunity, generate beneficial metabolite, activate acidic protease activity, participate in organism metabolism, and prevent harmful substance generation.
At present, the abuse of antibiotics, chemical synthetic drugs and hormones in feed and livestock breeding causes serious consequences, including that the hormone content in livestock and poultry products is over-standard, the drug residue is serious, human health can be damaged after people ingest the drugs, the quality of the livestock and poultry products is also reduced, and consumers are distrusted and eliminated by the market. In addition, long-term use of antibiotics can increase drug resistance of bacteria and drug-resistant strains, so that the bacteria are difficult to remove, and the safety of human beings is seriously threatened. Experts of the world health organization think that various drug-resistant germs generated by livestock and poultry can be transmitted to people through ingestion, and diseases which are difficult to cure can be generated, thus harming human health. Meanwhile, the use of a large amount of antibiotics and synthetic antibacterial drugs increases the drug residue, enhances the drug resistance of the strains, influences the immunity of animals and is easy to be infected. The pediococcus acidilactici has good stress resistance and bacteriostatic property as probiotics, can inhibit pathogenic microorganisms, is harmless to human bodies, can be used as an additive of animal feed, is called as a green novel feed additive, and is a safe strain which is allowed to be used in a feed additive variety catalog (2013) (the No. 2045 bulletin of the Ministry of agriculture of the people's republic of China). Wherein Pediocin (Pediocin) produced by Pediococcus acidilactici has inhibiting and killing effects on Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Clostridium botulinum and Clostridium perfringens. Pediocin has wide pH tolerance and tolerance to low and high temperature.
Disclosure of Invention
In view of the above, the invention aims to provide a Pediococcus acidilactici (HEW-AP 27) strain with strong stress resistance and probiotics and application thereof.
In order to achieve the purpose of the invention, the Pediococcus acidilactici HEW-AP27 provided by the invention is a Pediococcus acidilactici strain separated from intestinal contents of healthy piglets, and is determined to be Pediococcus acidilactici by colony morphology observation, physiological and biochemical characteristics, molecular biological identification and the like. The strain has the advantages of acid resistance, cholate resistance, high temperature resistance, strong acidity production and stress resistance, and has an inhibiting effect on pathogenic bacteria.
The Pediococcus acidilactici HEW-AP27 provided by the invention has been deposited in the general microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, with the address of microorganism institute of China academy of sciences, 3, institute of China academy of sciences, No.1, North Cheng West Lu, No.1, of the area facing the sun, Beijing, the postal code is 100101), the deposition number is CGMCC NO.15419, and the Pediococcus acidilactici is classified and named.
The invention also provides a culture method of the pediococcus acidilactici HEW-AP27, which comprises the following steps:
1) streak-culturing strain HEW-AP27 on a plate, culturing at 40 deg.C for 48h, picking single colony from the plate, culturing in 100mL seed culture medium at 40 deg.C for 24h with shaking at 180r/min to obtain HEW-AP27 first-grade seed solution with activity of 2.5 × 109CFU/mL。
2) Inoculating the strain HEW-AP27 primary seed liquid into a 50L fermentation tank according to the inoculation amount of 0.5-5% (V/V), performing secondary fermentation for 5-15h to obtain strain HEW-AP27 secondary seed liquid;
3) inoculating the secondary seed liquid of strain HEW-AP27 into 5000L fermenter according to 0.5-5% (V/V) for three-stage fermentation for 20-40 hr to obtain strain HEW-AP27 fermentation liquid with viable count of 3.0 × 1010CFU/mL；
4) Centrifuging the third fermentation liquid by a disc centrifuge to obtain strain HEW-AP27 active bacterial sludge (most of the bacterial sludge is microbial thallus, and a small part of the bacterial sludge is unconsumed culture medium and produced metabolite), with activity of 5.5 × 1011CFU/g。
Wherein, the seed culture medium comprises the following components in percentage by weight: 0.5-2.5% of sucrose, 0.5-1.5% of galactose, 0.1-1.2% of yeast powder, 0.5-1.5% of tryptone, 0.5-1.5% of beef extract, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.01-0.1% of calcium carbonate, 0.01-0.15% of manganese sulfate, 0. 800.05-0.15% of tween and the balance of water; pH5.8-7.2.
Preferably: 1.2% of sucrose, 0.6% of galactose, 0.4% of yeast powder, 0.75% of tryptone, 0.75% of beef extract, 0.08% of magnesium sulfate, 0.05% of dipotassium phosphate, 0.05% of calcium carbonate, 0.05% of manganese sulfate, 800.1% of tween and the balance of water; pH 6.5. + -. 0.2.
Wherein, the common conditions of the second-level fermentation culture and the third-level fermentation culture are as follows: the liquid loading amount is 35-70%, the fermentation temperature is 30-45 ℃, and the stirring speed is 80-150 r/min. Preferably: the liquid loading amount is 55%, the fermentation temperature is 40 ℃, and the stirring speed is 100 r/min.
Centrifuging the three-stage fermentation liquid by a disc centrifuge to obtain strain HEW-AP27 active bacterial sludge with activity of 5.5 × 1011CFU/g。
The secondary seed culture medium (in weight percent) is: 0.5-1.5% of glucose, 1.0-2.5% of sucrose, 0.5-2.0% of yeast extract, 0.5-2.0% of beef extract, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.05-1.02% of calcium carbonate, 0.05-0.5% of sodium chloride, 0.01-0.2% of manganese sulfate, 0. 800.05-0.15% of tween and the balance of water; pH5.8-7.2. Preferably: 1.2% of glucose, 1.5% of sucrose, 0.8% of yeast extract, 1.4% of beef extract, 0.06% of magnesium sulfate, 0.04% of dipotassium phosphate, 0.2% of calcium carbonate, 0.1% of sodium chloride, 0.04% of manganese sulfate, 800.05% of tween and the balance of water; pH 6.5. + -. 0.2.
The three-stage fermentation medium (in weight percentage) is as follows: 0.5-2.0% of cane sugar, 0.5-2.0% of molasses, 0.5-2.0% of brown sugar, 0.5-2.5% of yeast extract, 0.1-2.0% of corn steep liquor dry powder, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.01-0.2% of manganese sulfate, 78-0.15% of Tween 800.05 and the balance of water; pH5.8-7.2. Preferably: 1.0 percent of cane sugar, 0.8 percent of molasses, 0.5 percent of brown sugar, 0.75 percent of yeast extract, 0.8 percent of corn steep liquor dry powder, 0.07 percent of magnesium sulfate, 0.05 percent of dipotassium phosphate, 0.1 percent of manganese sulfate, 800.11 percent of Tween and the balance of water; pH 6.5. + -. 0.2.
In addition, the invention provides a preparation method of the pediococcus acidilactici preparation, which comprises the following steps:
active bacteria mud of pediococcus acidilactici and a stabilizing protective agent are mixed according to the mass ratio of 0.8: 1.2 mixing in a stirring tank for 20min, fully and uniformly mixing, and then adding water to adjust the humidity to 36% to obtain wet bacteria powder; putting the wet powder into a granulator for granulation to ensure that the granularity is 25 meshes, and then drying the wet powder by a granulation coating machine of a cyclone fluidized bed, wherein the air inlet temperature is controlled to be 60 +/-2 ℃, the air outlet temperature is controlled to be 32 +/-2 ℃, and the wet powder is dried for 1 hour; starting the side-spraying coating device, spraying the coating liquid to the material in the rotational flow state at a speed of 20mL/min to form a coating film layer, controlling the air inlet temperature to be 50 +/-2 ℃ and the atomization pressure to be 0.15MPa, and obtaining the 25-mesh pediococcus acidilactici viable bacteria preparation.
Wherein the stabilizing protectant: 2-10% of galactomannan, 10-20% of dextrin, 1-8% of L-sodium glutamate, 8-25% of trehalose, 5-10% of sodium alginate, 10-45% of corn starch, 1-10% of xanthan gum, 1-10% of microcrystalline cellulose, 0.1-10% of polyvinylpyrrolidone, 1-15% of glycerol, 1-5% of vegetable oil and 20-50% of water. Preferably: 3.2% of galactomannan, 10.3% of dextrin, 2.8% of L-sodium glutamate, 9.3% of trehalose, 6.2% of sodium alginate, 18% of corn starch, 4.5% of xanthan gum, 2.4% of microcrystalline cellulose, 1.5% of polyvinylpyrrolidone, 5.6% of glycerol, 2.2% of vegetable oil and 34% of water; sequentially adding the above components into water, stirring for 15min, and mixing completely.
Wherein the coating agent solution: 1-10% of corn starch, 1-5% of hydroxypropyl methyl cellulose phthalate, 2-20% of sodium alginate, 1-8% of carboxymethyl cellulose, 1-5% of Arabic gum, 2-10% of sodium alginate, 1-10% of carrageenan, 2-8% of polyacrylic resin, 1-15% of fructo-oligosaccharide, 2-8% of isomalto-oligosaccharide, 1-10% of skimmed milk powder, 2-20% of maltodextrin and 10-30% of water. Preferably: 8.3% of corn starch, 3.5% of hydroxypropyl methylcellulose phthalate, 8.5% of sodium alginate, 4.6% of carboxymethyl cellulose, 3% of Arabic gum, 6.6% of sodium alginate, 5.8% of carrageenan, 4.4% of polyacrylate resin, 5.6% of fructo-oligosaccharide, 4.2% of isomalto-oligosaccharide, 8.5% of skimmed milk powder, 12% of maltodextrin and 25% of water; sequentially adding the above components into water, stirring for 15min, and mixing completely.
In addition, the invention further provides application of the pediococcus acidilactici HEW-AP27 in production of weaned piglets.
In addition, the invention also provides application of the pediococcus acidilactici HEW-AP27 in laying hen breeding.
The pediococcus acidilactici (Lactobacillus buchneri) HEW-AP27 provided by the invention has the following microbiological characteristics: the colony of the strain HEW-AP27 is small and circular on an MRS plate, the diameter of the colony is 0.8-1.4mm, the colony is white, the surface is smooth and opaque, the colony is raised, the edge is neat and easy to pick up, the colony morphology is shown in figure 1, the microscopic morphology is shown in figure 2, the colony is gram-positive, spherical, spore-free, tetrad or pair-wise arranged, not arranged in a chain, not moving and facultative anaerobic, the suitable growth temperature range is 4-50 ℃, the optimal growth temperature is 15-45 ℃, the growth pH is 3.5-9.8, and the optimal pH is 4.5-8.5; some physiological and biochemical properties are shown in Table 1.
TABLE 1 part of the physiological and biochemical characteristics of Strain HEW-AP27
Note: "+" indicates positive reaction; "-" indicates negative reaction.
The pediococcus acidilactici HEW-AP27 has remarkable antibacterial performance, can effectively inhibit the antagonistic performance of enteropathogenic escherichia coli, pasteurella multocida, proteus vulgaris, pseudomonas aeruginosa, haemophilus, vibrio cholerae, staphylococcus aureus, pseudomonas aeruginosa, salmonella typhi, salmonella choleraesuis, salmonella gallinarum, salmonella pullorum and salmonella typhimurium, and the results are shown in Table 4, and the strain HEW-AP27 can be known to grow and propagate pathogenic bacteria such as enteropathogenic escherichia coli, pasteurella multocida, proteus, pseudomonas aeruginosa, haemophilus, vibrio cholerae, staphylococcus aureus, pseudomonas aeruginosa, salmonella typhi, salmonella choleraesuis, salmonella gallinarcoleorum, salmonella pullorum, salmonella typhimurium and the like.
The pediococcus acidilactici HEW-AP27 has strong acid production performance, can resist acid, and can still maintain the survival rate of 92 percent after being treated in the artificial gastric juice with the pH value of 1.5 for 3 hours.
The pediococcus acidilactici HEW-AP27 has strong bile salt resistance, and can still maintain 72% of survival rate after being treated in artificial intestinal juice (containing 3% of bile salt) for 3 hours.
The pediococcus acidilactici HEW-AP27 has strong high-temperature resistance, and can still keep 95 percent of survival rate after being treated in water bath at 85 ℃ for 30 min.
The pediococcus acidilactici HEW-AP27 has strong adhesion performance, and the adhesion coefficient reaches 64 CFU/cell.
The pediococcus acidilactici HEW-AP27 has certain resistance to some antibiotics, such as profofloxacin, norfloxacin, olaquindox, ofloxacin, vancomycin, kanamycin, cephalomycin VI, cefixime, gentamicin, furazolidone, amikacin, polymyxin B, colistin sulfate, streptomycin, bacitracin, enramycin, trimethoprim, compound sulfamethoxazole and quinocetone, and has certain resistance.
The pediococcus acidilactici HEW-AP27 can be used for preparing a probiotic agent.
The pediococcus acidilactici preparation prepared by the pediococcus acidilactici HEW-AP27 can be applied to weaned pig production and laying hen breeding.
The invention has the following beneficial effects:
1. the pediococcus acidilactici HEW-AP27 has obvious probiotics, can obviously inhibit the antagonistic performance of enteropathogenic escherichia coli, pasteurella multocida, proteus vulgaris, pseudomonas aeruginosa, haemophilus, vibrio cholerae, staphylococcus aureus, pseudomonas aeruginosa, salmonella typhi, salmonella choleraesuis, salmonella gallinarum, salmonella pullorum and salmonella typhimurium, and has broad-spectrum antibacterial property as shown in Table 4, and the results show that the strain HEW-AP27 has the growth and propagation of pathogenic bacteria such as enteropathogenic escherichia coli, pasteurella multocida, proteus, pseudomonas aeruginosa, haemophilus, vibrio cholerae, staphylococcus aureus, pseudomonas aeruginosa, salmonella typhi, salmonella choleraesuis, salmonella gallinarcotiana pullorum, salmonella typhimurium and the like.
2. The pediococcus acidilactici HEW-AP27 has excellent microbiological characteristics, remarkable probiotics and stress resistance and super-strong fermentation performance, can resist acid, and can keep more than 92% of activity; the product is resistant to bile salt and can keep more than 72% of activity; high temperature resistance and 95% of activity can be maintained; the adhesive has strong adhesiveness and the adhesive coefficient reaches 64CFU/cell, and is more suitable for the requirements of feed industry and livestock breeding industry.
3. The pediococcus acidilactici HEW-AP27 is applied to the production of weaned pigs, the pediococcus acidilactici preparation is added into the feed of the weaned pigs, the skin and hair of the weaned pigs are bright, the bodies of the weaned pigs are strong and beautiful, the activity is active, the average daily gain (P is less than 0.05) can be obviously improved, the average daily gain is improved by 14.39% compared with a control group, the feed conversion ratio, the diarrhea rate and the death rate (P is less than 0.05) are obviously reduced, and the feed conversion ratio, the diarrhea rate and the death rate. The addition of the pediococcus acidilactici preparation into the piglet feed is shown to significantly improve the daily gain of the piglets in the experimental group, significantly reduce the feed conversion ratio, promote the growth and feed conversion rate of the weaned piglets, and significantly reduce the diarrhea rate and the death rate, thereby improving the pig raising benefit. After the pediococcus acidilactici preparation is added, the content of lactic acid bacteria in pig manure is remarkably higher than that of a control group (P is less than 0.05), and the content of escherichia coli, staphylococcus aureus and salmonella is remarkably lower than that of the control group (P is less than 0.05), so that the pediococcus acidilactici can improve the content of beneficial bacteria in the intestinal tract of weaned pigs, reduce the content of harmful bacteria such as escherichia coli, staphylococcus aureus and salmonella, and maintain the balance of intestinal flora. It is possible that organic acid generated by pediococcus acidilactici can effectively reduce the pH value of the intestinal tract and can also inhibit the growth of pathogenic bacteria in the intestinal tract such as escherichia coli.
4. The pediococcus acidilactici HEW-AP27 is applied to the breeding of laying hens, after the pediococcus acidilactici preparation is added into the feed of the laying hens, the average laying rate of the laying hens is kept above 91%, and the laying rate of each stage of the laying hens is obviously improved compared with that of a control group (P is less than 0.05); the feed-egg ratio of each stage of the test group is obviously reduced (P is less than 0.05); the egg weight of each stage of the test group is obviously increased by 1.0-1.5 g (P < 0.05). The addition of the pediococcus acidilactici preparation can obviously improve the quality of eggs, the gloss, the eggshell thickness, the Haw's unit and the yolk color of the eggs are obviously superior to those of a control group, and the pediococcus acidilactici preparation can effectively replace antibiotic additives and is suitable for large-scale popularization and use.
Drawings
FIG. 1 is a colony morphology of Pediococcus acidilactici HEW-AP27 on MRS medium in example 1;
FIG. 2 is a gram stain plot of the Pediococcus acidilactici strain HEW-AP27 of example 1;
FIG. 3 is a phylogenetic tree of the Pediococcus acidilactici strain HEW-AP27 of example 1;
FIG. 4 is a graph showing the pH change of fermentation of Pediococcus acidilactici HEW-AP 27;
FIG. 5 is the acid production curve of Pediococcus acidilactici HEW-AP 27.
Detailed Description
The present invention is explained below with reference to examples, which are merely illustrative of the present invention. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
Example 1 separation, screening, identification and preservation of Pediococcus acidilactici HEW-AP27
1. Primary screening of strains
Taking 1g of intestinal contents of healthy piglets to perform enrichment culture in 100mL of MRS liquid culture medium for 8h, processing the cultured enrichment culture solution in a water bath kettle at 85 ℃ for 30min, carrying out partition streaking on the processed enrichment culture solution on an MRS plate, carrying out inverted culture in an incubator at 37 ℃ for 24-48h, observing the colony morphology, selecting the colony which has better growth and has the typical colony morphology of the lactic acid bacteria to perform pure culture, and preserving for later use.
2. Strain re-screening
The strains which are gram-positive, catalase-negative and non-spore-forming can be primarily determined as lactobacillus, 58 strains are respectively numbered as HEW-AP1, HEW-AP2, HEW-AP3 and … … HEW-AP58, and screening is carried out according to the following method.
The performance of only strain HEW-AP27 is described below, but the performance of the remaining strains is not as good as that of strain HEW-AP 27.
1) Preparation of fermentation broth of strain HEW-AP27
Streak-culturing strain HEW-AP27 on a plate, culturing at 40 deg.C for 48h, picking single colony from the plate, culturing in 100mL seed culture medium at 40 deg.C for 24h with shaking at 180r/min to obtain HEW-AP27 first-grade seed solution with activity of 2.5 × 109CFU/mL. Inoculating the strain HEW-AP27 first-grade seed liquid to 50L for fermentation according to the inoculation amount of 1% (V/V)And (4) performing secondary fermentation in the tank for 7 hours to obtain a secondary seed solution of the strain HEW-AP 27. Inoculating the strain HEW-AP27 secondary seed liquid into a 5000L fermentation tank according to 1% (V/V), performing three-stage fermentation for 24h to obtain strain HEW-AP27 three-stage fermentation liquid with viable count of 3.0 × 1010CFU/mL。
Wherein, the seed culture medium comprises the following components in percentage by weight: 0.5-2.5% of sucrose, 0.5-1.5% of galactose, 0.1-1.2% of yeast powder, 0.5-1.5% of tryptone, 0.5-1.5% of beef extract, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.01-0.1% of calcium carbonate, 0.01-0.15% of manganese sulfate, 0. 800.05-0.15% of tween and the balance of water; pH5.8-7.2.
Preferably: 1.2% of sucrose, 0.6% of galactose, 0.4% of yeast powder, 0.75% of tryptone, 0.75% of beef extract, 0.08% of magnesium sulfate, 0.05% of dipotassium phosphate, 0.05% of calcium carbonate, 0.05% of manganese sulfate, 800.1% of tween and the balance of water; pH 6.5. + -. 0.2.
Wherein, the common conditions of the second-level fermentation culture and the third-level fermentation culture are as follows: the liquid loading amount is 35-70%, the fermentation temperature is 30-45 ℃, and the stirring speed is 80-150 r/min. Preferably: the liquid loading amount is 55%, the fermentation temperature is 40 ℃, and the stirring speed is 100 r/min.
Centrifuging the three-stage fermentation liquid by a disc centrifuge to obtain strain HEW-AP27 active bacterial sludge with activity of 5.5 × 1011CFU/g。
The secondary seed culture medium (in weight percent) is: 0.5-1.5% of glucose, 1.0-2.5% of sucrose, 0.5-2.0% of yeast extract, 0.5-2.0% of beef extract, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.05-1.02% of calcium carbonate, 0.05-0.5% of sodium chloride, 0.01-0.2% of manganese sulfate, 0. 800.05-0.15% of tween and the balance of water; pH5.8-7.2. Preferably: 1.2% of glucose, 1.5% of sucrose, 0.8% of yeast extract, 1.4% of beef extract, 0.06% of magnesium sulfate, 0.04% of dipotassium phosphate, 0.2% of calcium carbonate, 0.1% of sodium chloride, 0.04% of manganese sulfate, 800.05% of tween and the balance of water; pH 6.5. + -. 0.2.
The three-stage fermentation medium (in weight percentage) is as follows: 0.5-2.0% of cane sugar, 0.5-2.0% of molasses, 0.5-2.0% of brown sugar, 0.5-2.5% of yeast extract, 0.1-2.0% of corn steep liquor dry powder, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.01-0.2% of manganese sulfate, 78-0.15% of Tween 800.05 and the balance of water; pH5.8-7.2. Preferably: 1.0 percent of cane sugar, 0.8 percent of molasses, 0.5 percent of brown sugar, 0.75 percent of yeast extract, 0.8 percent of corn steep liquor dry powder, 0.07 percent of magnesium sulfate, 0.05 percent of dipotassium phosphate, 0.1 percent of manganese sulfate, 800.11 percent of Tween and the balance of water; pH 6.5. + -. 0.2.
2) Determination of high temperature resistance
Taking 10mL of HEW-AP27 tertiary fermentation liquid, treating in a test tube at 85 ℃ water bath for 30min, respectively detecting the activity (viable count) before and after treatment, and calculating the survival rate, as shown in Table 2. The results in Table 2 show that the survival rate of HEW-AP27 reaches 95%, and the HEW-AP27 has strong high-temperature resistance.
TABLE 2 survival of Pediococcus acidilactici HEW-AP27 after high temperature treatment
| Bacterial strains | CK(CFU/mL) | High temperature treatment (CFU/mL) | Survival rate/%) |
| Strain HEW-AP27 | 3.0×1010 | 2.85×1010 | 95 |
3) Acid production Performance measurement
Inoculating strain HEW-AP27 into MRS liquid culture medium at an inoculum size of 1% (volume ratio), culturing at 40 deg.C for 24h, measuring pH every 2h, and detecting lactic acid content in the fermentation broth by HPLC, as shown in FIG. 4 and FIG. 5. It can be known that the pH value of the strain HEW-AP27 gradually decreases along with the prolonging of the time in the fermentation process, the content of the lactic acid also gradually increases along with the prolonging of the time, the yield of the lactic acid reaches up to 15.8g/L in 24h of fermentation, and the pH value is lowest and is 3.52.
4) Determination of acid resistance
The number of viable bacteria was determined after treating 1mL of the prepared strain HEW-AP27 tertiary fermentation broth in 9mL of artificial gastric juice (pH1.5, pH2.5, pH3.5) at different pH values for 1h, 2h and 3h, respectively, and the results are shown in Table 3. As can be seen from Table 3, the survival rate of the strain HEW-AP27 in artificial gastric juice with pH1.5, pH2.5 and pH3.5 after being respectively treated for 1 hour is more than 96%, the survival rate of the strain HEW-AP27 after being respectively treated for 2 hours is more than 95%, the survival rate of the strain HEW-AP is respectively treated for 3 hours, and the survival rate is more than 92%, which indicates that the strain HEW-AP27 has high acid resistance, can resist gastric acid and can successfully reach the intestinal tract to play a role.
Wherein, the preparation of the artificial gastric juice comprises the following steps: diluting 100g/L hydrochloric acid 16.4mL with distilled water to pH of 1.5, 2.5 and 3.5, respectively, dissolving 100mL diluted hydrochloric acid solution with pepsin 1g to obtain artificial gastric juice, and sterilizing with microporous membrane (0.22 μm).
TABLE 3 survival of Pediococcus acidilactici HEW-AP27 after treatment in simulated gastric fluid
5) Determination of the bile salt resistance
After 1mL of the prepared strain HEW-AP27 tertiary fermentation liquid is respectively treated in 9mL of artificial intestinal juice with different concentrations of bile salts (0.3%, 1%, 1.5%, 2%, 3%), 1h, 2h and 3h, the number of viable bacteria is detected, and the result is shown in Table 4. As can be seen from Table 4, the survival rate of the strain HEW-AP27 in the artificial intestinal juice containing 0.3%, 1%, 1.5%, 2% and 3% of bile salts after being respectively treated for 1h is more than 82%, the survival rate of the strain HEW-AP27 in the artificial intestinal juice after being respectively treated for 2h is more than 76%, and the survival rate of the strain HEW-AP27 in the artificial intestinal juice after being respectively treated for 3h is more than 72%, which shows that the strain has high bile salt resistance, can tolerate the bile salts in the intestinal juice, can preserve the activity and plays a role.
Wherein, the preparation of the artificial intestinal juice comprises the following steps: a1 g/L solution was prepared with pancreatin, and bile salt (0.3 wt%, 1 wt%, 1.5 wt%, 2 wt%, 3 wt%) was added to the solution, adjusted to pH 8.0 with 10 wt% NaOH, and then sterilized by filtration through a 0.45 μm microfiltration membrane.
TABLE 4 survival of Pediococcus acidilactici HEW-AP27 after treatment in Artificial intestinal juice
6) Determination of bacteriostatic Properties
The antagonistic performance of the strain HEW-AP27 on enteropathogenic escherichia coli, pasteurella multocida, proteus vulgaris, pseudomonas aeruginosa, haemophilus, vibrio cholerae, staphylococcus aureus, pseudomonas aeruginosa, salmonella typhi, salmonella choleraesuis, salmonella gallinarum, salmonella pullorum and salmonella typhimurium is detected, and the results are shown in Table 5, and the strain HEW-AP27 has strong inhibitory effect on 13 pathogenic bacteria of enteropathogenic escherichia coli, pasteurella multocida, proteus, pseudomonas aeruginosa, haemophilus, vibrio cholerae, staphylococcus aureus, pseudomonas aeruginosa, salmonella typhi, salmonella choleraesuis, salmonella gallinarum, salmonella pullorum and salmonella typhimurium and has strong antibacterial performance.
TABLE 5 bacteriostatic effect of Pediococcus acidilactici HEW-AP27 on putrefying bacteria
| Pathogenic bacteria | Bacteriostatic diameter (mm) |
| Enteropathogenic E.coli | ++++ |
| Pasteurella multocida | ++ |
| Proteobacteria | + |
| Pseudomonas aeruginosa | +++ |
| Haemophilus | ++ |
| Vibrio cholerae | ++++ |
| Staphylococcus aureus | +++ |
| Pseudomonas aeruginosa | ++ |
| Salmonella typhi | +++ |
| Salmonella choleraesuis | ++ |
| Salmonella gallinarum | +++ |
| Salmonella pullorum | +++ |
| Salmonella typhimurium | ++++ |
| Bacillus anthracis | ++ |
| Listeria monocytogenes | ++ |
Note: a greater number of "+" indicates greater bacteriostatic properties
7) Measurement of adhesion
Washing activated Pediococcus acidilactici HEW-AP27 with PBS (phosphate buffered saline, commercially available) for 3 times to obtain 108CFU/mL of the bacterial suspension, HT-29 cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% heat-inactivated Fetal Calf Serum (FCS) purchased from Gibco, USA, 1 wt% L-glutamine and 1 wt% penicillin-streptomycin, and cultured in a humid environment containing 5 wt% carbon dioxide. The cultured HT-29 cells were washed 3 times with a D-hanks balanced salt solution of Zrbioise, then digested by adding 6 to 7 drops of a mixture of 0.25% Trypsin and EDTA (0.25 wt% Trypsin-EDTA reagent from Gibco), the state of cell digestion was observed under a microscope until the cells became round and did not join together and began to slowly drop off, and 2mL of RPMI-1640 cell culture solution from Gibco, USA was added to terminate the digestion. Then using RPMI-1640 cell culture solution to make cell suspension (5X 10)4one/mL), 2mL of cell suspension was added to each well of a six-well plate previously loaded with a coverslip, 37 ℃, CO2Culturing in an incubator. After the cells are attached to the wall, the cells are washed 2 times by sterile PBS solution, and 1mL of pediococcus acidilactici HEW-AP27 bacterial liquid (containing thalli 1X 10) is added into each hole8one/mL) and 1mL of RPMI-1640 cell culture medium at 37 ℃ with CO2Culturing in an incubator for 2 h. Washing with sterile PBS, fixing with 10% formaldehyde for 0.5h, performing gram staining and microscopic examination, randomly selecting 20 fields, and calculating the total number of bacteria adhered to the cells in the fields and the number of the cells. The result shows that the adhesion coefficient of the strain is 64CFU/cell, which indicates that the strain has stronger adhesion capability. The probiotic bacteria can pass through and be fine with intestinal epitheliumCell receptor adhesion, space-occupying colonization, pathogenic bacteria adhesion prevention, pathogenic bacteria inhibition and intestinal flora balance regulation.
8) Antibiotic resistance assay
0.1mL of fermentation liquid of the strain HEW-AP27 is taken to be uniformly coated on an MRS plate, antibiotic drug sensitive paper sheets are respectively placed on the MRS plate containing HEW-AP27 and are cultured for 24-48h at 40 ℃, the diameter of the inhibition zone is observed and measured, and the result is shown in Table 6. It is known that Pediococcus acidilactici HEW-AP27 is sensitive to antibiotics such as amoxicillin, cephalothin, cephalosporin IV, cefotaxime, prohexadionene, clindamycin, doxycycline, rifampin, penicillin, clarithromycin, mezlocillin, oxacillin, azithromycin, tetracycline, roxithromycin, erythromycin, geithromycin, chloramphenicol, carbenicillin, oxytetracycline, and aureomycin; sensitive to cephalomycin V, lincomycin, furazolidone and nosiheptide poisoning; the compound norfloxacin and flufenamic acid has low sensitivity to profofloxacin, norfloxacin, olaquindox, ofloxacin, vancomycin, kanamycin, cephalomycin VI, cefixime, gentamicin, furazolin, amikacin, polymyxin B, colistin sulfate, streptomycin, bacitracin, enramycin, trimethoprim, compound sulfamethoxazole and quinocetone, and has certain drug resistance.
TABLE 6 antibiotic resistance results of Pediococcus acidilactici HEW-AP27
| Antibiotic | Diameter/mm of bacteriostatic circle | Degree of sensitivity | Antibiotic | Diameter of zone of inhibition/mm | Degree of | |
| Propofloxacin | ||||||
| 4 | Hyposensitivity | Penicillin | 26 | Extreme sensitivity | ||
| Norfloxacin hydrochloride | 5 | Hyposensitivity | Tetramine carba | 5 | Hyposensitivity | |
| Olaquindox | 9 | Hyposensitivity | Clarithromycin | 22 | | |
| Ofloxacin | ||||||
| 7 | Hyposensitivity | Mezlocillin | 20 | | ||
| Vancomycin | ||||||
| 7 | Hyposensitivity | Benzoxazole penicillin | 25 | Extreme sensitivity | ||
| Amoxicillin | 20 | Gao Min | Azithromycin | 24 | | |
| Kanamycin | ||||||
| 6 | | Tetracycline derivatives | 16 | Gao Min | ||
| Cefthiophene | 25 | Extreme sensitivity | Roxithromycin | 29 | Extreme sensitivity | |
| Cephalosporin V | 12 | Mianmin | Erythromycin | 24 | Extreme sensitivity | |
| Cephalosporin IV | 15 | Gao Min | Gitamycin | 22 | Extreme sensitivity | |
| Cephalosporin VI | 5 | | Chloromycetin | 18 | Gao Min | |
| Cefaminothiazine oxime | 22 | Extreme | Polymyxin B | 4 | Hyposensitivity | |
| Precistine (named Fenpribi) | 23 | Extreme sensitivity | Colistin sulfate | 9 | | |
| Cefixime | ||||||
| 7 | | Streptomycin | 6 | Hyposensitivity | ||
| Lincomycin | 12 | Mianmin | Bacitracin | 5 | Hyposensitivity | |
| Gentamicin | 9 | Hyposensitivity | Enramycin derivative | 8 | Hyposensitivity | |
| Clindamycin | 25 | | Trimethoprim | 7 | Hyposensitivity | |
| Doxycycline | 21 | | Carboxybenomycins | 18 | Gao Min | |
| Rifampicin | 21 | Extreme | Compound sulfamethoxazole | 4 | Hyposensitivity | |
| Furazolidones | 12 | Mianmin | Oxytetracycline | 27 | Extreme sensitivity | |
| Effective remedy for dysentery | 9 | Hyposensitivity | Aureomycin | 22 | Extreme sensitivity | |
| Nosiheptide | 13 | Mianmin | Quinocetone | 9 | Hyposensitivity |
9) Acute toxicity assay
Acute toxicity tests are carried out by referring to the maximum tolerated dose of GB15193.3-2003, and the results show that all mice do not have toxic or death phenomena, which indicates that the maximum tolerated dose MTD of the pediococcus acidilactici HEW-AP27 is more than 15000mg/kg, and the mice can be determined to be non-toxic according to the classification standard.
3. Morphological characteristics
The colony of the strain HEW-AP27 is small and round on an MRS plate, the diameter of the colony is 0.8-1.4mm, the colony is white, the surface is smooth and opaque, the colony is raised, the edge is neat and easy to pick up, the colony morphology is shown in figure 1, the microscopic morphology is shown in figure 2, the colony is gram-positive, spherical, spore-free, tetrad or pair-wise arranged, not arranged in a chain, not moving and facultative anaerobic, the growth suitable temperature range is 4-50 ℃, the optimal growth temperature is 15-45 ℃, the growth pH is 3.5-9.8, and the optimal pH is 4.5-8.5.
4. Physiological and biochemical characteristics
The strain species are identified by performing physiological and biochemical tests on the strain HEW-AP27 according to the handbook of identifying common bacteria systems. The results showed that strain HEW-AP27 was Pediococcus acidilactici. The physiological and biochemical properties of this strain are shown in Table 7.
TABLE 7 part of the physiological and biochemical characteristics of Pediococcus acidilactici HEW-AP27
5. Characteristics of molecular biology
Extracting DNA genome of strain HEW-AP27 by using bacterial DNA kit, obtaining 16S rDNA gene fragment thereof by PCR amplification, recovering PCR product, completing sequencing by bioengineering (Shanghai) limited company, performing Blast comparison analysis on the obtained sequence in GenBank, finding that homology of strain HEW-AP27 and Pediococcus acidilactici reaches 99.99%, and finally determining HEW-AP27 as Pediococcus acidilactici by combining morphological characteristics and physiological and biochemical characteristics of the strain.
The 16S rDNA sequence of the strain HEW-AP27 is shown in a sequence table. The sequence of 16S rDNA of the strain HEW-AP27 is subjected to Blast comparison with the known sequence in GenBank, the 16S rDNA of the related species is obtained from a database, a phylogenetic tree (figure 3) is constructed by utilizing Mega 6.0 software, and as can be seen from the phylogenetic tree shown in figure 3, the strain HEW-AP27 and Pediococcus acidilactici (Pediococcus acidilactici) are gathered in the same branch, and the similarity reaches 100%. And comprehensively determining that the strain belongs to the Pediococcus by the cell morphology, physiological and biochemical characteristics, 16S rDNA sequence and other experimental data of the strain, and finally determining the strain as Pediococcus acidilactici.
The Pediococcus acidilactici HEW-AP27 obtained above was deposited in China general microbiological culture Collection center (CGMCC for short, with the address of 100101, China academy of sciences institute of microbiology 3, Navy No.1, North Chen Xilu, No. 3, Kyoho, Beijing) on 7.3.2018, and the deposition number is CGMCC NO. 15419.
EXAMPLE 2 preparation of Pediococcus acidilactici preparation
The mass ratio of the pediococcus acidilactici HEW-AP27 bacterial mud obtained in example 1 to the stabilizing protective agent is 0.8: 1.2 mixing in a stirring tank for 20min, fully and uniformly mixing, and then adding water to adjust the humidity to 36% to obtain wet bacteria powder; putting the wet powder into a granulator for granulation to ensure that the granularity is 25 meshes, and then drying the wet powder by a granulation coating machine of a cyclone fluidized bed, wherein the air inlet temperature is controlled to be 60 +/-2 ℃, the air outlet temperature is controlled to be 32 +/-2 ℃, and the wet powder is dried for 1 hour; starting the side-spraying coating device, spraying the coating liquid to the material in the rotational flow state at a speed of 20mL/min to form a coating film layer, controlling the air inlet temperature to be 50 +/-2 ℃ and the atomization pressure to be 0.15MPa, and obtaining the 25-mesh pediococcus acidilactici viable bacteria preparation.
The stabilizing protective agent comprises the following components in percentage by weight: 2-10% of galactomannan, 10-20% of dextrin, 1-8% of L-sodium glutamate, 8-25% of trehalose, 5-10% of sodium alginate, 10-45% of corn starch, 1-10% of xanthan gum, 1-10% of microcrystalline cellulose, 0.1-10% of polyvinylpyrrolidone, 1-15% of glycerol, 1-5% of vegetable oil and 20-50% of water. Preferably: galactomannan 3.2%, dextrin 10.3%, L-sodium glutamate 2.8%, trehalose 9.3%, sodium alginate 6.2%, corn starch 18%, xanthan gum 4.5%, microcrystalline cellulose 2.4%, polyvinylpyrrolidone 1.5%, glycerol 5.6%, vegetable oil 2.2%, and water 34%; sequentially adding the above components into water, stirring for 15min, and mixing completely.
The coating agent solution comprises the following components in percentage by weight: 1-10% of corn starch, 1-5% of hydroxypropyl methyl cellulose phthalate, 2-20% of sodium alginate, 1-8% of carboxymethyl cellulose, 1-5% of Arabic gum, 2-10% of sodium alginate, 1-10% of carrageenan, 2-8% of polyacrylic resin, 1-15% of fructo-oligosaccharide, 2-8% of isomalto-oligosaccharide, 1-10% of skimmed milk powder, 2-20% of maltodextrin and 10-30% of water. Preferably: 8.3% of corn starch, 3.5% of hydroxypropyl methylcellulose phthalate, 8.5% of sodium alginate, 4.6% of carboxymethyl cellulose, 3% of Arabic gum, 6.6% of sodium alginate, 5.8% of carrageenan, 4.4% of polyacrylate resin, 5.6% of fructo-oligosaccharide, 4.2% of isomalto-oligosaccharide, 8.5% of skimmed milk powder, 12% of maltodextrin and 25% of water; sequentially adding the above components into water, stirring for 15min, and mixing completely.
The prepared pediococcus acidilactici preparation is stored at room temperature for 12 months, the activity of the pediococcus acidilactici is monitored every month, the result is shown in table 8, the activity of the pediococcus acidilactici preparation can still reach more than 90% after the pediococcus acidilactici preparation is stored at room temperature for 12 months, and the stability of the pediococcus acidilactici preparation is good.
TABLE 8 Pediococcus acidilactici preparation storage Property measurement
Example 3 application of Pediococcus acidilactici preparation in weaned pig production
200 weaned piglets which are healthy, have similar body weight and sex (8.8 +/-0.36) and are the same in day age are selected for the test, and are randomly divided into 2 groups (a control group and a test group), wherein each group has 5 repetitions, each repetition has 20 repetitions, and the control group is fed with basic ration (the formula is shown in table 9); the other group was a test group, which was fed with basal diet plus Pediococcus acidilactici preparation (addition amount 10)6CFU/kg piglet feed). The test period is 1 month, and all piglets in the test period are subjected to free feeding, free drinking and conventional immunization.
The fasting weight of piglets is weighed at the beginning and the end of the experiment, the health, diarrhea and death conditions of the piglets are observed every day, the feed consumption, the diarrhea and the death conditions of the piglets are recorded, the diarrhea rate, the death rate, the average daily feed intake, the average daily gain and the feed-meat ratio are calculated (see table 10), and when the experiment is ended, fresh excrement samples are randomly collected from each group, and the viable counts of lactic acid bacteria, escherichia coli, staphylococcus aureus and salmonella in the excrement samples are respectively measured (table 11).
TABLE 9 basic ration ratio and Nutrition level (in weight percent of the total weight of the ingredients)
TABLE 10 influence of Pediococcus acidilactici preparations on weaned piglet production Performance
Note: the difference of the capital letters of the shoulder marks of the same person is obvious (P <0.05)
The piglet skin and hair in the experimental period are bright, and the piglet skin and hair are body-building, active and movable. As can be seen from Table 10, the average daily gain of the test group is significantly higher than that of the control group, which is improved by 14.39% (P <0.05), the feed-meat ratio is significantly reduced by 17.74% (P <0.05), and the diarrhea rate and the mortality rate are respectively reduced by 5.98% and 3.2%. The addition of the pediococcus acidilactici preparation into the piglet feed is shown to significantly improve the daily gain of the piglets in the experimental group, significantly reduce the feed conversion ratio, promote the growth and feed conversion rate of the weaned piglets, and significantly reduce the diarrhea rate and the death rate, thereby improving the pig raising benefit.
TABLE 11 influence of Pediococcus acidilactici preparations on the faecal microbial flora of weaned piglets
Note: the difference of the capital letters of the shoulder marks of the same person is obvious (P <0.05)
As can be seen from Table 11, the content of lactic acid bacteria in the pig manure of the test group is significantly higher than that of the control group (P is less than 0.05), and the content of Escherichia coli, Staphylococcus aureus and Salmonella is significantly lower than that of the control group (P is less than 0.05), which indicates that pediococcus acidilactici can increase the content of beneficial bacteria in the intestinal tract of the weaned pig, reduce the content of harmful bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella, and maintain the intestinal flora balance. It is possible that organic acid generated by pediococcus acidilactici can effectively reduce the pH value of the intestinal tract and can also inhibit the growth of pathogenic bacteria in the intestinal tract such as escherichia coli.
Example 3 application of Pediococcus acidilactici preparation in laying hen farming
2000 healthy 24-week-old kalanchoe brown laying hens with similar body weight are selected and randomly divided into 2 groups (a control group and a test group), each test group is provided with 10 repetitions, each experiment group is provided with 10 cages, and each cage is provided with 10 chickens. Control group was basal diet (see table 12); the test group is basal diet + Pediococcus acidilactici preparation (addition amount is 100 g/t). The experimental period is 24-47 weeks, and experimental chicken feeding management and immunization program refer to the laying hen feeding management manual. The method comprises the steps of routine daily management, cage culture, free food intake and drinking. The illumination, temperature and humidity in the house are strictly carried out according to the conventional feeding management requirements. During the test period, the health and egg laying condition of the laying hens are observed daily, the egg laying number, the egg weight, the feed intake and the egg breakage number are recorded, and the feed-egg ratio, the egg laying rate, the breakage rate and the death and culling rate of the laying hens are calculated (see tables 13 and 14).
TABLE 12 basic ration formulas and nutritional levels (in weight percent of the total weight of the components)
As can be seen from table 13, in the whole experimental period, after the pediococcus acidilactici preparation was added to the layer feed, the average laying rate of the layer was maintained at 91% or more, and the laying rate of each stage of the layer was significantly increased (P < 0.05); the feed-egg ratio of each stage of the test group is obviously reduced (P is less than 0.05); the egg weight of each stage of the test group is obviously increased by 1.0-1.5 g (P < 0.05).
TABLE 13 influence of Pediococcus acidilactici preparations on the production Performance of egg-laying hens
Note: the difference of the same-person shoulder-marked letters is obvious (P <0.05)
Randomly extracting 10 eggs from each of the control group and the test group, namely 100 eggs in each group, and testing the egg quality of the extracted egg sample by using an egg quality tester. As can be seen from Table 14, the addition of the Pediococcus acidilactici preparation can significantly improve the quality of the eggs, the quality indexes of the eggs are obviously better than those of the control group in terms of gloss, eggshell thickness, Haugh unit and yolk color, and the Pediococcus acidilactici preparation can effectively replace antibiotic additives and is suitable for large-scale popularization and use.
TABLE 14 influence of Pediococcus acidilactici preparations on egg quality
Note: the difference of the same-person shoulder-marked letters is obvious (P <0.05)
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Beijing Haowo Biotechnology Co., Ltd
<120> Pediococcus acidilactici HEW-AP27 and application thereof
<130> 180289-I-CP-NZJ
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1451
<212> DNA
<213> Pediococcus acidilactici)
<400> 1
atgcaagtcg aacgaacttc cgttaattga ttatgacgtg cttgcactga atgagatttt 60
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ataacacctg gaaacagatg ctaataccgt ataacagaga aaaccgcctg gttttctttt 180
aaaagatggc tctgctatca cttctggatg gacccgcggc gcattagcta gttggtgagg 240
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actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360
caagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta aagctctgtt 420
gttaaagaag aacgtgggtg agagtaactg ttcacccagt gacggtattt aaccagaaag 480
ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat 540
ttattgggcg taaagcgagc gcaggcggtc ttttaagtct aatgtgaaag ccttcggctc 600
aaccgaagaa gtgcattgga aactgggaga cttgagtgca gaagaggaca gtggaactcc 660
atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 720
ggtctgtaac tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg 780
tagtccatgc cgtaaacgat gattactaag tgttggaggg tttccgccct tcagtgctgc 840
agctaacgca ttaagtaatc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaagaa 900
ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac 960
cttaccaggt cttgacatct tctgccaacc taagagatta ggcgttccct tcggggacag 1020
aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080
caacgagcgc aacccttatt actagttgcc agcattcagt tgggcactct agtgagactg 1140
ccggtgacaa accggaggaa ggtggggacg acgtcaaatc atcatgcccc ttatgacctg 1200
ggctacacac gtgctacaat ggatggtaca acgagtcgcg aaaccgcgag gtttagctaa 1260
tctcttaaaa ccattctcag ttcggactgt aggctgcaac tcgcctacac gaagtcggaa 1320
tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380
gcccgtcaca ccatgagagt ttgtaacacc caaagccggt ggggtaacct tttaggagct 1440
agccgtctaa g 1451
Claims (14)
1. A strain of Pediococcus acidilactici (HEW-AP 27) is preserved in the China general microbiological culture Collection center on 3, 7 and 2018, and the preservation number is CGMCC 15419.
2. The method for culturing Pediococcus acidilactici HEW-AP27 according to claim 1, comprising the steps of:
1) streak-culturing strain HEW-AP27 on a plate at 40 deg.C for 48h, picking single colony from the plate, shake-culturing in 100mL seed culture medium at 40 deg.C for 24h at 180r/min to obtain HEW-AP27 first-grade seed solution with viable count of 2.5 × 109CFU/mL；
2) Inoculating the strain HEW-AP27 primary seed liquid into a 50L fermentation tank according to the inoculation amount of 0.5-5% (V/V), performing secondary fermentation for 5-15h to obtain strain HEW-AP27 secondary seed liquid;
3) inoculating the secondary seed liquid of strain HEW-AP27 into 5000L fermenter according to 0.5-5% (V/V) for three-stage fermentation for 20-40h to obtain HEW-AP27 three-stage fermentation liquid with viable count of 3.0 × 1010CFU/mL；
4) Centrifuging the three-stage fermentation liquid by a disc centrifuge to obtain strain HEW-AP27 active bacterial sludge with viable count of 5.5 × 1011CFU/g。
3. The method according to claim 2, wherein the seed medium consists of the following components in percentage by weight: 0.5-2.5% of sucrose, 0.5-1.5% of galactose, 0.1-1.2% of yeast powder, 0.5-1.5% of tryptone, 0.5-1.5% of beef extract, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.01-0.1% of calcium carbonate, 0.01-0.15% of manganese sulfate, 0. 800.05-0.15% of tween and the balance of water; pH5.8-7.2.
4. The method according to claim 3, wherein the seed medium consists of the following components in percentage by weight: 1.2% of sucrose, 0.6% of galactose, 0.4% of yeast powder, 0.75% of tryptone, 0.75% of beef extract, 0.08% of magnesium sulfate, 0.05% of dipotassium phosphate, 0.05% of calcium carbonate, 0.05% of manganese sulfate, 800.1% of tween and the balance of water; pH 6.5. + -. 0.2.
5. The culture method according to claim 2, wherein the conditions for the secondary fermentation and the tertiary fermentation are that the liquid loading is 35-70% (v/v), the fermentation temperature is 30-45 ℃, and the stirring speed is 80-150 r/min.
6. The culture method according to claim 2, wherein the secondary medium consists of the following components in percentage by weight: 0.5-1.5% of glucose, 1.0-2.5% of sucrose, 0.5-2.0% of yeast extract, 0.5-2.0% of beef extract, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.05-1.02% of calcium carbonate, 0.05-0.5% of sodium chloride, 0.01-0.2% of manganese sulfate, 0. 800.05-0.15% of tween and the balance of water; pH5.8-7.2; the third-stage culture medium comprises the following components in percentage by weight: 0.5-2.0% of cane sugar, 0.5-2.0% of molasses, 0.5-2.0% of brown sugar, 0.5-2.5% of yeast extract, 0.1-2.0% of corn steep liquor dry powder, 0.01-0.2% of magnesium sulfate, 0.01-0.15% of dipotassium phosphate, 0.01-0.2% of manganese sulfate, 78-0.15% of Tween 800.05 and the balance of water; pH5.8-7.2.
7. The culture method according to claim 6, wherein the secondary medium consists of the following components in percentage by weight: 1.2% of glucose, 1.5% of sucrose, 0.8% of yeast extract, 1.4% of beef extract, 0.06% of magnesium sulfate, 0.04% of dipotassium phosphate, 0.2% of calcium carbonate, 0.1% of sodium chloride, 0.04% of manganese sulfate, 800.05% of tween and the balance of water; pH 6.5. + -. 0.2. The third-stage culture medium comprises the following components in percentage by weight: 1.0 percent of cane sugar, 0.8 percent of molasses, 0.5 percent of brown sugar, 0.75 percent of yeast extract, 0.8 percent of corn steep liquor dry powder, 0.07 percent of magnesium sulfate, 0.05 percent of dipotassium phosphate, 0.1 percent of manganese sulfate, 800.11 percent of Tween and the balance of water; pH 6.5. + -. 0.2.
8. A preparation method of a pediococcus acidilactici preparation is characterized by comprising the following steps:
mixing the active bacterial mud of the pediococcus acidilactici according to claim 1 and a stabilizing protective agent according to a mass ratio of 0.8: 1.2 mixing in a stirring tank for 20min, fully and uniformly mixing, and then adding water to adjust the humidity to 36% to obtain wet bacteria powder; putting the wet powder into a granulator for granulation to ensure that the granularity is 25 meshes, and then drying the wet powder by a granulation coating machine of a cyclone fluidized bed, wherein the air inlet temperature is controlled to be 60 +/-2 ℃, the air outlet temperature is controlled to be 32 +/-2 ℃, and the wet powder is dried for 1 hour; starting the side-spraying coating device, spraying the coating liquid to the material in the rotational flow state at a speed of 20mL/min to form a coating film layer, controlling the air inlet temperature to be 50 +/-2 ℃ and the atomization pressure to be 0.15MPa, and obtaining the 25-mesh pediococcus acidilactici viable bacteria preparation.
9. The preparation method according to claim 8, wherein the stabilizing protective agent consists of the following components in percentage by weight: 2-10% of galactomannan, 10-20% of dextrin, 1-8% of L-sodium glutamate, 8-25% of trehalose, 5-10% of sodium alginate, 10-45% of corn starch, 1-10% of xanthan gum, 1-10% of microcrystalline cellulose, 0.1-10% of polyvinylpyrrolidone, 1-15% of glycerol, 1-5% of vegetable oil and 20-50% of water.
10. The preparation method according to claim 9, wherein the stabilizing protective agent consists of the following components in percentage by weight: galactomannan 3.2%, dextrin 10.3%, L-sodium glutamate 2.8%, trehalose 9.3%, sodium alginate 6.2%, corn starch 18%, xanthan gum 4.5%, microcrystalline cellulose 2.4%, polyvinylpyrrolidone 1.5%, glycerol 5.6%, vegetable oil 2.2%, and water 34%.
11. The preparation method according to claim 8, wherein the coating agent solution consists of the following components in percentage by weight: 1-10% of corn starch, 1-5% of hydroxypropyl methyl cellulose phthalate, 2-20% of sodium alginate, 1-8% of carboxymethyl cellulose, 1-5% of Arabic gum, 2-10% of sodium alginate, 1-10% of carrageenan, 2-8% of polyacrylic resin, 1-15% of fructo-oligosaccharide, 2-8% of isomalto-oligosaccharide, 1-10% of skimmed milk powder, 2-20% of maltodextrin and 10-30% of water.
12. The method according to claim 11, wherein the coating agent solution comprises the following components in percentage by weight: 8.3 percent of corn starch, 3.5 percent of hydroxypropyl methylcellulose phthalate, 8.5 percent of sodium alginate, 4.6 percent of carboxymethyl cellulose, 3 percent of Arabic gum, 6.6 percent of sodium alginate, 5.8 percent of carrageenan, 4.4 percent of polyacrylic resin, 5.6 percent of fructo-oligosaccharide, 4.2 percent of isomalto-oligosaccharide, 8.5 percent of skim milk powder, 12 percent of maltodextrin and 25 percent of water.
13. A pediococcus acidilactici preparation obtained by the production method according to any one of claims 8 to 12.
14. Use of the pediococcus acidilactici preparation of claim 13 in weaned pig production or laying hen breeding.
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| CN110093289B (en) * | 2019-05-05 | 2020-12-04 | 西南大学 | Pediococcus acidilactici and application thereof |
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