CN116083262A - Lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property and preparation and application of preparation thereof - Google Patents
Lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property and preparation and application of preparation thereof Download PDFInfo
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- CN116083262A CN116083262A CN202210793310.8A CN202210793310A CN116083262A CN 116083262 A CN116083262 A CN 116083262A CN 202210793310 A CN202210793310 A CN 202210793310A CN 116083262 A CN116083262 A CN 116083262A
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Abstract
Lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property and preparation and application of preparation thereof belong to the technical field of microorganisms. The invention relates to lactobacillus plantarumLactobacillus plantarum）The FF34 has wide carbon and nitrogen sources for fermentation, has amylase activity and can utilize starchy carbon sources. The nitrite is fast in degradation and has antagonism to various aquatic common pathogenic bacteria, and the FF34 single bacterial agent is obtained through preparation or the FF34 composite bacterial agent is prepared through mixed fermentation with various bacillus. The FF34 single microbial inoculant or the composite microbial inoculant is used for fermenting feed raw materials such as fish, shrimp and crab feed, cotton meal and the like, the nutrition components of the feed are obviously improved, and the obtained fermented feed is rich in probiotics, acid soluble protein and active small peptide, tanninThe content of anti-nutritional factors such as free gossypol, phytic acid and the like is reduced; FF34 microbial inoculum is added into the feed to promote the intestinal tissue growth of aquatic animals, improve liver function, improve antioxidant activity, improve the growth performance of the cultivated animals and reduce feed coefficient. The FF34 composite microbial inoculum is immobilized and then put into the culture water body, so that the water quality can be continuously improved.
Description
Technical Field
The invention relates to a lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property, a single bacterial preparation thereof, a mixed fermentation culture method of the lactobacillus plantarum strain and bacillus, a preparation method and application of a composite microbial agent of the lactobacillus plantarum strain and the single bacterial preparation, and belongs to the technical field of microorganisms.
Background
Lactobacillus plantarum is a gram-positive lactobacillus species which is widely found in nature and is a human gastrointestinal tract probiotic group. The research shows that the lactobacillus plantarum has various beneficial effects on human bodies, such as immunity function regulation, chronic metabolic disease regulation, antagonism of pathogenic bacteria infection, intestinal function regulation, mental and nerve function regulation and the like. Because lactobacillus plantarum has good physiological characteristics and probiotic functions, lactobacillus plantarum is widely applied in the fields of food industrial production, medical care and the like. In recent years, along with the implementation and promotion of the policy of feed resistance prohibition, the urgent demands of fish meal soybean meal reduction replacement and feed development of substitute food are increasing, the application research of lactobacillus in animal cultivation is increasing, and lactobacillus plantarum is used as a catalogue variety of national microbial feed additives, and the application of silage fermentation of ruminants such as initial cattle and sheep is expanded to the research of beneficial application in the field of aquaculture. At present, the research reports that the beneficial effects of lactobacillus plantarum in aquaculture are mainly expressed as follows: (1) regulating water quality by splashing lactobacillus plantarum; (2) Through feeding with mixed bait or fermenting feed, the gastrointestinal tract environment of the aquaculture animals is improved, the growth of harmful intestinal bacteria is inhibited, the nutrition absorption of the feed is promoted, the resistance of the feed to pathogenic bacteria is enhanced, and the growth of the aquaculture animals is promoted.
At present, lactobacillus has not been applied to large scale in aquaculture production practice, and lactobacillus plantarum has limited application as a beneficial lactobacillus in aquaculture, mainly because of (1) high use cost. Lactic acid bacteria, including lactobacillus plantarum, are strict anaerobic bacteria or facultative anaerobic bacteria, have no spores, are poor in environmental stress resistance, are easy to inactivate, have high requirements for nutrition during strain proliferation and growth, have large nitrogen source demand, and the carbon source used for high-density thallus culture is mainly saccharides and cannot utilize cheap starch agricultural byproducts such as wheat middlings and the like. In addition, large-scale high-density pure-breed lactobacillus culture needs anaerobic fermentation equipment and anaerobic environment conditions, and the culture cost is high. Compared with aerobic bacteria such as bacillus subtilis, the general aerobic fermentation tank is adopted for the aerobic fermentation of the bacillus, the fermentation system is mature, the volume of the fermentation tank can reach more than 50 tons, the pure lactobacillus fermentation needs a special anaerobic system, the anaerobic fermentation tank is small in scale and cannot reach the volume scale of the aerobic fermentation, so that the pure lactobacillus living bacteria preparation lacks scale effect, and the preparation has high manufacturing cost and high use cost. (2) The aquaculture in China has various types, complex aquaculture varieties and regional environments, the applicable lactobacillus plantarum excellent strain is very lacking, the strain with aquatic pathogenic bacteria antagonistic property is less in source, and the low-cost large-scale culture method is lacking, so that the requirement of the strain serving as an antibiotic substitute can not be met. (3) lack of multifunctional novel lactobacillus plantarum. At present, the aquaculture is mainly in a high-density intensive still water aquaculture mode, the aquaculture density is high, and high-protein feeds are fed. As the utilization rate of the feed for the cultured animals is low, for example, the utilization rate of the feed protein is only about 30 percent, most of nitrogen and phosphorus in the feed are discharged into the culture water along with residual baits and excreta, so that the ecological environment of the culture water is deteriorated, the self-cleaning and regulating capacity of a pond is lost, the concentration of ammonia nitrogen and nitrite in the water exceeds the standard, the health of the cultured animals is endangered, the autoimmune capacity of the cultured animals is reduced, bacterial diseases are frequently generated, and great harm is brought to the cultivation production. Therefore, development of multifunctional novel lactobacillus plantarum strains is urgently needed, purification and regulation functions of reducing bad water quality factors such as nitrite are exerted, pathogenic bacteria are antagonized, the utilization rate of feed is improved, and the growth of cultured animals is promoted. (4) The current demand of fish meal soybean meal reduction replacement and feed development is urgent, and bacillus and lactobacillus are good candidate strains in the aspect of improving and improving the feeding nutritive value of plant protein resources such as cotton meal, rapeseed meal, mulberry leaves and the like by means of the growth metabolism of probiotics. However, the strain applied to the fermented feed at present is single, the compatibility and the synergistic effect among the strains are weak, and the effect of regulating and controlling the inflammation of the intestinal tract function restoration and aquatic animals is poor. (5) At present, although research reports on improving the growth performance of aquaculture animals by fermenting feed with lactobacillus plantarum or adding lactobacillus plantarum into the feed exist, the research on the mechanisms of nutrition utilization, metabolic influence and the like of lactobacillus plantarum on fish feed is lacking, so that popularization and application of lactobacillus plantarum are influenced. (6) The lactobacillus plantarum is facultative anaerobe, when the lactobacillus plantarum is applied in a splashing mode, the lactobacillus plantarum has poor oxygen tolerance, is easy to inactivate in an oxygen-containing aquaculture water environment, has short effective time, is easy to run off along with water when the free lactobacillus plantarum changes water in a pond, and needs to be added again, so that the aquaculture cost is increased.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum strain (Lactobacillus plantarum) FF34 with aquatic pathogen antagonistic property, a single-bacterium culture method thereof, a mixed culture method of FF34 and bacillus complex, a single-bacterium agent, a preparation method of the complex agent and application of the complex agent.
The lactobacillus plantarum FF34 has facultative anaerobism, has tolerance to oxygen, can ferment and utilize carbon and nitrogen sources widely, has amylase activity, and can directly utilize starchy carbon sources. The lactobacillus plantarum FF34 can rapidly degrade nitrite under the anoxic condition, has antagonism to aquatic common pathogenic bacteria such as Lestonia anguillarum, edwardsiella, vibrio alginolyticus, aeromonas hydrophila, escherichia coli and the like, and has remarkable inhibition effect to Vibrio alginolyticus, edwardsiella, escherichia coli and Aeromonas hydrophila.
The FF34 single-fungus aqua or powder is obtained through strain activation, immobilized fermentation culture and preparation; the lactobacillus plantarum FF34 has good biocompatibility with various beneficial bacillus, such as bacillus subtilis AB90008-15 or bacillus amyloliquefaciens JSSW-LA-07-1 or clostridium butyricum JSIM-MCB20040312 or bacillus thuringiensis JSSW-BP44 with aquatic pathogenic bacteria antagonistic property, and can be respectively subjected to co-fermentation co-culture with one of the bacillus, so as to obtain the FF34 composite microbial inoculum.
The lactobacillus plantarum FF34 single microbial inoculum or composite microbial inoculum is used for fermenting feed raw materials such as cotton meal, bean meal and the like, novel leaf feed raw materials such as mulberry leaves and the like and fish, shrimp and crab feed, can obviously improve the nutrition components and quality of the feed raw materials, is rich in probiotics, acid soluble proteins and active small peptides, and reduces the content of anti-nutritional factors such as tannin, free gossypol, phytic acid and the like; the lactobacillus plantarum FF34 single microbial agent or compound microbial agent is added into basic ration or high-starch feed, so that intestinal tissue growth of the aquaculture animals can be promoted, liver functions can be improved, antioxidant activity can be improved, growth performance of the aquaculture animals can be obviously improved, and feed coefficients can be reduced. After being adsorbed and immobilized by light stones, activated carbon or ceramsite, the lactobacillus plantarum FF34 composite microbial inoculum is put into a culture water body, can degrade the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content of the water body, inhibit the growth of aquatic pathogenic bacteria and continuously improve the water quality of the culture pond.
The technical scheme of the invention is that lactobacillus plantarum FF34 is classified and named as lactobacillus plantarum (lactobacillus plantarum) and is preserved in China Center for Type Culture Collection (CCTCC), and addresses: the preservation number CCTCC NO: m20211221.
The lactobacillus plantarum strain FF34 provided by the invention has the advantages that gram positive bacteria are rod-shaped after microscopic examination, spores are not generated, the strain belongs to non-strict anaerobic bacteria, the growth temperature range is wide, and the strain can grow at 15-45 ℃. Anaerobic culture is carried out on MRS flat plate for 48 hours, the diameter of the colony grown on the surface is 1.0-3.0 mm, the colony is round, the surface is smooth, moist, opaque, milky white, and no pigment is produced.
The lactobacillus plantarum FF34 belongs to non-strict anaerobe, facultative anaerobe, oxygen tolerance and homotype fermentation of saccharides to generate probiotic DL-lactic acid, can adapt to the water body and bottom mud environment of an aquaculture pond, can ferment and utilize wide carbon and nitrogen sources, has amylase activity and can directly utilize starchy carbon sources. The lactobacillus plantarum FF34 can rapidly degrade nitrite under the anoxic condition, has antagonism to aquatic common pathogenic bacteria such as Lestonia anguillarum, edwardsiella, vibrio alginolyticus, aeromonas hydrophila, escherichia coli and the like, and has remarkable inhibition effect to Vibrio alginolyticus, edwardsiella, escherichia coli and Aeromonas hydrophila.
Lactobacillus plantarum FF34 is prepared into single-bacterium aqua or powder by preserving strain activation, high-density immobilized fermentation culture in a fermenter and preparation; the lactobacillus plantarum FF34 can be respectively mixed, fermented and co-cultured with bacillus subtilis AB90008-15 or bacillus amyloliquefaciens JSSW-LA or bacillus coagulans JSSW-07-1 or one of bacillus pumilus JSSW-BP44 or clostridium butyricum JSIM-MCB20040312 with aquatic pathogenic bacteria antagonistic property to obtain the functional composite microbial inoculum. The lactobacillus plantarum FF34 single microbial inoculum or composite microbial inoculum is used for fermenting feed raw materials such as cotton meal, bean meal and the like, novel leaf feed raw materials such as mulberry leaves and the like and fish, shrimp and crab feed, can obviously improve the nutrition components of the feed raw materials, and the obtained fermented feed is rich in probiotics, acid soluble proteins and active small peptides, and has reduced content of anti-nutritional factors such as tannin, free gossypol, phytic acid and the like; the lactobacillus plantarum FF34 single microbial agent or compound microbial agent is added into basic ration or high-starch feed, so that intestinal tissue growth of the aquaculture animals can be promoted, liver functions can be improved, antioxidant activity can be improved, growth performance of the aquaculture animals can be obviously improved, and feed coefficients can be reduced. After the lactobacillus plantarum FF34 composite microbial inoculum is adsorbed and immobilized by light stones, activated carbon or ceramsite, the composite microbial inoculum is put into a culture water body to degrade the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content of the water body, inhibit the growth of aquatic pathogenic bacteria and continuously improve the water quality of the culture pond.
In summary, lactobacillus plantarum FF34 is a multifunctional microorganism strain capable of improving the nutritive value of feed, improving the growth performance of aquaculture animals and improving the water quality of an aquaculture pond.
The method for preparing the single bacterial agent with aquatic pathogenic bacteria antagonistic property by the lactobacillus plantarum FF34 comprises the following steps:
(1) Activating strains:
freeze-drying preserved strain of the bacteria-free lactobacillus plantarum FF34 is inoculated into a test tube filled with MRS broth, subjected to stationary culture at 30-35 ℃ for 24-48 hours, transferred into an MRS broth triangular flask, and subjected to culture and activation at 30-35 ℃ for 20-48 hours; repeated activation2-3 times of microscopic examination and counting, when the concentration of the thallus reaches 10 9 CFU/mL is used as seed liquid;
MRS broth composition in g/L: 10 parts of casein enzyme digest, 10 parts of beef extract powder, 4 parts of yeast extract powder, 2 parts of citric acid triammonium, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose and 1.08 parts of tween-80, and preparing the beef extract powder and the yeast extract powder by constant volume with distilled water, wherein the pH value is 5.7+/-0.2;
(2) Immobilized fermentation culture:
preparing a triangular flask seed culture solution: inoculating the seed liquid obtained in the step (1) into a 3L triangular flask filled with an immobilized fermentation culture medium according to the volume ratio of 1% -5%, standing and culturing for 24-40 h at 30-35 ℃ until the thallus concentration reaches 10% 9 CFU/mL is used as a seed culture solution of the triangular flask; the mixture is put into a common aerobic fermentation tank for primary and secondary immobilized fermentation, and the amplification and the culture are carried out step by step, namely: 3L triangular flask, 500L fermenter, 10T fermenter, and sterile air pressure feeding method for inoculation;
primary immobilization culture: inoculating the immobilized culture seed liquid into a 500L fermentation tank with the inoculum size of 1-5% by volume, wherein the liquid volume in the fermentation tank is 80-90% by volume, the sterile air is kept under pressure of 0.01-0.05 Mpa, intermittent stirring is carried out, the rotating speed is 80-100 r/min, the culture is finished when the temperature is 30-35 ℃ for 20-40 h and the pH is 4.5-5.5, and the first-stage immobilized culture liquid is obtained, wherein lactobacillus plantarum FF34 thalli is adsorbed and fixed in gel small particles formed by calcium alginate and silicon dioxide, and the concentration of viable bacteria is more than or equal to 5 multiplied by 10 9 CFU/mL；
And (3) secondary immobilization culture: the primary immobilized culture solution is delivered by sterile air pressure, the inoculation amount of 1 to 5 percent of the volume ratio is connected into a 10T fermentation tank, the liquid amount of the fermentation tank is 70 to 90 percent of the volume ratio, the sterile air is kept under the pressure of 0.01 to 0.05Mpa, the intermittent stirring is carried out, the rotating speed is 80 to 100r/min, the culture is carried out for 20 to 40 hours at the temperature of 30 to 38 ℃, when the pH value of the fermentation solution is reduced to 4.0 to 5.5, the culture is finished, the secondary immobilized culture solution is obtained, wherein lactobacillus plantarum FF34 thalli is adsorbed and fixed in gel small particles formed by calcium alginate and silicon dioxide, and the concentration of the thalli is more than or equal to 5 multiplied by 10 9 CFU/mL；
The immobilized fermentation medium consists of the following components in g/L: 10-40 parts of carbon source, 3-15 parts of nitrogen source, 0.2-5 parts of dipotassium hydrogen phosphate, 0-0.2 part of magnesium sulfate, 0-0.05 part of manganese sulfate, 5-40 parts of calcium carbonate, 3-10 parts of sodium alginate and 1-10 parts of silicon dioxide, and sterilizing for 30min at 121 ℃ by using tap water with constant volume; wherein the carbon source comprises one or more of wheat middling, corn starch, dextrin, molasses, glucose, fructose syrup and the like. The nitrogen source comprises one or more of fish meal peptone, tryptone, soybean peptone, soybean protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal;
(3) Preparation of lactobacillus plantarum FF34 single bacterial agent with aquatic pathogenic bacteria antagonistic property:
(A) Preparation of lactobacillus plantarum FF34 single-fungus aqua: filling the secondary immobilized culture solution obtained in the step (2) to obtain a lactobacillus plantarum FF34 single-fungus water aqua, wherein the concentration of FF34 viable bacteria in the fungus water aqua is more than or equal to 5.0x10 9 CFU/mL；
(B) Preparation of Lactobacillus plantarum FF34 single-strain powder: centrifuging the secondary immobilized culture solution obtained in the step (2) to collect wet thalli, wherein the mass ratio is as follows: wet thalli, calcium carbonate and dextrin are mixed according to the proportion of silicon dioxide=1:0.1-2:0.5-2:0.01, and lactobacillus plantarum FF34 bacterial powder is obtained through spray drying or vacuum drying, wherein the concentration of FF34 viable bacteria in the bacterial powder is not lower than 1 multiplied by 10 10 CFU/g；
The method for preparing the composite microbial inoculum by mixed fermentation co-culture of the lactobacillus plantarum FF34 and the bacillus comprises the following steps:
(1) The bacillus mixed fermentation culture with the strain FF34 is as follows: aerobic bacillus, facultative aerobic bacillus and anaerobic bacillus, namely, the aquatic pathogenic bacteria antagonistic property:
bacillus subtilis AB90008-15, the antagonistic properties of which are shown in patent number ZL 201911117444.2; the strain is preserved in China general microbiological culture Collection center (CGMCC) No.18684, and the preservation date is 2019, 10, 15.
Or bacillus amyloliquefaciens JSSW-LA, the antagonistic properties of which are described in patent No. ZL 201510733305.8; deposited in China center for type culture Collection, accession number: CCTCC NO is M2015602, and the preservation date is 2015, 10 months and 12 days.
Or bacillus coagulans JSSW-LA-07-1, the antagonistic properties of which are shown in patent ZL 201510057403.4, are preserved in the China general microbiological culture Collection center, with the preservation number: CGMCC No.10182, the preservation date is 2014, 12 and 15.
Or clostridium butyricum JSIM-MCB20040312, see patent ZL201110083328.0 for its bacteriostasis properties see paper Chen Qiugong et al, study of the biological characteristics of probiotic butyric acid bacteria CB-7 [ J ]. Anhui agriculture science, 2011, 39 (10): 5922-5925 ", and is preserved in China general microbiological culture Collection center with a preservation number of CGMCC NO.1647 and a preservation date of 2006 of 3 months and 10 days.
Or bacillus Zhangzhou JSSW-BP44, see patent application No. 202010066397.X, deposited in China center for type culture collection, deposit No. CCTCC NO:2019989, the date of preservation is 2019, 12, 2.
(2) FF34 and the bacillus mixed fermentation described above are performed in two steps: the first step of fermentation is to inoculate the bacillus into a mixed fermentation culture medium after the bacillus is subjected to activation culture by an inclined plane or a triangular flask, wherein the inoculation amount is 1-10% of the volume ratio, the culture temperature is 30-37 ℃, the culture is carried out for 24-48 hours, and spores in the bacteria are subjected to microscopic examination, namely the first step of fermentation is finished; the second fermentation step is that the pH value of the fermentation liquor obtained in the first fermentation step is adjusted to 5.0-5.4 by hydrochloric acid, then activated lactobacillus plantarum FF34 is inoculated, the inoculation amount is 1-10% by volume, the culture temperature is 30-35 ℃, intermittent stirring is carried out, the fed-batch concentration is 20% (w/v) glucose or 30% calcium carbonate or ammonia water, the pH value of the fermentation liquor is controlled to 4.5-5.0, the fermentation liquor is cultured for about 24 hours, the mixed fermentation liquor of FF34 and bacillus is obtained, the coexistence of lactobacillus and bacillus is observed by microscopic examination, and the concentration of FF34 viable bacteria in the mixed fermentation liquor is more than or equal to 1.0X 10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
the composition of the mixed fermentation medium is calculated in g/L: wheat middling 0-50, peptone 0.1-5, yeast extract 0-5, sodium acetate 0-3, corn starch 0-10, ammonium sulfate 0-2, sodium chloride 0-5, calcium carbonate 0-5, monopotassium phosphate 0-0.5, magnesium sulfate 0-0.5, manganese sulfate 0-0.3, sodium alginate 0-5, tap water constant volume, pH 7.0, sterilizing at 121 ℃ for 30min;
(3) Preparation of lactobacillus plantarum FF34 composite microbial inoculum:
(A) Directly filling the mixed fermentation liquid of the lactobacillus plantarum FF34 and the bacillus obtained in the step (2) to obtain a lactobacillus plantarum FF34 composite bacterial aqua, wherein the concentration of the FF34 viable bacteria in the composite bacterial aqua is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
(B) And (3) centrifuging the lactobacillus plantarum FF34 and bacillus mixed fermentation liquid obtained in the step (2) to collect wet thalli, wherein the mass ratio is as follows: mixing wet thalli and calcium carbonate, wherein the silicon dioxide is=1:0.1-3:0.01, and spray drying or vacuum drying to obtain the composite bacterial powder of the lactobacillus plantarum FF34, wherein the concentration of FF34 viable bacteria in the composite bacterial powder is more than or equal to 5 multiplied by 10 9 CFU/g, bacillus viable bacteria concentration is more than or equal to 5.0X10 9 CFU/g。
The Lactobacillus plantarum FF34 single microbial inoculant prepared by the steps is applied to the composite microbial inoculant:
(1) The lactobacillus plantarum FF34 single microbial inoculum or composite microbial inoculum is used for fermenting feed raw materials such as fish, shrimp and crab feed or cotton meal, bean pulp, mulberry leaves and the like, 0.1-5% of the lactobacillus plantarum FF34 single microbial inoculum or composite microbial inoculum is mixed with the feed or feed raw materials according to the mass percentage, water is added for adjusting the water content of the feed to 25-40%, and the feed is subjected to closed fermentation at 28-35 ℃ for 3-5 days. After the feed or the feed raw material is fermented by lactobacillus plantarum FF34 single bacteria or composite bacteria, no mildew exists, the nutrition components of the feed or the feed raw material for fish, shrimp and crab are obviously improved, the obtained fermented feed is rich in probiotics, acid soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free gossypol, phytic acid and the like is reduced.
(2) The lactobacillus plantarum FF34 single microbial agent or the composite microbial agent is added into basic ration or high-starch feed according to the mass percentage of 0.05-1%, so that intestinal tissue growth of the aquaculture animals can be promoted, liver functions can be improved, antioxidant activity can be improved, growth performance of the aquaculture animals can be obviously improved, and feed coefficients can be reduced.
(3) Uniformly mixing the lactobacillus plantarum FF34 compound bacterial agent aqua with sodium alginate with the mass volume ratio of 2-4%, adding pumice, activated carbon or ceramsite, soaking and adsorbing for 1-4 h, taking out, soaking in 2-3% calcium chloride solution, curing and crosslinking for 1-10 h at 4-8 ℃ to obtain lactobacillus plantarum FF34 compound bacterial immobilized bacteria spheres (blocks), wherein the concentration of bacterial bodies in the bacteria spheres (blocks) is not lower than 5 multiplied by 10 8 CFU/g. The lactobacillus plantarum FF34 composite bacteria immobilized bacteria balls (blocks) are put into a culture water body and continuously used for more than 20 days, so that the contents of ammonia nitrogen, nitrite, total phosphorus and total nitrogen in the water body can be continuously degraded, the growth of aquatic pathogenic bacteria such as aeromonas is inhibited, and the water quality of a culture pond is continuously improved.
Measurement of bacteriostasis of lactobacillus plantarum FF 34: the pathogenic bacteria and the lactobacillus plantarum FF34 bacterial liquid are jointly inoculated into LB broth, so that the concentration of FF34 initial bacteria in the culture medium is 10 5 ～10 6 CFU/mL, pathogenic bacteria initial bacteria concentration of 10 5 ～10 7 CFU/mL, stationary culture at 30 ℃ for 24 hours, sampling and counting at 0 hours and 24 hours, and observing antagonism of FF34 on pathogenic bacteria. The results show that the composition has antagonism to aquatic common pathogenic bacteria such as Lestonia anguillarum, edwardsiella, vibrio alginolyticus, aeromonas hydrophila and Escherichia coli, and the like, wherein the composition has remarkable inhibition effect to Vibrio alginolyticus, edwardsiella, escherichia coli and Aeromonas hydrophila.
Measurement of the ability of lactobacillus plantarum FF34 to degrade nitrite: packaging 50 mL/test tube of MRS broth culture medium, sterilizing with 2mol/L sodium nitrite solution at 121deg.C for 15min, adding 2mol/L sodium nitrite solution 0.1mL into each test tube filled with MRS broth culture medium, inoculating 5mL of activated Lactobacillus plantarum FF34 bacterial suspension, inoculating 5mL of sterile distilled water into control group, standing at 35deg.C for 20h, sampling for 0h, 7h and 20h, centrifuging for 10min at 4000r/min, collecting supernatant, and detecting nitrous nitrogen concentration with America Hash reagent. The data were statistically analyzed using SPSS18.0, and all results were averaged.+ -. Standard error
The independent sample T-test comparison tests for differences between groups. Under anaerobic condition, the nitrite nitrogen can be obviously degraded in 20 hours, and the degradation rate reaches 96 percent.
The invention has the beneficial effects that:
(1) The common lactobacillus (including lactobacillus plantarum) has complex nutrition requirements and lacks amylase activity, and not only needs glucose, fructose or maltose and other monosaccharides or disaccharides as carbon sources, but also needs amino acids, peptides, nucleic acid derivatives, salts, fatty acids or fatty acid esters, and each species has special nutrition requirements, and the characteristics cause the limitation of sources of raw materials for large-scale culture of beneficial lactobacillus, so that the cost of the culture medium is increased. The lactobacillus plantarum FF34 has amylase activity, can directly utilize starchy carbohydrate, has wide available carbon source and nitrogen source, is different from common lactobacillus plantarum (including common lactobacillus plantarum), and leads the available carbon source of the lactobacillus plantarum FF34 to be expanded from glucose, fructose, maltose and other monosaccharides or disaccharides to starchy carbohydrate, thereby leading low-cost wheat middling, cotton meal, rapeseed meal, soybean meal and other agricultural and sideline products to be directly used as the carbon source and the nitrogen source for the multiplication culture of the FF34 and greatly reducing the cost of the culture medium.
(2) The lactobacillus plantarum FF34 is a facultative lactobacillus, has good tolerance to dissolved oxygen, and can grow without strict anaerobic conditions, so that the lactobacillus plantarum FF34 can be cultivated on a large scale by adopting a common aerobic fermentation tank, but the pressure of the aerobic fermentation tank is maintained, and a sterile air pressure conveying and seed transferring mode is adopted between a seed tank and the fermentation tank, so that the risk of high dissolved oxygen still exists in the fermentation cultivation process, and the growth of the FF34 is inhibited. The invention adopts a high-density low-cost immobilized fermentation and spread cultivation method, and adopts a cultivation method of sodium alginate immobilized package thalli while fermentation in a common aerobic fermentation tank, utilizes the acid production characteristics of starch or saccharides to dissolve calcium carbonate in a culture medium to release free calcium ions, forms calcium alginate gel with sodium alginate in the culture medium, packages thalli in calcium alginate gel small particles, isolates dissolved oxygen to transfer and protect the growth activity of thalli, thereby obtaining high-concentration thalli, and the fermentation liquid Lactobacillus plantarum FF34 strain concentration is more than 5×10 9 CFU/mL; then the FF34 strain concentration is obtained by spray drying to be more than 1 multiplied by 10 10 The CFU/g high-concentration bacterial powder preparation further reduces the use cost of the lactobacillus plantarum FF34 aqua and bacterial powder preparation, and is beneficial to popularization and application of the lactobacillus plantarum FF34 bacterial agent in ecological breeding of livestock and poultry.
(3) The lactobacillus plantarum FF34 has good compatibility with various beneficial bacillus and can be used for multi-strain mixed fermentation co-culture. The growth of the lactobacillus plantarum FF34 has low nutrition requirements, can utilize a wide range of carbon sources and nitrogen sources, and can directly utilize starchy carbohydrate. Because the starchy carbon sources such as wheat middling and the like are low-cost carbon sources suitable for bacillus, the characteristics of the starchy carbon sources can be directly utilized by the lactobacillus plantarum FF34, so that the FF34 can be mixed and fermented with various bacillus for culture, the bacillus type coverage is wide, and the bacillus is aerobic bacillus, facultative aerobacillus and anaerobic bacillus, such as bacillus subtilis AB90008-15 (aerobic bacteria) or bacillus amyloliquefaciens JSSW-LA (aerobic bacteria) or bacillus coagulans JSSW-LA-07-1 (facultative aerobacteria) or clostridium butyricum JSIM-MCB20040312 (anaerobic bacteria) or bacillus Zhuzhou JSSW-BP44 (facultative aerobacteria) with aquatic pathogen antagonistic characteristics are involved. When FF34 and bacillus are co-cultured in a mixed fermentation medium, after the bacillus forms spores, the spores are in a dormant state by adjusting the pH, then FF34 is continuously inoculated, and residual nutrient substances in the fermentation medium are fully utilized to carry out FF34 proliferation culture, so that the composite microbial inoculum containing high-concentration bacillus (the spore rate is more than or equal to 90 percent) and lactobacillus plantarum FF34 is finally obtained, the fermentation cost of the composite microbial inoculum is reduced, and the emission of fermentation waste liquid is reduced.
(4) The lactobacillus plantarum FF34 has antagonism to aquatic common pathogenic bacteria such as Lestonia anguillarum, edwardsiella, vibrio alginolyticus, aeromonas hydrophila and Escherichia coli, and has remarkable inhibition effect to Vibrio alginolyticus, edwardsiella, escherichia coli and Aeromonas hydrophila. The lactobacillus plantarum FF34 can be mixed, fermented and co-cultured with a plurality of bacillus with aquatic pathogenic bacteria antagonistic property such as bacillus subtilis AB90008-15, bacillus amyloliquefaciens JSSW-LA or bacillus coagulans JSSW-LA-07-1 or clostridium butyricum JSIM-MCB20040312, and the obtained composite microbial inoculum further enriches the types of bacteria antagonistic aquatic pathogenic bacteria, enhances the disease control effectiveness of aquatic animals and promotes the healthy growth of the aquatic animals.
(5) After lactobacillus plantarum FF34 single or composite bacteria ferments the feed, the feed is mildew-free, the fermented feed is rich in probiotics, the nutritional ingredients of the feed are obviously improved, the soluble protein is increased by 42.38-116.31%, the acid soluble protein is increased by 118.54-218.53%, the active small peptide is increased by 42.14-168.41%, and the content of anti-nutritional factors such as phytic acid, tannin, free gossypol and the like is obviously reduced. The preliminary researches on the mechanisms of the lactobacillus plantarum FF34 on the aspects of nutrition utilization, metabolic influence and the like of fish feed show that: the lactobacillus plantarum FF34 single microbial agent or the composite microbial agent is added into basic ration or high-starch feed according to the mass percentage of 0.05-1%, so that intestinal tissue growth of the aquaculture animals can be promoted, liver functions can be improved, antioxidant activity can be improved, growth performance of the aquaculture animals can be obviously improved, and feed coefficients can be reduced.
(6) The lactobacillus plantarum FF34 composite microbial inoculum is immobilized by light stones, activated carbon or ceramsite, and then is put into a culture water body, compared with the microbial inoculum which is directly sprayed and not immobilized, the immobilized microbial inoculum is not easy to be deactivated and lost in an oxygen-containing culture water body environment, the effective time is longer than 20 days, the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content of the water body can be continuously degraded, the growth of aquatic pathogenic bacteria is inhibited, and the water quality of a culture pond is improved.
Preservation of biological Material samples
(1) Lactobacillus plantarum FF34, classified and named lactobacillus plantarum (lactobacillus plantarum), is preserved in the China center for type culture collection, CCTCC for short, address: the preservation number of the Chinese university of Wuhan is CCTCC NO: m20211221, storage date 2021, 10, 11.
Drawings
FIG. 1 Lactobacillus plantarum and high starch addition liver oil red 0 staining sections of young megalobrama amblycephala. Panel A is a 15WM group (x 200 times) of oil red O stained sections, panel B is a 35WM+LAB group (x 200 times) of oil red O stained sections, and panel C is a 35WM group (x 200 times) of oil red O stained sections.
FIG. 2 shows a Neighbor-Joining phylogenetic tree constructed by using Enterococcus faecalis (Y18293.1) as an outer branch based on the 16S rRNA gene sequence alignment result of the strain FF-34.
Detailed Description
Example 1: screening of bacterial species
Collecting pond muddy water in a pond for culturing the crabs in the mountain and town of the Mars in the area of the Bin-free city, adding normal saline, grinding to prepare homogenate, taking a pipetting gun to suck 0.1mL, and coating the pipetting gun on an MRS solid culture medium for anaerobic culture at 25-35 ℃. Well-grown colonies were selected according to colony size and repeatedly inoculated and screened until a uniform single colony was obtained, designated as FF34.
Example 2: identification of species
(1) Morphological characteristics: lactobacillus plantarum strain FF34, gram staining is positive, after MRS plate coating, bacterial colony grown for 48h anaerobic culture at 35 ℃ is raised, the surface is smooth, fine, white, opaque, diameter 1-3 mm, deep bacterial colony of MRS pouring culture is lens or diamond, bacterial colony at the bottom of plate is flat and round, the bacterial colony is observed by microscope (16 x 100), the bacterial colony is in short rod shape, both ends are in round and slightly square shape, are arranged in pairs or short chain shape, (0.9-1.2) mu m x (2-4) mu m, no flagella and can move.
(2) Biochemical characteristics:
TABLE 1 physiological and biochemical characteristics of strain FF-34-enzyme activity and carbon source oxidation
+: a positive reaction; -: a negative reaction; w: weak positive reaction
TABLE 2 physiological and biochemical characteristics of strain FF-34-acid production Using carbon Source
+: a positive reaction; -: a negative reaction; weak positive response W
(3) 16S rRNA sequence analysis and phylogenetic tree construction:
the 16S rRNA gene sequence of the lactobacillus plantarum strain FF34 is shown as SEQ ID NO. 1.
Sequencing results of the Lactobacillus plantarum strain FF34 gene sequence: the 16S rRNA gene sequence amplified by the strain is subjected to homology search at NCBI through Blast, and the 16S rRNA gene sequence of lactobacillus is searched out as a result, a phylogenetic tree of the strain is constructed by adopting a adjacency method, and the isolated strain belongs to the same branch on the phylogenetic tree as lactobacillus plantarum Lactobacillus plantarum subsp.plantarum ATCC 14917 (accession number: ACGZ 01000098). The isolated strain was identified as lactobacillus plantarum Lactobacillus plantarum (see figure 2) in combination with morphological and physiological biochemical characteristics.
Example 3: measurement of bacteriostasis ability of lactobacillus plantarum FF34 on aquatic pathogenic bacteria
And (3) inoculating pathogenic bacteria and lactobacillus plantarum FF34 bacterial liquid into an LB culture medium together, standing at 30 ℃ for culturing for 24 hours, sampling and counting for 0 hour and 24 hours, and observing antagonism of lactobacillus plantarum FF34 on the pathogenic bacteria. The measurement results are shown in Table 3:
TABLE 3 Co-culture assay of Lactobacillus plantarum FF34 and pathogenic bacteria
Under the co-culture condition, the lactobacillus plantarum FF34 has the strongest inhibition capability to vibrio alginolyticus and Edwardsiella, has weaker inhibition capability to escherichia coli and aeromonas hydrophila and has the weaker inhibition capability to Lestonia anguillarum. Lactobacillus plantarum FF34 completely inhibits the growth of Vibrio alginolyticus at 24h, so that the proliferation of Edwardsiella is reduced by about 2 orders of magnitude, and the proliferation of Escherichia coli and Aeromonas hydrophila are respectively reduced by one order of magnitude.
Example 4: determination of nitrite degrading ability of Lactobacillus plantarum FF34
The results in Table 4 show that the lactobacillus plantarum FF34 nitrite reductase has high activity, can rapidly degrade nitrite nitrogen under anaerobic conditions, has extremely obvious degradation in 20 hours, and has a degradation rate of 82.30 +/-0.50% in 7 hours and a degradation rate of 96.00+/-0.01% in 20 hours.
TABLE 4 ability of Lactobacillus plantarum FF34 to degrade nitrous nitrogen
| Grouping | Control group | FF34 test group |
| Time | Nitrosamine mg/L | Nitrosamine mg/L |
| 0h | 135.22±2.34 Aa | 135.89±2.34 Aa |
| 7h | 135.69±0.55 Aa | 24.05±0.99 Bb |
| 20h | 134.31±0.20 Aa | 5.22±1.24 Cb |
| 20h nitronitrogen degradation rate | 1.12±1.37％ b | 96.00±0.01％ a |
Note that: different lowercase letters in the same row indicate that the difference is significant (P < 0.05), and different uppercase letters in the same column indicate that the difference is significant (P < 0.05).
Example 5 preparation of Lactobacillus plantarum FF34 Mono-bacterial preparation with aquatic pathogen antagonistic Properties
1) Activating strains:
freeze-drying preserved strain of the bacteria-free lactobacillus plantarum FF34 is inoculated into a test tube filled with MRS broth, subjected to stationary culture at 30-35 ℃ for 24-48 hours, transferred into an MRS broth triangular flask, and subjected to culture and activation at 30-35 ℃ for 20-48 hours; repeatedly activating for 2-3 times, performing microscopic examination, and counting until the concentration of the bacterial cells reaches 10 9 CFU/mL is used as seed liquid;
MRS broth composition in g/L: 10 parts of casein enzyme digest, 10 parts of beef extract powder, 4 parts of yeast extract powder, 2 parts of citric acid triammonium, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose and 1.08 parts of tween-80, and preparing the beef extract powder and the yeast extract powder by constant volume with distilled water, wherein the pH value is 5.7+/-0.2;
(2) Immobilized fermentation culture:
preparing a triangular flask seed culture solution: inoculating the seed liquid obtained in the step (1) into a 3L triangular flask filled with an immobilized fermentation culture medium according to the volume ratio of 1% -5%, standing and culturing for 24-40 h at 30-35 ℃ until the thallus concentration reaches 10% 9 When CFU/mL is used as a seed culture solution of a triangular flask, the seed culture solution is connected into a common aerobic fermentation tank for primary and secondary immobilized fermentation and gradual amplification and culture, namely: 3L triangular flask, 500L fermenter, 10T fermenter, and sterile air pressure feeding method for inoculation;
Primary immobilization culture: inoculating the immobilized culture seed liquid into a 500L fermentation tank with the inoculum size of 1-5% by volume, wherein the liquid volume in the fermentation tank is 80-90% by volume, the sterile air is kept under pressure of 0.01-0.05 Mpa, intermittent stirring is carried out, the rotating speed is 80-100 r/min, the culture is finished when the temperature is 30-35 ℃ for 20-40 h and the pH is 4.5-5.5, and the first-stage immobilized culture liquid is obtained, wherein lactobacillus plantarum FF34 thalli is adsorbed and fixed in gel small particles formed by calcium alginate and silicon dioxide, and the concentration of viable bacteria is more than or equal to 5 multiplied by 10 9 CFU/mL；
And (3) secondary immobilization culture: the primary immobilized culture solution is delivered by sterile air pressure, the inoculation amount of 1 to 5 percent of the volume ratio is connected into a 10T fermentation tank, the liquid amount of the fermentation tank is 70 to 90 percent of the volume ratio, the sterile air is kept under the pressure of 0.01 to 0.05Mpa, the intermittent stirring is carried out, the rotating speed is 80 to 100r/min, the culture is carried out for 20 to 40 hours at the temperature of 30 to 38 ℃, when the pH value of the fermentation solution is reduced to 4.0 to 5.5, the culture is finished, the secondary immobilized culture solution is obtained, wherein lactobacillus plantarum FF34 thalli is adsorbed and fixed in gel small particles formed by calcium alginate and silicon dioxide, and the concentration of the thalli is more than or equal to 5 multiplied by 10 9 CFU/mL；
The immobilized fermentation medium consists of the following components in g/L: 10-40 parts of carbon source, 3-15 parts of nitrogen source, 0.2-5 parts of dipotassium hydrogen phosphate, 0-0.2 part of magnesium sulfate, 0-0.05 part of manganese sulfate, 5-40 parts of calcium carbonate, 3-10 parts of sodium alginate and 1-10 parts of silicon dioxide, and sterilizing for 30min at 121 ℃ by using tap water with constant volume; wherein the carbon source comprises one or more of wheat middling, corn starch, dextrin, molasses, glucose, fructose syrup and the like. The nitrogen source comprises one or more of fish meal peptone, tryptone, soybean peptone, soybean protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal.
(3) Preparation of lactobacillus plantarum FF34 single-strain preparation:
(A) Preparation of lactobacillus plantarum FF34 single-fungus aqua: filling the secondary immobilized culture solution obtained in the step (2) to obtain a lactobacillus plantarum FF34 aqua, wherein the concentration of FF34 viable bacteria in the lactobacillus plantarum aqua is more than or equal to 5.0x10 9 CFU/mL；
(B) Preparation of Lactobacillus plantarum FF34 single-strain powder: centrifuging the secondary immobilized culture solution obtained in the step (2) to collect wet thalli, wherein the mass ratio is as follows: wet thalli, calcium carbonate and dextrin are mixed according to the proportion of silicon dioxide=1:0.1-2:0.5-2:0.01, and lactobacillus plantarum FF34 bacterial powder is obtained through spray drying or vacuum drying, wherein the concentration of FF34 viable bacteria in the bacterial powder is not lower than 1 multiplied by 10 10 CFU/g。
Example 6: lactobacillus plantarum FF34 and clostridium butyricum JSIM-MCB20040312 (collection number CGMCC No. 1647) mixed fermentation co-culture and preparation of composite microbial inoculum thereof
(1) Strain activation and seed liquid culture in triangular flask
(A) Bacterial activation of clostridium butyricum JSIM-MCB20040312 and culture of a triangular flask seed solution:
aseptically opening clostridium butyricum JSIM-MCB20040312 freeze-dried and preserved strains, inoculating a test tube filled with an activation culture medium, carrying out standing anaerobic culture for 18-24 hours at 30-37 ℃, then transferring the culture medium into a fresh activation culture medium according to the inoculum size of 1-10% of the volume ratio, culturing for 20-24 hours at 30-37 ℃, carrying out microscopic examination, when more than 90% of the strains form spores, namely, maturing, repeatedly activating for 2-3 times, inoculating seed bacterial suspension into a plurality of 3L triangular flasks filled with seed culture medium according to the inoculum size of 1-10% of the volume ratio, carrying out standing culture for 20-24 hours in a constant temperature incubator with the volume ratio of 80-90% of the triangular flask liquid, and carrying out microscopic examination, when more than 90% of the strains form spores, namely, obtaining the triangular flask seed liquid;
Clostridium butyricum JSIM-MCB 20040312 activation and seed Medium composition in g/L:
3-5 parts of yeast extract, 5-20 parts of beef extract, 5-20 parts of peptone, 5-10 parts of glucose, 1-2 parts of soluble starch, 5 parts of sodium chloride, 3-5 parts of sodium acetate, 0.5-1 part of cysteine hydrochloride, 1-7 parts of calcium carbonate, and preparing with deionized water, wherein the pH value is 6.0-7.5;
(B) Strain activation of lactobacillus plantarum FF34 and triangular flask seed liquid culture
Freeze-drying preserved strain of the bacteria-free lactobacillus plantarum FF34 is inoculated into a test tube filled with MRS broth, subjected to stationary culture at 30-35 ℃ for 24-48 hours, transferred into a small triangular flask of the MRS broth, and subjected to culture and activation at 30-35 ℃ for 20-48 hours; repeatedly activating for 2-3 times, performing microscopic examination, counting, and obtaining the bacterial concentration of 10 9 CFU/mL is used as seed liquid, a plurality of 3L triangular flasks filled with MRS fermentation culture medium are inoculated with 1-5% of inoculation amount by volume ratio, the liquid filling amount is 90% by volume ratio, static culture is carried out for 24-40 h at 30-35 ℃, and when the thallus concentration reaches 10% 9 CFU/mL is used as a seed solution of the triangular flask;
MRS broth composition in g/L: 10 parts of casein enzyme digest, 10 parts of beef extract powder, 4 parts of yeast extract powder, 2 parts of citric acid triammonium, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose and 1.08 parts of tween-80, and preparing the beef extract powder and the yeast extract powder by constant volume with distilled water, wherein the pH value is 5.7+/-0.2;
(2) Mixed fermentation co-culture
The mixed fermentation of FF34 and clostridium butyricum JSIM-MCB 20040312 is carried out in two steps: and (3) fermentation: inoculating the clostridium butyricum JSIM-MCB 20040312 fermentation tank seed liquid obtained in the step (A) into a 500L fermentation tank filled with a mixed fermentation medium according to the volume ratio of 1-10%, wherein the liquid loading amount is 70-80% (V/V), the temperature is 30-37 ℃, and the fermentation is carried out for 24-48 hours, wherein spores are formed in microscopic bacteria, namely the first fermentation is finished; second step of fermentation, the pH of the fermentation liquor obtained in the first step is adjusted to 5.0-5.4 by hydrochloric acid, and then the seed liquor of the lactobacillus plantarum FF34 triangle bottle obtained in the step (B) is inoculatedThe seed quantity is 1-10% of the volume ratio, the culture temperature is 30-35 ℃, intermittent stirring is carried out, the fed-batch concentration is 20% (w/v) glucose or 30% calcium carbonate or ammonia water, the pH of the fermentation liquor is controlled to be 4.5-5.0, the culture is carried out for about 24 hours, the mixed fermentation liquor of FF34 and bacillus is obtained, microscopic examination shows that the lactobacillus and the spore coexist, and the concentration of FF34 viable bacteria in the mixed fermentation liquor is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
the composition of the mixed fermentation medium is calculated in g/L: wheat middling 0-50, peptone 0.1-5, yeast extract 0-5, sodium acetate 0-3, corn starch 0-10, ammonium sulfate 0-2, sodium chloride 0-5, calcium carbonate 0-5, monopotassium phosphate 0-0.5, magnesium sulfate 0-0.5, manganese sulfate 0-0.3, sodium alginate 0-5, tap water constant volume, pH 7.0, sterilizing at 121 ℃ for 30min;
(3) Preparation of lactobacillus plantarum FF34 composite microbial inoculum:
(A) Directly filling the mixed fermentation liquor of the lactobacillus plantarum FF34 obtained in the step (2) and the clostridium butyricum JSIM-MCB 2004031 to obtain a lactobacillus plantarum FF34 composite bacterial aqua, wherein the concentration of the viable bacteria of the FF34 in the composite bacterial aqua is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
(B) And (2) centrifuging the mixed fermentation liquor of the lactobacillus plantarum FF34 and the clostridium butyricum JSIM-MCB 2004031 obtained in the step (2) to collect wet thalli, wherein the mass ratio is as follows: mixing wet thalli and calcium carbonate, wherein the silicon dioxide is=1:0.1-3:0.01, and spray drying or vacuum drying to obtain lactobacillus plantarum FF34 composite microbial inoculum powder, wherein the concentration of FF34 viable bacteria in the microbial inoculum powder is more than or equal to 5 multiplied by 10 9 CFU/g, bacillus viable bacteria concentration is more than or equal to 5.0X10 9 CFU/g。
EXAMPLE 7 Lactobacillus plantarum FF34 and Bacillus subtilis AB90008-15 (collection number CGMCC No. 18684) mixed fermentation co-culture and preparation of composite microbial inoculum
(1) Strain activation and seed liquid culture in triangular flask
(A) Bacterial activation and seed preparation of bacillus subtilis AB 90008-15:
aseptically starting a freeze-drying tube of bacillus subtilis AB90008-15 to store strains, inoculating the strains to bran nutrition agar, culturing for 24-48 hours at the temperature of 30-37 ℃, then transferring to a bran nutrition agar slope, repeatedly activating for 2-3 times, performing microscopic examination, when more than 90% of the bacillus subtilis forms spores, namely the spores are mature, scraping lawn, and preparing bacterial suspension serving as bacillus subtilis seed liquid by using aseptic water;
Bran nutrient agar slant culture medium composition is calculated in g/L: peptone 10, beef extract 3, naCl 5, bran 10, agar 15-20, and distilled water to constant volume, wherein the pH value is 7.0-7.2;
(B) Strain activation of lactobacillus plantarum FF34 and triangular flask seed liquid culture
Freeze-drying preserved strain of the bacteria-free lactobacillus plantarum FF34 is inoculated into a test tube filled with MRS broth, subjected to stationary culture at 30-35 ℃ for 24-48 hours, transferred into a small triangular flask of the MRS broth, and subjected to culture and activation at 30-35 ℃ for 20-48 hours; repeatedly activating for 2-3 times, performing microscopic examination, counting, and obtaining the bacterial concentration of 10 9 Taking CFU/mL as seed liquid, inoculating 1-5% of inoculum size into a plurality of 3L triangular flasks filled with immobilized fermentation culture medium, standing at 30-35deg.C for 24-40 hr until thallus concentration reaches 10% 9 CFU/mL is used as a seed solution of the triangular flask;
MRS broth composition in g/L: 10 parts of casein enzyme digest, 10 parts of beef extract powder, 4 parts of yeast extract powder, 2 parts of citric acid triammonium, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose and 1.08 parts of tween-80, and preparing the beef extract powder and the yeast extract powder by constant volume with distilled water, wherein the pH value is 5.7+/-0.2;
the immobilized fermentation medium consists of the following components in g/L: 10-40 parts of carbon source, 3-15 parts of nitrogen source, 0.2-5 parts of dipotassium hydrogen phosphate, 0-0.2 part of magnesium sulfate, 0-0.05 part of manganese sulfate, 5-40 parts of calcium carbonate, 3-10 parts of sodium alginate and 1-10 parts of silicon dioxide, and sterilizing for 30min at 121 ℃ by using tap water with constant volume; wherein the carbon source comprises one or more of wheat middling, corn starch, dextrin, molasses, glucose, fructose syrup and the like. The nitrogen source comprises one or more of fish meal peptone, tryptone, soybean peptone, soybean protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal.
(2) Mixed fermentation co-culture
The mixed fermentation of FF34 and bacillus subtilis AB90008-15 is carried out in two steps: and (3) fermentation: inoculating the bacillus subtilis seed bacterial suspension obtained in the step (A) into a 500L fermentation tank filled with a fermentation medium according to the inoculum size of 1-10% by volume ratio for primary seed tank culture, wherein the liquid filling amount is 50-60% by volume ratio and the ventilation volume is 15m 3 And/h, the pot pressure is 0.05-0.1 Mpa, the rotating speed is 100-150 r/min, the temperature is 30-37 ℃, the aeration culture is carried out for 24-48 h, and spores are formed in microscopic bacteria, namely, the first-step fermentation is finished; the second step of fermentation, firstly, regulating pH of fermentation liquor to 5.0-5.4 by using hydrochloric acid, then, inoculating lactobacillus plantarum FF34 triangular flask seed liquor obtained in the step (B), wherein the inoculum size is 1-10% by volume, the culture temperature is 30-35 ℃, intermittent stirring is carried out, the fed-batch concentration is 20% (w/v) glucose or 30% calcium carbonate or ammonia water, the pH of the fermentation liquor is controlled to 4.5-5.0, the fermentation liquor is cultured for about 24 hours, the mixed fermentation liquor of FF34 and bacillus is obtained, the coexistence of lactobacillus and spores is detected by a mirror, and the concentration of FF34 viable bacteria in the mixed fermentation liquor is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
the composition of the mixed fermentation medium is calculated in g/L: wheat middling 0-50, peptone 0.1-5, yeast extract 0-5, sodium acetate 0-3, corn starch 0-10, ammonium sulfate 0-2, sodium chloride 0-5, calcium carbonate 0-5, monopotassium phosphate 0-0.5, magnesium sulfate 0-0.5, manganese sulfate 0-0.3, sodium alginate 0-5, tap water constant volume, pH 7.0, sterilizing at 121 ℃ for 30min;
(3) Preparation of lactobacillus plantarum FF34 composite microbial inoculum:
(A) Directly filling the mixed fermentation liquor of the lactobacillus plantarum FF34 obtained in the step (2) and the bacillus subtilis AB90008-15 to obtain a lactobacillus plantarum FF34 composite bacterial aqua, wherein the concentration of the viable bacteria of the FF34 in the composite bacterial aqueous solution is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
(B) Separating the mixed fermentation liquid of the lactobacillus plantarum FF34 obtained in the step (2) and the bacillus subtilis AB9008-15Collecting wet thalli from the heart, mixing the wet thalli with calcium carbonate and silicon dioxide with the ratio of 1:0.1-3:0.01, and performing spray drying or vacuum drying to obtain lactobacillus plantarum FF34 composite microbial inoculum powder, wherein the concentration of FF34 viable bacteria in the microbial inoculum powder is more than or equal to 5 multiplied by 10 9 CFU/g, bacillus viable bacteria concentration is more than or equal to 5.0X10 9 CFU/g。
Example 8: results of lactobacillus plantarum FF34 composite microbial inoculant fermented feed
Mixing the lactobacillus plantarum FF34 composite microbial inoculant powder prepared in the example 7 with freshwater shrimp feed, crab feed, lobster feed and cottonseed meal respectively according to the additive amount of 0.1-5% by mass, adding water to adjust the water content of the materials to 25-40%, respectively filling fermentation bags (commercially available) with the volume capacity of 5L according to 1 kg/bag, taking freshwater shrimp feed, crab feed, lobster feed and cottonseed meal fermented without adding microbial inoculant powder as a control, sealing and fermenting for 3-5 days at 28-35 ℃ in the fermentation bags in each group of 3 parallel, wherein the fermentation results are shown in the following table 5, the data are statistically analyzed by SPSS18.0, and all the results are obtained by using average value +/-standard error 
The independent sample T-test is shown comparing the differences between groups.
Table 5 results of Lactobacillus plantarum FF34 composite microbial inoculant shrimp and crab feed
Note that: * Indicating significant differences (p < 0.05).
The results in Table 5 show that after freshwater shrimp feed, crab feed and crayfish feed are fermented by lactobacillus plantarum FF34 composite microbial inoculum, the fermented feed is rich in beneficial lactobacillus and bacillus, the nutrition value of the feed is remarkably improved, wherein the content of soluble protein is respectively increased by 116.31%, 92.57%, 42.38%, the content of acid soluble protein is 218.53%, 155.21%, 118.41%, and the content of small peptide is respectively increased by 45.20%, 76.13% and 168.41%.
TABLE 6 results of fermentation of cottonseed meal with Lactobacillus plantarum FF34 Complex inoculant
Note that: * Indicating significant differences (p < 0.05).
Table 6 the results show that: after the composite bacterial agent of the lactobacillus plantarum FF34 ferments the cotton meal, the cotton meal is rich in probiotics such as lactobacillus, wherein the lactobacillus is greatly proliferated and the concentration is up to 10 9 Bacillus in CFU/g and FF34 composite microbial inoculum fermented cotton dreg can reach 10 7 CFU/g. In the process of fermenting the cotton seed meal, the lactobacillus plantarum FF34 composite microbial inoculum inhibits the growth of mould and prevents the cotton seed meal from mildew after water is added. After the lactobacillus plantarum FF34 compound bacteria ferments the cotton dregs, the nutrition components of the cotton dregs are improved, the contents of crude protein, soluble protein, acid soluble protein and active small peptide of the cotton dregs are obviously improved (p is less than 0.05), the content of the soluble protein is improved by 85.13%, the content of the acid soluble protein is improved by 155.42%, the content of the active small peptide is improved by 42.14%, and the content of the crude protein of the fermented cotton dregs is improved by 9.02%, but the difference is not obvious (p is more than 0.05). In addition, after the lactobacillus plantarum FF34 composite microbial inoculum ferments the cotton dregs, the content of anti-nutritional factors such as phytic acid, tannin, free gossypol and the like in the cotton dregs can be obviously reduced (p is less than 0.05), the phytic acid is reduced by 63.74%, the tannin is reduced by 50.61%, and the free gossypol is reduced by 54.17%.
Example 9: lactobacillus plantarum FF34 is added into the feed for the growth of young megalobrama amblycephala and the utilization of the feed
In the embodiment, fish meal, bean pulp, rapeseed meal and cotton pulp are taken as protein sources, soybean oil is taken as fat sources, 2 groups of test feeds are prepared, namely a conventional control feed group (CN) and a lactobacillus plantarum FF34 additive group LAB, wherein the LAB group is a lactobacillus plantarum FF34 single-strain preparation prepared in the embodiment 5 is added into the conventional control feed (CN) according to the mass percentage of 0.01-1%, so that the concentration of FF34 strain is 10 6 cfu/g feed. The crude protein content in each feed group was 32.5%, the crude fat content was 6.2%, and the total feed weight was 17.4kJ/Kg. The conventional control feed comprises the following components in percentage by mass: fish meal 6.4%, bean pulp 25%4%, 16.2% of rapeseed meal, 15.3% of cotton meal, 16.6% of wheat starch, 5.7% of rice bran, 5.8% of wheat bran, 3.1% of soybean oil, 1.0% of monocalcium phosphate, 1.0% of premix, 0.5% of vitamin C, 0.4% of choline chloride, 2.1% of microcrystalline cellulose and 0.5% of bentonite.
After 120 megalobrama amblycephala juvenile fish (initial weight 13.5+/-0.5 g) with healthy constitution and uniform specification are selected and temporarily cultured for one week, 6 outdoor net cages (1 m multiplied by 1 m) are randomly divided, 20 tails of each net cage are divided into 2 groups, 3 repeats of each group are carried out, and the apparent satiety is carried out 3 times per day (8:00, 12:00 and 17:00) for 8 weeks. The water temperature is kept at 28-31 ℃ during the cultivation period, the dissolved oxygen is more than or equal to 7mg/L, the ammonia nitrogen is less than or equal to 0.1mg/L, the pH value is 7.3-7.8, and the cultivation water quality condition is measured every week by adopting a natural illumination period (12L: 12D). After the 8-week culture test, the test was fasted for 24 hours, and the growth index, organ index and blood index were measured.
The growth performance index was calculated as follows:
weight Gain Ratio (WGR) (%) =100× (Wt-W0)/W0;
specific Growth Rate (SGR) (%/d) =100× (ln Wt-ln W0)/t;
bait coefficient (FCR) =f/(Wt-W0);
wherein: w0 is the initial weight (g); wt is the final weight (g); t is the number of days of feeding (d); f is the total amount of feed intake (air-dried basis) (g).
Serum blood Glucose (GLU), cholesterol (TC), triglyceride (TG), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), high Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) levels were measured using a Shengzheri fully automatic Biochemical Analyzer (BS-400Q 2080, shengzhen, china) using kits all purchased from Shengzheri Co., ltd. The data of CN control group and LAB (lactobacillus plantarum test group) are statistically analyzed by SPSS18.0, and all the results are obtained by mean value + -standard error
The independent sample T-test is shown to compare the differences between the groups, indicating significant differences between the two groups, at a test level P < 0.05. The results are shown in Table 7.
TABLE 7 influence of Lactobacillus plantarum FF34 added to feed on the growth and blood physiology of young megalobrama amblycephala
| Index (I) | CN (control group) | LAB (Lactobacillus plantarum test group) |
| Growth performance index | ||
| WGR，％ | 126.87±3.85 | 155.48±2.69* |
| SGR，％/d | 1.64±0.04 | 11.88±0.02* |
| FCR | 2.18±0.06 | 11.89±0.05* |
| Serum biochemical index | ||
| GLU，mmol/L | 9.93±0.56 | 7.14±0.70* |
| LDL，mmol/L | 0.61±0.06 | 0.66±0.03 |
| HDL，mmol/L | 1.04±0.09 | 0.64±0.02* |
| TG，mmol/L | 1.43±0.11 | 11.87±0.07* |
| TC，mmol/L | 5.83±0.20 | 7.03±0.17* |
| ALT，mmol/L | 4.74±0.37 | 2.78±0.27* |
| AST，mmol/L | 185.01±13.34 | 142.32±6.81* |
Note that: * Indicating significant differences (p < 0.05).
Table 7 the results show that: add 10 6 The cfu/g lactobacillus plantarum FF34 remarkably improves the weight gain rate (P is less than 0.05) and the specific growth rate (P is less than 0.05), remarkably reduces the feed coefficient (P is less than 0.05), and improves the utilization and growth performance of the young megalobrama amblycephala for the feed. Alanine aminotransferases and aspartate aminotransferases are indicators of liver metabolic function, and high density lipoproteins are capable of transporting cholesterol and promoting cholesterol metabolism. General gallbladder fixationAlcohol is an important raw material for synthesizing physiologically active substances such as epinephrine, bile acid, vitamin D, and the like. The results of the application example show that the addition of lactobacillus plantarum can obviously reduce the contents of blood sugar, alanine transferase and aspartic aminotransferase in blood (P is less than 0.05), which indicates that the lactobacillus plantarum has the functions of improving liver metabolism and reading and improving the physiological health of the blood of megalobrama amblycephala. Meanwhile, the increase of lipid substances such as total cholesterol and the like also suggests that the addition of lactobacillus plantarum FF34 can promote the metabolism of lipid substances in fish bodies and provide precursor substances for the lipid metabolism of livers and organisms.
Example 10: influence of Lactobacillus plantarum FF34 added into high-starch feed on utilization and growth performance of megalobrama amblycephala juvenile fish high-starch feed
In the embodiment, 2 groups of test feeds, namely a high starch control feed (HWM) group and a high starch+Lactobacillus plantarum FF34 (HWM+LAB) group, are prepared by taking fish meal, bean pulp, rapeseed meal and cottonseed meal as protein sources and soybean oil as fat sources, wherein 0.01-1% by mass of the Lactobacillus plantarum FF34 single-strain preparation prepared in the embodiment 5 is added into the high starch control feed (HWM) to obtain the (HWM+LAB) group, and the concentration of FF34 strain is 10 6 cfu/g feed. The crude protein content in each feed group was 32.5%, the crude fat content was 6.2%, the carbohydrate content was 43%, and it was always 17.4kJ/Kg. The conventional control feed comprises the following components in percentage by mass: 6.4% of fish meal, 21.6% of bean pulp, 16.2% of rapeseed meal, 15.3% of cotton pulp, 33.2% of wheat starch, 3.9% of soybean oil, 1.0% of monocalcium phosphate, 1.0% of premix, 0.5% of vitamin C, 0.4% of choline chloride and 0.5% of bentonite.
After 120 megalobrama amblycephala juvenile fish (initial weight 13.5+/-0.5 g) with healthy constitution and uniform specification are selected and temporarily cultured for one week, the megalobrama amblycephala juvenile fish is randomly divided into 6 outdoor net cages (1 m multiplied by 1 m), 20 tails of each net cage are divided into 2 groups, 3 repeats of each group, and 3 times (8:00, 12:00 and 17:00) of apparent satiety feeding test feed are carried out every day for 8 weeks. The water temperature is kept at 28-31 ℃ during the cultivation period, the dissolved oxygen is more than or equal to 7mg/L, the ammonia nitrogen is less than or equal to 0.1mg/L, the pH value is 3-7.8, and the cultivation water quality condition is measured every week by adopting a natural illumination period (12L: 12D). After the 8-week culture test, the animals were fasted for 24 hours, and the growth index and organ index were measured.
The growth performance index was calculated as follows:
weight Gain Ratio (WGR) (%) =100× (Wt-W0)/W0;
specific Growth Rate (SGR) (%/d) =100× (ln Wt-ln W0)/t;
bait coefficient (FCR) =f/(Wt-W0);
Intestinal body ratio (VI) =100×w i /W b
Wherein: w0 is the initial weight (g); wt is the final weight (g); t is the number of days of feeding (d); f is the total amount of feed intake (air-dried basis) (g); wb is the final body weight (g) of each fish; wi is the intestinal weight (g) of each fish.
In this example, HWM (high starch control diet) and HWM+LAB (high starch diet+FF 34 test) data were statistically analyzed using SPSS18.0, and all results were averaged.+ -. Standard error
The independent samples were tested for differences between groups, indicating significant differences between the two groups, test level P < 0.05, and the results are shown in table 8.
TABLE 8 influence of Lactobacillus plantarum FF34 added to high starch feed on the growth of young megalobrama amblycephala and utilization of high starch feed
| Index (I) | HWM (high starch control feed group) | HWM+LAB (high starch feed+ |
| Last weight, g | 38.26±0.61 | 42.60±1.56* |
| WGR，％ | 139.59±7.50 | 189.48±5.03* |
| SGR，％/d | 1.74±0.06 | 2.12±0.03* |
| FCR | 2.13±0.12 | 1.45±0.03* |
| Intestinal body ratio | 3.02±0.22 | 3.75±0.18* |
Note that: * Indicating significant difference (p < 0.05)
Megalobrama amblycephala is one of large freshwater fishes in China, has relatively weak tolerance and utilization capacity to carbohydrate, and excessive carbohydrate in feed can cause sugar intolerance phenomenon of fishes, so that growth is blocked. The results in Table 8 show that the addition of lactobacillus plantarum FF34 into the high-starch feed significantly improves the growth performance and feed utilization efficiency of young megalobrama amblycephala, the average individual weight of the young megalobrama amblycephala is significantly improved by 11.3 percent (P < 0.05), the weight gain rate is significantly improved by 35.7 percent (P < 0.05), the specific growth rate is significantly improved by 21.8 percent (P < 0.05), and the feed coefficient is significantly reduced by 31.9 percent (P < 0.05). And the lactobacillus plantarum FF34 is added into the high-starch feed to improve the intestinal volume ratio index, promote the growth and development of intestinal tracts, and obviously improve the specific gravity of the intestinal tracts and individuals by 24.2 percent (P is less than 0.05).
Example 11: influence of Lactobacillus plantarum FF34 added into high-starch feed on liver antioxidation of young megalobrama amblycephala
In this example, fish meal, soybean meal, rapeseed meal and cotton meal are usedAs protein source and soybean oil as fat source, 2 groups of test feeds, namely high starch feed (HCH) and high starch feed+Lactobacillus plantarum FF34 (HCH+LAB), are prepared, wherein the high starch feed (HCH) is used as a control group, the Lactobacillus plantarum FF34 single-strain preparation prepared in the example 5 is added into the high starch feed (HCH) according to the mass percentage of 0.01% -1%, namely (HCH+LAB) test group, and the FF34 strain concentration is 10 6 cfu/g feed. The crude protein content was 33.4%, crude fat was 6.5% and carbohydrate content was 47% for each test group. The conventional control feed composition was: 7% of fish meal, 23% of bean pulp, 15% of rapeseed meal, 12% of cotton pulp, 36% of wheat starch, 4% of soybean oil, 1.0% of monocalcium phosphate, 1.0% of premix, 0.5% of choline chloride and 0.5% of bentonite.
After 120 megalobrama amblycephala juvenile fish (initial weight 13.1+/-0.4 g) with healthy constitution and uniform specification are selected and temporarily cultured for one week, the megalobrama amblycephala juvenile fish is randomly divided into 6 outdoor net cages (1 m multiplied by 1 m), 20 tails of each net cage are divided into 2 groups, 3 repeats of each group are carried out, and the test feed is fed for 3 times (8:00, 12:00 and 17:00) per day in apparent satiety for 8 weeks. The water temperature is kept at 26-30 ℃ during the cultivation period, the dissolved oxygen is more than or equal to 7mg/L, the ammonia nitrogen is less than or equal to 0.1mg/L, the pH is 6, 9-7.5, and the cultivation water quality condition is measured every week by adopting a natural illumination period (12L: 12D). After the 8-week culture test is finished, the fish is fasted for 24 hours, and the oxidation resistance index of the liver of the fish is measured. The total antioxidant capacity (TAOC), catalase (CAT), total superoxide dismutase (SOD), reduced Glutathione (GSH) and Malondialdehyde (MDA) content of the liver are measured by using a kit of Nanjing institute of biological engineering.
In this example, the data of HCH (high starch feed control feed group) and HCH+LAB (high starch feed+Lactobacillus plantarum FF34 test group) were analyzed statistically using SPSS18.0, and all results were averaged.+ -. Standard error
The independent samples were tested for differences between groups, indicating significant differences between the two groups, test level P < 0.05, and the results are shown in table 9.
TABLE 9 influence of Lactobacillus plantarum FF34 added to high starch feed on liver antioxidant of young megalobrama amblycephala
The fish is not tolerant to high level sugar in the feed naturally, and excessive carbohydrate in the feed can reduce the growth performance and immunity of the fish, cause sugar metabolism disorder, show the sugar intolerance phenomenon of the fish and cause the oxidation resistance of the liver to be weakened. Resulting in impaired antioxidant function of the liver. Table 9 the results show that: the addition of lactobacillus plantarum FF34 to high starch feed significantly increases liver GSH, CAT and SOD levels (P < 0.05), so it is speculated that lactobacillus plantarum FF34 may utilize the rich starch in the feed to increase cholesterol levels to promote bile acid circulation metabolism, activate liver antioxidant capacity, and maintain growth and health.
Example 12: influence of Lactobacillus plantarum FF34 added into high-starch feed on fat deposition of muscle and liver of young megalobrama amblycephala
In the embodiment, 3 groups of test feeds, namely control feed (15 WM), high-starch feed (35 WM) and high-starch feed+lactobacillus plantarum FF34 (35WM+LAB) are prepared by taking fish meal, bean pulp, rapeseed meal and cottonseed meal as protein sources and soybean oil as fat sources, and the lactobacillus plantarum FF34 single-strain preparation prepared in the embodiment 5 is added into the high-starch feed (35 WM) according to the mass percentage of 0.01-1%, namely the 35WM+LAB test group, wherein the concentration of FF34 strain is 10 6 cfu/g feed, feed formulation is shown in Table 10.
Table 10 feed formulation composition of this example
After 180 megalobrama amblycephala juvenile fish (initial weight 13.6+/-0.7 g) with healthy constitution and uniform specification are selected and temporarily cultured for one week, the megalobrama amblycephala juvenile fish is randomly divided into 9 outdoor net cages (1 m multiplied by 1 m), 20 tails of each net cage are divided into 3 groups, 3 repeats of each group are carried out, and the apparent satiety of each group is carried out 3 times per day (8:00, 12:00 and 17:00) for 8 weeks. The water temperature is kept at 25-30 ℃ during the cultivation period, the dissolved oxygen is more than or equal to 7mg/L, the ammonia nitrogen is less than or equal to 0.1mg/L, the pH is 6.9-7.5, and the cultivation water quality condition is measured every week by adopting a natural illumination period (12L: 12D). After the 8-week culture test is finished, the fish is fasted for 24 hours, the muscle fat content of the fish is measured, and lipid drop deposition in the liver is analyzed. The analysis of the nutritional components of the muscle is determined by reference to the national standard specification method. Drying in an oven at 105 ℃ to constant weight by adopting a normal pressure drying method to calculate dry matter content; the crude protein content was determined by Kjeldahl method (GB/T6432-1994); crude fat content was determined by Soxhlet extraction (GB/T6433-1994); the coarse ash content was determined by the 560℃firing method (GB/T6438-1992). Liver tissue pieces fixed in 4% paraformaldehyde were removed, frozen sections of liver were prepared, stained with oil red O, and analyzed for oil red O lipid drop area using Image J software. In this example, the data comparison between 15WM (control feed), 35WM (high starch feed) and 35WM+LAB (high starch feed+Lactobacillus plantarum FF 34) was tested using one-factor analysis of variance turkey's test at a level of P < 0.05 and the results are shown in Table 11.
The fish cannot tolerate high level sugar in the feed naturally, excessive carbohydrate in the feed can cause glycolipid metabolic disturbance, excessive deposition of lipid in fish body, oxidative stress injury and disease resistance reduction. In fig. 1, the oil red O-stained pictures of lipid droplets in liver tissue sections show that the deposition of lipid droplets in liver tissue of megalobrama amblycephala fed with 35WM group feed is significantly increased, while the addition of lactobacillus plantarum (35wm+lab) to fed high starch feed reduces the lipid droplet area in liver. Further analysis of megalobrama amblycephala muscle fat and liver fat drop areas (Table 11) found that the high starch group (35 WM) significantly increased the amount of crude fat and liver fat drop deposition (P < 0.05) in the muscle compared to the basal control group (15 WM). The addition of lactobacillus plantarum FF34 (35WM+LAB) in the high-starch feed can obviously reduce the crude fat content of the megalobrama amblycephala body muscle (P is less than 0.05), and has no obvious difference with the control group; while liver lipid droplet deposition in the 35WM+LAB group, while higher than in the control group, was significantly lower than in the high starch group (P < 0.05).
FIG. 1 Lactobacillus plantarum and high starch addition liver oil red 0 staining sections of young megalobrama amblycephala. Panel A is a 15WM group (x 200 times) of oil red O stained sections, panel B is a 35WM+LAB group (x 200 times) of oil red O stained sections, and panel C is a 35WM group (x 200 times) of oil red O stained sections. In the figure, red represents lipid droplets, and blue represents nuclei.
TABLE 11 Effect of high starch added Lactobacillus plantarum FF34 on muscle fat content and liver fat droplet deposition of megalobrama amblycephala juvenile fish
| Grouping | 15WM | 35WM | 35WM+LAB |
| Muscle fat content% | 4.88±0.61 a | 6.59±0.35 b | 4.75±0.48 a |
| Liver fat drop deposition% | 2.50±0.64 a | 28.48±3.01 c | 20.48±0.92 b |
Note that: the same lowercase letters of the same row indicate that the difference is not significant (p > 0.05), and the different lowercase letters of the same row indicate that the difference is significant (p < 0.05).
Claims (4)
1. Lactobacillus plantarum FF34, classified and named as Lactobacillus plantarumlactobacillus plantarum) Preserving in China Center for Type Culture Collection (CCTCC), address: the preservation number CCTCC NO: m20211221, the preservation date is 2021, 10, 11.
2. The method for preparing the FF34 single-fungus preparation with aquatic pathogen antagonistic property by the strain FF34 as claimed in claim 1, which is characterized by comprising the following steps:
(1) Activating strains:
freeze-drying preserved strain of the bacteria-free lactobacillus plantarum FF34 is inoculated into a test tube filled with MRS broth, subjected to stationary culture at 30-35 ℃ for 24-48 hours, transferred into an MRS broth triangular flask, and subjected to culture and activation at 30-35 ℃ for 20-48 hours; repeatedly activating for 2-3 times, performing microscopic examination, and counting until the concentration of the bacterial cells reaches 10 9 CFU/mL is used as seed liquid;
MRS broth composition in g/L: 10 parts of casein enzyme digest, 10 parts of beef extract powder, 4 parts of yeast extract powder, 2 parts of citric acid triammonium, 5 parts of sodium acetate, 0.2 parts of magnesium sulfate, 0.05 parts of manganese sulfate, 2 parts of dipotassium hydrogen phosphate, 20 parts of glucose and 1.08 parts of tween-80, and preparing the beef extract powder and the yeast extract powder by constant volume with distilled water, wherein the pH value is 5.7+/-0.2;
(2) Immobilized fermentation culture:
preparing a triangular flask seed culture solution: inoculating the seed liquid obtained in the step (1) into a 3L triangular flask filled with an immobilized fermentation culture medium according to the volume ratio of 1% -5%, standing and culturing for 24-40 h at 30-35 ℃ until the thallus concentration reaches 10% 9 CFU/mL is used as a seed culture solution of the triangular flask;
amplifying and expanding cultivation: the triangular flask seed culture solution is connected into a common aerobic fermentation tank for primary and secondary immobilized fermentation and gradual amplification and expansion culture, namely: 3L triangular flask, 500L fermenter, 10T fermenter, and sterile air pressure feeding method for inoculation;
primary immobilization culture: inoculating the triangular flask seed culture solution into a 500L fermentation tank with 1-5% of inoculation amount by volume ratio, wherein the volume ratio of the fermentation tank liquid is 80-90%, the sterile air is kept under pressure of 0.01-0.05 Mpa, intermittent stirring is carried out, the rotating speed is 80-100 r/min, the culture is finished when the temperature is 30-35 ℃ for 20-40 h and the pH is 4.5-5.5, and the culture is finished, thus obtaining a first-stage immobilized culture solution, wherein lactobacillus plantarum FF34 thalli is adsorbed and immobilized in gel small particles formed by calcium alginate and silicon dioxide, and the concentration of viable bacteria is more than or equal to 5 multiplied by 10 9 CFU/mL；
And (3) secondary immobilization culture: the primary immobilized culture solution is delivered by sterile air pressure, the inoculation amount of 1 to 5 percent of the volume ratio is connected into a 10T fermentation tank, the liquid amount of the fermentation tank is 70 to 90 percent of the volume ratio, the sterile air is kept under the pressure of 0.01 to 0.05Mpa, the intermittent stirring is carried out, the rotating speed is 80 to 100r/min, the culture is carried out for 20 to 40 hours at the temperature of 30 to 38 ℃, when the pH value of the fermentation solution is reduced to 4.0 to 5.5, the culture is finished, the secondary immobilized culture solution is obtained, wherein lactobacillus plantarum FF34 thalli is adsorbed and fixed in gel small particles formed by calcium alginate and silicon dioxide, and the concentration of the thalli is more than or equal to 5 multiplied by 10 9 CFU/mL；
The immobilized fermentation medium consists of the following components in g/L: 10-40 parts of carbon source, 3-15 parts of nitrogen source, 0.2-5 parts of dipotassium hydrogen phosphate, 0-0.2 part of magnesium sulfate, 0-0.05 part of manganese sulfate, 5-40 parts of calcium carbonate, 3-10 parts of sodium alginate and 1-10 parts of silicon dioxide, and sterilizing for 30min at 121 ℃ by using tap water with constant volume; wherein the carbon source comprises one or more of wheat middling, corn starch, dextrin, molasses, glucose and fructose syrup; the nitrogen source comprises one or more of fish meal peptone, tryptone, soybean peptone, soybean protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal;
(3) Preparation of lactobacillus plantarum FF34 single bacterial agent with aquatic pathogenic bacteria antagonistic property:
(A) Preparation of lactobacillus plantarum FF34 single-fungus aqua: filling the secondary immobilized culture solution obtained in the step (2) to obtain a lactobacillus plantarum FF34 single-fungus water agent, wherein the concentration of FF34 viable bacteria in the single-fungus water agent is more than or equal to 5.0x10 9 CFU/mL；
(B) Preparation of Lactobacillus plantarum FF34 single-strain powder: centrifuging the secondary immobilized culture solution obtained in the step (2) to collect wet thalli, wherein the mass ratio is as follows: wet thalli, calcium carbonate and dextrin are mixed according to the proportion of silicon dioxide=1:0.1-2:0.5-2:0.01, and lactobacillus plantarum FF34 bacterial powder is obtained through spray drying or vacuum drying, wherein the concentration of FF34 viable bacteria in the bacterial powder is not lower than 1 multiplied by 10 10 CFU/g。
3. The method for preparing the mixed fermentation culture and composite microbial inoculum of the strain FF34 and bacillus as claimed in claim 1 is characterized by comprising the following steps:
(1) The bacillus mixed fermentation culture with the strain FF34 is as follows: bacillus subtilis AB90008-15, bacillus amyloliquefaciens JSSW-LA, bacillus coagulans JSSW-LA-07-1, clostridium butyricum JSIM-MCB20040312 or one of bacillus thuringiensis JSSW-BP44 with aquatic pathogen antagonistic property;
(2) FF34 is mixed fermented with the above bacillus: the method comprises two steps, wherein the first fermentation step is to perform activation culture on the bacillus through an inclined plane or a triangular flask, then inoculate the bacillus into a mixed fermentation culture medium, wherein the inoculum size is 1-10% by volume, the culture temperature is 30-37 ℃, and the culture is performed for 24-48 hours, and spores in the bacteria are subjected to microscopic examination, namely the first fermentation step is finished; the second fermentation step is that the pH of the fermentation liquor obtained in the first fermentation step is adjusted to 5.0-5.4 by hydrochloric acid, then activated lactobacillus plantarum FF34 is inoculated, the inoculation amount is 1-10% by volume, the culture temperature is 30-35 ℃, intermittent stirring is carried out, the pH of the fermentation liquor is controlled to 4.5-5.0 by feeding glucose with w/v concentration of 20% or calcium carbonate or ammonia water with concentration of 30%, the fermentation liquor is cultured for 24 hours, the mixed fermentation liquor of FF34 and bacillus is obtained, microscopic examination can be carried out to ensure that lactobacillus and bacillus coexist, and the concentration of FF34 viable bacteria in the mixed fermentation liquor is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
the composition of the mixed fermentation medium is calculated in g/L: wheat middling 0-50, peptone 0.1-5, yeast extract 0-5, sodium acetate 0-3, corn starch 0-10, ammonium sulfate 0-2, sodium chloride 0-5, calcium carbonate 0-5, monopotassium phosphate 0-0.5, magnesium sulfate 0-0.5, manganese sulfate 0-0.3, sodium alginate 0-5, tap water constant volume, pH 7.0, sterilizing at 121 ℃ for 30min;
preparation of lactobacillus plantarum FF34 composite microbial inoculum:
(A) Directly filling the mixed fermentation liquid of the lactobacillus plantarum FF34 and the bacillus obtained in the step (2) to obtain a lactobacillus plantarum FF34 composite bacterial aqua, wherein the concentration of the live bacteria FF34 in the composite bacterial aqua is more than or equal to 1.0x10 9 CFU/mL, bacillus viable bacteria concentration is more than or equal to 5.0X10 8 CFU/mL, the spore rate is more than or equal to 90%;
(B) And (3) centrifuging the lactobacillus plantarum FF34 and bacillus mixed fermentation liquid obtained in the step (2) to collect wet thalli, wherein the mass ratio is as follows: mixing wet thalli and calcium carbonate, wherein the silicon dioxide is=1:0.1-3:0.01, and spray drying or vacuum drying to obtain lactobacillus plantarum FF34 composite bacterial powder, wherein the concentration of FF34 viable bacteria in the composite bacterial powder is more than or equal to 5 multiplied by 10 9 CFU/g, bacillus viable bacteria concentration is more than or equal to 5.0X10 9 CFU/g。
4. The application of the lactobacillus plantarum FF34 single microbial inoculant and the composite microbial inoculant prepared by the method of claims 2 and 3, which are characterized in that: (1) The lactobacillus plantarum FF34 single microbial inoculum or composite microbial inoculum is used for fermenting fish, shrimp and crab feed, cotton meal and rapeseed meal feed raw materials according to the addition amount of 0.1-5% by mass, the novel mulberry leaf feed raw materials can obviously improve the nutrition components of the fish, shrimp and crab feed or feed raw materials, the obtained fermented feed is rich in probiotics, acid soluble protein and active small peptide, and the content of tannins, free gossypol and phytic acid anti-nutrition factors is reduced; (2) The lactobacillus plantarum FF34 single microbial agent or the composite microbial agent is added into basic ration or high-starch feed according to the mass percentage of 0.05-1%, so that intestinal tissue growth of the aquaculture animals can be promoted, liver function is improved, antioxidant activity is improved, growth performance of the aquaculture animals is obviously improved, and feed coefficient is reduced; (3) Uniformly mixing the lactobacillus plantarum FF34 compound bacterial agent aqua with sodium alginate with the mass volume ratio of 2-4%, adding pumice, activated carbon or ceramsite, soaking and adsorbing for 1-4 h, taking out, soaking in 2-3% calcium chloride solution, curing and crosslinking for 1-10 h at 4-8 ℃ to obtain lactobacillus plantarum FF34 compound bacterial immobilized bacteria spheres or blocks, wherein the concentration of bacterial bodies in the bacteria spheres or blocks is not lower than 5 multiplied by 10 8 CFU/g, lactobacillus plantarum FF34 composite bacteria immobilized bacteria balls or blocks are put into the culture water body, so that the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content of the water body can be degraded, growth of aquatic pathogenic bacteria is inhibited, and water quality of the culture pond is continuously improved.
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