CN116790409A - Lactobacillus reuteri and preparation and application of microecological preparation thereof - Google Patents
Lactobacillus reuteri and preparation and application of microecological preparation thereof Download PDFInfo
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- CN116790409A CN116790409A CN202310285695.1A CN202310285695A CN116790409A CN 116790409 A CN116790409 A CN 116790409A CN 202310285695 A CN202310285695 A CN 202310285695A CN 116790409 A CN116790409 A CN 116790409A
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- 229940111202 pepsin Drugs 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- KFQYTPMOWPVWEJ-INIZCTEOSA-N rotigotine Chemical compound CCCN([C@@H]1CC2=CC=CC(O)=C2CC1)CCC1=CC=CS1 KFQYTPMOWPVWEJ-INIZCTEOSA-N 0.000 description 1
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- 150000004666 short chain fatty acids Chemical class 0.000 description 1
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Abstract
The invention discloses lactobacillus reuteri and preparation and application of a microecological preparation thereof. The lactobacillus reuteri (Lactobacillus reuteri) is named as SAMMRS-1G; the culture is preserved in China Center for Type Culture Collection (CCTCC) No. M20221519. The strain is derived from aquatic animal sources, has broad-spectrum antagonism to animal breeding pathogenic bacteria, especially aquatic pathogenic bacteria, and can inhibit vibrio, putrefying bacteria, monad, escherichia coli, staphylococcus aureus, salmonella and the like. The microecological preparation of the strain has the characteristics of remarkable efficacy, good stability and strong stress resistance. The microecological preparation has strong capability of preventing and treating pathogenic bacteria, and has the characteristics of probiotics and prebiotics; the preparation is not affected by temperature, and the antibacterial activity is not destroyed by protease, lipase and amylase in host animals, so that the preparation has stronger stability; and has better tolerance capability and certain tolerance to gastric juice, intestinal juice and bile salts.
Description
Technical Field
The invention belongs to the technical field of application microorganisms, and particularly relates to lactobacillus reuteri and preparation and application of a microecological preparation of lactobacillus reuteri.
Background
China is the first large aquiculture country in the world, and the culture yield is about 70% of the total yield of aquiculture in the world. However, with the gradual expansion of the aquaculture scale and the continuous improvement of the intensive degree in China, the aquaculture water quality is continuously deteriorated due to bait residues, excrement accumulation and the like in the aquaculture process, diseases of the aquaculture industry are aggravated, and main pathogens of aquatic animals comprise bacteria, viruses, parasites and the like, wherein the disease loss of the aquatic animals caused by bacterial diseases accounts for 58%, and is the most serious factor causing the economic loss of the aquaculture. Common pathogenic bacteria of bacterial diseases in aquaculture mainly comprise aeromonas, vibrio, shewanella and the like, which often cause large-scale outbreak of diseases related to aquaculture animals, bring great loss to the aquaculture industry which is vigorously developed, and greatly limit healthy and sustainable development of aquaculture in China.
In order to effectively prevent and treat diseases easily caused by fishes and shrimps and promote the rapid growth of organisms, the phenomenon of abusing antibiotics is common to farmers, a large number of resistant strains and resistant genes are induced to generate, and the method has a great threat to food safety and public health. Aiming at the problem of antibiotic resistance, three coping strategies of immune control, chinese herbal medicine cultivation and microecological cultivation are mainly available at present. Compared with the other two species, the microecological culture has stronger applicability, the microecological preparation adheres and colonizes intestinal dominant flora such as lactobacillus and the like in the animal intestinal tract, and the biological activity of pathogenic bacteria can be effectively inhibited through biological oxygen deprivation and organic acid generated by self metabolism, bacteriocin and other biological active components, so that the method is beneficial to improving the defensive capability of the organism to diseases and enhancing the resistance and stability of an intestinal microecological system. However, the commercialized lactobacillus bacteriocins are limited to only a few types of pediocin PA-1 and Nisin, etc., because many lactobacillus bacteriocins have a narrow pH range, unstable heat, narrow bacteriostasis spectrum or relatively low yield, and the antibacterial lactobacillus is applied to less antibacterial and disease prevention in aquaculture, such as the invention patent CN 113604387A, which only has salt tolerance, can be used for preventing and treating pathogenic bacteria in aquaculture, but is derived from pig manure, and has no bacteriostasis performance and related test of systemic aquatic pathogenic bacteria, so that whether the lactobacillus bacteriocins are really suitable for preventing and treating the pathogenic bacteria in aquaculture cannot be known.
Therefore, most of lactobacillus used for aquaculture at present is not derived from water-host animals, the probiotics used for aquaculture are unstable, and the application of lactobacillus reuteri in aquaculture is less, so that lactobacillus reuteri with high-efficiency broad-spectrum capability of inhibiting aquatic pathogenic bacteria is separated and screened and applied to aquaculture, meanwhile, a high-density culture method of the strain is further optimized, the effective viable count is improved, the use method of the lactobacillus reuteri in the field of aquaculture is standardized, and the lactobacillus reuteri has important practical significance for gradually realizing little-to-no-resistance aquaculture in the aquaculture industry.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides lactobacillus reuteri and preparation and application of a microbial inoculum thereof. The lactobacillus reuteri screened from aquatic animal sources, especially from fish and shrimp intestinal tracts, has broad-spectrum antagonism to aquatic pathogenic bacteria, and can inhibit vibrio, putrefying bacteria, monad, colibacillus, staphylococcus aureus, salmonella and the like. The application of the lactobacillus reuteri preparation in the prevention and treatment of aquaculture diseases provided by the invention has the advantages of high antibacterial efficiency, quick response and no side effect, and can be used for replacing antibiotics in aquaculture industry.
The invention provides lactobacillus reuteri, which is named as follows: lactobacillus reuteri (Lactobacillus reuteri), strain SAMMRS-1G; the 16S RNA sequence of the strain is submitted to GenBank database with accession number OM149729 and is preserved to China center for type culture Collection (China, accession number: eight paths of the university of Lopa nationality in Wuchang district of Wuhan, hubei province have the preservation number of CCTCC NO: M20221519.
The invention also provides application of the lactobacillus reuteri SAMMRS-1G in preventing and controlling pathogenic bacteria in animal cultivation.
The invention also provides application of the lactobacillus reuteri SAMMRS-1G in preventing and controlling pathogenic bacteria in aquaculture.
In some embodiments, the pathogenic bacteria is escherichia coli, salmonella, staphylococcus aureus, unicona, putrefying bacteria, or vibrio.
Specifically, the monad is aeromonas hydrophila or pseudoalteromonas; the putrefying bacteria are Shewanella putrefying bacteria; the vibrio is Vibrio harveyi, vibrio alginolyticus, vibrio parahaemolyticus, vibrio cholerae or Vibrio vulnificus.
The invention also provides a microecological preparation which contains the lactobacillus reuteri SAMMRS-1G or fermentation liquor thereof as an active ingredient.
The invention also provides a preparation method of the lactobacillus reuteri SAMMRS-1G microecological preparation, which comprises the following steps:
s1, strain activation: inoculating the Lactobacillus reuteri SAMMRS-1G strain on MRS slant culture medium for culturing, strictly performing aseptic operation, and culturing at 32-39deg.C for 18-30 hr;
s2, primary seed culture: inoculating the lactobacillus reuteri SAMMRS-1G inclined plane obtained in the step S1 into a primary seed liquid culture medium, and standing and culturing for 12-18 h at the temperature of 32-39 ℃ to obtain primary seed liquid;
s3, culturing secondary seeds: the first-level seed liquid obtained in the step S2 is inoculated into a second-level seed culture medium according to the volume ratio of 3-5%, and is subjected to stationary culture for 8-12 h at the temperature of 32-39 ℃ to obtain the second-level seed liquid;
s4, liquid fermentation culture: preparing a fermentation medium according to the volume of 60-80% of the fermentation tank, introducing steam, and sterilizing the material at 121 ℃; cooling to 32-39 ℃, inoculating the secondary seed liquid obtained in the step S3 into a fermentation medium according to the volume ratio of 1-3%, and carrying out anaerobic fermentation for 6-14 h under the conditions of 32-39 ℃ and pH 4.0-5.5 at the rotating speed of 100r/min and the nitrogen pressure of 0.05Mpa under the condition of maintaining the pressure of nitrogen, thereby obtaining the lactobacillus reuteri SAMMRS-1G microecologics.
The effective viable count of the obtained lactobacillus reuteri SAMMRS-1G microecological preparation can reach 1.0 to 1.5X10 10 CFU/mL。
The beneficial effects of the invention are as follows:
1. the lactobacillus reuteri SAMMRS-1G provided by the invention is derived from aquatic animals, and has good capability of inhibiting aquatic pathogenic bacteria growth, good acid resistance, bile salt resistance and adhesion performance and good probiotics performance through screening.
2. At present, little research is carried out on lactobacillus reuteri in aquaculture, and the lactobacillus reuteri SAMMRS-1G provided by the invention has the effects of inhibiting aquatic pathogenic bacteria, improving growth performance and feed utilization rate, regulating intestinal flora, recovering intestinal barrier, improving the ecological environment of aquaculture and the sensory quality of aquatic products, and prolonging shelf life. The concrete steps are as follows:
(1) The inhibition capability is strong, the antibacterial activity is not affected by temperature, and the antibacterial activity is not damaged by protease, lipase and amylase in a host animal body, so that the antibacterial agent has stronger stability;
(2) The method has strong adhesion capability to mucous membrane mucus and intestinal mucosa epithelial cells of a host, and the generated short chain fatty acid can promote the increase of goblet cell number and mucin 1 and 2 expression, and quickly restore intestinal barrier so as to regulate intestinal flora and restore the intestinal barrier;
(3) The metabolic prebiotics and various enzyme substances can improve intestinal villus height, promote digestion and absorption of nutrient substances by a host, and improve growth performance and feed utilization rate;
(4) The method can effectively reduce the concentration of nitrite, ammonia nitrogen, hydrogen sulfide and the like in the water quality of cultivation, inhibit the reproduction and growth of harmful microorganisms in the water body, purify the water quality and improve the ecological environment of cultivation;
(5) Can effectively control the generation of putrefying bacteria and organic amines, effectively reduce pseudomonas and hydrogen sulfide-producing bacteria in the product during the storage period, improve the sensory quality of aquatic products and prolong the shelf life.
3. The lactobacillus reuteri SAMMRS-1G microecological preparation provided by the invention has the characteristics of remarkable efficacy, good stability and strong stress resistance. The microecological preparation has strong capability of preventing and treating pathogenic bacteria, and has the characteristics of probiotics and prebiotics; the preparation is not affected by temperature, and the antibacterial activity is not destroyed by protease, lipase and amylase in host animals, so that the preparation has stronger stability; and has better tolerance capability and certain tolerance to gastric juice, intestinal juice and bile salts.
In addition, the application technical scheme of the lactobacillus reuteri (Lactobacillus reuteri) SAMMRS-1G in aquaculture disease control is not reported yet.
Preservation description
Lactobacillus reuteri (Lactobacillus reuteri), strain SAMMRS-1G; has been deposited to China center for type culture Collection, accession number: eight paths of the university of Lopa nationality in Wuchang district of Wuhan, hubei province have the preservation number of CCTCC NO: M20221519.
Drawings
FIG. 1 shows the colony morphology of the bacterium Lactobacillus reuteri SAMMRS-1G (a) and the microscopic examination (1000).
FIG. 2 is the biological properties of Lactobacillus reuteri SAMMRS-1G.
FIG. 3 is a growth curve of Lactobacillus reuteri SAMMRS-1G.
FIG. 4 shows the bacteriostatic ability of the Lactobacillus reuteri SAMMRS-1G fermentation broth against aquatic pathogenic bacteria,
wherein, a is pseudoalteromonas; b Aeromonas hydrophila; c salmonella; d, escherichia coli; e Vibrio parahaemolyticus; f Vibrio alginolyticus; g Vibrio harveyi; h spoiling Shewanella; i Vibrio vulnificus.
Detailed Description
The invention provides preparation and application of lactobacillus reuteri and a microecological preparation thereof, and a person skilled in the art can refer to the content of the invention and properly improve the technological parameters, and the preparation and application are regarded as being included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Example 1 screening and identification of Lactobacillus reuteri SAMMRS-1G Strain for controlling aquatic pathogenic bacteria
1. Sample collection
And selecting apparent healthy carps, marchans, penaeus vannamei and sea cucumbers from a certain aquaculture farm in Dalian city as experimental study objects. In the laboratory, the aquatic animals were surface sterilized and their intestinal tracts (foregut, midgut and hindgut) were homogenized in a sterile manner for later use in experiments.
2. Isolation of strains
Diluting the above homogenate with appropriate amount of MRS culture medium to obtain stock solution, subjecting the stock solution to 10-time gradient dilution with sterile physiological saline, collecting stock solution and 100 μl of each of the multiple dilutions, and respectively coating on MRS solid culture medium (containing 1% CaCO) 3 ) Anaerobic culturing at 30 deg.c for 2d until single colony grows, observing and recording colony morphology and characteristic, picking different colony with calcium ring in MRS solid culture medium, streaking and pure culturing the single colony until single colony with homogeneous morphology and size grows, picking single colony and inoculating into liquid MRS culture medium, anaerobic culturing at 30 deg.c for 2d. The cultured liquid bacteria can be subjected to glycerol sterilization and stored in a refrigerator at-80 ℃ for later use.
3. Screening of strains
The common pathogenic bacteria of aquatic products mainly have waterAeromonas, vibrio harveyi and vibrio parahaemolyticus are used as indicator bacteria for screening. The oxford cup method is adopted: pouring 15-18 mL of sterilized nutrient agar culture base culture dish, standing for solidification. The bacterial suspension is added into nutrient agar culture medium with the temperature of 45-50 ℃ to prepare the culture medium with the final concentration of 0.2 multiplied by 10 8 Sucking 5mL of cfu/mL on a plate for solidification to form a bacterial layer, wherein the bacterial content of each plate is 1 multiplied by 10 8 cfu. Placing 4 oxford cups on a culture dish at equal intervals, respectively injecting 200 mu L of strain fermentation liquor into the oxford cups by using a pipetting gun, adding the same volume of MRS liquid culture medium as a blank control, standing for 30min, placing the dish in a constant temperature incubator, culturing for 24h at 30 ℃, measuring by using a vernier caliper, and recording the diameter of a bacteriostasis ring. Functional probiotics were screened by in vitro bacteriostasis tests. The facultative anaerobic lactic acid bacteria with the maximum bacteriostasis diameter of the 3 pathogenic bacteria and more than 15.0mm are selected. The isolated facultative anaerobic lactic acid bacteria strain was streaked onto blood agar plates containing 5% (v/v) of commercial sterile sheep blood and anaerobically cultured at 30℃for two days, and this strain had no hemolytic activity and was labeled as SAMMRS-1G.
4. Identification of strains
(1) Morphological features
The strain SAMMRS-1G had a single colony diameter of about 1 to 3mm on an MRS solid plate, and the colony was white in color, smooth and matt in surface, slightly convex in the middle, opaque and neat in edge (FIG. 1 (a)). The strain SAMMRS-1G was gram stained and the cells were in the form of stubbles rounded at both ends under a 10 x 100 microscope, with a smoother surface, free of spores, free of flagella, free of movement, gram positive bacteria (fig. 1 (b)).
(2) Molecular biological identification
Molecular identification is carried out on the strain SAMMRS-1G obtained by screening, genomic DNA is extracted, primer sequences are 27F, AGAGTTTGATCCMTGGCTTAG and 1490 2R, GGTTACCTTGTTACGACTT, 16S rDNA is amplified, and the amplified strain SAMMRS-1G is submitted to sequencing of biological engineering (Shanghai). The total length of the SAMMRS-1G sequence is 1473bp, and the sequencing result is shown as SEQ ID No. 1.
SEQ ID No.1 is shown below:
1ggctcaggat gaacgccggc ggtgtgccta atacatgcaa gtcgtacgca ctggcccaac
61tgattgatgg tgcttgcacc tgattgacga tggattacca gtgagtggcg gacgggtgag
121taacacgtag gtaacctgcc ccggagcggg ggataacatt tggaaacaga tgctaatacc
181gcataacaac aaaagccaca tggcttttgt ttgaaagatg gctttggcta tcactctggg
241atggacctgc ggtgcattag ctagttggta aggtaacggc ttaccaaggc gatgatgcat
301agccgagttg agagactgat cggccacaat ggaactgaga cacggtccat actcctacgg
361gaggcagcag tagggaatct tccacaatgg gcgcaagcct gatggagcaa caccgcgtga
421gtgaagaagg gtttcggctc gtaaagctct gttgttggag aagaacgtgc gtgagagtaa
481ctgttcacgc agtgacggta tccaaccaga aagtcacggc taactacgtg ccagcagccg
541cggtaatacg taggtggcaa gcgttatccg gatttattgg gcgtaaagcg agcgcaggcg
601gttgcttagg tctgatgtga aagccttcgg cttaaccgaa gaagtgcatc ggaaaccggg
661cgacttgagt gcagaagagg acagtggaac tccatgtgta gcggtggaat gcgtagatat
721atggaagaac accagtggcg aaggcggctg tctggtctgc aactgacgct gaggctcgaa
781agcatgggta gcgaacagga ttagataccc tggtagtcca tgccgtaaac gatgagtgct
841aggtgttgga gggtttccgc ccttcagtgc cggagctaac gcattaagca ctccgcctgg
901ggagtacgac cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcggtgga
961gcatgtggtt taattcgaag ctacgcgaag aaccttacca ggtcttgaca tcttgcgcta
1021accttagaga taaggcgttc ccttcgggga cgcaatgaca ggtggtgcat ggtcgtcgtc
1081agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gttactagtt
1141gccagcatta agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg
1201acgacgtcag atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt
1261acaacgagtc gcaagctcgc gagagtaagc taatctctta aagccgttct cagttcggac
1321tgtaggctgc aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc
1381gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg agtttgtaac
1441gcccaaagtc ggtggcctaa cctttatgga ggg
nucleotide homology alignment was performed with NCBI's Gen-Bank (http:// NCbi.nlm.fox/BLAST) database and Lactobacillus reuteri in the database, with sequence homology of 100.00%, and the bacterium was determined to be Lactobacillus reuteri (Lactobacillus reuteri) and designated Lactobacillus reuteri SAMMRS-1G. The 16SRNA sequence of the strain is submitted to GenBank database with accession number OM149729 and is preserved to China center for type culture Collection (China, accession number: eight paths of the university of Lopa nationality in Wuchang district of Wuhan, hubei province have the preservation number of CCTCC NO: M20221519.
Example 2 Luo Yishi Properties of Lactobacillus SAMMRS-1G
1. Lactobacillus reuteri SAMMRS-1G optimal pH assay
The initial pH of the liquid medium of the activated Lactobacillus reuteri SAMMRS-1G strain was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0 and 7.0 with HCl and NaOH, respectively. The number of viable bacteria was measured after stationary culture at 39℃for 24 hours at an inoculum size of 3%, and the optimum culture pH was found to be 5.0 as shown in FIG. 2-1.
2. Determination of the optimal temperature of Lactobacillus reuteri SAMMRS-1G
The activated Lactobacillus reuteri SAMMRS-1G strain was subjected to stationary culture at 25℃at 28℃at 31℃at 35℃at 39℃at 42℃for 24 hours at an inoculum size of 3%, and the number of viable bacteria was determined to give an optimum culture temperature of 39℃as shown in FIGS. 2-2.
3. Lactobacillus reuteri SAMMRS-1G test for simulating gastrointestinal environment of fish and shrimp-tolerance to gastric acid
Adjusting the pH of MRS liquid culture medium to 7.0, 6.0, 5.0, 4.0, 3.0, 2.0 with 1M hydrochloric acid or NaOH respectively10mL of culture media with different pH values are taken, fresh lactobacillus reuteri suspension cultured for 12h is inoculated into the bacterial liquid according to the inoculation amount of 3 percent, anaerobic culture is carried out for 0, 2 and 4h at 37 ℃, and MRS without pH adjustment is used as a control. The viable count before and after each treatment was counted as plate colonies, and the survival rate of lactobacillus reuteri was measured. The calculation formula of the survival rate is as follows: survival (%) =n t /N 0 X 100. In the above, N 0 Viable count (CFU/mL) for 0h of test strain; n (N) t Viable count (CFU/mL) for 4h of treatment for the test strain. The results showed that the survival rate was 90.7.+ -. 2.1% at pH3.0, and that pH5.0 was the optimum pH as shown in FIGS. 2-3. The enterogastric environment of the fishes and the shrimps is weak acid, and the pH is 3.0-6.0, so that the lactobacillus reuteri can be well adapted to the enterogastric environment of the fishes and the shrimps.
4. Test of Lactobacillus reuteri SAMMRS-1G for simulating gastrointestinal environment-bile salt tolerance of fishes and shrimps
The method is the same as described above. The bile salt content in the MRS culture medium of the test group is 0g/L, 1.0g/L, 2.0g/L, 3.0g/L and 6.0g/L respectively. Inoculating fresh lactobacillus reuteri suspension cultured for 12 hours into the bacterial liquid according to an inoculum size of 1 percent, carrying out anaerobic culture for 0, 2 and 4 hours at 37 ℃, measuring the viable count of the lactobacillus reuteri, and calculating the survival rate. The results showed that the survival rate was 85.3.+ -. 2.5% at a bile salt content of 3.0g/L, as shown in FIGS. 2-4.
5. Determination of the growth curve of Lactobacillus reuteri SAMMRS-1G
Inoculating into MRS liquid culture medium according to 3% inoculum size, and standing at 39deg.C for culturing. The number of viable bacteria was measured by sampling every 2 hours. The results show (FIG. 3), luo Yishi Lactobacillus SAMMRS-1G strain enters the logarithmic phase at 4h, enters the platform phase at 14h, and the maximum viable count reaches 1×10 8 CFU/mL。
6. Tolerance of lactobacillus reuteri SAMMRS-1G under conditions of different sodium chloride concentrations
Using MRS as basic culture medium, adding 0g, 1.0g, 2.0g, 3.0g, 4.0g and 6.0g sodium chloride into every 100mL basic culture medium to prepare culture mediums with salinity of 0%, 1.0%, 2.0%, 3.0%, 4.0% and 6.0%, respectively, adjusting pH to 5.0, and sterilizing at 121deg.C under high temperature and high pressure. Inoculating fresh lactobacillus reuteri suspension cultured for 12 hours into the bacterial liquid according to the inoculation amount of 3 percent, carrying out anaerobic culture for 0, 2 and 4 hours at 37 ℃, measuring the viable count of the lactobacillus reuteri, and calculating the survival rate. The result shows that the survival rate of the treated water for 4 hours under the condition of 6.0 percent of salinity is still more than 80.5+/-1.4 percent.
In summary, the optimal culture temperature, pH and growth curve of the SAMMRS-1G4 further provide a theoretical basis for the fermentation process optimization of the SAMMRS-1G; the SAMMRS-1G has better tolerance to the gastrointestinal environment of fishes and shrimps, and the tolerance is more than 85 percent; the salinity of seawater is generally 3.5%, and the SAMMRS-1G also shows good activity when being treated for 4 hours under the condition of high salinity of 6.0%. These properties make lactobacillus reuteri SAMMRS-1G a potential to be a probiotic added to aquatic animal diets and resistant to stress and can be effectively utilized.
Example 3 Luo Yishi preparation Process of Lactobacillus samMRS-1G microecological preparation
S1, strain activation: the lactobacillus reuteri SAMMRS-1G strain is inoculated on an MRS slant culture medium for culture, and is strictly sterilized for culture for 24 hours at the temperature of 32-39 ℃. The slant culture medium: 4.0g of tryptone, 10.0g of beef extract, 8.0g of yeast powder, 10.0g of glucose, 0.5g of L-cysteine hydrochloride, 5.0g of sodium acetate, 2.0g of sodium citrate, 2.0g of Tween-801.0g of dipotassium hydrogen phosphate, 2.0g of magnesium sulfate, 0.05g of manganese sulfate, 3.0g of calcium carbonate, 18.0g of agar powder, 1000mL of purified water and pH value of 6.4-7.0;
s2, primary seed culture: inoculating the lactobacillus reuteri SAMMRS-1G inclined plane obtained in the step S1 into a primary seed liquid culture medium, and performing stationary culture for 18 hours at 32-39 ℃, preferably 39 ℃ in a triangular flask to obtain primary seed liquid; primary seed medium: 4.0g of tryptone, 10.0g of beef extract, 8.0g of yeast powder, 10.0g of glucose, 0.5g of L-cysteine hydrochloride, 5.0g of sodium acetate, 2.0g of sodium citrate, 2.0g of Tween-801.0g of dipotassium hydrogen phosphate, 2.0g of magnesium sulfate, 0.05g of manganese sulfate, 3.0g of calcium carbonate, 1000mL of purified water and pH value of 6.4-7.0;
s3, culturing secondary seeds: the first-level seed liquid obtained in the step S2 is inoculated into a second-level seed culture medium according to the volume ratio of 3 percent, and is subjected to stationary culture for 8 hours at the temperature of 32-39 ℃ to obtain the second-level seed liquid; the secondary seed culture medium is the same as the primary seed culture medium;
s4, liquid fermentation culture: preparing a fermentation medium according to 80% of the volume of a fermentation tank, wherein the fermentation medium comprises the following components: 5% of yeast powder, 1% of peptone, 8% of sucrose, 0.02% of manganese sulfate, 0.02% of magnesium sulfate, 0.2% of diammonium phosphate, 0.5% of anhydrous sodium acetate and 0.1% of Tween-80; introducing steam, and sterilizing the material at 121deg.C for 30min. Cooling to 32-39deg.C, preferably 39deg.C, inoculating the secondary seed solution obtained in step S3 into fermentation tank at 1% volume ratio, maintaining pressure of nitrogen gas at 0.05Mpa and rotation speed of 100r/min under the conditions of 32-39deg.C, preferably 39deg.C and pH5.0, and anaerobic fermenting for 10h to obtain Luo Yishi lactobacillus SAMMRS-1G microecological preparation with effective viable count of 1.2X10 10 CFU/mL of Lactobacillus reuteri SAMMRS-1G broth.
EXAMPLE 4 Luo Yishi evaluation of antibacterial Capacity of Lactobacillus samMRS-1G fermentation broth against aquatic pathogenic bacteria
1. Antibacterial ability of lactobacillus reuteri SAMMRS-1G fermentation liquor to aquatic pathogenic bacteria
Preparation of aquatic pathogenic bacteria suspension: vibrio vulnificus (Vibrio vulnificus) BNCC336860, vibrio parahaemolyticus (Vibrio parahaemolyticus) ATCC17802, vibrio harveyi (Vibrio harveyi) ATCC BAA-1117, vibrio alginolyticus (Vibrio alginolyticus) ATCC33787, vibrio cholerae (Vibrio cholerae) BNCC232030, shewanella putrefying (Shewanella putrefaciens) ATCC BAA-1097, pseudomonas Pseudoalteromonas sp.) BNCC336860, aeromonas hydrophila (Aeromonas hydrophila) BNCC336453, staphylococcus aureus (Staphylococcus aureus) CICC10384, salmonella (Salmonella typhimurium) CICC21513, escherichia coli (E.coli) O78 are respectively removed from the lyophilization tubes to the corresponding optimum medium plates for resuscitation and activation; single colony is selected and inoculated in a liquid culture medium, shake culture is carried out at 37 ℃ until the logarithmic phase, and a plate counting method is adopted for bacterial counting. The bacterial liquid is preserved in a refrigerator at 4 ℃ for standby, and is diluted to 1 multiplied by 10 by normal saline before use 8 CFU/mL。
Bacteriostasis experiment: pouring 15-18 mL of sterilized optimal agar culture base culture dish by oxford cup method, standing for solidification. Adding the pathogen suspension into the most suitable agar culture with the temperature kept between 45 and 50 DEG CThe final concentration of the mixture in the matrix is 0.2 multiplied by 10 8 Sucking 5mL of cfu/mL on a plate for solidification to form a bacterial layer, wherein the bacterial content of each plate is 1 multiplied by 10 8 cfu. On a culture dish, 4 oxford cups were placed at equal intervals, 200. Mu.L of the SAMMRS-1G broth of Lactobacillus reuteri obtained in example 4 was poured into each oxford cup with a pipette, and after standing for 30min, the dish was placed in a constant temperature incubator, incubated at 37℃for 18 to 24 hours, the diameter of the inhibition zone was measured and recorded with a vernier caliper, and the results were shown in FIG. 4 and Table 1.
2. Influence of temperature on bacteriostasis of Luo Yishi lactobacillus SAMMRS-1G fermentation liquor
Centrifuging the lactobacillus reuteri SAMMRS-1G fermentation liquor to obtain supernatant, respectively performing constant temperature treatment at room temperature, 60, 80 and 121 ℃ (sterilizing pot damp heat) for 30min, rapidly cooling to room temperature in a water bath, taking the aquatic pathogenic bacteria as indicator bacteria, measuring and recording the diameter of a bacteriostasis zone by using a vernier caliper, and respectively calculating the ratio of the bacteriostasis zone of the room temperature, 60, 80 and 121 ℃ to the supernatant at room temperature. The results are shown in Table 1 and are labeled SAMMRS-1G supernatant (room temperature), treatment samples 2-60, treatment samples 2-80 and treatment samples 2-121, respectively.
3. Effect of organic acids (pH) on the bacteriostatic Properties of Luo Yishi Lactobacillus SAMMRS-1G fermentation broth
In order to eliminate the interference of acidic substances, the pH value is regulated to 6.0 by using a sodium hydroxide solution, the aquatic pathogenic bacteria are used as indicator bacteria, the diameter of a bacteriostasis zone is measured and recorded by using a vernier caliper, and the ratio of the bacteriostasis zone to the supernatant of the room temperature SAMMRS-1G is calculated. Labeled treatment sample 3, the results are shown in Table 1.
4. Sensitivity of Lactobacillus reuteri SAMMRS-1G fermentation broth to protease
Taking lactobacillus reuteri SAMMRS-1G fermentation supernatant, reacting with porcine pepsin (final concentration 2 mg/mL) and bovine trypsin (final concentration 2 mg/mL) at 37 ℃ for 1h, wherein the pH is 2.5 and 8.0 when the supernatant is acted, and regulating the pH to 5.0 by using NaOH and HCl solution after the supernatant is acted. And measuring and recording the diameter of the inhibition zone by using a vernier caliper by taking the aquatic pathogenic bacteria as indicator bacteria, and calculating the inhibition zone ratio with the room temperature SAMMRS-1G supernatant. Labeled treatment sample 4-1 and treatment sample 4-2, respectively, and the results are shown in Table 1.
5. Effect of Hydrogen peroxide on the bacteriostatic Properties of Lactobacillus reuteri SAMMRS-1G fermentation broth
In order to eliminate the interference of hydrogen peroxide, 1.5mg/mL catalase is added into the fermentation supernatant, the aquatic pathogenic bacteria are taken as indicator bacteria, the diameter of a bacteriostasis zone is measured and recorded by a vernier caliper, and the ratio of the bacteriostasis zone to the room temperature SAMMRS-1G supernatant is calculated. Labeled treatment sample 5, the results are shown in Table 1.
TABLE 1 Luo Yishi antibacterial ability of Lactobacillus SAMMRS-1G fermentation broth against aquatic pathogenic bacteria
TABLE 1 Luo Yishi antibacterial Capacity of Lactobacillus samMRS-1G fermentation broth against aquatic pathogenic bacteria (follow-up)
Common pathogenic bacteria of bacterial diseases in aquaculture mainly comprise aeromonas, vibrio, shewanella and the like, and the healthy and sustainable development of the aquaculture industry in China is greatly limited. Wherein aeromonas hydrophila is the main pathogenic bacteria of bacterial sepsis and bacterial enteritis of fish and shrimp; vibrio alginolyticus is one of main pathogenic bacteria of the outbreak diseases of culture varieties such as litopenaeus vannamei, grouper, variegated abalone and the like; vibrio parahaemolyticus is a pathogen causing early death syndrome of prawns and acute hepatopancreatic necrosis syndrome of prawns, and is commonly found in marine fishes, shrimps, crabs, shellfish and other aquatic products; vibrio harveyi is an important pathogen causing epidemic diseases of aquatic animals, particularly marine fish; vibrio vulnificus and Vibrio cholerae are the main pathogenic bacteria of acute diarrhea or septicemia transmitted to humans through aquatic animals; the common putrefying bacteria Shewanella, pseudomonas and the like in water are easy to cause putrefaction in the storage process of prawns or aquatic animals. The lactobacillus reuteri SAMMRS-1G fermentation liquor has remarkable inhibition capability on aquatic pathogenic bacteria, and the diameters of inhibition zones are not less than 15.0, so that the treatment effect can be quickly achieved.
Most lactic acid bacteria produce bacteriocins or bacterioids with a narrow antimicrobial spectrum that inhibit only a few bacteria of similar species to the producing bacteria. The SAMMRS-1G metabolized reuterin is a broad-spectrum effective antibacterial substance, can inhibit the growth of aquatic pathogenic bacteria, and has stable antibacterial effect. The results show that the SAMMRS-1G fermentation liquor has no influence on the inhibition of aquatic pathogenic bacteria, other cultured animal pathogenic bacteria and the protease in the stomach and intestinal tracts of animals, most of the antibacterial activity of the supernatant after treatment is over 90% of the antibacterial capacity of the supernatant at room temperature, and the antibacterial activity is not destroyed by pH and catalase to lose efficacy. The bacteriostatic ability of the fermentation broth after neutralization of the organic acid is slightly affected, which means that the organic acid is also a small part of substances with the function of inhibiting the bacteria of SAMMRS-1G, most bacteriocins are polypeptides or protein substances, the antibacterial property of the bacteriocins is usually destroyed by protease, and the antibacterial substances metabolized by the SAMMRS-1G have stronger stability.
Application example 1 Luo Yishi lactobacillus SAMMRS-1G microecological preparation in aquaculture-prawn
1. Test sample
Test sample 1: example 3 lactobacillus reuteri SAMMRS-1G microecological preparation prepared by fermentation, the stock solution was sprayed onto the surface of commercial feed, dried at room temperature for 5 hours and stored in a refrigerator at 4 ℃;
test sample 2: EXAMPLE 3 Lactobacillus reuteri SAMMRS-1G microecologics prepared by fermentation, centrifuging at 4000r/min for 30min, and spraying the supernatant onto the surface of commercial feed.
2. Test grouping and method:
1) Grouping: selecting 120 litopenaeus vannamei (1.0+/-0.06 g) with similar specifications, randomly dividing the litopenaeus vannamei into 4 groups, wherein 3 groups are parallel, and culturing the litopenaeus vannamei in an indoor circulating water cluster box, namely: the blank control group is fed with conventional commercial feed, the disease control group is fed with conventional commercial feed, the test group 1 is fed with prepared test sample 1, and the test group 2 is fed with prepared test sample 2.
2) The method comprises the following steps: and (3) carrying out growth performance and in-vivo vibrio index measurement after 8 weeks of group feeding, wherein pathogenic bacteria are not infected by a disease control group during the growth performance index measurement period, and the disease control group is fed normally. After 8 weeks, the pathogenic control group, test group 1 and test group 2 were each added with mixed pathogenic bacteria (pathogenic bacteria of example 3 were mixed, and the seawater contained pathogenic bacteria 1X 10) 6 CFU/mL), statistical survival 2 after 1 week of feeding.
3. And (3) cultivation management: prior to the start of the experiment, selected prawns were acclimatized with conventional commercial feed for one week. All shrimps were kept in fresh seawater (salinity, 5% per mill) at 28.+ -. 2 ℃ and pH 7.5-7.9, continuously aerated, and water was changed by 60% per day. The daily feeding amount is 10% of the weight. 3 feeds were made 7:00, 11:00 and 19:00 a day, with each feed rate (ratio of feed amount to total daily feed amount) being 35%, 20% and 45%, respectively. The undried feed and the undried excrement are removed every day, the growth and the feeding condition of the shrimps are observed, the shrimps are fished out in time after being found out, weighed and recorded, and the test is carried out for 9 weeks.
4. Investigation indexes:
1) Growth performance: after the end of the 8 week trial, the total number and weight of shrimp in each aquarium was recorded and survival rate 1 (SR 1), weight Gain Rate (WGR) and Specific Growth Rate (SGR), bait coefficient (FCR) were assessed.
Survival 1 (SR 1)/% = (survival mantissa after 8 weeks/initial mantissa) ×100
Weight Gain Ratio (WGR)/% =100× (W) t -W 0 )/W 0
Specific Growth Rate (SGR) =100× (lnW) t -lnW 0 )/t
Bait coefficient (FCR) =fi/(Wt-W) 0 )
Wherein W is 0 Is the initial weight of the prawns in each aquarium; w (W) t The wet weight of the prawns in each water cluster box is the wet weight of the prawns in each water cluster box at the end of the test; t is the duration of the test (56 days in this application); FI represents the average total amount g of feed ingested by each prawn.
2) Vibrio count: 9 prawns (3 prawns in parallel) were randomly treated in each group, viscera of the prawns were dissected under aseptic condition and homogenized, diluted by multiple times, spread on TCBS agar plates, cultured at 28deg.C for 2 days, and viable count was obtained. In the pre-test, pathogenic bacteria of viscera of the prawn, vibrio is a main pathogenic bacteria, and is an exponential difference of the sum of other pathogenic bacteria. In order to highlight the prevention and treatment effect of the Luo Yishi lactobacillus SAMMRS-1G microecological preparation on pathogenic bacteria, representative vibrio is selected, and the number of vibrio in the prawn body is counted.
3) Immune index: lysozyme (LZM), phenol Oxidase (PO), alkaline phosphatase (ALP) and superoxide dismutase (SOD) were measured using the Nanjing institute of biological engineering kit.
4) Survival rate after addition of mixed pathogenic bacteria 2 (SR 2) to water.
Survival rate 2 (SR 2)/% = (survival mantissa after 9 weeks/(survival mantissa after 8 weeks-vibrio count treatment mantissa) ×100
5. Results and analysis:
TABLE 2 influence of Lactobacillus reuteri SAMMRS-1G on shrimp culture
The test result shows that the lactobacillus reuteri SAMMRS-1G fermentation liquor can improve the growth rate of the Litopenaeus vannamei under the normal feeding condition, obviously improve the weight gain rate and the specific growth rate, reduce the bait coefficient, obviously reduce the number of vibrios in the prawn body, and have a certain prevention effect on the occurrence of vibrios. After the pathogenic infection experiment was performed at week 9, the survival rate of the pathogenic control group decreased by 55.56, indicating that the model was effective. Compared with the infection group, the lactobacillus reuteri SAMMRS-1G fermentation liquid can effectively treat the infection of aquatic pathogenic bacteria after being mixed with the feed, and the survival rate is improved by 41.27 percent.
It is worth noting that the various indices of the supernatant (test group 2) after centrifugation by lactobacillus reuteri SAMMRS-1G were not significantly different from those of test group 1. Therefore, in practical application, the newly produced lactobacillus reuteri SAMMRS-1G microecological preparation has the best effect, and after long-term placement, the effective viable count in the preparation can be reduced to a certain extent, but the effect on the prawn culture is not obvious, so that the defect of unsatisfactory using effect caused by the fact that the traditional lactobacillus is not resistant to storage is overcome.
TABLE 3 Luo Yishi influence of Lactobacillus SAMMRS-1G on the immunocompetence of prawns
The immune system of the prawn is imperfect, and the prawn lacks specific immunity, and mainly depends on nonspecific immune defense to kill pathogens and improve the resistance to diseases. When the shellfish is injured or is invaded by bacteria, the phenol oxidase generates melanin and other high-activity substances through oxidation reaction, so that the active substances can effectively kill the metaprotein, promote the healing of host wounds and play an important role in the shrimp defense system; lysozyme is an alkaline protein, and can greatly help phagocytes kill external pathogenic bacteria; meanwhile, SOD is used as one of important antioxidant enzymes, can remove oxygen free radicals in the prawn body, improves the anti-stress capability, plays a role in immune disease resistance, and alkaline phosphatase is an important detoxification system, is closely related to phosphorus metabolism, can rapidly transport and ingest substances in organisms, and is an enzyme substance essential for immune disease resistance of organisms. In the application example, the feed spray Luo Yishi lactobacillus SAMMRS-1G fermentation liquor can obviously improve the activity of lysozyme and alkaline phosphatase of litopenaeus vannamei and the activity of phenol oxidase and superoxide dismutase. As shown in Table 3, the supernatant of Lactobacillus reuteri SAMMRS-1G used in test group 2 was not significantly different from that in test group 1 in terms of the immunocompetence index. Therefore, luo Yishi lactobacillus SAMMRS-1G can effectively improve the immunity of the prawns, and provides a part of theoretical basis for preventing and treating pathogenic bacteria of the prawns and improving the survival rate.
In conclusion, the lactobacillus reuteri SAMMRS-1G can obviously improve the survival rate and the growth performance of the prawns, reduce the bait coefficient, prevent and treat frequent diseases and increase the economic benefit.
Application example 2 Luo Yishi application of lactobacillus SAMMRS-1G microecological preparation in aquaculture-fish
1. Test sample
Test sample 1: example 3 lactobacillus reuteri SAMMRS-1G microecological preparation prepared by fermentation, the stock solution was sprayed onto the surface of commercial feed, dried for 5 hours at room temperature and stored in a refrigerator at 4 degrees celsius;
test sample 2: EXAMPLE 3 Lactobacillus reuteri SAMMRS-1G microecologics prepared by fermentation, centrifuging at 4000r/min for 30min, and spraying the supernatant onto the surface of commercial feed.
2. And (3) test design:
after 2 weeks of the acclimation of the garrupa with similar specifications, 90 individuals with healthy, nondestructive, active and average weight (21.00+/-0.76 g) are selected as test fishes, and are randomly divided into 3 groups of 3 parallel groups, and are cultivated in an indoor water tank, namely: the blank control group is fed with conventional commercial feed, the test group 1 is fed with the prepared test sample 1, and the test group 2 is fed with the prepared test sample 2. Feeding for 2 times (8:30 and 18:30) per day, feeding for 30min, weighing the residual feed in each net cage of the mobile phone after drying, and determining the feed intake. Continuously aerating during the cultivation test, wherein the dissolved oxygen concentration is more than or equal to 6mg/L, the water temperature is 30+/-2 ℃, the pH is 8.0+/-0.2, and the cultivation period is 56d.
3. Sample collection and index determination
Stopping feeding 24 hours before sample collection, and randomly grabbing growth performance data such as weight and the like of 9 garrupa with garrupa from each net cage of each group. The viscera are taken after surface sterilization, dissected and homogenized under aseptic condition, spread on TCBS agar plates after multiple dilution, cultured for 2 days at 28 ℃, and counted for vibrios in vivo by viable bacteria.
Survival rate (SR,%) = (survival mantissa/initial mantissa) ×100
Weight gain rate (WGR,%) =100× (W t -W 0 )/W 0
Specific growth rate (SGR,%/d) =100× (lnW) t -lnW 0 )/t
Bait coefficient (FCR) =fi/(Wt-W) 0 )
Liver index (HSI,%) =100×visceral weight/body weight
Full of fertilizer (CF, g/cm) 3 )＝100×(W t /L 3 )
Wherein W is 0 Is the initial weight of the prawns in each aquarium; w (W) t The wet weight of the prawns in each water cluster box is the wet weight of the prawns in each water cluster box at the end of the test; t is the duration of the test (56 days in this application); FI represents the average total feeding bait amount g of each prawn, and L represents the body length.
4. Results and analysis:
TABLE 4 Luo Yishi influence of Lactobacillus samMRS-1G on grouper cultivation
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The grouper is always cultivated in the first three places of sea water fish, and vibriosis is one of common epidemic diseases. As can be seen from the test results, compared with the control group, the final average weight, the weight gain rate, the specific growth rate and the liver index of each group of the added lactobacillus reuteri SAMMRS-1G fermentation liquor and the supernatant are obviously improved, the fullness is higher than that of the control group, the bait coefficient is lower than that of the control group, and the number of vibrios in the body of the grouper can be obviously reduced. The effect of the SAMMRS-1G fermentation liquor is slightly better than that of the supernatant liquor, and mainly because the lactobacillus reuteri can decompose proteins, saccharides, fat and other substances in the feed, the digestibility of the feed is improved, and the thalli also contains a large amount of nutrient substances and thalli proteins, so that the requirements of aquatic animal growth are met. The substances such as the rotigotine and the organic acid produced by the SAMMRS-1G metabolism can effectively inhibit pathogenic bacteria such as vibrio and the like, improve the disease resistance of hosts, and have no obvious difference in the supernatant inhibition effect of the live bacteria fermentation liquor.
Claims (10)
1. Lactobacillus reuteri (Lactobacillus reuteri) SAMMRS-1G with the preservation number of CCTCC NO: M20221519.
2. Use of lactobacillus reuteri SAMMRS-1G according to claim 1 for controlling pathogenic bacteria in animal farming.
3. Use of lactobacillus reuteri SAMMRS-1G according to claim 1 for controlling pathogenic bacteria in aquaculture.
4. Use according to claim 2 or 3, wherein the pathogenic bacteria are escherichia coli, salmonella, staphylococcus aureus, pseudomonas, putrefying bacteria or vibrio.
5. The use according to claim 4, wherein the monad is aeromonas hydrophila or pseudoalteromonas; the putrefying bacteria are Shewanella putrefying bacteria; the vibrio is Vibrio harveyi, vibrio alginolyticus, vibrio parahaemolyticus, vibrio cholerae or Vibrio vulnificus.
6. A microecological preparation comprising the Lactobacillus reuteri SAMMRS-1G or a fermentation broth thereof according to claim 1 as an active ingredient.
7. The preparation method of the lactobacillus reuteri SAMMRS-1G microecological preparation is characterized by comprising the following steps:
s1, strain activation: inoculating the Lactobacillus reuteri SAMMRS-1G strain on MRS slant culture medium for culturing, strictly performing aseptic operation, and culturing at 32-39deg.C for 18-30 hr;
s2, primary seed culture: inoculating the lactobacillus reuteri SAMMRS-1G inclined plane obtained in the step S1 into a primary seed liquid culture medium, and standing and culturing for 12-18 h at the temperature of 32-39 ℃ to obtain primary seed liquid;
s3, culturing secondary seeds: the first-level seed liquid obtained in the step S2 is inoculated into a second-level seed culture medium according to the volume ratio of 3-5%, and is subjected to stationary culture for 8-12 h at the temperature of 32-39 ℃ to obtain the second-level seed liquid;
s4, liquid fermentation culture: preparing a fermentation medium according to the volume of 60-80% of the fermentation tank, introducing steam, and sterilizing the material at 121 ℃; cooling to 32-39 ℃, inoculating the secondary seed liquid obtained in the step S3 into a fermentation medium according to the volume ratio of 1-3%, and carrying out anaerobic fermentation for 6-14 h under the conditions of 32-39 ℃ and pH 4.0-5.5 at the rotating speed of 100r/min and the nitrogen pressure of 0.05Mpa under the condition of maintaining the pressure of nitrogen, thereby obtaining the lactobacillus reuteri SAMMRS-1G microecologics.
8. Method for the preparation of lactobacillus reuteri SAMMRS-1G microecologics according to claim 7, characterized in that the fermentation medium of step S4: 5% of yeast powder, 1% of peptone, 8% of sucrose, 0.02% of manganese sulfate, 0.02% of magnesium sulfate, 0.2% of diammonium phosphate, 0.5% of anhydrous sodium acetate and 0.1% of Tween-80.
9. Method for the preparation of lactobacillus reuteri SAMMRS-1G microecologics according to claim 7, characterized in that said slant medium: 4.0g of tryptone, 10.0g of beef extract, 8.0g of yeast powder, 10.0g of glucose, 0.5g of L-cysteine hydrochloride, 5.0g of sodium acetate, 2.0g of sodium citrate, 2.0g of Tween-801.0g of dipotassium hydrogen phosphate, 2.0g of magnesium sulfate, 0.05g of manganese sulfate, 3.0g of calcium carbonate, 18.0g of agar powder, 1000mL of purified water and pH value of 6.4-7.0;
the primary seed culture medium or the secondary seed culture medium: 4.0g of tryptone, 10.0g of beef extract, 8.0g of yeast powder, 10.0g of glucose, 0.5g of L-cysteine hydrochloride, 5.0g of sodium acetate, 2.0g of sodium citrate, 2.0g of Tween-801.0g of dipotassium hydrogen phosphate, 2.0g of magnesium sulfate, 0.05g of manganese sulfate, 3.0g of calcium carbonate, 1000mL of purified water and pH value of 6.4-7.0.
10. The method for preparing the SAMMRS-1G microecological preparation of lactobacillus reuteri according to claim 7, wherein the effective viable count of the obtained SAMMRS-1G microecological preparation of lactobacillus reuteri can reach 1.0 to 1.5X10 10 CFU/mL。
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| CN114891705A (en) * | 2022-07-11 | 2022-08-12 | 东北农业大学 | Acid-producing Min pig source lactobacillus reuteri and culture method and application thereof |
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| CN116333910A (en) * | 2022-09-23 | 2023-06-27 | 华中农业大学 | Culture medium and method for promoting lactobacillus reuteri growth |
| CN117801989A (en) * | 2023-12-19 | 2024-04-02 | 广东杰昇生命科技有限公司 | Preparation method of acid and alkali resistant biological preparation for resisting ulcerative colitis |
| CN117801989B (en) * | 2023-12-19 | 2024-05-14 | 广东杰昇生命科技有限公司 | Preparation method of acid and alkali resistant biological preparation for resisting ulcerative colitis |
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