CN115181688B - Lactobacillus acidophilus strain and fermentation method and application thereof - Google Patents
Lactobacillus acidophilus strain and fermentation method and application thereof Download PDFInfo
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- CN115181688B CN115181688B CN202210654999.6A CN202210654999A CN115181688B CN 115181688 B CN115181688 B CN 115181688B CN 202210654999 A CN202210654999 A CN 202210654999A CN 115181688 B CN115181688 B CN 115181688B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
The invention discloses lactobacillus acidophilus and application thereof, belongs to the technical field of microorganism application, and relates to a microorganism strain, a fermentation method and application. The invention takes a lactobacillus acidophilus from the intestinal canal of large yellow croaker as an initial strain, the lactobacillus acidophilus has an inhibition effect on alpha-glucosidase and alpha-amylase, and a new strain with a stronger inhibition effect on the alpha-glucosidase and the alpha-amylase is obtained through ultraviolet-nitrosoguanidine combined mutagenesis treatment and screening. The strain is preserved in China general microbiological culture Collection center, with the preservation number: cgmccno.17717. The strain is added into fish feed or used for fermenting the fish feed, blood sugar of fish after feeding can be effectively reduced, fatty liver symptoms can be relieved, and simultaneously, growth performance and activity of liver superoxide dismutase, catalase and lysozyme in serum can be improved.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to a lactobacillus acidophilus strain and a fermentation method and application thereof.
Background
The starch plays a double role of expansion and adhesion in the processing process of the aquatic puffed feed, can form a certain hardness of the puffed feed and increase the stability of the feed in water, and meanwhile, the starch has a low price compared with the protein raw material, so that the starch is widely applied to the aquatic feed. However, carnivorous fishes have poor utilization ability of carbohydrates such as starch, and continuous hyperglycemia is caused after eating high-sugar feed, which is harmful to life and health. Thus, there is a conflict between the intolerance of carnivorous fish to carbohydrates and the reliance of the feed industry on carbohydrates. Therefore, the breeding of probiotics with the function of regulating blood sugar has important theoretical and production significance for relieving sugar metabolism syndrome of carnivorous fishes caused by excessive starch in feed.
The development and utilization of probiotics fermented feed has become a popular field in the feed industry. After the feed is fermented by probiotics, the digestion, absorption and utilization effects can be enhanced, the feed utilization rate can be improved, and the growth performance and the activity of liver superoxide dismutase, catalase and lysozyme in serum can be improved. The probiotics mainly comprise three major categories of lactobacillus, bacillus and saccharomycetes. The lactobacillus acidophilus belongs to the genus lactobacillus of the family lactobacillus, has strong acid resistance, can grow and reproduce in an acidic environment where other lactobacillus cannot survive, and is widely applied to probiotic preparations.
The method for relieving the hyperglycemia of the fish by utilizing the lactobacillus acidophilus microbial feed and the related technology are not reported through the search of related documents and patents at home and abroad.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a novel lactobacillus acidophilus strain.
The lactobacillus acidophilus CGMCC No.17717 provided by the invention is obtained by combining ultraviolet-nitrosoguanidine with mutation breeding by a lactobacillus acidophilus from the intestinal canal of large yellow croaker, and has strong capacity of inhibiting alpha-glucosidase and alpha-amylase, and the inhibition rate of fermentation liquor on the alpha-glucosidase and the alpha-amylase is 82% and 78% respectively.
The lactobacillus acidophilus provided by the invention is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at the address: the collection number of the microbiological institute of China academy of sciences is CGMCC No.17717, and the North Chen Xili No.1, 3 of the Chaoyang area of Beijing city.
Further, the 16S rRNA sequence of the lactobacillus acidophilus is shown in a sequence table SEQ ID No. 1.
The invention also provides a microecological preparation containing the lactobacillus acidophilus. Specifically, the microecological preparation contains fermentation liquor or bacterial powder of the lactobacillus acidophilus.
The invention also provides a feed containing the lactobacillus acidophilus or a microecological preparation thereof.
Further, the content of Lactobacillus acidophilus in the feed was 1×10 9 ～1×10 11 CFU/g。
The invention also provides a fermentation method of the lactobacillus acidophilus fermentation liquid, which comprises the following steps:
fermentation medium components and content: glucose 1%, whey powder 2%, soybean meal 2%, sodium chloride 0.5%, magnesium sulfate 0.05%;
inoculating the seed solution of lactobacillus acidophilus into a fermentation tank filled with a fermentation medium according to the inoculation amount of 3-10%, wherein the temperature is 35-37 ℃, the rotating speed is 200-300 rpm, the tank pressure is 0.05Mpa, and the ventilation ratio is as follows: 1:0.8, the number of the bacteria to be alive reaches 10 10 And (3) obtaining the fermentation liquor of lactobacillus acidophilus after CFU/mL is higher than the CFU/mL.
Further, the lactobacillus acidophilus bacterial powder is vacuum freeze-dried bacterial powder or spray-dried bacterial powder.
Further, the viable count in the lactobacillus acidophilus bacterial powder is not less than 1×10 11 CFU/g。
The invention also provides application of the lactobacillus acidophilus or the microecological preparation containing the lactobacillus acidophilus in improving liver sugar stress and fatty liver of fish.
Specifically, the lactobacillus acidophilus or the microecological preparation thereof is prepared into a drug or feed for improving liver sugar stress and/or fatty liver of fish.
The invention also provides application of the lactobacillus acidophilus or the microecological preparation containing the lactobacillus acidophilus in inhibiting alpha-glucosidase and/or alpha-amylase.
Specifically, the lactobacillus acidophilus or the microecological preparation thereof is prepared into an inhibitor of alpha-glucosidase and/or alpha-amylase.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a novel lactobacillus acidophilus strain, and the inhibition rate of the strain to alpha-glucosidase and alpha-amylase is divided into 82% and 78%. Experiments prove that the strain can promote the growth of fish, improve the enzyme activities of liver superoxide dismutase (SOD), catalase (CAT) and Lysozyme (LZM) in serum, and reduce the blood sugar content after eating. The lactobacillus acidophilus bacterial powder can effectively relieve liver sugar stress and fatty liver of fish.
Drawings
FIG. 1 is a phylogenetic tree of Lactobacillus acidophilus CGMCC No.1771716S rDNA.
Fig. 2 is a microscopic view of liver tissue sections of micropterus salmoides.
FIG. 3 is a microscopic view of liver tissue sections of large yellow croaker.
Detailed Description
Example 1: screening of alpha-glucosidase and alpha-amylase inhibitor producing strains
Isolation of strains: taking the middle section of the intestinal canal of the large yellow croaker, cutting the intestinal wall by using sterile scissors, flushing chyme on the mucous membrane by using sterile physiological saline, scraping the mucous membrane in the intestinal canal by using a sterile glass slide, and collecting and carrying out gradient dilution. Selecting 10 -1 、10 -2 、10 -3 Respectively taking 100 mu L of the dilution liquid, uniformly coating the dilution liquid on an MRS agar plate, culturing for 20-24 hours at 37 ℃, picking single bacterial colony, carrying out streak purification, repeating the operation for 3-5 times until a pure culture of the single bacterial strain is obtained, and preserving the purified single bacterial colony in a glycerol tube for later use.
Screening of alpha-glucosidase and alpha-amylase inhibitor producing strains: after streaking for 24 hours, the purified single colonies were inoculated into MRS liquid medium for 12 hours. And (3) performing primary screening on the strain producing the alpha-amylase inhibitor by adopting a perforation method. Mixing the strain fermentation liquor with alpha amylase, spotting the mixture into the holes of a starch flat plate, judging the existence and activity of amylase inhibitor in a screened sample according to the existence or the size of starch hydrolysis rings on the flat plate, wherein the smaller the transparent ring is, the larger the inhibition activity is.
The strain producing the alpha-glucosidase inhibitor is screened by adopting a 4-nitrophenyl-alpha-D-glucopyranoside (PNPG) method, p-nitrophenol (PNP) with specific light absorption is produced under the catalysis of the alpha-glucosidase according to PNPG, and the activity of the enzyme inhibitor is detected based on the change of absorbance.
Preparing a sample to be tested: the strain was activated 3 times on MRS agar plates, single colonies were picked up and inoculated in MRS liquid test tubes, and after 10 hours of incubation at 37℃the strains were transferred to MRS liquid test tubes at 1% of the inoculum size and incubated for 12 hours. Centrifuging the cultured bacterial liquid for 10min at the rotating speed of 6000r/min, and taking the supernatant as a sample to be detected.
Determination of the inhibition ratio of lactic acid bacteria to alpha-glucosidase: the reactants were added in the order of metering in Table 1, and 50. Mu.L of P-nitrophenol-. Alpha. -D-glucopyranose, at a concentration of 20mmol/L, was added to a total volume of 210. Mu.L of the system with 50. Mu.L of LPBS buffer (0.1 mol/L, pH=6.8)Incubating the mixture at 37deg.C for 10min, adding 20U/mL of 30 μL alpha-glucosidase solution, reacting for 20min, adding 50 μL (1 mL) 1mol/LNa 2 CO 3 As a reaction stopping solution, the light absorption value of the reaction solution is measured at 405nm, the light absorption value is in direct proportion to the free quantity of p-nitrophenol PNP, and a PBS solution with the pH value of 6.8 and the concentration of 0.1mol/L is adopted as a blank control of the alpha-glucosidase solution and the sample to be detected in the reaction system. After the reaction, the inhibition of the lactobacillus on the alpha-glucosidase is calculated, and the experiment is performed in three times in parallel, and the average value is obtained. The inhibition rate calculation formula is as follows:
α -glucosidase inhibition rate (%) = [1- (a-B)/(C-D) ]x100%, wherein:
a is the measured absorbance (sample) of a sample to be tested containing an alpha-glucosidase solution
B is the measured absorbance value (sample blank) without alpha-glucosidase solution but with the sample to be tested
C is the measured absorbance containing the alpha-glucosidase solution but no sample (negative control)
D is the measured absorbance value (negative blank) of the sample to be tested without the alpha-glucosidase solution
Table 1 metering and sequencing of the addition of the reactants (unit: μL)
Alpha-amylase inhibition assay: accurately measuring 0.25mL of a sample to be measured, mixing with 1mg/mL of alpha-amylase solution in an equal volume, carrying out water bath at 37 ℃ for 10min, adding 0.5mL of 1.5% soluble starch solution, carrying out water bath at 37 ℃ for 5min, adding 1mL of DNS solution, carrying out boiling water bath for 5min, rapidly cooling to room temperature, diluting 10 times, standing for 30min, measuring an OD 540nm value at a wavelength of 540nm, and marking as a V sample. The OD 540nm value measured with phosphate buffer (distilled water) at pH 6.8 instead of the test solution was designated as Vmax; the OD 540nm value measured by using distilled water instead of the test solution and the alpha-amylase solution is recorded as Vmin; the OD 540nm value measured by using distilled water instead of alpha-amylase solution is recorded as V root, and the inhibition rate R of the sample to the alpha-amylase is calculated by the following calculation formula:
experimental results: the strain 300, which was initially isolated from the intestinal tract, was designated JC1-JC300, wherein JC10 has an inhibitory effect on alpha-glucosidase and alpha-amylase with an inhibitory rate of 35% and 30%. The bacterial colony of the strain on the MRS flat plate is white, convex, wet, neat in edge, opaque and about 2-3 mm in diameter. The thalli are in a chain rod shape under a microscope, gram staining is positive, and spores and flagellum are not produced.
Using lactobacillus specific primers: 5'-gga aac aga tgc taa tac cg-3' and 5'-cac cgc tac aca tgg ag-3' PCR identification of the screened strains was performed. Template DNA was extracted according to the bacterial DNA extraction kit protocol. Referring to the method of QB/T5165-2017 'technical guidelines for identifying lactobacillus for food', JC10 is initially identified as a strain of lactobacillus.
Example 2: mutagenesis breeding of lactobacillus JC10
The original strain is lactobacillus acidophilus JC10, and ultraviolet-nitrosoguanidine combined mutagenesis is carried out on the lactobacillus acidophilus JC10, and the specific process is as follows:
measurement of growth curves: lactobacillus acidophilus JC10 was activated twice and inoculated into 500mL of liquid medium for cultivation at 37℃and OD was measured every two hours. And drawing a growth curve by taking time as an abscissa and OD as an ordinate. And simultaneously, measuring the concentration of bacteria by using a spiral automatic inoculator.
Preparation of bacterial suspension: activating the original strain twice, inoculating into 500mL liquid culture medium, culturing at 37deg.C for shake culture for 9-10 hr to logarithmic phase according to growth curve, centrifuging to collect thallus, washing with sterile physiological saline for 3 times, and making into 10 7 CFU/mL～10 8 CFU/mL bacterial suspension is used for standby.
Ultraviolet mutagenesis mortality: 3-5 mL of the bacterial suspension is placed in a sterile culture dish with the diameter of 6cm, placed under a 20W ultraviolet lamp, irradiated for 28cm, and respectively irradiated for 10s,20s,40s,60s, 120s and 240s under stirring. Appropriate dilutions were made according to mutagenesis time, 100. Mu.L of each plate was plated, and 3 replicates were made for each dilution. Diluting the stock solution to 10 -6 、10 -7 、10 -8 As a control, 3 replicates were run per dilution. Culturing at 37 deg.c for 18-24 hr. Mortality was calculated and the results are shown in the following table.
TABLE 2 ultraviolet mutagenesis mortality
| Mutagenesis time/s | 10s | 20s | 40s | 60s | 120s | 240s | Control |
| Mortality/% | 30.8 | 71.2 | 83.3 | 87.5 | 99.6 | 99.9 | - |
Ultraviolet mutation breeding: and (3) selecting a mutagenesis time with the mortality rate of 70-85%, carrying out ultraviolet mutagenesis on the original strain, placing 3-5 mL of bacterial suspension in a sterile culture dish with the diameter of 6cm, placing under a 20W ultraviolet lamp, irradiating for 28cm, inoculating the mutagenized bacterial liquid in a fresh liquid culture medium at 37 ℃ for 18-24 h under the condition that the irradiation time is the mutagenesis time with the mortality rate of 70-85%, namely 20s and 40s under the stirring condition. After the bacterial liquid becomes turbid, the bacterial liquid is automatically inoculated and separated by a spiral, and 100-300 strains with large bacterial colony morphology and initial strain change are selected and compared with the survival rate of mutation. 10-60 strains with higher survival rate were stored at 4℃and assayed for alpha-glucosidase and alpha-amylase inhibitory activity (as in example 1), with strain JM40 having the best performance of both enzymes having 68% and 59% inhibitory activity, respectively.
Nitrosoguanidine mutagenesis mortality: activating excellent mutant strain JM40 subjected to ultraviolet mutation breeding to obtain 10 7 CFU/mL～10 8 CFU/mL bacterial suspension at 10mg NTG: preparing nitrosoguanidine solution at a ratio of 1m L acetone, and treating the bacterial suspension at 37 deg.C and 100r/min at a concentration of 0.3g/L for 30min, 40min, 50min and 60min respectively. Appropriate dilutions were made according to mutagenesis time, 100. Mu.L of each plate was plated, and 3 replicates were made for each dilution. Diluting the stock solution to 10 -6 、10 -7 、10 -8 As a control, 3 replicates were run per dilution. Culturing at 37 deg.c for 18-24 hr. Mortality was calculated and the results are shown in the following table.
TABLE 3 nitrosoguanidine mutagenesis lethality
| Mutagenesis time/min | 30 | 40 | 50 | 60 | Control |
| Mortality/% | 69.1 | 79.6 | 92.4 | 99.9 | - |
And (3) nitrosoguanidine mutation breeding: selecting mutagenesis time with the mutagenesis mortality of 70% -85%, and carrying out nitrosoguanidine mutagenesis on a starting strain according to 10mg NTG:1m L acetone, and treating the bacterial suspension at 37 ℃ and 100r/min for 40min at a concentration of 0.3 g/L. Inoculating the bacterial liquid after mutagenesis into a fresh liquid culture medium, and culturing for 18-24 h at 37 ℃. After the bacterial liquid becomes turbid, the bacterial liquid is automatically inoculated and separated by a spiral, and 100-200 strains with large bacterial colony morphology and initial strain change are selected and compared with the survival rate of mutation. Preserving 10-60 strains with high survival rate at 4 ℃, and then measuring the inhibition activity of alpha-glucosidase and alpha-amylase. Among them, the strain SN4 showed a good performance, and the inhibition rates for alpha-glucosidase and alpha-amylase were classified into 82% and 78%.
The bacterial 16S universal primers 5'-gagagtttgatcctggctcag-3' and 5'-cggctaccttgttacgactt-3' were used to amplify the 16S rDNA fragment of strain SN 4. Template DNA was extracted according to the bacterial DNA extraction kit protocol. Reaction system 20 μl:10 XPCR buffer 2. Mu.L, primers (20. Mu. Mol/L) each 0.5. Mu. L, dNTP (2.5 mmol/L) 2. Mu. L, taq enzyme (5U/. Mu.L), template DNA 1. Mu.L, water make up to 20. Mu.L. Reaction conditions: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, and extension at 72℃for 5min were performed for 30 cycles.
After 16S rDNA sequencing of strain SN4, the obtained gene sequence (SEQ ID No.1 of the sequence Listing) was Blast-aligned with the 16S rDNA sequence in GenBank, and a phylogenetic tree was constructed with Mega4.0 software (FIG. 1). The results showed that the strain had the highest homology (99%) with Lactobacillus acidophilus (MT545145.1 Lactobacillus acidophilus strain 4768) published in GenBank, and was identified as Lactobacillus acidophilus.
The obtained lactobacillus acidophilus SN4 is preserved in the China general microbiological culture Collection center, address: the collection number of the microbiological institute of China academy of sciences is CGMCCNo.17717.
Example 3: lactobacillus acidophilus CGMCC No.17717 bacterial powder preparation (vacuum freeze drying)
Activating the frozen lactobacillus acidophilus CGMCC No.17717 in an MRS agar plate for three times, picking single bacterial colony, inoculating the single bacterial colony into a 10mL deep MRS liquid test tube, culturing for 10 hours at 37 ℃, transferring the single bacterial colony into a 250mLMRS liquid culture medium with 1% inoculum size, and culturing for 8-10 hours to obtain seed liquid; inoculating the obtained seed liquid into a 5L fermentation tank for culture according to the inoculation amount of 5-10%; maintaining the pH value of the fermentation liquor to be 6.0-6.5 in the fermentation process, and supplementing the feed liquor when the pH value exceeds a set value; when the pH value is lower than a set value, any one of alkaline neutralizer ammonia water, KOH and NaOH is automatically added; fermenting for 10-14 hours at the temperature of 37-39 ℃ and the rotating speed of 100rpm, wherein the fermentation process is not aerated; stopping fermentation when the pH of the added liquid is not reduced or the light absorption value (namely OD 600) of the fermentation liquid at the wavelength of 600nm is not increased, so as to obtain lactobacillus acidophilus fermentation liquid;
the fermentation broth prepared above was centrifuged at 5000rpm at 4℃for 10min with a high-speed refrigerated centrifuge. And (5) centrifuging and discarding the supernatant to obtain bacterial mud. The bacterial sludge is stirred with 5.0% sucrose, 1.0% sodium glutamate and 1.0% ascorbic acid aqueous solution, and then 50% skimmed milk powder is added. Freezing in a refrigerator at-80deg.C, and vacuum freeze drying in a vacuum freeze dryer for 2 timesTaking out, pulverizing, and sieving with 60 mesh sieve for 4-30 hr. The viable count is 1×10 11 CFU/g of bacterial powder.
Example 4: lactobacillus acidophilus CGMCC No.17717 bacterial powder preparation (spray drying)
The preparation method of lactobacillus acidophilus CGMCC No.17717, namely a spray drying method, comprises the following steps:
plate culture rejuvenation: inoculating lactobacillus acidophilus strain on MRS plate culture medium, culturing at 37deg.C for 14 hr to rejuvenate the strain and form single colony;
preparing primary seed liquid: picking single colony from the flat plate, inoculating into a triangular flask (100 mL/250 mL) filled with MRS liquid culture medium, and placing into a shaking table at 37 ℃ for culturing for about 10 hours at 100rpm with the OD600nm being above 0.7 to obtain first-stage seed liquid;
preparation of secondary seeds: inoculating 5mL of the primary seed liquid prepared in the step 2) into a triangular flask (inoculating 2 bottles) filled with 500mL of MRS liquid culture medium, and culturing for about 7 hours at 37 ℃ on a shaking table at 100rpm, wherein the OD600nm is above 0.7, so as to obtain a secondary seed liquid;
preparation of lactobacillus acidophilus fermentation liquor: inoculating the secondary seed liquid prepared in the step 3) into a seed liquid containing 0.5M according to the inoculation amount of 3-10% 3 In a fermentation tank of a fermentation medium, the temperature is 37 ℃, the rotating speed is 250rpm, the tank pressure is 0.05Mpa, and the ventilation ratio is as follows: 1:0.8, culturing for 20h, sampling and measuring the number of viable bacteria in each milliliter of fermentation broth every 2h, and waiting for the number of viable bacteria to reach 10 10 And stopping fermentation when the CFU/mL is above, so as to obtain lactobacillus acidophilus fermentation liquor.
The fermentation medium is as follows: glucose 1%, whey powder 2%, soybean meal 2%, sodium chloride 0.5%, magnesium sulfate 0.05%;
preparation of lactobacillus acidophilus bacterial powder: adding 15% porous starch and 10% cyclodextrin into the fermentation broth prepared in step 4, mixing, spray drying at 125 deg.C for air intake and 50 deg.C for air exhaust, and rotating atomizer at 15000-18000 rpm to obtain viable bacteria with water content of 4% 1×10 11 CFU/g of bacterial powder.
Example 5: application of lactobacillus acidophilus CGMCC No.17717 in preparation of microorganism feed for Japanese weever
TestThe place is the Zhaoan cultivation base. The test is purchased from a base by the Micropterus salmoides, and individuals with consistent specification, strong physique, active ingestion and health and no disease are selected as test materials. The formula of the basic feed for the weever is as follows: 4.7% of domestic fish meal, 14.7% of soybean meal, 19.6% of rapeseed meal, 21.2% of cottonseed meal, 21.2% of rice bran, 12.5% of corn gluten meal, 1.8% of calcium hydrophosphate, 2.1% of secondary meal, 0.6% of mineral premix, 0.5% of vitamin premix, 0.4% of choline chloride, 0.4% of salt and 0.3% of adhesive. Mixing lactobacillus acidophilus powder with basic feed raw material according to 5% adding amount, pulverizing, concocting, plasmid, cooling, etc., and processing into microorganism feed for Lasiosphaera Seu Calvatia with lactobacillus acidophilus content of 1×10 10 CFU/g. The test is divided into two groups, 4 repeats are carried out in each group, 30 weever is bred in a feeding cylinder with consistent specification, the two feeds are respectively used for feeding, the test group is added with lactobacillus acidophilus bacterial powder feed, the control group is taken as basic feed, and the culture is carried out for 10 weeks. After the test is finished, the related indexes are measured and calculated, the death condition is observed and recorded in the whole course, and the pathological observation of the liver tissue section is carried out under a microscope.
The test indexes are as follows:
weight gain ratio WGR (%) = (end average weight-initial average weight) ×100/initial average weight;
survival rate SR (%) =number of final remaining fish tails×100/initial fish tails;
feed coefficient FCR = feed/(final fish weight-initial fish weight);
superoxide dismutase (SOD), catalase (CAT) and Lysozyme (LZM) in serum in liver pancreas.
The results are shown in Table 4: compared with a control group of basic feed, the lactobacillus acidophilus bacterial powder added into the feed can promote the growth of the weever, improve the enzyme activities of liver superoxide dismutase (SOD), catalase (CAT) and Lysozyme (LZM) in serum, and reduce the blood sugar content after eating. The observation results of the liver tissue sections are shown in fig. 2, and the liver color of the micropterus salmoides added with lactobacillus acidophilus powder in the feed is bright red, the cell nucleus is uniformly distributed, and the lipid drops are smaller. The liver of the control group is dark red, the nuclei are pressed and deformed to different degrees, the nuclei are aggregated, and lipid droplets are large. This shows that the lactobacillus acidophilus powder can effectively relieve liver sugar stress and fatty liver of fish.
TABLE 4 influence of Lactobacillus acidophilus powder on growth and immune index of California weever
Example 6: application of lactobacillus acidophilus CGMCC No.17717 in preparation of large yellow croaker fermented feed
The test site is in Zhaoan culture base. The large yellow croaker for the test is purchased from a culture base of the large yellow croaker of Ningde, and individuals with consistent specification, strong physique, active feeding and health and no disease are selected as test materials. The formula of the basic feed for the large yellow croaker is as follows: 20% of white fish meal; 30% of red fish meal; 10% of peeled soybean meal; corn gluten meal 5%; spraying dry blood cell powder 4%; gu Wan powder 4%; 0.5% of a dolphin 1; 2% of squid paste; 0.8% of calcium dihydrogen phosphate; multidimensional 1% for fish; 0.7% of multi-ore for fish; 2% of soybean lecithin oil; 10% of alpha-starch; 6% of refined seawater fish oil; 4% of zeolite powder. Mixing lactobacillus acidophilus powder with basic feed raw material according to 5% adding amount, pulverizing, concocting, plasmid, cooling, etc., and processing into large yellow croaker feed with lactobacillus acidophilus content of 1×10 10 CFU/g. The test is divided into two groups, 4 repeats are carried out on each group, 30 large yellow croakers are bred in a breeding pond with consistent specifications, two feeds are respectively used for feeding, the test group is added with lactobacillus acidophilus bacteria powder feed, the control group is basic feed, and the breeding test is carried out for 8 weeks. After the test is finished, the related indexes are measured and calculated, the death condition is observed and recorded in the whole course, and the pathological observation of the liver tissue section is carried out under a microscope.
The results are shown in Table 5: compared with a control group of basic feed, the growth speed, the conversion rate and the survival rate of the large yellow croaker added with lactobacillus acidophilus powder in the feed are obviously improved, and the enzyme activities of liver superoxide dismutase (SOD), catalase (CAT) and Lysozyme (LZM) in serum are also superior to those of the control group, so that the blood sugar content is obviously reduced after eating. The observation results of liver tissue sections are shown in figure 3, and the liver of the large yellow croaker added with lactobacillus acidophilus powder is bright red, the cell nuclei are uniformly distributed, and the lipid drops are smaller. The liver of the control group is dark red, the cell nuclei are compressed and deformed and aggregated to different degrees, the cell nuclei disappear, and the lipid droplets are large. The lactobacillus acidophilus bacterial powder can promote the growth of the large yellow croaker, improve the immunity of the large yellow croaker, regulate the sugar metabolism and effectively relieve the liver sugar stress and fatty liver of fish.
TABLE 5 influence of Lactobacillus acidophilus powder on growth and immune index of Large yellow croaker
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Sequence listing
<110> Fujian Dabei farmer with aquatic technologies group Co., ltd
Beijing Dabei Agricultural Technology Group Co., Ltd.
Jiujiang Dabei aquatic technologies Co.Ltd
<120> Lactobacillus acidophilus strain, fermentation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213> Lactobacillus acidophilus (Lactobacillus acidophilus)
<400> 1
tgcttagacg gctccttccc gaagttaggc caccggcttt gggcattgca gactcccatg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcgtg ctgatccgcg 120
attactagcg attccagctt cgtgcagtcg agttgcagac tgcagtccga actgagaaca 180
gctttaagag attcgcttgc cttcgcaggc ttgctcctcg ttgtactgtc cattgtagca 240
cgtgtgtagc ccaggtcata aggggcatga tgacttgacg tcatccccac cttcctccgg 300
tttgtcaccg gcagtctcat tagagtgccc aacttaatgc tggcaactaa tgacaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac agccatgcac 420
cacctgtctt agtgtccccg aagggaactc cgtatctcta cggattgcac tagatgtcaa 480
gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca attcctttga gtttcaacct tgcggtcgta ctccccaggc ggagtgctta 600
atgcgttagc tgcagcactg agaggcggaa acctcccaac acttagcact catcgtttac 660
ggcatggact accagggtat ctaatcctgt tcgctaccca tgctttcgag cctcagcgtc 720
agttgcagac cagagaaccc cctttcgcca ctggtgttct tccatatatc tacgcattcc 780
accgctacac atgggagttc cactctcctc ttctgcactc aagaaaaaac agtttccgat 840
gcagttcctc ggttaagccc gagggctttc acatcagact tattcttccg cctgcgctcg 900
ctttacgccc aataaatccg gacaacgctt gccacctacg tattaccgcg gctgctggca 960
cgtagttagc cgtgactttc tggttgatta ccgtcaaata aaggccagtt actacctcta 1020
tccttcttca ccaacaacag agctttacga tccgaaaacc ttcttcactc acgcggcgtt 1080
gctccatcag actttcgtcc attgtggaag attccctact gctgcctccc gtaggagttt 1140
gggccgtgtc tcagtcccaa tgtggccgat cagtctctca actcggctat gcatcattgc 1200
cttggtaggc cgttacccta ccaactagct aatgcaccgc ggggccatcc catagcgaca 1260
gcttacgccg ccttttataa gctgatcatg cgatctgctt tcttatccgg tattagcacc 1320
tgtttccaag tggtatccca gactatgggg caggttcccc acgtgttact cacccatccg 1380
ccgctcgcgt tcccaacgtc atcaccgaag tgaatctgtt ggttcagctc gctcgactgc 1440
atgtattagc a 1451
Claims (10)
1. Lactobacillus acidophilusLactobacillus acidophilus) The bacterial strain is characterized in that the preservation number is CGMCC No.17717.
2. A probiotic comprising lactobacillus acidophilus as claimed in claim 1.
3. The probiotic formulation according to claim 2, characterized in that it contains a fermentation broth or a bacterial powder of said lactobacillus acidophilus.
4. A microecological preparation according to claim 3, wherein the preparation method of the lactobacillus acidophilus fermentation broth comprises the following steps: inoculating the seed liquid of lactobacillus acidophilus into a fermentation tank filled with a fermentation medium according to an inoculum size of 3-10%, wherein the temperature is 35-37 ℃, the rotating speed is 200-300 rpm, the tank pressure is 0.05Mpa, and the ventilation ratio is as follows: 1:0.8, the number of the bacteria to be alive reaches 10 10 CFU/mL is used for obtaining the fermentation liquor of lactobacillus acidophilus, wherein the components of the fermentation medium are as follows: glucose 1%, whey powder 2%, soybean meal 2%, sodium chloride 0.5%, magnesium sulfate 0.05% and the balance of water.
5. The microecological preparation according to claim 3, wherein the lactobacillus acidophilus powder is vacuum freeze-dried or spray-dried, and the viable count in the lactobacillus acidophilus powder is not less than 1 x 10 11 CFU/g。
6. A fish feed comprising the lactobacillus acidophilus of claim 1 or the microecological formulation of claim 3.
7. The fish feed according to claim 6, wherein the content of lactobacillus acidophilus in the feed is 1 x 10 9 ~1×10 11 CFU/g。
8. Use of lactobacillus acidophilus as claimed in claim 1 or a probiotic formulation as claimed in claim 3 or 4 in the manufacture of a medicament for ameliorating hepatic glucose stress and/or fatty liver in fish.
9. Use of lactobacillus acidophilus as claimed in claim 1 or of a probiotic formulation as claimed in claim 3 or 4 for the preparation of an alpha-glucosidase and/or alpha-amylase inhibitor.
10. Use of lactobacillus acidophilus as claimed in claim 1 or of the probiotic formulation as claimed in claim 3 or 4 in the preparation of fish feed.
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