CN107828688B - Butyric acid-resistant clostridium butyricum with strong stress resistance and application thereof - Google Patents
Butyric acid-resistant clostridium butyricum with strong stress resistance and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to a butyric acid-resistant clostridium butyricum with strong stress resistance and application thereof. The Clostridium butyricum is Clostridium butyricum (Clostridium butyricum) LXKJYB-1, and the preservation number is as follows: CCTCC NO: M2017485. The strain LXKJYB-1 has the characteristics of high fermentation level, high organic acid yield, capability of inhibiting various intestinal pathogens, high tolerance to gastrointestinal fluids, bile salts and damp heat and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of microbial fermentation, and particularly relates to a butyric acid-resistant clostridium butyricum with strong stress resistance and application thereof in replacing antibiotics.
Background art:
clostridium butyricum is a completely anaerobic gram-positive bacillus, is a normal flora of human and animal intestinal tracts, and also exists in soil, cheese and natural yogurt; the flagella of the whole body has motility; the spore is eccentric or secondary (endogenous), is circular or elliptical, and has no spore outer wall and accessory silk. The clostridium butyricum spore structure can resist the severe environment of the gastrointestinal tract of animals and can tolerate the high-temperature and high-humidity process in the feed processing process; the clostridium butyricum is only sensitive to a few antibiotics such as novobiocin, vancomycin and tetracycline, has strong drug resistance to other various antibiotics, and has good application prospect.
The clostridium butyricum can prevent the abnormal proliferation of pathogenic bacteria and putrefying bacteria in the intestinal tract, and can promote the proliferation and breeding of beneficial intestinal flora, thereby correcting the disorder of the intestinal flora, reducing the generation of enterotoxin, enhancing the immune function of animals by active substances generated by the metabolism, promoting the growth of the animals, improving the production performance of the animals, degrading feed by enzymes generated by the metabolism, improving the nutrition of the feed, improving the intestinal environment and promoting the propagation of the beneficial bacteria by organic acids such as butyric acid, acetic acid and the like generated by the metabolism, inhibiting the proliferation of harmful bacteria, improving the palatability of the feed and the like; it can also be metabolized in intestinal tract to generate various enzymes, amino acids, B vitamins, vitamin K and the like, and has other health care functions for supplementing nutrient substances for the host; the clostridium butyricum can decompose harmful substances such as amines and the like, and improve the culture environment; the hydrogen generated by the important metabolism can repair the damage of the liver and the kidney.
Currently, clostridium butyricum is allowed to be added into feed as a feed additive, and has an anti-substitution effect in non-resistant breeding as a substitute product of anti-diarrhea antibiotics, and even has an obvious effect compared with a plurality of growth-promoting antibiotics. However, the application of clostridium butyricum is seriously influenced due to the low fermentation level, so that how to reduce the production cost of clostridium butyricum is urgent, and the method for screening high-yield and strong-stress resistance clostridium butyricum strains through mutation breeding is a main way for popularizing clostridium butyricum products.
The fermentation process of the clostridium butyricum is metabolized to generate a large amount of organic acids such as butyric acid, acetic acid and the like, the butyric acid and the acetic acid are used as main probiotic substances to determine the probiotic effect of the clostridium butyricum, but the large amount of the butyric acid and the acetic acid has great influence on the fermentation level and the spore rate of the clostridium butyricum, and the screening of the clostridium butyricum with high butyric acid resistance and high stress resistance is an important way for solving the existing fermentation level and application of the clostridium butyricum, so that the screening of the clostridium butyricum strain capable of resisting acid stress has important significance. Whether the clostridium butyricum can resist heat or not in high-temperature treatment in the application process of the clostridium butyricum and in the extreme environment after entering the gastrointestinal tract of animals is an important index for screening the clostridium butyricum and is also a measurement index with high and low application value. Therefore, the screening of clostridium butyricum strains with strong stress resistance and high fermentation level is an important way for the wide popularization and application of clostridium butyricum.
The invention content is as follows:
in order to solve the technical problems, the invention screens out a Clostridium butyricum mutant strain with high butyric acid resistance and stress resistance, wherein the strain is Clostridium butyricum (Clostridium butyricum) LXKJYB-1 which is preserved in the China center for type culture collection in 2017, 9 and 8 months, and the address is as follows: china, wuhan university, zip code 430072, the accession number is: CCTCC NO: M2017485.
The LXKJYB-1 is obtained by taking Clostridium butyricum (Clostridium butyricum) LXKJ-1 as an initial strain through ARTP room temperature pressure plasma mutagenesis, and bacterial colonies of the strain are circular, milky white and protruded; the spore is elliptical and the spore is thick. After mutagenesis, LXKJYB-1 has higher stress resistance and antibacterial activity than LXKJ-1, and the fermentation level is greatly improved compared with that of the initial strain.
The method comprises the following specific steps:
(1) butyric acid resistance: compared with the butyric acid resistance of the initiator LXKJ-1, the resistance is improved by three times, and 40g/L sodium butyrate can be tolerated;
(2) the fermentation capacity is that after the strain LXKJYB-1 is fermented, the number of viable bacteria in the system reaches 7.19 × 109cfu/mL, the spore rate reaches 98.79%;
(3) antibacterial activity: the inhibition rate of the strain on pathogenic bacteria such as escherichia coli is improved by 35-52% compared with that of the original strain; the effect of promoting the proliferation of probiotics such as bifidobacterium, lactobacillus and the like in the animal body is obviously enhanced compared with the original strain.
(4) The stress resistance is as follows: the high temperature resistance and the gastrointestinal tract extreme environment resistance are enhanced compared with the strain.
The invention also provides a probiotic preparation prepared by adopting the butyric acid-resistant clostridium butyricum, and the preparation method comprises the following steps:
(1) carrying out fermentation culture on clostridium butyricum LXKJYB-1 to obtain clostridium butyricum LXKJYB-1 fermentation liquor;
(2) after the fermentation liquor is centrifuged, the freeze-dried powder is crushed to prepare the freeze-dried powder, and the freeze-dried powder can be directly freeze-dried without adding any protective agent due to high spore rate, cold resistance and strong thermal property.
The application effect is as follows: the addition of 0.01-0.08% of clostridium butyricum LXKJYB-1 freeze-dried powder into the daily ration can improve the survival rate of cherry valley meat ducks, promote growth and improve daily gain; meanwhile, the feed conversion ratio, the diarrhea rate and the death rate are reduced; increasing the number of lactic acid bacteria and bifidobacteria in the cecum of the cherry valley meat duck and reducing the number of escherichia coli; the strain is more prominent than the original strain in the aspects of promoting growth and particularly reducing diarrhea rate, so that the strain has stronger application value.
Has the advantages that:
1. the clostridium butyricum mutant strain provided by the invention solves the problem that the fermentation level is relatively low due to poor butyric acid tolerance of the traditional strain, the fermentation level of the clostridium butyricum screened by the invention is improved by 3-4 times compared with that of the original control strain, and the viable count reaches 7.19 × 10 after anaerobic fermentation for 24 hours9cfu/mL, the spore rate is 98.79%, the fermentation level is greatly improved, and the economic benefit is improved.
2. The probiotic preparation prepared by the invention has improved butyric acid resistance. In the metabolic process of clostridium butyricum, the pH of the growth environment is reduced by the metabolism of butyric acid, harmful bacteria are not favorably grown in a lower acid environment, the growth of the indigenous clostridium butyricum is inhibited, and the number of viable bacteria and spores is reduced. According to the invention, a single colony of the mutagenized clostridium butyricum is inoculated into a seed culture solution containing sodium butyrate with a certain concentration and cultured for 12-15h, and the sodium butyrate resistance of the mutagenized clostridium butyricum is improved by more than three times compared with that before mutagenesis, the maximum bearing concentration of the original 18g/L sodium butyrate is improved to 40g/L, and the OD is obtained after the single colony of the mutagenized clostridium butyricum is cultured for 24h in a culture medium containing 40g/L sodium butyrate600Still 1.432 can be achieved.
3. The stress resistance of the clostridium butyricum screened by the invention is obviously improved compared with that of an initial strain, the survival rate is kept at 89 +/-2.2% after the clostridium butyricum is treated for 15min at 90 ℃, the survival capability in artificial gastric juice and intestinal juice is stronger, the survival rate is kept at 98.4 +/-2.4% after the clostridium butyricum is treated for 1h in the artificial gastric juice, the survival rate is kept at 98.0 +/-2.3% after the clostridium butyricum is treated for 2h in the artificial intestinal juice, and the strain can be directly applied to livestock breeding to produce non-resistant livestock and poultry products.
4. In the practical application process, the clostridium butyricum provided by the invention has improved inhibition capacity on pathogenic bacteria, has inhibition rates on escherichia coli, salmonella, listeria monocytogenes, shigella and staphylococcus aureus of 84.34%, 75.53%, 73.07%, 69.25% and 80.03%, and has stronger application value.
5. The capacity of producing organic acid by clostridium butyricum provided by the invention is improved, and the yield of butyric acid and acetic acid respectively reaches 12.19g/L and 3.06g/L after fermentation culture for 20-24h, so that the probiotic effect of clostridium butyricum is improved.
6. In the invention, the fermentation medium used by the mutagenized strain is low in price and is more suitable for large-scale production in factories.
Description of the drawings:
FIG. 1 growth curve of Clostridium butyricum LXKJ-1;
FIG. 2 Clostridium butyricum LXKJ-1 lethal Curve
FIG. 3 the passage stability of Clostridium butyricum LXKJYB-1 and the strain LXKJ-1.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The culture medium of the invention is as follows:
RCM liquid medium: 3g of yeast extract, 10g of beef extract, 10g of tryptone, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of sodium acetate trihydrate, 0.15g of cysteine hydrochloride, 1000ml of distilled water, pH: 7.1
TPY medium: 10g of tryptone, 5g of soytone, 3g of yeast powder, 10g of monthly peptone, 10g of glucose, 0.3g of L-cysteine hydrochloride, 2.5g of dipotassium hydrogen phosphate, 0.3g of sodium thioglycolate, 3g of sodium chloride, 20g of agar, 1000mL of distilled water and pH of 6.5
Activation and seed liquid culture medium: RCM medium.
The slant culture medium is: 1% of yeast extract, 1% of glucose, 2% of agar and 6.5-7.0 of pH.
The first-order seed culture medium is as follows: 2-5% of ammonium sulfate, 2-5% of corn flour, 2-5% of yeast extract, 1-3% of urea, 0.5-3% of dipotassium hydrogen phosphate and 6.5-7.0 of pH;
the secondary seed culture medium is as follows: 2-5% of ammonium sulfate, 2-5% of corn flour, 2-5% of yeast extract, 1-3% of urea, 0.5-3% of dipotassium hydrogen phosphate, 6.5-7.0% of pH and 0.5-1% of calcium carbonate;
the culture medium of the third-stage fermentation tank is as follows: 2-5% of ammonium sulfate, 2-5% of corn flour, 2-5% of yeast extract, 1-3% of urea, 0.5-3% of dipotassium hydrogen phosphate, 6.5-7.0% of pH and 0.5-1% of calcium carbonate;
all the above media were steam sterilized (121 ℃, 30min) and cooled before use.
Example 1 mutagenic culture of LXKJYB-1
(1) Suspension preparation:
activating Clostridium butyricum LXKJ-1 strain preserved in glycerol tube, selecting single colony to inoculate in RCM liquid culture medium, culturing to 8-10h according to growth curve, collecting bacterial liquid at logarithmic phase, centrifuging, collecting thallus, washing with normal saline for two to three times, preparing mutagenesis suspension, and performing mutagenesis, wherein the concentration of the thallus in the mutagenesis liquid is 2 × 106cfu/ml. The growth curve is shown in FIG. 1.
(2) Making lethal Curve
The mutation treatment dosage or time not only affects the lethality, but also can significantly affect the mutation efficiency, and effective screening in a proper mutation efficiency interval is the key point of strain breeding. The study first performed plasma jet treatment of the starting strain at different times: the mutagenesis time is as follows: 15s, 30s, 45s, 60s, 90s, 120s, 150s, 180s, setting: radio frequency power (W): 115, 115; distance (mm) between plasma emission source and sample stage: 2; gas flow rate (L/min): 10.
diluting the mutagenized bacterial suspension to 10 with normal saline-3，10-4And adding 100 mu L of sample, coating the sample on a counting TPY culture medium, carrying out anaerobic culture at 37 ℃ for 24h, recording data after mature bacterial colonies grow on a flat plate, measuring the lethality according to the mutagenesis time, and drawing a lethality curve according to the mutagenesis lethality.
The bacteria suspension is mutagenized by an ARTP normal-temperature room pressure plasma mutagenesis technology, the damage effect of the normal-temperature room pressure plasma on the starting bacterial strain is obvious, and the lethality rate is gradually increased along with the increase of the plasma jet treatment time. The treatment time is 90s, and about 90% of the thallus is killed; when the treatment time reaches more than 120s, the lethality of the strain approaches 100%. According to literature reports, when the lethality is about 90%, the mutagenesis treatment has strong mutagenesis effect on cells, so the mutagenesis time adopts 90s treatment time (the lethality is about 90%) to carry out mutagenesis on the strains. The lethality curve is shown in FIG. 2.
(3) Primary screening of butyric acid-resistant strains:
the colonies grown after 90s mutagenesis were numbered separately and stored in glycerin tube. Activating the preserved strain into RCM liquid culture medium, inoculating into primary screening culture medium containing sodium butyrate with different concentrations, and culturing at 37 deg.C for 24 hr. By UV spectrophotometer at OD600And respectively measuring the maximum light absorption value, recording, selecting the strains with the resistance higher than that of the original strains, and transferring the strains to a slant culture medium for storage.
Primary screening of culture medium: peptone 2%, yeast extract powder 2%, dipotassium hydrogen phosphate 0.05%, and sodium chloride 0.05%;
sodium butyrate addition amount: sodium butyrate of 1-40g/L is added.
(4) Re-screening:
transferring the strains with strong butyric acid resistance obtained by primary screening for five generations, and then performing shake flask fermentation and re-screening: and (3) taking the number of viable bacteria of the initial strain as a control, comparing the fermentation level, culturing for 20 hours under the anaerobic condition at the temperature of (37 +/-1) DEG C, and determining the number of viable bacteria and spores in the fermentation liquid. The strains with higher viable count were selected and the rescreened results are shown in table 2.
Re-screening the culture medium: 2-5% of ammonium sulfate, 2-5% of corn flour, 2-5% of yeast extract, 1-3% of urea, 0.5-3% of dipotassium hydrogen phosphate and 6.5-7.0 of pH.
(5) Results and analysis:
under the condition of the optimal mutagenesis time of 90s, 228 strains with good growth vigor are obtained in total, and after sodium butyrate screening, strains with high sodium butyrate tolerance are obtained, and 11 strains with high positive mutation rate (%) -positive mutant strain number divided by total screened strain number multiplied by 100%) are obtained in total. The results of preliminary screening are shown in Table 1
TABLE 1 Primary screening results after mutagenesis treatment of Clostridium butyricum LXKJ-1
TABLE 2 Clostridium butyricum LXKJ-1 after mutagenesis treatment and repeated screening
The results show that 11 butyric acid-resistant strains obtained by primary screening can be seen, and the secondary screening result shows that the fermentation level of the 11 strains is improved to different degrees compared with the original strain LXKJ-1, wherein one strain is remarkably improved in fermentation level, the number of the strain is LXKJYB-1, the fermentation level of the strain LXKJYB-1 is 3-4 times higher than that of an initial strain LXKJ-1, and the passage stability is more stable than that of the initial strain. The results of the subculture stability are shown in FIG. 3.
Example 2 Effect of different fermentation media on fermentation levels of butyric acid-resistant high-producing Strain LXKJYB-1
Inoculating frozen LXKJYB-1 in a glycerol tube into an activation culture medium, standing at 37 ℃ for anaerobic culture for 18 h; inoculating the activated clostridium butyricum LXKJYB-1 to a seed culture medium, wherein the inoculation amount is 5%, and carrying out anaerobic culture at 37 ℃ for 24 hours to obtain a seed solution.
Fermentation medium 1: 2% of ammonium sulfate, 2% of corn flour, 2% of yeast extract, 1% of urea, 0.5% of dipotassium hydrogen phosphate, 6.5-7.0% of pH and 0.5% of calcium carbonate; dissolving with deionized water, stirring with glass rod, packaging, sterilizing, and sterilizing at 121 deg.C for 20 min;
fermentation medium 2: 4% of ammonium sulfate, 4% of corn flour, 4% of yeast extract, 2% of urea, 1.5% of dipotassium hydrogen phosphate, 6.5-7.0% of pH and 0.7% of calcium carbonate; dissolving with deionized water, stirring with glass rod, packaging, sterilizing, and sterilizing at 121 deg.C for 20 min;
fermentation medium 3: 5% of ammonium sulfate, 5% of corn flour, 5% of yeast extract, 5% of urea, 3% of dipotassium hydrogen phosphate, pH 6.5-7.0 and 1% of calcium carbonate; dissolving with deionized water, stirring with glass rod, packaging, sterilizing, and sterilizing at 121 deg.C for 20 min;
respectively inoculating LXKJYB-1 seed liquid into the fermentation culture medium 3 according to the inoculation amount of 7 percent, filling gauze with kraft paper, sealing, placing into an anaerobic culture box, exhausting gas three times by using anaerobic equipment, and using N2Replacing air in the triangular flask, culturing at 37 ℃ for 24h, and detecting the viable count and the spore count of the clostridium butyricum. The results of the comparison are shown in Table 3.
TABLE 3 Effect of different fermentation media on the fermentation level of the Strain LXKJYB-1
The results show that the fermentation medium 2 is more suitable for improving the fermentation level of the strain LXKJYB-1.
Example 3 culture of Clostridium butyricum (Clostridium butyricum) LXKJYB-1
(1) Activation and seed culture
Respectively inoculating the frozen LXKJYB-1 and LXKJ-1 strains in a glycerol tube into an activation culture medium, standing at 37 ℃ and carrying out anaerobic culture for 18 h; the activated clostridium butyricum LXKJYB-1 and LXKJ-1 are respectively inoculated into a seed culture medium, the inoculum size is 5 percent, and the seed solution is obtained by anaerobic culture at 37 ℃ for 24 hours.
(2) First order seed culture
Respectively inoculating the LXKJYB-1 and LXKJ-1 seed solutions activated in the step (1) into a primary seed culture medium according to the inoculation amount of 5%, anaerobically culturing at 37 ℃ for 12h for amplification to obtain a primary seed solution, and comparing the viable count and the spore count after the culture is finished, wherein the viable count and the spore count are shown in Table 4.
(3) Second stage seed culture
Respectively inoculating the primary seed liquid of the clostridium butyricum LXKJ-1 and LXKJYB-1 obtained in the step (2) into a secondary seed culture medium according to the inoculation amount of 5%, performing anaerobic culture at 37 ℃, culturing for 12 hours to serve as a secondary seed liquid, and comparing the viable count and the spore count after the culture is finished, as shown in the following table 4.
(4) Three-stage fermenter culture
And (3) respectively inoculating the secondary seed liquid of the clostridium butyricum LXKJ-1 and LXKJYB-1 obtained in the step (3) into a culture medium of a three-stage fermentation tank according to the inoculation amount of 5%, culturing for 24h at 37 ℃ in a nitrogen environment, obtaining the clostridium butyricum LXKJ-1 after the culture is finished, and simultaneously comparing the viable bacteria number, the spore number and the acid-producing capacity with the culture of the mutagenized strain LXKJYB-1 to obtain the results shown in tables 4 and 5.
TABLE 4 comparison of fermentation levels of Strain LXKJ-1 with Strain LXKJYB-1
TABLE 5 comparison of acid production levels of Strain LXKJ-1 with Strain LXKJYB-1
(5) Preparation of freeze-dried powder
And (3) centrifugally collecting thalli: centrifuging 12000rmp of the obtained LXKJ-1 and LXKJYB-1 three-stage fermentation culture solution, freeze-drying for 24h, and pulverizing to obtain lyophilized powder.
Because of high spore rate, strong cold resistance and heat resistance, the freeze drying can be directly carried out without adding any protective agent.
The viable count and spore count of Clostridium butyricum LXKJ-1 in the lyophilized powder are 5.32 × 1011cfu/g, spore rate: 95.79 percent.
The viable count and spore count of Clostridium butyricum LXKJYB-1 in the lyophilized powder are 1.41 × 1012cfu/g, spore rate: 99.23 percent.
Example 4 comparative stress resistance test of strains LXKJYB-1 and LXKJ-1
(1) Heat resistance (Damp-Heat)
Respectively taking down 1mL of culture which has completely formed spores at the last stage of growth of clostridium acidocaldarius, centrifuging at 3000rpm for 15min, washing with PBS twice, resuspending, respectively placing in water bath at 60 ℃, 80 ℃ and 90 ℃ for 15min, then placing in ice-water mixture at 0 ℃ for cooling, and determining the survival rate by a gradient dilution plate method, wherein 3 repeats are used per sample. The results are shown in Table 6.
TABLE 6 comparison of the Heat resistance of the Strain LXKJ-1 with that of the Strain LXKJYB-1
From the above results, it can be seen that the strain LXKJ-1 and the strain LXKJYB-1 can tolerate 100% treatment at 80 ℃ for 15min, but the heat resistance of the strain LXKJYB-1 is slightly higher than that of the starting strain LXKJ-1 after treatment at 90 ℃ for 15 min.
(2) Tolerance of artificial gastric juice and artificial intestinal juice (prepared according to Chinese pharmacopoeia)
Simulated gastric juice tolerance test: taking 1ml of final-growth culture, centrifuging at 4 deg.C and 3000rpm for 15min, washing twice with PBS, resuspending, and adding 107cfu/mL is inoculated in artificial gastric juice, evenly mixed by shaking, incubated in water bath at 37 ℃, sampled after 1h, viable count is carried out by using a gradient dilution plate method, and 3 replicates/sample are obtained.
Artificial intestinal juice tolerance: the final growth culture 1ml at 4 degrees C, 3000rpm centrifugation for 15min, PBS washing two times after heavy suspension, with l07cfu/mL was inoculated in artificial intestinal fluid for 2h and sampled for viable count, 3 replicates/sample, using a gradient dilution plate method. The results are shown in Table 7.
TABLE 7 comparison of Strain LXKJ-1 with Strain LXKJYB-1 resistant to Artificial gastric juice and Artificial intestinal juice
From the above results, it can be seen that both the strain LXKJ-1 and the strain LXKJYB-1 can tolerate the environments of artificial gastric juice and artificial intestinal juice, but the strain LXKJYB-1 has stronger tolerance in artificial gastric juice and intestinal juice.
(3) Bile salt tolerance
Adding pig bile salt into RCM liquid culture medium at mass ratio of 0.20%, 0.40%, 0.60%, 0.80%, 1.0%, 121 deg.C, and sterilizing for 15 min. Inoculating according to 1%, culturing in anaerobic incubator, observing thallus growth brother, 3 replicates per sample. ("+ + + +" indicates good growth, "+" indicates minor growth, "+" indicates slight growth, "-" indicates no growth) the results are shown in Table 8.
TABLE 8 comparison of Strain LXKJ-1 with Strain LXKJYB-1 for Swine bile salt resistance
From the results, it can be seen that the strain LXKJ-1 and the strain LXKJYB-1 can both tolerate a certain concentration of bile salt, and the strain LXKJYB-1 has stronger tolerance in a high-concentration bile salt environment.
Example 5 comparison of the in vitro bacteriostatic Activity of strains LXKJYB-1 and LXKJ-1
The method for measuring escherichia coli, salmonella, listeria monocytogenes, shigella and staphylococcus aureus by using a turbidity method has the following steps:
(1) and centrifuging fermentation liquor of the clostridium butyricum LXKJ-1 and the mutagenized strain LXKJYB-1, and collecting clear liquid for later use.
(2) The target bacteria are diluted to 10 degrees in a gradient way8cfu/mL, 0.5mL was inoculated into 9mL of LB liquid medium.
(3) 0.5ml of each of the centrifugal clear liquid of the strain LXKJ-1 and the centrifugal clear liquid of the strain LXKJYB-1 is respectively added into an LB liquid culture medium inoculated with a target bacterium and cultured for 18h at 37 ℃ in an incubator. OD of each culture tube was measured using LB medium as a blank medium600. The inhibition ratios were calculated in turn and the results are shown in Table 9.
TABLE 9 comparison of the in vitro bacteriostatic ability of the strain LXKJ-1 with that of the strain LXKJYB-1
The results show that the fermentation liquor of the strain LXKJ-1 and the strain LXKJYB-1 has certain bacteriostatic activity on target bacteria such as escherichia coli, staphylococcus aureus and the like, the fermentation liquor of the strain LXKJYB-1 has stronger bacteriostatic ability, especially has very strong inhibitory activity on escherichia coli and staphylococcus aureus, and the strain is suitable for being used as a strain for preventing and treating livestock diarrhea caused by the escherichia coli and applied to the breeding industry.
Example 6 comparison of the Effect of Clostridium butyricum LXKJYB-1 and LXKJ-1 on the production of cherry valley ducks
(1) The experiment adopts bacterial strain LXKJYB-1 and original bacterial strain LXKJ-1 microbial inoculum after the mutation of clostridium butyricum (the viable bacteria of clostridium butyricum in per gram of preparation is more than or equal to 2.0 × 108cfu/g)。
(2) Selecting 1-day-old cherry valley meat ducks for testing, and averagely dividing the cherry valley meat ducks into a control group and a test group. The control group is fed with basic daily ration, the test group is added with clostridium butyricum preparation in the amount of 100g/t, 200g/t, 400g/t and 800g/t, and the test period is 42 days.
(3) And (3) measuring the growth performance: at the first day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks after the beginning of the test, 50 cherry valley ducks in each group are randomly selected and weighed, and the weight of each group of cherry valley ducks is calculated by taking the average value. And recording the material consumption in the early stage and the later stage of the test, and calculating the average daily gain, the average daily feed intake and the feed-meat ratio of the cherry valley ducks.
(4) Diarrhea and mortality: diarrhea and mortality were recorded during feeding for each group and diarrhea and mortality were calculated.
(5) Determination of the number of flora in feces: collecting 1.0g fresh feces sample from each group, and diluting to 10 times by 10 times gradient dilution method-8Selecting three serial dilutions, respectively coating on differential medium plates such as EMB, MRS, BBL, etc., with the inoculum size of 100 μ L/9cm plate, making 3 dilutions in parallel, and culturing at 37 deg.C for 24-48 h. Mainly detects the contents of escherichia coli, salmonella, lactobacillus and bifidobacterium in the excrement. The test results are shown in tables 10 and 11.
TABLE 10 influence of Clostridium butyricum LXKJ-1 and LXKJYB-1 on growth Performance of cherry valley ducks
TABLE 11 influence of Clostridium butyricum LXKJ-1 and LXKJYB-1 on cecal microorganisms of cherry valley duck
The addition of 0.01-0.08% of clostridium butyricum LXKJ-1 preparation and LXKJYB-1 preparation in daily ration has the effects of improving the survival rate of cherry valley duck, promoting growth, improving daily gain, reducing feed conversion ratio, diarrhea rate and death rate, increasing the number of lactic acid bacteria and bifidobacteria in cecum of cherry valley duck and reducing the number of escherichia coli, but the promotion effect of clostridium butyricum LXKJYB-1 on lactic acid bacteria is improved by 11-55% compared with LXKJ-1, the inhibition capability on animal pathogenic bacteria and salmonella is improved by 20-50%, and the effect on reducing diarrhea rate and death rate is obviously better than that of the strain LXKJ-1, so the strain LXKJYB-1 is more suitable for promoting growth and resisting diarrhea, and the microecological preparation for improving the culture production level is applied to the culture of the cherry valley duck.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (3)
1. The clostridium butyricum is characterized in that the clostridium butyricum is Clostridium butyricum (Clostridium butyricum) LXKJYB-1 with the preservation number as follows: CCTCC NO: M2017485.
2. Use of clostridium butyricum according to claim 1 for the preparation of a probiotic bacterial agent.
3. Use of clostridium butyricum according to claim 1 for the preparation of animal feed.
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