CN115704002B - Clostridium butyricum CC02001 and application thereof - Google Patents
Clostridium butyricum CC02001 and application thereof Download PDFInfo
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- CN115704002B CN115704002B CN202110918935.8A CN202110918935A CN115704002B CN 115704002 B CN115704002 B CN 115704002B CN 202110918935 A CN202110918935 A CN 202110918935A CN 115704002 B CN115704002 B CN 115704002B
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- clostridium butyricum
- feed additive
- clostridium
- butyricum
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- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention discloses clostridium butyricum CC02001 and application thereof, and belongs to the technical field of microecology. The clostridium butyricum CC02001 is obtained by separating and screening from the intestinal tracts of weaned pigs, and the strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC 20170. Clostridium butyricum CC02001 has stronger stress resistance and in-vitro antibacterial capacity. As one of feed additive strains, the microbial inoculum is applied to animal feed, can adjust the balance of animal intestinal flora and enhance the proliferation of beneficial bacteria; can enhance animal immunity and prevent tumor occurrence; can produce B vitamins and other probiotics and has health care effect on animals. Because of the resistance to various antibiotics such as streptomycin, the compound can be used as veterinary drugs and has great potential for replacing antibiotics.
Description
Technical Field
The invention belongs to the technical field of microecology, and particularly relates to clostridium butyricum CC02001 and application thereof.
Background
Clostridium butyricum (Clostridium butyricum), also known as Clostridium butyricum, belongs to the family Bacillus, genus Clostridium, gram-positive, sporulated, an obligate anaerobic bacterium, resistant to adverse conditions, was first discovered and reported in 1933 by the doctor of the university of Qianye medical science, by the uterus. Clostridium butyricum is widely found in the intestinal tract, faeces and soil of humans and animals. Clostridium butyricum was isolated from human feces and soil by doctor KINGI MIYAIRI in the 1935 russian microbiological institute, and then the anaerobically cultured filtrate was found to contain less fatty acids, and had a very strong intestinal function, which inhibited the proliferation of pathogenic bacteria in the intestinal tract, and promoted the growth of beneficial bacteria such as bifidobacteria and lactobacilli in the intestinal tract. Clostridium butyricum introduced from Russian microbiological institute in the academy of sciences of Heilongjiang in 1992 and colonized in China made a great contribution to the study of beneficial microorganisms in China.
Clostridium butyricum has stronger stability and can generate endophytic spores, so the clostridium butyricum can resist heat and acid, is not influenced by gastric acid, digestive enzyme, bile acid and the like in vivo, and therefore, the clostridium butyricum can be preserved for three years in an in vitro room temperature dry environment through the digestive tract without inactivation, the viable bacteria are not obviously reduced, and the function is not invalid. And has a certain resistance to antibiotics such as penicillin, ampicillin, streptomycin, gentamicin, etc., so that the preparation can be used together with the antibiotics to improve the curative effect. And a large number of experimental results prove that clostridium butyricum has no toxic or side effect and is widely applied to medicines, functional health-care foods and animal feed additives.
Clostridium butyricum plays a good role in pig industry, and in the stage of suckling pigs and piglets, the clostridium butyricum grows fast, the hair color is bright, the diarrhea rate is obviously reduced, the weight gain of the suckling pigs and piglets is improved by 8-10% compared with a control group, and the feed conversion ratio is reduced by 5-7%; on growing and fattening pigs, the feed intake is increased, the weight is increased quickly, the hair color is good, and the manure is formed well. Early weaning technology is commonly adopted in the modern pig industry, and diarrhea of weaned piglets is a key factor influencing production performance, and according to statistics, economic losses caused by diarrhea of piglets in China are up to tens of millions of yuan each year, wherein escherichia coli in diarrhea caused by bacterial infection is one of the most main pathogens. Coli is a common bacterium in the digestive tract and is early parasitic in the colon of piglets.
Studies have shown that fecal flora of healthy humans is known to be dominated by anaerobic bacteria, and bifidobacteria are known to be one of the most common normal flora in humans and animals. Zhang Darong and other studies prove that the dysbacteriosis of the patient with the irritable bowel syndrome is shown by that the total anaerobic bacteria bifidobacteria and lactobacillus in the main proportion of the fecal flora are reduced, the clostridium with potential pathogenicity in the small proportion of the intestinal flora is obviously increased, and the bifidobacteria, the anaerobic bacteria, the escherichia coli, the enterococcus and the lactobacillus in the fecal flora are not obviously different from healthy people after the clostridium butyricum treatment, and the clinical symptoms are correspondingly obviously improved. Zhang Xue in vitro biological antagonism research of clostridium butyricum preparation on intestinal pathogenic bacteria shows that clostridium butyricum and bifidobacterium can obviously inhibit the growth and reproduction of escherichia coli, typhoid bacillus and shigella dysenteriae in vitro.
With the development of society and the continuous development of science and technology, side effects brought by the long-term use of antibiotics such as bacterial resistance, unbalanced intestinal flora, drug residues and the like are increasingly prominent, so that the biological product which can replace the antibiotics is urgently needed to be developed for meeting the production requirements of the breeding industry and guaranteeing the life health of human beings, and the microecological preparation is a safer biological preparation which is involved by various researchers and is expected to replace the antibiotics due to the excellent characteristics.
Disclosure of Invention
In a first aspect of the invention, clostridium butyricum CC02001 is obtained by separating and screening clostridium butyricum (Clostridium butyricum) from intestinal tracts of healthy weaned pigs in a large-scale pig farm, the strain is CC02001 and is preserved in China general microbiological culture collection center (academy of sciences) with a preservation address of CGMCC 20170 and a preservation date of 2020.07.16.
In a second aspect the invention provides the use of clostridium butyricum CC02001 in a formulation for the prevention and treatment of bacterial infectious diseases.
In a third aspect the invention provides the use of clostridium butyricum CC02001 in an immunity enhancing formulation.
The preparation can be a microecological preparation or a pharmaceutical preparation.
In a fourth aspect of the invention there is provided the use of clostridium butyricum CC02001 in a feed additive.
The feed additive can be used for preventing diarrhea of piglets and promoting growth of the piglets.
In a fifth aspect of the invention, there is also provided a feed additive comprising clostridium butyricum CC 02001.
The feed additive comprises clostridium butyricum CC02001, prebiotics, organic acid salt and a carrier.
The feed additive comprises the following components in parts by weight: clostridium butyricum CC02001 0.5-2 parts, prebiotics 10-15 parts, organic acid salt 100-300 parts, and carrier 1000 parts.
The viable count of clostridium butyricum CC02001 is 1 multiplied by 10 8～1×1010 CFU/g.
The prebiotics are one or more of xylo-oligosaccharide, fructo-oligosaccharide and isomaltooligosaccharide.
The organic acid salt is one or more of potassium sorbate, sodium benzoate, calcium propionate and sodium diacetate.
The carrier is one or more of maltodextrin, corncob powder, starch, medical stone, calcium carbonate, chaff powder, sepiolite powder, rice hull powder, zeolite powder and montmorillonite.
The preparation method of the feed additive comprises the following steps: the preparation method comprises the steps of respectively weighing raw materials according to the weight portion, uniformly mixing clostridium butyricum and prebiotics, adding organic acid salt, uniformly mixing, adding a carrier, and uniformly mixing all the raw materials.
The using method of the feed additive comprises the following steps: 50-200g (100 hundred million products) of the feed additive is added into each ton of feed.
Compared with the prior art, the invention has the beneficial effects that:
1. the clostridium butyricum CC02001 has strong stress resistance and in-vitro antibacterial capacity, has strong immunocompetence regulating capacity, can obviously improve proliferation and phagocytic activity of (RAW 264.7) cells, has excellent strain performance, has high research value and application value, and can be applied to microecological preparations and pharmaceutical preparations for preventing or treating bacterial infectious diseases and improving immunity.
2. According to the invention, clostridium butyricum CC02001 is applied to a feed additive, and acts together with prebiotics to increase the number of beneficial bacteria in the intestinal tract, and organic acid salt is added to inhibit the growth of harmful bacteria in the intestinal tract, so that the effects of improving the immunity of piglets, preventing bacterial diarrhea of the piglets after weaning and promoting the growth of the piglets are achieved.
3. Clostridium butyricum CC02001 is used as one of feed additive strains, is applied to animal feed, can adjust the balance of animal intestinal flora, and enhances the proliferation of beneficial bacteria; can enhance animal immunity and prevent tumor occurrence; can produce B vitamins and other probiotics and has health care effect on animals. Because of the resistance to various antibiotics such as streptomycin, the compound can be used as veterinary drugs and has great potential for replacing antibiotics.
Drawings
FIG. 1 is a graph showing the microscopic examination results of the strains in example 1 and example 2 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
Example 1 isolation, screening and identification of Clostridium butyricum CC02001
Experimental materials: piglet intestinal tract content without any antibiotics and microecological products, clostridium intensified medium (RCM medium), test pigs; tryptone-sulfite-cycloserine agar base (TSC), 9mL sterile water tube.
The experimental steps are as follows:
(1) And (3) colony separation and purification: adding 1g of intestinal content sample into a 100mL enrichment medium blue cap bottle, carrying out water bath at 80 ℃ for 10min, immediately cooling with water, placing into a constant temperature incubator at 37 ℃ for enrichment culture for 3 days, selecting a culture solution with a large amount of bubbles generated, further carrying out enrichment in the enrichment medium, carrying out passage for 5 times, sampling for gas chromatography analysis and detection, and transferring a fermentation solution with butyric acid generated into a separation medium for continuous fermentation culture. Taking 1mL of the enriched fermentation broth, adding the fermentation broth into a 9mL sterile water test tube, uniformly mixing, and sequentially carrying out 10-time gradient dilution.
Respectively taking 0.1mL of supernatant in test tubes with dilution gradients of 10 -5、10-6、10-7, respectively coating the supernatant on the surface of a TSC agar medium, and making 3 gradients in parallel; the supernatant from the 10 -3、10-4 dilution gradient test tube was dipped in a sterile loop and each gradient was repeated 2 times on three sections of the TSC agar medium surface. After anaerobic cultivation for 48h at 37℃the black single colonies on the medium were found, visualized and recorded.
The enrichment medium comprises the following components: 10g/L of tryptone, 10g/L of beef extract, 5g/L of glucose, 5g/L of sodium chloride, 3g/L of yeast extract, 3g/L of sodium acetate, 1g/L, L g/L of soluble starch and 0.5g/L of cysteine hydrochloride.
The isolation medium, i.e., TSC agar medium composition, includes: 15.0g of tryptone, 5.0g of soybean peptone, 5.0g of yeast powder, 1.0g of sodium metabisulfite, 1.0g of ferric ammonium citrate, 15.0g of agar, 1000mL of distilled water and pH value of 7.6+/-0.2 (25 ℃).
The strain to be detected is subjected to gram staining, and the gram positive bacteria contains a special complex of nuclear protein magnesium salt and polysaccharide, can be firmly combined with a complex of iodine and crystal violet, is not easy to decolorize, has low combination degree of a negative bacteria complex, is poor in dye adsorption and is easy to decolorize. And observing the gram staining condition under an optical microscope, and judging the gram staining condition to be gram-positive bacteria or gram-negative bacteria. And morphological features of the cells were initially observed.
(2) Resistance plate detection strain: clostridium butyricum is a gram positive bacterium that can be grown on media containing 50. Mu.g/mL streptomycin, while gram negative bacteria do not. And (3) culturing the purified strain for 40 hours at 37 ℃ by three dividing lines on a resistance culture medium, observing whether a colony exists on a plate, and observing whether the colony is identical with an inoculated strain after microscopic examination.
(3) And (3) carrying out 16s rDNA sequence analysis and identification on 8 strains obtained after the resistance detection.
Experimental results:
8 single colonies are obtained through total separation from intestinal tracts, after the resistance experiment is carried out on 8 strains of bacteria, the same colonies among different gradients of the same culture medium are removed, and the final separation result is as follows: the TSC agar medium has 4 bacteria numbered T, T1, T2, and T3, respectively. These 4 strains were subjected to microscopic examination, and as a result, as shown in FIG. 1, more pronounced clostridia were observed.
After sequencing 4 strains of bacteria, a new strain of clostridium butyricum, clostridium butyricum CC02001 (shown as CC02001 in figure 1) is screened by sequence comparison. The base sequence shown by the sequencing result of the 16S rDNA sequence of clostridium butyricum CC02001 is shown as SEQ ID NO. 1.
SEQ ID NO.1：
cccggtgggg ctcttaccat gcagtcgagc gatgaagctc cttcgggagt ggattagcgg
cggacgggtg agtaacacgt gggtaacctg cctcatagag gggaatagcc tttcgaaagg
aagattaata ccgcataaga ttgtagtacc gcatggtaca gcaattaaag gagtaatccg
ctatgagatg gacccgcgtc gcattagcta gttggtgagg taacggctca ccaaggcgac
gatgcgtagc cgacctgaga gggtgatcgg ccacattggg actgagacac ggcccagact
cctacgggag gcagcagtgg ggaatattgc acaatggggg aaaccctgat gcagcaacgc
cgcgtgagtg atgacggtct tcggattgta aagctctgtc tttagggacg ataatgacgg
tacctaagga ggaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc
aagcgttgtc cggatttact gggcgtaaag ggagcgtagg tggatattta agtgggatgt
gaaatacccg ggcttaacct gggtgctgca ttccaaactg gatatctaga gtgcaggaga
ggaaaggaga attcctagtg tagcggtgaa atgcgtagag attaggaaga ataccagtgg
cgaaggcgcc tttctggact gtaactgaca ctgaggctcg aaagcgtggg gagcaaacag
gattagatac cctggtagtc cacgccgtaa acgatgaata ctaggtgtag gggttgtcat
gacctctgtg ccgccgctaa cgcattaagt attccgcctg gggagtacgg tcgcaagatt
aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggt ttaattcgaa
gcaacgcgaa gaaccttacc tagacttgac atctcctgaa ttactctgta atggaggaag
ccacttcggt ggcaggaaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg
ttgggttaag tcccgcaacg agcgcaaccc ttattgttag ttgctaccat ttagttgagc
actctagcga gactgcccgg gttaaccggg aggaaggtgg ggatgacgtc aaatcatcat
gccccttatg tctagggcta cacacgtgct acaatggtcg gtacaatgag atgcaacctc
gcgagagtga gcaaaactat aaaaccgatc tcagttcgga ttgtaggctg aaactcgcct
acatgaagct ggagttgcta gtaatcgcga atcagaatgt cgcggtgaat acgttcccgg
gccttgtaca caccgcccgt cacaccatga gagttggcaa tacccaaagt tcgtgagcta
accgcaagga ggcagcgact aagtagtccg g。
EXAMPLE 2 stress resistance study of Clostridium butyricum CC02001
Experimental materials: sterile normal saline, test tube, pig bile salt, diluted hydrochloric acid, artificial gastric juice, artificial intestinal juice, TSC liquid culture medium and TSC solid culture medium
Experimental strain: clostridium butyricum CC02001, clostridium butyricum 1 (T1), clostridium butyricum 2 (T2), clostridium butyricum 3 (T3)
The experimental steps are as follows: and (3) carrying out passage activation on 4 clostridium butyricum for 2 generations, and obtaining the experimental strain bacterial liquid after bacterial liquid is turbid. Centrifuging 4 bacterial solutions at 3500rpm for 10min at 4deg.C, removing supernatant, collecting bacterial sludge, detecting viable count of 4 bacterial sludge, and diluting bacterial count of 4 bacterial sludge to 1×10 10 CFU/mL with 0.85% physiological saline.
(1) Comparison of heat resistance of 4 Clostridium butyricum
Respectively taking bacterial mud of 4 clostridium butyricum after centrifugation, washing with PBS for two times, respectively placing in water baths at 60 ℃, 80 ℃ and 90 ℃ for 10min after re-suspension, then placing in an ice-water mixture at 0 ℃ for cooling, and measuring the survival rate by a gradient dilution plate method, wherein the survival rate is 3 repeats/sample. The measurement results are shown in Table 1.
TABLE 1 comparison of Heat resistance of four Clostridium butyricum
Note that: the upper right hand corner shoulder letters a, b in the table: the same letters indicate that the difference is not significant (P > 0.05) and the different letters indicate that the difference is significant (P < 0.05).
The results in Table 1 show that the 4 Clostridium butyricum are 100% tolerant to 60℃for 10min, but treated at 80℃and 90℃for 10min respectively, clostridium butyricum CC02001 strain has slightly higher heat tolerance than the other 3 strains and a significant difference (P < 0.05).
(2) Acid resistance study of 4 Clostridium butyricum
The 4 clostridium butyricum strains were inoculated after 2 hours of treatment in 5 TSC liquid media each having a pH of 1.0 to 5.0 with HCl, and after 48 hours of incubation at 37 ℃, absorbance (OD 600nm) was measured, and survival was determined qualitatively. The results are shown in Table 2.
TABLE 2 acid resistance study results of four Clostridium butyricum
The results in table 2 show that 4 clostridium butyricum strains cannot survive in the environment of ph1.0, the survival rate is between 60% and 90% in the environment of ph 2.0-3.0, wherein the survival rate of clostridium butyricum CC02001 strain is highest, the survival rate is 69.23% in the environment of ph2.0, and the difference between the clostridium butyricum strains and other 3 strains is obvious (P < 0.05); the survival rate at pH3.0 is 81.04%, the difference between the survival rate and the strain of clostridium butyricum 2 and clostridium butyricum 3 is obvious (P < 0.05), and compared with clostridium butyricum 1, the survival rate tends to be improved; compared with three strains of clostridium butyricum 1, clostridium butyricum 2 and clostridium butyricum 3, the survival rates of the three strains are highest at the pH of 4.0 and the pH of 5.0, the survival rates of clostridium butyricum CC02001 strain and clostridium butyricum 2 are obviously different (P < 0.05) when the survival rates reach 96.01 percent and 98.17 percent respectively and the survival rates of the three strains are 4.0 and 4.0, and the survival rates of the three strains and clostridium butyricum 2 and the three strains of clostridium butyricum 3 are obviously different (P < 0.05) when the pH value is 5.0. Thus, clostridium butyricum CC02001 has better acid resistance.
(3) Bile salt resistance study of 4 Clostridium butyricum strains
Pig bile salt is added into TSC liquid culture medium, and the mass fractions are respectively 0.1%, 0.3%, 0.5%, 0.7%,121 ℃ and 30min for sterilization. Inoculating 4 bacterial strains diluted into bacterial count of 1 multiplied by 10 10 CFU/mL according to 1% (volume ratio), placing into an anaerobic culture box (adding a bag of anaerobic gas producing bag), placing into an incubator, culturing at 37 ℃ for 6h, detecting bacterial count of each solution, and calculating survival rate, wherein the number of bacterial strains is 3. The measurement results are shown in Table 3.
TABLE 3 results of study on bile salt resistance of four Clostridium butyricum
As is clear from the results in table 3, the survival rates of clostridium butyricum CC02001 were the highest at both 0.1% and 0.3%, respectively, and 89.24% and 75.02%, but the differences were not significant (P > 0.05) compared to clostridium butyricum 1, and the differences were significant (P < 0.05) compared to clostridium butyricum 2 and clostridium butyricum 3; when the concentration of bile salt is 0.5% and 0.7%, the survival rate of clostridium butyricum CC02001 is highest, which is 71.01% and 59.23% respectively, and the difference between the clostridium butyricum CC02001 and other 3 strains is obvious (P < 0.05), which indicates that clostridium butyricum CC02001 has stronger bile salt tolerance.
(4) Artificial gastric juice and artificial intestinal juice tolerance study of 4 clostridium butyricum strains
Artificial gastric juice tolerance experiment: taking 1mL of culture at the end of growth, centrifuging at 4 ℃ and 3500rpm for 10min, re-suspending after washing twice by PBS, inoculating 10 7 cfu/mL into artificial gastric juice, shaking and mixing uniformly, incubating in a water bath at 37 ℃ for 1h, sampling, and counting viable bacteria by using a gradient dilution plate method, wherein the number of viable bacteria is 3.
The artificial gastric juice is prepared according to Chinese pharmacopoeia, namely 16.4ml of dilute hydrochloric acid is taken, about 800ml of water and 10g of pepsin are added, and after shaking, the water is added to be weighed and released into 1000ml, thus obtaining the artificial gastric juice. Wherein, the diluted hydrochloric acid is diluted to 1000mL by adding water into 234mL of concentrated hydrochloric acid, and the diluted hydrochloric acid with the concentration of 9.5-10.5% is obtained.
Artificial intestinal juice tolerance: 1mL of the culture at the end of growth was centrifuged at 3500rpm at 4℃for 10min, washed twice with PBS, resuspended, inoculated in artificial intestinal juice at 0 7 cfu/mL for 2 hours, sampled, and subjected to viable count by a gradient dilution plate method for 3 replicates/sample. The measurement results are shown in Table 4.
The artificial intestinal juice is prepared according to Chinese pharmacopoeia, namely, 6.8g of monopotassium phosphate is taken, 500ml of water is added to dissolve the monopotassium phosphate, and 0.1mol/l of sodium hydroxide solution is used for regulating the pH value to 6.8; and (3) taking 10g of pancreatin, adding a proper amount of water to dissolve, mixing the two solutions, and adding water to dilute to 1000ml to obtain the pancreatic enzyme.
TABLE 4 comparison of artificial gastric juice and artificial intestinal juice tolerance of four Clostridium butyricum strains
From the above experimental results, it is known that the strain clostridium butyricum CC02001 shows good tolerance to acid, pig bile salts, artificial gastric juice and artificial intestinal juice compared with clostridium butyricum 1, clostridium butyricum 2 and clostridium butyricum 3, which means that clostridium butyricum CC02001 has good stress resistance.
Example 3 Clostridium butyricum CC02001 and other 3 Clostridium butyricum antibacterial test
Experimental materials: TSC liquid culture medium, TSC solid culture medium, LB liquid culture medium, LB solid culture medium and culture dish
Experimental strain: clostridium butyricum CC02001, clostridium butyricum 1, clostridium butyricum 2, clostridium butyricum 3
Indicating strains: coli standard strain, staphylococcus aureus standard strain, salmonella (salmonella intestinal subspecies CICC 10982), clostridium perfringens (CICC 22949 = ATCC 13124)
The experimental steps are as follows:
(1) And respectively activating the experimental strain and the indicating strain for two generations to obtain experimental strain bacterial liquid and indicating strain bacterial liquid.
(2) Pouring the culture medium of the vegetable agar based on a sterile plate, placing 4-5 sterile oxford cups on the surface of the bottom agar after cooling, solidifying and solidifying, uniformly mixing activated strain indicating bacterial liquid and the culture medium according to the volume ratio of 1:100 when the TSC solid culture medium is cooled to about 50 ℃, pouring the mixture into the culture plate to prepare the solid indication culture medium of 4 indicator bacteria, and removing the oxford cups by using forceps after cooling and solidifying the bacteria-containing culture medium to prepare uniform oxford cup holes.
(3) The supernatant of each experimental strain activation solution (obtained by centrifugation at 3500rpm for 10min at 4 ℃) is injected into oxford cup holes, 100uL of each hole is obtained, and 4 experimental strains are parallel. The dishes were placed in a 37℃incubator for 16h. The experimental results are shown in table 5:
Table 5 evaluation results of antibacterial Properties of test strains
The sizes of the antibacterial circle diameters of clostridium butyricum CC02001, clostridium butyricum 1, clostridium butyricum 2 and clostridium butyricum 3 on indicator bacteria such as escherichia coli and staphylococcus aureus can indicate respective antibacterial effects, and the larger the antibacterial circle diameter is, the better the antibacterial effect is. As shown in the experimental results of Table 5, the antibacterial effect of clostridium butyricum CC02001 on escherichia coli, staphylococcus aureus, salmonella and clostridium perfringens is stronger than that of other 3 clostridium butyricum, and the antibacterial circle has obvious difference (P < 0.05) with other 3 clostridium butyricum, so that clostridium butyricum CC02001 has excellent antibacterial effect and can be used for preparing a preparation for preventing and treating bacterial infectious diseases.
EXAMPLE 4 immunomodulatory Activity of Clostridium butyricum CC02001
Experimental materials: mouse mononuclear/macrophage strain RAW264.7 in vitro cell model, DMEM culture medium, TSC liquid culture medium, griess reagent, 0.1% (m/v) neutral red solution, cell lysate and the like
Experimental strain: clostridium butyricum CC02001, clostridium butyricum 1, clostridium butyricum 3
The experimental steps are as follows:
(1) Activation and pretreatment of strains
3 Strains of clostridium butyricum are inoculated into TSC liquid culture medium, and are used for viable bacteria counting (gradient dilution coating method) and cell experiment after two generations of continuous activation. Strains cultured to logarithmic phase were centrifuged at 3500rpm for 10min at 4℃to collect the cells, which were then resuspended in PBS and diluted, and then adjusted to working concentration using DMEM complete medium.
(2) Effect of Clostridium butyricum 3 on proliferation, phagocytic Activity, NO and cytokine secretion of RAW264.7 cells (TNF-. Alpha., IFN-. Gamma., and IL-10)
The method is characterized in that a mouse mononuclear/macrophage strain RAW264.7 in-vitro cell model is adopted, clostridium butyricum CC02001, clostridium butyricum 1 and clostridium butyricum 3 are taken as experimental objects, a DMEM culture medium is taken as a blank control, and the influence of 3 clostridium butyricum on proliferation, phagocytic activity, NO and cytokine (TNF-alpha, IFN-gamma and IL-10) secretion of RAW264.7 cells, namely the effect of the clostridium butyricum on the proliferation, the phagocytic activity and the NO and cytokine secretion of the RAW264.7 cells are respectively determined by adopting an MTT method, a neutral red method, a Griess method and an ELISA kit method, so that the probiotic clostridium butyricum strain with potential immunoregulatory activity is preliminarily determined. The experimental results are shown in Table 6.
TABLE 6 influence of different strains on mouse mononuclear/macrophage strain RAW264.7 in vitro cells
Note that: the upper right hand corner marks a, b of each group indicate significant differences between each treatment group (P < 0.05), with the same letters indicating insignificant differences (P > 0.05).
Macrophages are not only widely distributed in the body, but also are one of the important lines of defense of the body against infection by pathogenic microorganisms, playing an extremely important role in natural immunity. The macrophage ingests various pathogenic microorganisms to form phagosome, and then fuses with lysosomes to form phagosome, and under the action of various enzymes, the antigenic foreign matters are killed or digested through various ways, so that the aim of protecting the organism from infection is achieved.
As shown in table 6, clostridium butyricum CC02001 significantly (P < 0.05) promoted proliferation of RAW264.7 cells, increased phagocytic activity, and significantly increased secretion of NO, TNF- α, IFN- γ, and IL-10 by RAW264.7 cells. The clostridium butyricum CC02001 has outstanding immunocompetence regulating capability, so that the clostridium butyricum CC02001 has great potential in the preparation of the microecological preparation for improving the immunity.
Example 5 preparation of feed additive
The following materials were weighed and mixed in the following order: mixing clostridium butyricum CC02001 0.5g and xylooligosaccharide 10g, adding calcium propionate 100g after uniform mixing, adding corncob powder to 1000g, and uniformly mixing to obtain the feed additive.
Example 6 preparation of feed additive
The following materials were weighed and mixed in the following order: mixing clostridium butyricum CC02001 2g and fructo-oligosaccharide 15g, adding sodium benzoate 300g after uniform mixing, adding maltodextrin to 1000g, and uniformly mixing to obtain the feed additive.
Example 7 preparation of feed additive
The following materials were weighed and mixed in the following order: mixing clostridium butyricum CC02001 1g and isomaltooligosaccharide 13g, adding 200g of sodium diacetate after uniformly mixing, adding starch to 1000g, and uniformly mixing to obtain the feed additive.
EXAMPLE 8 Effect of the feed additive of the invention on the growth performance of weaned piglets
Experimental materials: a feed additive prepared according to example 7, 1000kg per 1kg of feed mix
Experimental animals: ternary weaned piglet of 30 days of age
Experimental grouping and management: the weaned pigs with the average healthy weight of about 7kg are randomly divided into 3 groups, 3 replicates of each group and 15 pigs of each replicate, the control group is only fed with basic ration, the test group 1 (gentamicin group) is added with gentamicin (60 mg/kg) in the basic ration, the test group 2 (feed additive group) is added with 1 millof the feed additive of the invention on the basis of the basic ration, and other experimental conditions are unchanged and the feed is fed for 21d.
The results are shown in Table 7:
TABLE 7 Effect of feed additives on growth performance of weaned piglets
| Project | Control group | Test group 1 | Test group 2 |
| Head number (head) | 45 | 45 | 45 |
| Initial average weight (kg) | 7.52±0.37a | 7.60±0.31a | 7.58±0.21a |
| Ending average weight (kg) | 13.77±1.15b | 15.86±1.04a | 15.53±0.56a |
| Daily gain (g) | 298±1.14c | 393±0.56a | 389±1.12b |
| Daily average feed intake (g) | 462±2.34c | 543±1.54a | 507±2.16b |
| Feed to meat ratio | 1.59±0.07a | 1.48±0.07b | 1.50±0.06b |
| Diarrhea rate | 35.52％±0.02a | 11.27％±0.01b | 13.02％±0.01b |
As shown in Table 7, the daily gain of the test group was increased by 31.88% compared with the control group, and the feed return rate (feed conversion ratio) was increased by 6.92% compared with the control group. The diarrhea rate of the piglets in the test group 1 and the test group 2 is reduced by 68.27 percent and 63.34 percent compared with that in the control group, the difference is obvious (P <0.05 and P < 0.05), but the difference between the test group 2 and the test group 1 is not obvious (P > 0.05), and the diarrhea of the weaned piglets is effectively relieved. The result shows that the invention can effectively maintain the intestinal health of weaned pigs, relieve weaning stress, reduce diarrhea and improve the production performance of the pigs, can be used for preparing feed additives or medicaments for preventing or treating bacterial diarrhea of the pigs, and has great popularization and application values.
The method can be realized by the upper and lower limit values of the interval and the interval value of the process parameters (such as temperature, time and the like), and the examples are not necessarily listed here.
The invention may be practiced without these specific details, using any knowledge known in the art.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and are not limiting. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the appended claims.
Sequence listing
<110> Beijing family Bo Biotech Co., ltd
<120> Clostridium butyricum CC02001 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1411
<212> DNA
<213> Clostridium butyricum
<400> 1
cccggtgggg ctcttaccat gcagtcgagc gatgaagctc cttcgggagt ggattagcgg 60
cggacgggtg agtaacacgt gggtaacctg cctcatagag gggaatagcc tttcgaaagg 120
aagattaata ccgcataaga ttgtagtacc gcatggtaca gcaattaaag gagtaatccg 180
ctatgagatg gacccgcgtc gcattagcta gttggtgagg taacggctca ccaaggcgac 240
gatgcgtagc cgacctgaga gggtgatcgg ccacattggg actgagacac ggcccagact 300
cctacgggag gcagcagtgg ggaatattgc acaatggggg aaaccctgat gcagcaacgc 360
cgcgtgagtg atgacggtct tcggattgta aagctctgtc tttagggacg ataatgacgg 420
tacctaagga ggaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 480
aagcgttgtc cggatttact gggcgtaaag ggagcgtagg tggatattta agtgggatgt 540
gaaatacccg ggcttaacct gggtgctgca ttccaaactg gatatctaga gtgcaggaga 600
ggaaaggaga attcctagtg tagcggtgaa atgcgtagag attaggaaga ataccagtgg 660
cgaaggcgcc tttctggact gtaactgaca ctgaggctcg aaagcgtggg gagcaaacag 720
gattagatac cctggtagtc cacgccgtaa acgatgaata ctaggtgtag gggttgtcat 780
gacctctgtg ccgccgctaa cgcattaagt attccgcctg gggagtacgg tcgcaagatt 840
aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggt ttaattcgaa 900
gcaacgcgaa gaaccttacc tagacttgac atctcctgaa ttactctgta atggaggaag 960
ccacttcggt ggcaggaaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1020
ttgggttaag tcccgcaacg agcgcaaccc ttattgttag ttgctaccat ttagttgagc 1080
actctagcga gactgcccgg gttaaccggg aggaaggtgg ggatgacgtc aaatcatcat 1140
gccccttatg tctagggcta cacacgtgct acaatggtcg gtacaatgag atgcaacctc 1200
gcgagagtga gcaaaactat aaaaccgatc tcagttcgga ttgtaggctg aaactcgcct 1260
acatgaagct ggagttgcta gtaatcgcga atcagaatgt cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccatga gagttggcaa tacccaaagt tcgtgagcta 1380
accgcaagga ggcagcgact aagtagtccg g 1411
Claims (8)
1. Clostridium butyricum (Clostridium butyricum) CC02001 is characterized in that the preservation number of the strain is CGMCC No. 20170.
2. Use of clostridium butyricum (Clostridium butyricum) CC02001 according to claim 1 in the manufacture of a formulation for the prevention or treatment of bacterial infectious diseases in which the bacteria are escherichia coli, staphylococcus aureus, salmonella or clostridium perfringens.
3. The use according to claim 2, wherein the formulation is a microecological formulation.
4. Use of clostridium butyricum (Clostridium butyricum) CC02001 according to claim 1 for the preparation of a feed additive.
5. The use according to claim 4, wherein the feed additive is a feed additive for preventing diarrhea in piglets and promoting growth of piglets.
6. A feed additive, characterized in that the feed additive comprises clostridium butyricum (Clostridium butyricum) CC02001 according to claim 1.
7. The feed additive according to claim 6, wherein the feed additive comprises the following components in parts by weight: clostridium butyricum CC02001 0.5-2 parts, prebiotics 10-15 parts, organic acid salt 100-300 parts, and carrier 1000 parts.
8. The feed additive of claim 7, wherein the prebiotic is one or more of xylo-oligosaccharide, fructo-oligosaccharide and isomalto-oligosaccharide; the organic acid salt is one or more of potassium sorbate, sodium benzoate, calcium propionate and sodium diacetate; the carrier is one or more of maltodextrin, corncob powder, starch, medical stone, calcium carbonate, chaff powder, sepiolite powder, rice hull powder, zeolite powder and montmorillonite.
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| CN104164377A (en) * | 2014-04-08 | 2014-11-26 | 浙江大学 | Clostridium butyricum and its application |
| WO2016068186A1 (en) * | 2014-10-29 | 2016-05-06 | 東亜薬品工業株式会社 | Intestinal muscle layer thinning preventing/treating composition, diarrhea preventing/treating composition, and weaning stress alleviating composition |
| CN106479924A (en) * | 2016-10-31 | 2017-03-08 | 湖北绿雪生物科技有限公司 | The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application |
| CN107075460A (en) * | 2015-03-26 | 2017-08-18 | 韩国生命工学研究院 | With clostridium butyricum bacterial strain for strengthening immune and antiviral activity and application thereof |
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| CN104164377A (en) * | 2014-04-08 | 2014-11-26 | 浙江大学 | Clostridium butyricum and its application |
| WO2016068186A1 (en) * | 2014-10-29 | 2016-05-06 | 東亜薬品工業株式会社 | Intestinal muscle layer thinning preventing/treating composition, diarrhea preventing/treating composition, and weaning stress alleviating composition |
| CN107075460A (en) * | 2015-03-26 | 2017-08-18 | 韩国生命工学研究院 | With clostridium butyricum bacterial strain for strengthening immune and antiviral activity and application thereof |
| CN106479924A (en) * | 2016-10-31 | 2017-03-08 | 湖北绿雪生物科技有限公司 | The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application |
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| Effects of Clostridium butyricum on growth performance, metabonomics and intestinal microbial differences of weaned piglets;Jing Liang等;《BMC Microbiol》;参见全文 * |
| 日粮添加丁酸梭菌对断奶仔猪生长性能和腹泻率的影响;王海亮等;《国外畜牧学(猪与禽)》;参见全文 * |
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