CN115704002A - Clostridium butyricum CC02001 and application thereof - Google Patents
Clostridium butyricum CC02001 and application thereof Download PDFInfo
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- CN115704002A CN115704002A CN202110918935.8A CN202110918935A CN115704002A CN 115704002 A CN115704002 A CN 115704002A CN 202110918935 A CN202110918935 A CN 202110918935A CN 115704002 A CN115704002 A CN 115704002A
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- clostridium butyricum
- feed additive
- strain
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- clostridium
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Images
Abstract
The invention discloses clostridium butyricum CC02001 and application thereof, and belongs to the technical field of micro-ecology. The clostridium butyricum CC02001 is obtained by separating and screening weaned pig intestinal tracts, and the bacterial strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC 20170. Clostridium butyricum CC02001 has strong stress resistance and in-vitro bacteriostasis capacity. The strain serving as one of feed additive strains 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; can produce probiotic products such as B vitamins and the like, and has health-care effect on animals. Because of the resistance to various antibiotics such as streptomycin, the compound can be used as a veterinary drug and has great potential for replacing the 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 butyric acid bacteria belongs to the family bacillaceae, clostridium, gram-positive, has spores, is an obligate anaerobic bacterium, can resist adverse environment, and is first discovered and reported by womb nearly treating doctor of qianye medical university in japan in 1933. Clostridium butyricum is widely found in the intestinal tract, feces and soil of humans and animals. In 1935, doctor Kingi miyairii, russian institute of microbiology, isolated Clostridium butyricum from human feces and soil, and subsequently found that the filtrate of anaerobic culture contained less fatty acid, had a strong intestinal function, and it could inhibit the reproduction of pathogenic bacteria in the intestinal tract and promote the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus in the intestinal tract. The clostridium butyricum is introduced from Russian microbiology research institute by the department of sciences of Heilongjiang province in 1992, is planted in China, and makes a great contribution to the research of microorganism beneficial bacteria in China.
The clostridium butyricum has stronger stability, can generate endogenic spores, can resist heat and acid, is not influenced by gastric acid, digestive enzyme, bile acid and the like in vivo, is not inactivated by the digestive tract, can be stored for three years in an in vitro room temperature drying environment, has no obvious reduction of viable bacteria, and has no failure in function. And has certain resistance to antibiotics such as penicillin, ampicillin, streptomycin, gentamicin, etc., therefore, the preparation can be used together with antibiotics, thereby improving the curative effect. And a large number of experimental results prove that the clostridium butyricum has no toxic or side effect and is widely applied to medicines, functional health-care foods and animal feed additives.
The clostridium butyricum has good effect in the pig industry, and shows that the growth is fast, the hair color is bright, the diarrhea rate is obviously reduced in the piglet and piglet stage, the weight gain of the piglet and the piglet 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 excrement formation is good. Early weaning technology is generally adopted in the modern pig raising industry, and the diarrhea of weaned piglets is a key factor influencing the production performance, and according to statistics, the economic loss caused by the diarrhea of the piglets in China is up to millions of yuan every year, wherein escherichia coli in the diarrhea caused by bacterial infection is one of the most main pathogens. Coli is a common bacterium in the digestive tract, and is parasitic in the colon of piglets at an early stage.
Studies have shown that the faecal flora of healthy humans is known to be dominated by anaerobic bacteria, and that bifidobacteria are known to be one of the most common normal flora in humans and animals. Studies such as Zhang darong prove that flora imbalance in irritable bowel syndrome patients is represented by the fact that the quantity of total anaerobic bacteria bifidobacteria and lactobacillus in the main proportion in fecal flora is reduced, the proportion of the total anaerobic bacteria bifidobacteria and lactobacillus in the fecal flora is smaller, the potential pathogenic clostridium with smaller proportion in intestinal flora is obviously increased, after the treatment of clostridium butyricum, the bifidobacteria, the bacillus-free anaerobic bacteria, escherichia coli, enterococcus and lactobacillus in the fecal flora have no obvious difference with healthy people, and clinical symptoms are correspondingly and obviously improved. The research on the in vitro biological antagonism of the pathogenic bacteria in the intestinal tract by the clostridium butyricum preparation of Zhangxuepin and the like shows that the clostridium butyricum and the bifidobacteria can obviously inhibit the growth and the propagation of Escherichia coli, typhoid bacillus and dysentery bacillus in vitro.
With the progress of society and the continuous development of science and technology, side effects such as bacterial drug resistance, intestinal flora imbalance, drug residues and the like caused by the long-term use of antibiotics are increasingly remarkable, a safe and green biological product capable of replacing antibiotics is urgently needed to be developed in order to meet the production requirements of the breeding industry and guarantee the life health of human beings, and the microecological preparation is a safer biological preparation involved by various researchers and is expected to replace antibiotics due to the excellent characteristics of the microecological preparation.
Disclosure of Invention
The invention provides a Clostridium butyricum CC02001, which is obtained by separating, screening and classifying Clostridium butyricum (Clostridium butyricum) from intestinal tracts of healthy weaned piglets in a large-scale pig farm, is numbered as CC02001, is preserved in China general microbiological culture collection center, is preserved at China academy of sciences microorganism research institute (Beijing), is CGMCC 20170 and has the preservation date of 2020.07.16.
In a second aspect of the invention, there is provided the use of clostridium butyricum CC02001 in an agent for the prevention and treatment of bacterial infectious diseases.
In a third aspect of the invention there is provided the use of clostridium butyricum CC02001 in an immunity enhancing formulation.
The preparation can be a micro-ecological preparation or a pharmaceutical preparation.
In a fourth aspect of the invention, the invention provides an application of clostridium butyricum CC02001 in a feed additive.
The feed additive can be used for preventing piglet diarrhea and promoting piglet growth.
In the fifth aspect of the invention, the invention also provides a feed additive containing clostridium butyricum CC02001.
The feed additive comprises the components of clostridium butyricum CC02001, prebiotics, organic acid salt and a carrier.
The feed additive comprises the following components in parts by weight: 0.5-2 parts of clostridium butyricum CC02001, 10-15 parts of prebiotics, 100-300 parts of organic acid salt and 1000 parts of carrier.
The viable count of the clostridium butyricum CC02001 is 1 multiplied by 10 8 ～1×10 10 CFU/g。
The prebiotics are 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, rice husk powder, sepiolite powder, rice husk powder, zeolite powder and montmorillonite.
The preparation method of the feed additive comprises the following steps: weighing the raw materials according to the weight part ratio, uniformly mixing the clostridium butyricum and the prebiotics, adding the organic acid salt, uniformly mixing, adding the carrier, and uniformly mixing all the raw materials to obtain the feed additive.
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 bacteriostatic ability, also has strong immunocompetence regulating ability, can obviously improve the proliferation and phagocytosis activity of (RAW 264.7) cells, has excellent performance and high research value and application value, and can be applied to microecologics and pharmaceutical preparations for preventing or treating bacterial infectious diseases and improving immunity.
2. According to the invention, the clostridium butyricum CC02001 is applied to the feed additive, the number of beneficial bacteria in intestinal tracts is increased under the combined action of the clostridium butyricum CC02001 and prebiotics, and the growth of harmful bacteria in the intestinal tracts is inhibited by adding organic acid salt, so that the effects of improving the immunity of piglets, preventing bacterial diarrhea of weaned piglets and promoting the growth of the piglets are achieved.
3. The 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 enhance the multiplication of beneficial bacteria; can enhance animal immunity and prevent tumor; can produce probiotic products such as B vitamins and the like, and has health-care effect on animals. Because of the resistance to various antibiotics such as streptomycin, the compound can be used as a veterinary drug and has great potential for replacing the antibiotics.
Drawings
FIG. 1 is a graph showing microscopic examination results of the strains in examples 1 and 2 of the present invention.
Detailed Description
The invention is explained in further detail below with reference to the figures and the description of embodiments.
Example 1 isolation, screening and identification of Clostridium butyricum CC02001
Experimental materials: piglet intestinal contents, clostridium fortified medium (RCM medium), test pigs, which did not receive any antibiotics and microecological products; tryptone-sulfite-cycloserine agar base (TSC), 9mL sterile water test 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-covered bottle, carrying out water bath at 80 ℃ for 10min, immediately cooling with water, placing in a constant-temperature incubator at 37 ℃ for enrichment culture for 3 days, selecting a culture solution with a large amount of bubbles, further carrying out enrichment in the enrichment medium, carrying out gas chromatography analysis and detection after passage for 5 times, and transferring a fermentation solution with butyric acid to a separation medium for continuous fermentation culture. Taking 1mL of fermentation liquor of enrichment culture, adding the fermentation liquor into a 9mL sterile water test tube, uniformly mixing, and sequentially carrying out gradient dilution by 10 times.
Respectively taking the dilution gradient as 10 -5 、10 -6 、10 -7 Respectively coating 0.1mL of supernatant in the test tubes on the surface of a TSC agar medium, and making 3 gradients in parallel; the sterile inoculating loop is used for dipping and taking the dilution gradient to be 10 -3 、10 -4 The supernatants of the tubes were streaked onto three areas of the surface of the TSC agar medium, and 2 replicates were run for each gradient. After anaerobic culture for 48h at 37 ℃, finding out a black single colony on the culture mediumAnd (5) microscopic examination and recording.
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 of soluble starch and 0.5g/L of L-cysteine hydrochloride.
The TSC agar culture medium comprises the following components: tryptone 15.0g, soytone 5.0g, yeast powder 5.0g, sodium metabisulfite 1.0g, ferric ammonium citrate 1.0g, agar 15.0g, 1000mL distilled water, pH 7.6 + -0.2 (25 ℃).
Gram staining is carried out on a strain to be detected, gram-positive bacteria contain a special compound of nucleoprotein magnesium salt and polysaccharide, the compound can be firmly combined with a compound of iodine and crystal violet, and is not easy to decolor, and a compound of negative bacteria has low combination degree, poor dye adsorption and easy decoloration. The gram staining condition of the bacteria was observed under an optical microscope, and the bacteria were judged to be gram-positive bacteria or gram-negative bacteria. And the morphological characteristics of the cells were preliminarily observed.
(2) Resistant plate detection of strains: clostridium butyricum is a gram-positive bacterium that can grow on medium containing 50. Mu.g/mL streptomycin, whereas gram-negative bacteria do not. And (3) streaking the purified strain on a three-region resistant culture medium, culturing for 40h at 37 ℃, and observing whether a bacterial colony exists on a plate or not, wherein the bacterial colony is the same as the inoculated strain or not after microscopic examination.
(3) And (3) carrying out 16s rDNA sequence analysis and identification on 8 strains obtained after resistance detection.
The experimental results are as follows:
8 single colonies are obtained by co-separation in intestinal tracts, after 8 strains are subjected to a resistance experiment, 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 contains 4 kinds of bacteria, which are numbered T, T1, T2 and T3. As a result of microscopic examination of these 4 strains, a relatively distinct clostridia was observed as shown in FIG. 1.
After 4 strains of bacteria are sequenced, a new clostridium butyricum strain, namely clostridium butyricum CC02001 (shown as CC02001 in figure 1), is screened out through sequence comparison. The sequencing result of the 16S rDNA sequence of the clostridium butyricum CC02001 shows that the base sequence is shown in 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, dilute hydrochloric acid, artificial gastric juice, artificial intestinal juice, TSC liquid culture medium, and TSC solid culture medium
Experimental strains: clostridium butyricum CC02001, clostridium butyricum 1 (T1), clostridium butyricum 2 (T2), clostridium butyricum 3 (T3)
The experimental steps are as follows: and (4) subculturing and activating the 4 clostridium butyricum for 2 generations, and obtaining the bacterial liquid of the experimental strain, wherein the bacterial liquid is turbid. Centrifuging 4 bacterial solutions at 4 deg.C at 3500rpm for 10min, removing supernatant, collecting bacterial sludge, detecting viable count of 4 bacterial sludge, diluting the bacterial count of 4 bacterial sludge with 0.85% normal saline to 1 × 10 10 CFU/mL。
(1) Comparison of Heat resistance of 4 species of Clostridium butyricum
Respectively taking 4 strains of centrifuged bacterium mud of clostridium butyricum, washing and coating the bacterium mud with PBS twice, placing the bacterium mud in water bath at 60 ℃, 80 ℃ and 90 ℃ for 10min after resuspension, then placing the bacterium mud in ice-water mixture at 0 ℃ for cooling, and measuring the survival rate of the bacterium mud by a gradient dilution plate method for 3 repeats/sample. The results are shown in Table 1.
TABLE 1 comparison of the Heat resistance of four species of Clostridium butyricum
Note: letters a, b are marked on the upper right corner of the numbers in the table: the letters are identical for insignificant difference (P > 0.05) and the letters are different for significant difference (P < 0.05).
The results in table 1 show that 4 clostridium butyricum can tolerate 60 ℃ treatment for 10min by 100%, but the clostridium butyricum CC02001 strain has slightly higher heat resistance than the other 3 strains and has obvious difference (P < 0.05) after being treated for 10min at 80 ℃ and 90 ℃.
(2) Acid resistance study of 4 kinds of clostridium butyricum
In 5 TSC liquid media adjusted to pH1.0 to 5.0 with HCl, 4 Clostridium butyricum strains were inoculated after 2 hours of treatment and cultured at 37 ℃ for 48 hours, and the absorbance (OD) was measured 600nm ) And qualitatively determining the survival rate. The results are shown in Table 2.
TABLE 2 acid resistance study results of four species of Clostridium butyricum
The results in table 2 show that 4 clostridium butyricum strains can not survive under the environment of pH1.0, the survival rates under the environment of pH2.0-3.0 are all between 60% and 90%, wherein the clostridium butyricum CC02001 strain has the highest survival rate, the survival rate under the environment of pH2.0 is 69.23%, and the difference between the clostridium butyricum CC02001 strain and other 3 strains is obvious (P < 0.05); the survival rate is 81.04% under pH3.0, the difference between the two strains is obvious (P is less than 0.05), and the survival rate is improved compared with that of clostridium butyricum 1; compared with three strains of clostridium butyricum 1, clostridium butyricum 2, clostridium butyricum 3 and the like, the survival rates of the clostridium butyricum CC02001 strain are the highest at pH4.0 and pH5.0 and reach 96.01 percent and 98.17 percent respectively, the survival rate of the clostridium butyricum CC02001 strain is obviously different from that of the clostridium butyricum 2 (P < 0.05) at pH4.0, and the survival rate of the clostridium butyricum CC02001 strain is obviously different from that of the clostridium butyricum 2 strain (P < 0.05) at pH value of 5.0. Therefore, clostridium butyricum CC02001 has better acid resistance.
(3) Bile salt resistance study of 4 Clostridium butyricum strains
Adding pig bile salt into TSC liquid culture medium at mass ratio of 0.1%, 0.3%, 0.5%, 0.7%, sterilizing at 121 deg.C for 30 min. For dilution to the number of bacteria of 1 multiplied by 10 10 Inoculating 4 bacterial strains of CFU/mL according to a volume ratio of 1%, placing in an anaerobic culture box (adding a bag of anaerobic gas generating bag), placing in an incubator, culturing at 37 ℃ for 6h, detecting the bacterial count of each solution, calculating the survival rate, and repeating for 3 times/sample. Measurement ofThe results are shown in Table 3.
TABLE 3 results of the study of the bile salt resistance of four species of Clostridium butyricum
As can be seen from the results in table 3, the survival rates of clostridium butyricum CC02001 are highest at 0.1% and 0.3%, respectively 89.24% and 75.02%, but the differences are not significant (P > 0.05) compared with clostridium butyricum 1 and significant (P < 0.05) compared with clostridium butyricum 2 and clostridium butyricum 3; when the concentrations of the bile salts are 0.5% and 0.7%, the survival rates of the clostridium butyricum CC02001 are highest and are 71.01% and 59.23% respectively, and the differences between the clostridium butyricum CC02001 and other 3 strains are obvious (P < 0.05), which indicates that the clostridium butyricum CC02001 has stronger bile salt tolerance.
(4) Study on tolerance of artificial gastric juice and artificial intestinal juice of 4 clostridium butyricum strains
Simulated gastric juice tolerance test: 1ml of the final-stage culture was centrifuged at 3500rpm at 4 ℃ for 10min, washed twice with PBS and resuspended in 10 7 cfu/mL of the viable bacteria were inoculated in artificial gastric juice, shaken and mixed, incubated in a water bath at 37 ℃, sampled after 1 hour, and viable bacteria were counted by a gradient dilution plate method for 3 replicates/sample.
The artificial gastric juice is prepared according to Chinese pharmacopoeia, namely, taking 16.4ml of dilute hydrochloric acid, adding about 800ml of water and 10g of pepsin, shaking up, adding water, weighing and releasing to 1000ml to obtain the artificial gastric juice. Wherein the dilute hydrochloric acid is 234mL of concentrated hydrochloric acid, and the diluted hydrochloric acid is diluted to 1000mL by adding water to obtain the dilute hydrochloric acid with the concentration of 9.5-10.5%.
Artificial intestinal juice tolerance: 1ml of the final-stage culture was centrifuged at 3500rpm at 4 ℃ for 10min, washed twice with PBS, and resuspended at l0 7 cfu/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 4.
The artificial intestinal juice is prepared according to Chinese pharmacopoeia, namely, 6.8g of monopotassium phosphate is taken, 500ml of water is added for dissolving, and 0.1mol/l of sodium hydroxide solution is used for adjusting the pH value to 6.8; dissolving pancreatin 10g in water, mixing the two solutions, and diluting to 1000ml with water.
TABLE 4 comparison of the tolerance of the four Clostridium butyricum strains to artificial gastric juice and artificial intestinal juice
The experimental results are integrated, and compared with clostridium butyricum 1, clostridium butyricum 2 and clostridium butyricum 3, the strain clostridium butyricum CC02001 has good tolerance to acid, pig bile salt, artificial gastric juice and artificial intestinal juice, which indicates that the clostridium butyricum CC02001 has good stress resistance.
Example 3 Clostridium butyricum CC02001 and other 3 strains of Clostridium butyricum bacteriostasis test
Experimental materials: TSC liquid culture medium, TSC solid culture medium, LB liquid culture medium, LB solid culture medium and culture dish
Experimental strains: clostridium butyricum CC02001, clostridium butyricum 1, clostridium butyricum 2, clostridium butyricum 3
Indicating strains: escherichia coli standard strain, staphylococcus aureus standard strain, salmonella (Salmonella enterica subspecies CICC 10982), clostridium perfringens (CICC 22949= ATCC 13124)
The experimental steps are as follows:
(1) Respectively activating the experimental strain and the indicating strain for two generations to obtain the experimental strain bacterial liquid and the indicating strain bacterial liquid.
(2) Pouring agar culture based on an aseptic plate, after cooling and solidification, placing 4-5 aseptic Oxford cups on the surface of bottom agar by using aseptic tweezers, when the TSC solid culture medium is cooled to about 50 ℃, uniformly mixing the activated indication strain bacterial liquid and the culture medium according to the volume ratio of 1: 100, pouring the mixture into the culture dish to prepare a solid indication culture medium containing 4 indication bacteria, and after the bacteria-containing culture medium is cooled and solidified, removing the Oxford cups by using the tweezers to prepare uniform Oxford cup holes.
(3) Injecting the supernatant of the activation solution of each experimental strain (obtained by centrifuging at 3500rpm for 10min at 4 ℃) into the holes of the Oxford cup, wherein each hole is 100uL, and each experimental strain is 4 in parallel. The culture dish is placed in a constant temperature incubator at 37 ℃ for 16h. The results are shown in Table 5:
TABLE 5 evaluation results of antibacterial property of test strains
The diameters of the inhibition zones of clostridium butyricum CC02001, clostridium butyricum 1, clostridium butyricum 2 and clostridium butyricum 3 for indicator bacteria such as escherichia coli and staphylococcus aureus can show respective inhibition effects, and the larger the diameter of the inhibition zone is, the better the inhibition effect is. The experimental result in table 5 shows that the bacteriostatic effect of the clostridium butyricum CC02001 on escherichia coli, staphylococcus aureus, salmonella and clostridium perfringens is stronger than that of other 3 clostridium butyricum, and the bacteriostatic effect of the clostridium butyricum CC02001 is higher than that of the other 3 clostridium butyricum, and the bacteriostatic circle has significant difference (P < 0.05) with that of the other 3 clostridium butyricum, so that the clostridium butyricum CC02001 has excellent bacteriostatic 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 strains: 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 in a TSC liquid culture medium and used for viable bacteria counting (a gradient dilution coating method) and cell experiments after two continuous generations of activation. And (3) centrifuging the strain cultured to the logarithmic phase for 10min at 3500rpm under the condition of 4 ℃, collecting thalli, performing PBS (phosphate buffer solution) resuspension and dilution, and adjusting to the working concentration by using a DMEM (DMEM) complete culture medium.
(2) Effect of 3 strains of Clostridium butyricum on proliferation and phagocytic activity of RAW264.7 cells, and secretion amounts of NO and cytokines (TNF-alpha, IFN-gamma and IL-10)
The method comprises the steps of taking a mouse mononuclear/macrophage strain RAW264.7 in-vitro cell model, taking clostridium butyricum CC02001, clostridium butyricum 1 and clostridium butyricum 3 as experimental objects, taking a DMEM culture medium as a blank control, and respectively measuring the effects of the 3 strains of clostridium butyricum on the proliferation and phagocytosis activity of RAW264.7 cells, NO and the secretion amount of cytokines (TNF-alpha, IFN-gamma and IL-10) by adopting an MTT method, a neutral red method, a Griess method and an ELISA kit method, so as to preliminarily determine the probiotic clostridium butyricum strain with potential immunoregulatory activity. The results are shown in Table 6.
TABLE 6 Effect of different strains on in vitro cells of mouse monocyte/macrophage strain RAW264.7
Note: the upper right hand symbols a, b for each group indicate significant differences between treatment groups (P < 0.05) and the letters are the same indicating insignificant differences (P > 0.05).
Macrophages are not only widely distributed in the body, but also one of the important defense lines of the body against infection by pathogenic microorganisms, and play an extremely important role in natural immunity. The macrophage takes in various pathogenic microorganisms to form a phagosome, then the phagosome is fused with the lysosome to form the phagolysosome, and under the action of various enzymes, antigenic foreign matters are killed or digested through various ways, so that the aim of protecting organisms from being infected is fulfilled.
As can be seen from Table 6, clostridium butyricum CC02001 can significantly (P < 0.05) promote proliferation of RAW264.7 cells, improve phagocytic activity, and significantly improve the secretion of NO, TNF-alpha, IFN-gamma and IL-10 in RAW264.7 cells. The clostridium butyricum CC02001 has outstanding immunocompetence adjusting capacity, and therefore has great potential in preparation of immune microecologics.
EXAMPLE 5 preparation of feed additive
Weighing the following materials in the following amount (1 kg package) and mixing the materials in sequence: mixing 0.5g of clostridium butyricum CC02001 and 10g of xylo-oligosaccharide, adding 100g of calcium propionate after uniformly mixing, adding 1000g of corncob powder, and uniformly mixing to obtain the feed additive.
EXAMPLE 6 preparation of feed supplement
Weighing the following materials in the following amount (1 kg package) and mixing the materials in sequence: mixing clostridium butyricum CC02001 g and fructo-oligosaccharide 15g, adding 300g of sodium benzoate after uniformly mixing, adding maltodextrin to 1000g, and uniformly mixing to obtain the feed additive.
Example 7 preparation of feed additive
Weighing the following materials in the following amount (1 kg package) and mixing the materials in sequence: mixing clostridium butyricum CC02001 g and isomaltooligosaccharide 13g, adding sodium diacetate 200g 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 growth Performance of weaned piglets
Experimental materials: a feed supplement prepared according to example 7, 1000kg per 1kg of mix
Experimental animals: 30-day-old ternary weaned piglet
Experiment grouping and management: 135 weaned piglets with healthy average weight of about 7kg are randomly divided into 3 groups, each group has 3 repetitions, 15 pigs are repeated in each group, a control group only feeds basic daily ration, a test group 1 (gentamicin group) adds gentamicin (60 mg/kg) in the basic daily ration, a test group 2 (feed additive group) adds 1 per mill of the feed additive on the basis of the basic daily ration, and other test conditions are unchanged and the weaned piglets are fed for 21d.
The results are shown in Table 7:
TABLE 7 Effect of feed additives on growth Performance of weaned piglets
| Item | Control | Test group | 1 | |
| Number of heads (head) | 45 | 45 | 45 | |
| Initial weight (kg) | 7.52±0.37a | 7.60±0.31a | 7.58±0.21a | |
| End weight (kg) | 13.77±1.15b | 15.86±1.04a | 15.53±0.56a | |
| Daily average weight 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 conversion ratio | 1.59±0.07a | 1.48±0.07b | 1.50±0.06b | |
| Rate of diarrhea | 35.52％±0.02a | 11.27％±0.01b | 13.02％±0.01b |
As can be seen from Table 7, the daily average weight gain of the test group is 31.88% higher than that of the control group, and the feed conversion ratio (feed conversion ratio) is 6.92% higher than that of the control group. The diarrhea rate of piglets in the test group 1 and the test group 2 is respectively reduced by 68.27 percent and 63.34 percent compared with that in the control group, and the difference is significant (P <0.05 and P < 0.05), but the difference between the test group 2 and the test group 1 is not significant (P > 0.05), so that the diarrhea of weaned piglets is effectively relieved. The results show that the feed additive can effectively maintain the intestinal health of weaned pigs, relieve weaning stress, reduce diarrhea and improve the production performance of the piglets, can be used for preparing feed additives or medicaments for preventing or treating piglet bacterial diarrhea, and has great popularization and application values.
The method can be realized by upper and lower limit values of intervals of process parameters (such as temperature, time and the like) and interval values, and embodiments are not listed.
Those skilled in the art will recognize that the invention may be practiced without these specific details.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Beijing Kogyo 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 (9)
1. The clostridium butyricum CC02001 is characterized in that the preservation number of the strain is CGMCC 20170.
2. Use of clostridium butyricum CC02001 of claim 1 in the manufacture of a formulation for the prevention or treatment of bacterial infectious diseases.
3. Use of clostridium butyricum CC02001 of claim 1 in the manufacture of a formulation for enhancing immunity.
4. The formulation according to claim 2 or 3, wherein the formulation is a probiotic or a pharmaceutical formulation.
5. Use of clostridium butyricum CC02001 of claim 1 in the preparation of a feed additive.
6. The use according to claim 5, wherein the feed additive is a feed additive for preventing diarrhea and promoting growth of piglets.
7. A feed additive comprising clostridium butyricum CC02001 of claim 1.
8. The feed additive according to claim 7, wherein the feed additive comprises the following components in parts by weight: 0.5-2 parts of clostridium butyricum CC02001, 10-15 parts of prebiotics, 100-300 parts of organic acid salt and 1000 parts of carrier.
9. The feed additive according to claim 8, wherein the prebiotics are 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, rice husk powder, sepiolite powder, rice husk powder, zeolite powder and montmorillonite.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110918935.8A CN115704002B (en) | 2021-08-11 | Clostridium butyricum CC02001 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110918935.8A CN115704002B (en) | 2021-08-11 | Clostridium butyricum CC02001 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115704002A true CN115704002A (en) | 2023-02-17 |
| CN115704002B CN115704002B (en) | 2024-04-26 |
Family
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| JING LIANG等: "Effects of Clostridium butyricum on growth performance, metabonomics and intestinal microbial differences of weaned piglets", 《BMC MICROBIOL》 * |
| 王海亮等: "日粮添加丁酸梭菌对断奶仔猪生长性能和腹泻率的影响", 《国外畜牧学(猪与禽)》 * |
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