CN114214263A - Method for promoting clostridium butyricum to form spores, and microecological preparation and application thereof - Google Patents
Method for promoting clostridium butyricum to form spores, and microecological preparation and application thereof Download PDFInfo
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- CN114214263A CN114214263A CN202111629695.6A CN202111629695A CN114214263A CN 114214263 A CN114214263 A CN 114214263A CN 202111629695 A CN202111629695 A CN 202111629695A CN 114214263 A CN114214263 A CN 114214263A
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 132
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 230000001737 promoting effect Effects 0.000 title claims abstract description 22
- 238000000855 fermentation Methods 0.000 claims abstract description 87
- 230000004151 fermentation Effects 0.000 claims abstract description 87
- 239000000411 inducer Substances 0.000 claims abstract description 52
- 230000001580 bacterial effect Effects 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- 241000894006 Bacteria Species 0.000 claims abstract description 24
- 239000010802 sludge Substances 0.000 claims abstract description 20
- 230000008569 process Effects 0.000 claims abstract description 16
- 230000006698 induction Effects 0.000 claims abstract description 13
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 230000000415 inactivating effect Effects 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 31
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 26
- 230000015572 biosynthetic process Effects 0.000 claims description 23
- 239000000843 powder Substances 0.000 claims description 15
- 230000028070 sporulation Effects 0.000 claims description 15
- WJJMNDUMQPNECX-UHFFFAOYSA-N Dipicolinic acid Natural products OC(=O)C1=CC=CC(C(O)=O)=N1 WJJMNDUMQPNECX-UHFFFAOYSA-N 0.000 claims description 13
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 13
- 238000011218 seed culture Methods 0.000 claims description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 10
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- 235000010413 sodium alginate Nutrition 0.000 claims description 10
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- CABMTIJINOIHOD-UHFFFAOYSA-N 2-[4-methyl-5-oxo-4-(propan-2-yl)-4,5-dihydro-1H-imidazol-2-yl]quinoline-3-carboxylic acid Chemical compound N1C(=O)C(C(C)C)(C)N=C1C1=NC2=CC=CC=C2C=C1C(O)=O CABMTIJINOIHOD-UHFFFAOYSA-N 0.000 claims description 3
- 239000003674 animal food additive Substances 0.000 claims description 3
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- 230000002779 inactivation Effects 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 206010012735 Diarrhoea Diseases 0.000 abstract description 8
- 235000021050 feed intake Nutrition 0.000 abstract description 7
- 230000029087 digestion Effects 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
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- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
- 239000002068 microbial inoculum Substances 0.000 description 7
- 241000193830 Bacillus <bacterium> Species 0.000 description 6
- 230000003115 biocidal effect Effects 0.000 description 6
- 239000002054 inoculum Substances 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
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- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical group CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
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- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
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- 229960003022 amoxicillin Drugs 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
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- WCPGOAHESUQATA-UHFFFAOYSA-L calcium;pyridine-2,6-dicarboxylate Chemical compound [Ca+2].[O-]C(=O)C1=CC=CC(C([O-])=O)=N1 WCPGOAHESUQATA-UHFFFAOYSA-L 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 description 1
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- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 description 1
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- 238000011056 performance test Methods 0.000 description 1
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Classifications
-
- 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
- C12N3/00—Spore forming or isolating processes
-
- 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
-
- 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
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- 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
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
-
- 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 discloses a method for promoting clostridium butyricum to form spores, a microecological preparation and application thereof, and relates to the technical field of microbial preparations. The method for promoting the clostridium butyricum to form spores comprises the step of adding a spore-forming inducer in the fermentation culture process, wherein the components of the spore-forming inducer comprise inactivated clostridium butyricum bacterial sludge, and the inactivated clostridium butyricum bacterial sludge is obtained by inoculating clostridium butyricum into an induction culture medium for culture, inactivating and then carrying out solid-liquid separation. The method can effectively prevent partial bacteria which form spores firstly from releasing substances such as spore-producing delayed protein and the like to inhibit other bacteria from forming spores due to overlong spore forming process, further effectively improve the live bacteria quantity and the spore rate of the clostridium butyricum, solve the problem that the high bacteria quantity and the high spore rate of the clostridium butyricum are difficult to coexist, and greatly promote the storage, transportation and use of the clostridium butyricum; in addition, the microecological preparation prepared by the invention has obvious effect when being applied to piglet feeding, and can effectively improve the feed intake, promote digestion and reduce the occurrence of diarrhea.
Description
Technical Field
The invention relates to the technical field of microbial preparations, in particular to a method for promoting clostridium butyricum to form spores, a microbial ecological preparation and application thereof.
Background
The microecological preparation is one of substitute antibiotic products, and has the effects of regulating intestinal health, improving immunity, improving feed digestion utilization rate, reducing waste discharge and the like. The commonly used strains of the microecological preparation include bacillus subtilis, bacillus licheniformis, enterococcus faecalis, enterococcus faecium, lactobacillus acidophilus, lactobacillus plantarum, saccharomyces cerevisiae and the like, and the reports of preparing the microecological preparation by clostridium butyricum are less.
The clostridium butyricum is also called clostridium butyricum and is an obligate anaerobic gram-positive bacillus, the bacterium is straight rod-shaped or slightly bent and can generate spores, the middle of the bacterium expands into a clostridium when the spores are generated, and the clostridium butyricum is named clostridium butyricum because the main metabolite of the clostridium butyricum is butyric acid. Because the bacillus anaerobicus is anaerobic bacillus, the bacillus anaerobicus has stronger tolerance to gastric acid, bile acid and various feed antibiotics, and has wide application range.
The research of clostridium butyricum in China starts late, and at present, domestic clostridium butyricum preparations are low in yield, high in price and short in quality guarantee period, and the requirements of the market on clostridium butyricum are difficult to meet. The problems are mainly caused by the complex culture conditions of clostridium butyricum, immature technology and the like. In addition, the stress resistance of clostridium butyricum mainly comes from spore production capacity of clostridium butyricum, and how to improve the spore rate of clostridium butyricum is also a main factor restricting the production of clostridium butyricum, but clostridium butyricum is obligate anaerobe, culture conditions have high requirements on equipment, and products with high bacterial yield and high spore rate are difficult to produce under the condition of common equipment conditions, so that the clostridium butyricum cannot be popularized at present and the total yield is low.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for promoting clostridium butyricum to form spores, a microecological preparation and application thereof.
The invention is realized by the following steps:
in a first aspect, the invention provides a method for promoting clostridium butyricum to form spores, wherein a spore-forming inducer is added in a fermentation culture process, and the components of the spore-forming inducer comprise inactivated clostridium butyricum bacterial sludge which is obtained by inoculating clostridium butyricum into an induction culture medium for culture, inactivating and then carrying out solid-liquid separation.
In an alternative embodiment, the components of the sporulation inducer further comprise 2, 6-pyridinedicarboxylic acid and calcium carbonate;
preferably, the components of the spore-forming inducer comprise 4-5g/L of inactivated clostridium butyricum bacterial mud, 8-10g/L of 2, 6-dipicolinic acid and 15-18g/L of calcium carbonate according to concentration.
In an alternative embodiment, inoculating clostridium butyricum into an induction medium for culture comprises: inoculating the clostridium butyricum to the induction medium at a volume ratio of 1-2%, and performing static culture at 36-38 ℃ for 16-18 hours;
preferably, the inactivation comprises that the induction culture after the culture is finished is carried out for 20-40min in a boiling water bath, and no viable bacteria is detected;
preferably, the solid-liquid separation comprises centrifugation or filtration;
preferably, the inactivated Clostridium butyricum bacterial sludge is obtained by centrifugation at 8000-12000rpm for 8-12min and supernatant is removed.
In alternative embodiments, the sporulation inducer is added during the middle of the fermentation culture;
preferably, after the spore-forming inducer is added, the pH value of the system is adjusted to 3.5-4.0, the temperature is adjusted to 20-22 ℃, and the fermentation is finished after the culture is continued for 8-9 hours.
In alternative embodiments, adding a sporulation inducer during the fermentation culture comprises: inoculating clostridium butyricum into a seed culture medium for standing culture to obtain a seed solution; inoculating the seed liquid to a fermentation medium for propagation culture to obtain a fermentation liquid; adding the spore-forming inducer into the fermentation liquor, and continuously culturing until fermentation is completed; preferably, the addition amount of the sporulation inducer accounts for 8-10% of the volume ratio of the fermentation liquor.
In an alternative embodiment, the clostridium butyricum is inoculated into a seed culture medium and is subjected to static culture at 36-38 ℃ for 25-35 hours when the seed solution is prepared;
preferably, the inoculation amount of the clostridium butyricum accounts for 1-2% of the volume ratio of the seed culture medium;
preferably, the seed culture medium is RCM liquid culture medium.
In an alternative embodiment, when preparing the fermentation liquid, the seed liquid is inoculated into a fermentation medium and is subjected to static culture at 36-38 ℃ for 16-18 hours;
preferably, the composition of the fermentation medium comprises: 8-10g/L of glucose, 2-3g/L of yeast powder, 8-10g/L of peptone, 6-8g/L of corn flour, 8-10g/L of beef extract powder, 2-3g/L of sodium acetate, 1.8-2g/L of dipotassium hydrogen phosphate, 1.8-2g/L of ferrous sulfate, 4-5g/L of sodium alginate and 0.6-1g/L of agar;
preferably, the inoculation amount of the seed liquid accounts for 0.2-0.3% of the volume ratio of the fermentation medium;
preferably, the parameters when inoculating the seed liquid include: the temperature is 36-38 ℃, the rotating speed is 40-50rpm, and the ventilation is not carried out;
preferably, after the seed liquid is inoculated, the pH value of the fermentation medium is adjusted to be more than or equal to 6.5.
In a second aspect, the present invention provides a clostridium butyricum prepared by the method for promoting the formation of spores of clostridium butyricum according to any one of the preceding embodiments;
preferably, the spore rate of the clostridium butyricum is 97.33% -98.94%.
In a third aspect, the present invention provides a probiotic comprising clostridium butyricum according to the preceding embodiments.
In a fourth aspect, the present invention provides the use of a probiotic according to the preceding embodiments for the preparation of a feed additive.
The invention has the following beneficial effects:
according to the method for promoting the clostridium butyricum to form spores, the spore-forming inducer containing the inactivated clostridium butyricum bacterial mud is added in the fermentation process to promote all the spores to be quickly formed, a large amount of decomposed and dead bacteria can effectively prevent the bacteria which form the spores firstly release substances such as spore-forming delayed protein and the like to inhibit other bacteria from forming spores due to too long spore forming process, so that the live bacteria amount and the spore rate of the clostridium butyricum are effectively improved, the problem that the high bacteria amount and the high spore rate of the clostridium butyricum are difficult to coexist is solved, and the storage, the transportation and the use of the clostridium butyricum are greatly promoted; in addition, the microecological preparation prepared by the invention has obvious effect when being applied to piglet feeding, can effectively improve feed intake, promote digestion, reduce diarrhea, and replace antibiotics, and has better effect compared with the clostridium butyricum microbial inoculum prepared by conventional fermentation.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a method for promoting clostridium butyricum to form spores, which comprises the following steps:
s1, preparing seed liquid.
And inoculating clostridium butyricum into a seed culture medium for standing culture to obtain a seed solution.
Specifically, Clostridium butyricum was inoculated into a 1L conventional flask containing 800ml of liquid at a volume ratio of 1 to 2%, and incubated at 36 to 38 ℃ for 25 to 35 hours.
The seed culture medium in the shake flask can be RCM liquid culture medium, which is also called reinforced clostridial culture medium, and is used for enrichment culture and counting of clostridia. The specific components thereof can be referred to the prior art and are not specifically described in the application.
S2, expanding culture in a fermentation tank.
And carrying out expanding culture on the seed liquid to obtain fermentation liquid.
Specifically, the seed liquid is inoculated into a fermentation medium according to the volume ratio of 0.2-0.3%, and is subjected to static culture for 16-18 hours at the temperature of 36-38 ℃; when the seed liquid is inoculated, the temperature is controlled at 36-38 ℃, the rotating speed is controlled at 40-50rpm, and ventilation is not carried out. After the inoculation of the seed liquid is finished, the pH value of the fermentation medium is adjusted to be more than or equal to 6.5 by 10 percent sodium hydroxide solution, the pH value of the fermentation medium is continuously controlled to be more than or equal to 6.5 in the whole culture expanding and fermenting process, the temperature is controlled to be 36-38 ℃ in the fermenting process, the rotating speed is controlled to be 40-50rpm, and ventilation is not carried out.
The application is improved to clostridium butyricum's fermentation medium, and the composition of fermentation medium includes in this application: 8-10g/L of glucose, 2-3g/L of yeast powder, 8-10g/L of peptone, 6-8g/L of corn flour, 8-10g/L of beef extract powder, 2-3g/L of sodium acetate, 1.8-2g/L of dipotassium hydrogen phosphate, 1.8-2g/L of ferrous sulfate, 4-5g/L of sodium alginate and 0.6-1g/L of agar.
The sodium alginate is a natural polysaccharide, is usually used as a thickening agent in beverages and dairy products and has strong viscosity, the agar is also a natural polysaccharide and is also a thickening agent and a coagulating agent which are widely used in the food industry, the sodium alginate is usually used for preparing a solid culture medium in microbial culture, and the agar and the solid culture medium can both play a role in improving the viscosity of the culture medium. Because the culture mode of the invention only uses conventional equipment and adopts a liquid submerged culture mode to meet the requirement of the anaerobic property of the clostridium butyricum, the viscosity of the culture medium can be improved by adding sodium alginate and agar, and the culture medium can further play a role in blocking oxygen and reducing bottom dissolved oxygen. The corn flour and the beef extract powder are used as a delayed carbon source and a nitrogen source, are continuously subjected to enzymolysis in the fermentation process to provide for the growth of the thalli, are poor in solubility, provide attachments for the thalli, and can block partial oxygen. Ferrous sulfate is used as a reducing agent, so that the oxidation-reduction potential of the culture medium can be reduced, and the requirement of clostridium butyricum on dissolved oxygen is reduced.
This application improves through the component to the culture medium, can effectively promote the live bacteria count and the spore number of clostridium butyricum after the fermentation finishes.
And S3, promoting sporulation.
Adding a spore-forming inducer to promote the formation of spores of clostridium butyricum in the fermentation culture process, wherein the adding amount of the spore-forming inducer accounts for 8-10% of the volume ratio of the fermentation liquor, adjusting the pH value of a system to 3.5-4.0 after adding the spore-forming inducer, adjusting the temperature to 20-22 ℃, and continuously culturing for 8-9 hours to complete fermentation.
The components of the spore-forming inducer comprise inactivated clostridium butyricum bacterial mud, wherein the inactivated clostridium butyricum bacterial mud is obtained by inoculating clostridium butyricum into an induction culture medium for culture, inactivating and then carrying out solid-liquid separation.
Specifically, the preparation method of the inactivated clostridium butyricum bacterial sludge comprises the following steps: inoculating clostridium butyricum to an induction culture medium at the volume ratio of 1-2%, and standing and culturing at 36-38 ℃ for 16-18 hours; placing in boiling water bath for 20-40min, and performing solid-liquid separation after detecting no viable bacteria. There are various ways of solid-liquid separation, including but not limited to centrifugation or filtration; in the application, the solid-liquid separation is preferably carried out by adopting a centrifugal mode, the centrifugation is carried out for 8-12min at 8000-12000rpm, and the supernatant is removed to obtain the inactivated clostridium butyricum bacterial sludge.
As researches show that only part of the thalli can form spores when the bacillus colony forms the spores in the severe environment in nature, and the other part of the thalli can be disintegrated and die, substances such as spore production delay protein, spore production killing factors and the like can be released in the spore formation process of the bacillus, so that the formation of spores of other thalli is inhibited, the thalli are disintegrated and die, and the disintegrated and dead thalli can release similar spore production promoting factors to promote the formation of spores of other thalli and prevent the death of other thalli.
In the application, by taking the inactivated clostridium butyricum bacterial mud as a spore production inducer, the clostridium butyricum is inactivated after being cultured, a large amount of bacteria which are decomposed and dead can be obtained, and the quick formation of all spores is promoted by adding a large amount of bacteria which are decomposed and dead, so that the situation that the spores are partially formed at first and release substances such as spore production delay protein to inhibit other bacteria from forming spores due to the overlong spore forming process is prevented.
In addition, the components of the spore formation inducer of the present application further include 2, 6-pyridinedicarboxylic acid and calcium carbonate; due to the presence of a large amount of calcium 2, 6-pyridinedicarboxylate in the structure of clostridium butyricum spores, the present application may further accelerate the sporulation process by adding the components that make up the spores. Specifically, the spore-forming inducer comprises, by concentration, 4-5g/L of inactivated clostridium butyricum bacterial sludge, 8-10g/L of 2, 6-dipicolinic acid and 15-18g/L of calcium carbonate, and the preparation method comprises the step of directly adding the inactivated clostridium butyricum bacterial sludge, 8-10g/L of 2, 6-dipicolinic acid and calcium carbonate into a solvent to prepare the required concentration, wherein the solvent can comprise water.
In the application, the spore-forming inducer is added in the middle stage of fermentation culture; if the addition time is too late, the spore formation process of the clostridium butyricum begins, and spore production delay protein and other substances inhibiting the formation of spores are generated, so that part of thalli can not form spores finally, and the spore rate is low; if the adding time is too long, the thalli are still in the expanding culture stage, the germ amount is low due to the fact that the thalli are promoted to form spores in advance, and the formation of the spores is inhibited due to the fact that the carbon and nitrogen source still remains a lot, so that the spore rate is low.
In addition, the application also provides clostridium butyricum which is prepared by the method for promoting the clostridium butyricum to form spores; the spore rate is as high as 97.33% -98.94%.
Further, the application also provides a microecological preparation which comprises the clostridium butyricum. The microecological preparation can be widely applied to preparation of feed additives, can effectively improve feed intake, promote digestion, reduce diarrhea, and replace antibiotics, and has better effect compared with clostridium butyricum microbial inoculum obtained by conventional fermentation.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for promoting the formation of spores of clostridium butyricum, which comprises the following steps:
s1, preparing seed liquid.
And inoculating clostridium butyricum into a 1L conventional shake flask with the liquid containing 800ml of clostridium butyricum according to the volume ratio of 1%, and performing static culture at 36-38 ℃ for 30 hours to obtain clostridium butyricum seed liquid. The seed culture medium in the shake flask is RCM liquid culture medium.
S2, expanding culture in a fermentation tank.
Inoculating the seed solution into fermentation culture medium at volume ratio of 0.3%, wherein the temperature is controlled at 36-38 deg.C, the rotation speed is controlled at 50rpm, and aeration is not required. After the inoculation of the seed liquid is finished, the pH value of the fermentation medium is adjusted to be more than or equal to 6.5 by 10 percent sodium hydroxide solution, the pH value of the fermentation medium is continuously controlled to be more than or equal to 6.5 in the whole culture expanding and fermenting process, the temperature is controlled to be 36-38 ℃ in the fermenting process, the rotating speed is controlled to be 40-50rpm, ventilation is not carried out, and standing culture is carried out for 18 hours.
Wherein, the fermentation medium in the fermentation tank comprises the following components: 10g/L of glucose, 2g/L of yeast powder, 10g/L of peptone, 6g/L of corn flour, 10g/L of beef extract powder, 2g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate, 2g/L of ferrous sulfate, 5g/L of sodium alginate and 1g/L of agar.
And S3, promoting sporulation.
Adding 10% spore-forming inducer into the culture-expanding fermentation liquor, adjusting pH to 3.5-4.0, adjusting temperature to 20-22 deg.C, and culturing for 8-9 hr to complete fermentation.
Wherein, the spore-forming inducer is inactivated clostridium butyricum bacterial mud 5g/L, 2, 6-dipicolinic acid 10g/L and calcium carbonate 15 g/L. The preparation method of the inactivated clostridium butyricum bacterial sludge comprises the following steps: inoculating clostridium butyricum to an induction medium according to the volume ratio of 1%, and standing and culturing for 16 hours at 36-38 ℃; and (3) placing the mixture in a boiling water bath for 30min, centrifuging the mixture for 8-12min at 8000-12000rpm after detecting that no viable bacteria exist, and removing supernatant to obtain the inactivated clostridium butyricum bacterial sludge.
Example 2
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the fermentation medium in the fermentor in step S2 consists of: 8g/L of glucose, 3g/L of yeast powder, 8g/L of peptone, 8g/L of corn flour, 8g/L of beef extract powder, 3g/L of sodium acetate, 1.8g/L of dipotassium hydrogen phosphate, 1.8g/L of ferrous sulfate, 4g/L of sodium alginate and 0.6g/L of agar.
Example 3
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the adding proportion of the spore-forming inducer in the step S3 is as follows: 8 percent.
Example 4
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the sporulation inducer in step S3 is: 4g/L of inactivated clostridium butyricum bacterial mud, 8g/L of 2, 6-dipicolinic acid and 18g/L of calcium carbonate.
Example 5
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the inoculation ratio in step S1 was: 2 percent;
the inoculation ratio in step S2 was: 0.2 percent.
Example 6
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
in step S2, the fermentation broth is obtained after 16 hours of culture.
Comparative example 1
This comparative example is identical to example 1 in other step parameters, except that:
the fermentation medium in the fermentor in step S2 consists of: 10g/L of glucose, 2g/L of yeast powder, 10g/L of peptone, 6g/L of corn flour, 10g/L of beef extract powder, 2g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate and 2g/L of ferrous sulfate.
Comparative example 2
This comparative example is identical to example 1 in other step parameters, except that:
the fermentation medium in the fermentor in step S2 consists of: 10g/L of glucose, 2g/L of yeast powder, 10g/L of peptone, 6g/L of corn flour, 10g/L of beef extract powder, 2g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate, 5g/L of sodium alginate and 1g/L of agar.
Comparative example 3
This comparative example is identical to example 1 in other step parameters, except that:
in step S3, when the spore formation inducer is added, the pH and temperature are not adjusted.
Comparative example 4
This comparative example is identical to example 1 in other step parameters, except that:
the sporulation inducer in step S3 is: the inactivated clostridium butyricum bacterial sludge is 5 g/L.
Comparative example 5
This comparative example is identical to example 1 in other step parameters, except that:
the sporulation inducer in step S3 is: 10g/L of 2, 6-dipicolinic acid and 15g/L of calcium carbonate.
Comparative example 6
This comparative example is identical to example 1 in other step parameters, except that:
in step S3, no sporulation inducer is added.
Comparative example 7
This comparative example is identical to example 1 in other step parameters, except that:
in step S2, the fermentation broth is obtained after 12 hours of stationary culture.
Comparative example 8
This comparative example is identical to example 1 in other step parameters, except that:
in step S2, the fermentation broth is obtained after static culture for 22 hours.
Comparative example 9
This comparative example is identical to example 1 in other step parameters, except that:
in step S3, the spore formation inducer is added to the expanding culture broth in a volume ratio of 1%.
Comparative example 10
This comparative example is identical to example 1 in other step parameters, except that:
the inactivated clostridium butyricum bacterial sludge in the embodiment 1 is replaced by the inactivated clostridium butyricum bacterial sludge, and specifically, the preparation method of the inactivated clostridium butyricum bacterial sludge comprises the following steps: inoculating clostridium butyricum to an induction medium according to the volume ratio of 1%, and standing and culturing for 16 hours at 36-38 ℃; microscopic examination, when the spore formation rate is higher than 90%, centrifuging at 8000-12000rpm for 8-12min, and removing the supernatant to obtain Clostridium butyricum bacterial mud.
Comparative example 11
In the comparative example, the bacillus coagulans spore formation promoting method and the microecological preparation and the applied experimental method are adjusted to obtain the following components by referring to CN 202010780879.1:
s1, preparing seed liquid.
Inoculating clostridium butyricum into a 1L conventional shake flask with the liquid loading capacity of 800ml according to the volume ratio of 1%, standing and culturing for 30 hours at the temperature of 36-38 ℃, wherein the spore formation rate of the clostridium butyricum by staining microscopy is more than 90%, then carrying out water bath treatment at the temperature of 80-90 ℃ for 30-35min, and inactivating the thallus which does not form spores to obtain clostridium butyricum seed liquid. The seed culture medium in the shake flask is RCM liquid culture medium.
S2, and performing extended culture in a fermentation tank (the same as in example 1).
And S3, promoting sporulation.
Adding 10% spore-forming inducer into the culture-expanding fermentation liquor, adjusting pH to 3.5-4.0, adjusting temperature to 20-22 deg.C, and culturing for 8-9 hr to complete fermentation.
Wherein, the spore formation inducer is the spore formation inducer in the comparative example 9, namely 5g/L of inactivated clostridium butyricum bacterial mud, 10g/L of 2, 6-dipicolinic acid and 15g/L of calcium carbonate.
Experimental example I viable bacteria count
The live bacteria of clostridium butyricum prepared by fermentation in examples 1-6 and comparative examples 1-11 were counted, and the specific results are shown in table 1.
Wherein the spore number is the viable count after water bath for 10 minutes at 90 ℃, and the spore rate is the spore number/viable count 100%.
TABLE 1 viable cell count and spore count at the end of fermentation
As can be seen from the results in Table 1, the fermentation results were not greatly changed in the ranges of the recipes and the process variations in examples 2 to 6. As can be seen from the results of comparative examples 1-2 in Table 1, the amount of bacteria at the end of fermentation is greatly reduced when no sodium alginate or agar is added to the fermentation medium as a thickening agent or when no ferrous sulfate is added to the fermentation medium as a reducing agent; from the results of comparative example 3, it can be seen that the addition of the spore-forming inducer, without adjusting the pH and temperature, reduces the spore rate at the end of fermentation; from the results of comparative examples 4-5, it can be seen that the absence of 2, 6-dipicolinic acid and calcium carbonate in the spore production inducer, or the absence of inactivated clostridium butyricum bacterial sludge can greatly reduce the spore rate at the end of fermentation, especially the absence of inactivated clostridium butyricum bacterial sludge has a great influence on the spore rate and can also cause low bacterial yield due to death caused by no spore formation of part of the bacterial cells; it can be seen from the difference between the comparative example 6 and the comparative examples 4 to 5 and the example 1 that when only 2, 6-dipicolinic acid and calcium carbonate or only inactivated clostridium butyricum bacterial sludge is contained in the spore production inducer, the spore rate improving effect is far lower than that of the complete spore inducer, because the components in the spore production inducer can play a synergistic role in inducing clostridium butyricum to form spores so as to greatly promote the clostridium butyricum to form spores; as can be seen from the results of comparative example 7, when the spore inducer was added in advance, the amount of bacteria at the end of fermentation was reduced and the spore rate was also affected; as can be seen from the results of comparative example 8, delaying the addition of the spore inducer resulted in a large decrease in spore rate. As can be seen from the results of comparative example 9, decreasing the amount of spore formation inducer added results in a decrease in the spore rate at the end of fermentation; from the results of comparative example 10, it can be seen that when the clostridium butyricum added in the spore production inducer is not inactivated, the spore rate can be greatly reduced due to the addition of a large amount of clostridium butyricum thallus which is about to form spores; as can be seen from the results of comparative example 11, the spore rate was low when Clostridium butyricum spores were added as an inducer.
Experimental example II preparation of Clostridium butyricum inoculum
(1) Conventional fermentation group
Selecting clostridium butyricum bacterial liquid which is expanded and cultured by a fermentation tank, and diluting the expanded and cultured bacterial liquid with water until the bacterial quantity is 1.0 multiplied by 108And diluting the mixture by CFU/mL, adding a protective agent, uniformly mixing, subpackaging and sealing to obtain the clostridium butyricum inoculum of the conventional fermentation group.
Wherein the fermentation medium is RCM medium.
Wherein the fermentation process parameters are as follows: the inoculation amount of the seed liquid is 0.3%, the temperature is controlled to be 36-38 ℃, the rotating speed is 50rpm, aeration is not carried out, the pH value of the culture medium is regulated and controlled to be not lower than 6.5 by 10% of sodium hydroxide solution in the fermentation process, and the culture time is 24-28 hours.
The protective agent comprises the following components:
1 to 1.2 percent of sodium chloride, 0.1 to 0.2 percent of xanthan gum, 0.056 to 0.06 percent of dipotassium phosphate and 0.004 to 0.005 percent of monopotassium phosphate.
(2) Experimental fermentation group
The fermented bacterial liquid of example 1 was diluted with water to a bacterial count of 1.0X 108And diluting the mixture by CFU/mL, adding a protective agent, uniformly mixing, subpackaging and sealing to obtain the clostridium butyricum microbial inoculum of the experimental fermentation group. Wherein the protectant components are the same as those in conventional fermentation group.
(3) Experiment of feeding
Mixing 0.2% of aureomycin and 0.025% of amoxicillin with conventional feed, and using as antibiotic group; mixing 1% of a clostridium butyricum bacterial agent of a conventional fermentation group with a conventional feed and using the mixture as a conventional bacterial agent group; the clostridium butyricum microbial inoculum of 1 percent of experimental fermentation group is mixed with conventional feed and then used as an experimental microbial inoculum group.
Taking 120 healthy weaned piglets, randomly dividing into 4 groups, feeding the 3 groups of mixed feed respectively, feeding the other group of conventional feed, counting the initial weight of each group of piglets on day 1, measuring the weight and feed intake of the piglets for 1 time every day during the experiment period, and counting the final weight, feed-meat ratio, total number of diarrhea piglets and death number of each group of piglets during the experiment period on day 30. And (3) processing and calculating according to the data to obtain piglet production performance experimental results shown in the table 2.
Wherein, the calculation formula of the piglet diarrhea rate is as follows:
TABLE 2 piglet production performance test results of each treatment group
As can be seen from table 2, compared with the blank group, the daily gain and daily feed intake of the conventional clostridium butyricum fermented inoculum group are improved, the feed conversion ratio, diarrhea rate and mortality rate are reduced, the effect is close to that of the antibiotic group and is slightly better than that of the antibiotic group, the daily gain and daily feed intake of the experimental clostridium butyricum fermented inoculum group are significantly higher than those of the antibiotic group hosting the conventional clostridium butyricum fermented inoculum group, and the feed conversion ratio and diarrhea rate are also lower than those of the antibiotic group and the conventional clostridium butyricum fermented inoculum group.
In summary, the spore-forming inducer is added in the middle stage of fermentation, the process parameters are adjusted, and the fermentation medium is improved, so that the amount of the clostridium butyricum viable bacteria obtained by fermentation can reach 1.44 multiplied by 1010-1.70×1010CFU/mL, the spore rate can reach 97.33% -98.94%, has promoted the storage, transportation and use of clostridium butyricum greatly; in addition, the clostridium butyricum microbial inoculum prepared by the invention has obvious effect when being applied to piglet feeding, can effectively improve feed intake, promote digestion, reduce diarrhea, and replace antibiotics, and has better effect compared with the clostridium butyricum microbial inoculum prepared by conventional fermentation.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of promoting sporulation of clostridium butyricum, comprising: adding a spore-forming inducer in the fermentation culture process, wherein the components of the spore-forming inducer comprise inactivated clostridium butyricum bacterial sludge, and the inactivated clostridium butyricum bacterial sludge is obtained by inoculating clostridium butyricum into an induction culture medium for culture, inactivating and then carrying out solid-liquid separation.
2. The method of claim 1, wherein the spore forming inducer further comprises 2, 6-pyridinedicarboxylic acid and calcium carbonate;
preferably, the components of the spore-forming inducer comprise 4-5g/L of inactivated clostridium butyricum bacterial mud, 8-10g/L of 2, 6-dipicolinic acid and 15-18g/L of calcium carbonate according to concentration.
3. The method for promoting the sporulation of clostridium butyricum according to claim 1, wherein the inoculating clostridium butyricum into an induction medium for culture comprises: inoculating the clostridium butyricum to the induction medium at a volume ratio of 1-2%, and performing static culture at 36-38 ℃ for 16-18 hours;
preferably, the inactivation comprises that the induction culture after the culture is finished is carried out for 20-40min in a boiling water bath, and no viable bacteria is detected;
preferably, the solid-liquid separation comprises centrifugation or filtration;
preferably, the inactivated Clostridium butyricum bacterial sludge is obtained by centrifugation at 8000-12000rpm for 8-12min and supernatant is removed.
4. The method of claim 1, wherein the spore-forming inducer is added during the middle of the fermentation culture;
preferably, after the spore-forming inducer is added, the pH value of the system is adjusted to 3.5-4.0, the temperature is adjusted to 20-22 ℃, and the fermentation is finished after the culture is continued for 8-9 hours.
5. The method of claim 1, wherein the adding of the spore-forming inducer during the fermentation culture comprises: inoculating clostridium butyricum into a seed culture medium for standing culture to obtain a seed solution; inoculating the seed liquid to a fermentation medium for propagation culture to obtain a fermentation liquid; adding the spore-forming inducer into the fermentation liquor, and continuously culturing until fermentation is completed;
preferably, the addition amount of the sporulation inducer accounts for 8-10% of the volume ratio of the fermentation liquor.
6. The method for promoting the formation of spores of Clostridium butyricum of claim 5, wherein the Clostridium butyricum is inoculated into a seed culture medium and cultured at 36-38 ℃ for 25-35 hours;
preferably, the inoculation amount of the clostridium butyricum accounts for 1-2% of the volume ratio of the seed culture medium;
preferably, the seed culture medium is RCM liquid culture medium.
7. The method for promoting the formation of spores of clostridium butyricum of claim 5, wherein the seed solution is inoculated into the fermentation medium and subjected to static culture at 36-38 ℃ for 16-18 hours when the fermentation broth is prepared;
preferably, the composition of the fermentation medium comprises: 8-10g/L of glucose, 2-3g/L of yeast powder, 8-10g/L of peptone, 6-8g/L of corn flour, 8-10g/L of beef extract powder, 2-3g/L of sodium acetate, 1.8-2g/L of dipotassium hydrogen phosphate, 1.8-2g/L of ferrous sulfate, 4-5g/L of sodium alginate and 0.6-1g/L of agar;
preferably, the inoculation amount of the seed liquid accounts for 0.2-0.3% of the volume ratio of the fermentation medium;
preferably, the parameters when inoculating the seed liquid include: the temperature is 36-38 ℃, the rotating speed is 40-50rpm, and the ventilation is not carried out;
preferably, after the seed liquid is inoculated, the pH value of the fermentation medium is adjusted to be more than or equal to 6.5.
8. A clostridium butyricum prepared by the method for promoting the formation of spores of clostridium butyricum according to any one of claims 1 to 7;
preferably, the spore rate of the clostridium butyricum is 97.33% -98.94%.
9. A microecological formulation comprising the clostridium butyricum of claim 8.
10. Use of the probiotic of claim 9 for the preparation of a feed additive.
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| CN102220269A (en) * | 2011-05-16 | 2011-10-19 | 天津科技大学 | Clostridium butyricum and production method of clostridium butyricum feed additive |
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