CN110804564B - Preparation of clostridium butyricum powder and detection culture medium thereof - Google Patents
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Abstract
The invention belongs to the technical field of microbial detection and culture fermentation, and particularly relates to preparation of clostridium butyricum powder and a detection culture medium thereof. The clostridium butyricum powder is prepared by adopting the fermentation medium, the property of the fermentation liquid is stable, the thallus state is stable, the fermentation cost is obviously reduced by 50-60% compared with the common medium, the total production cost of the clostridium butyricum powder is reduced by 20-30%, and the viable bacteria concentration of clostridium butyricum in unit volume can be obviously improved. The method takes the self-made peptone as the carbon-nitrogen source, the viable bacteria content is accurately detected, the result is stable, the cost is low, the detection time is short, and the detection of one sample can be completed only within 10-15 min.
Description
Technical Field
The invention belongs to the technical field of microbial detection and culture fermentation, and particularly relates to preparation of clostridium butyricum bacterial powder and a detection culture medium thereof.
Background
Clostridium butyricum, also known as Gongrong bacterium and Clostridium butyricum, belongs to the genus Clostridium in bacteriological classification, and is an anaerobic heterotrophic gram-positive bacillus capable of producing butyric acid, lactic acid and acetic acid. At present, a TSC culture medium, a TSN culture medium or other self-made culture media are mainly used as a culture medium for detecting clostridium butyricum, and all related enterprises use enterprise standards which are self-designed by enterprises, and from the culture mode, a large amount of composite culture media or other culture media containing insoluble components are used, so that the post-treatment is difficult, and the cost is increased; from the perspective of culture effect, the method has the defects of low detection efficiency, poor accuracy, small universality and the like. Finally, the product is difficult to judge whether the product is true or false, and the product, namely the fish dragon, is mixed in the market, so that the popularization and the application of the clostridium butyricum are seriously influenced. In addition, a large amount of carbon and nitrogen sources are often used in the preparation process of the clostridium butyricum powder, if partial substitution is realized, the cost is greatly saved, and great benefits are generated for the popularization of the clostridium butyricum powder. Therefore, in order to reduce the detection cost, improve the detection efficiency and guarantee the rights and interests of consumers and the product quality, it is important to find a simple, rapid and effective method for detecting the number of the viable bacteria of the clostridium butyricum.
The Chinese patent application CN109022317A discloses a preparation method of clostridium butyricum bacterial powder, which is prepared by pretreating fermentation liquor, then adding a protective agent and finally performing spray drying on the fermentation liquor.
In conclusion, the existing methods for preparing clostridium butyricum bacterial powder generally have the defects of high cost, low accuracy and small universality, and the existing detection methods also generally have the problem of low detection efficiency.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a preparation method of clostridium butyricum bacterial powder and a detection culture medium thereof. The preparation method of the clostridium butyricum bacterial powder provided by the invention is used for preparing the key nitrogen source and carbon source in the fermentation process, so that the fermentation cost is greatly reduced, and the preparation of the high-content bacterial powder is realized.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of clostridium butyricum powder comprises the following steps:
s1, taking clostridium butyricum strains, inoculating the clostridium butyricum strains in a primary seed culture medium of a clostridium butyricum triangular flask, and standing in a incubator for anaerobic culture for 16 hours at 37 ℃ to obtain primary seed fermentation liquor; the seed culture medium comprises the following components in percentage by weight: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of ferrous chloride, 0.01 percent of calcium thioglycolate, 97.695 percent of water and 6.5 percent of pH value;
s2, secondary seed culture: inoculating the primary seed fermentation liquor obtained in the step S1 according to the volume of 3% of the secondary culture liquor, and standing in a incubator for anaerobic culture for 16h at 37 ℃ to obtain secondary seed fermentation liquor; the secondary culture solution comprises the following components in percentage by weight: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of chlorite, 0.01 percent of calcium thioglycolate, 97.695 percent of water and pH 6.5;
s3, three-stage seed culture: inoculating the secondary seed fermentation liquor obtained in the step S2 according to 3% of the volume of the tertiary culture liquor, and performing anaerobic culture in a fermentation tank at 37 ℃ for 24 hours to obtain tertiary seed liquor;
s4, fermentation culture: fermenting and inoculating the tertiary seed liquid obtained in the step S3 according to the volume of the fermentation liquid of 2%, carrying out anaerobic culture in a fermentation tank for 24h at 37 ℃, and finishing fermentation by using sodium hydroxide with the pH of 6.5 in the fermentation process until more than 98% of spores are formed to obtain primary thalli;
s5, cell concentration: heating the primary thalli obtained in the step S4 to 60 ℃, adjusting the pH value to 6.0 by using citric acid, naturally settling and concentrating for 4 hours to 60%, removing the supernatant, centrifuging by using a tubular or disc centrifuge at 8000rpm, and continuously collecting thalli to obtain bacterial sludge;
s6, adding a protective agent: and (4) mixing the bacterial sludge obtained in the step S5 according to the ratio of bacterial sludge: protective agent 1:8, mixing to obtain bacterial sludge containing a protective agent;
s7, spray drying to prepare powder: and (5) spray drying the bacterial sludge containing the protective agent obtained in the step (S6) at the air inlet temperature of 180 ℃ to obtain the protective agent.
Preferably, the culture medium in step S3 includes the following components and their weight percentages: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of ferrous chloride, 0.01 percent of calcium thioglycolate and 97.695 percent of water.
Preferably, the fermentation liquid in step S4 includes the following components and their weight percentages: 3 percent of soybean meal and 5 percent of corn flour, 0.05 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.05 percent of magnesium chloride, 0.05 percent of calcium chloride, 0.005 percent of ferrous chloride, 0.01 percent of calcium thioglycolate, 0.01 percent of sodium thioglycolate and 91.495 percent of water.
Preferably, the dipotassium phosphate, the calcium thioglycolate and the sodium thioglycolate in the fermentation liquor in the step S4 are prepared by adopting high pressure of 101 kPa; the bean pulp and corn flour are prepared by adopting a self-made compound enzyme for hydrolysis, and the specific process comprises the following steps: in 1 ton enzymolysis tank equipment, 100kg of soybean meal, 150kg of corn flour and 2 ton of water are fully stirred and uniformly mixed, then the mixture is heated to 85 ℃ for curing and fully dissolved, the temperature is reduced to 55 ℃, the pH value is 7.0, 5 ten thousand active unit neutral protease and 1.0 percent of 5 ten thousand active unit fungal alpha-amylase are added for maintaining for 4 hours, the pH value is adjusted to 7.5, 5 ten thousand active unit alkaline protease is added, and the temperature is maintained for 4 hours at 55 ℃.
Preferably, the protective agent in step S6 includes the following components and their weight percentages: 5% of maltodextrin, 5% of water-soluble starch, 0.1% of compound multivitamin and 89.9% of water.
The invention also provides a culture medium for detecting clostridium butyricum, wherein each liter of culture medium comprises the following components in parts by weight: 15 parts of self-made peptone, 5 parts of yeast powder, 10 parts of glucose, 3 parts of sodium thioglycolate, 0.5 part of L-cysteine salt, 2.5 parts of sodium chloride, 25 parts of agar and 0.001 part of resazurin.
Preferably, the preparation method of the homemade peptone comprises the following steps: 2.0 tons of soybean meal 100kg water are fully stirred and uniformly mixed, then the mixture is heated to 85 ℃ for curing and fully dissolved, the temperature is reduced to 55 ℃, the pH value is 7.0, 5 ten thousand active unit neutral protease is added for maintaining for 4 hours, the pH value is adjusted to 7.5, 5 ten thousand active unit alkaline protease is added for maintaining for 4 hours at the temperature of 55 ℃, and the enzymatic hydrolysate is injected into a spray dryer for spray drying to obtain the soybean meal.
The invention also provides a preparation method of the culture medium, which comprises the following operation processes: weighing the components according to the formula proportion, mixing, fully dissolving, adjusting the pH of the culture medium to 7.2 by using food-grade NaOH solution with the mass concentration of 1mol/L, and sterilizing at 121 ℃ for 20min to obtain the traditional Chinese medicine.
Compared with the prior art, the preparation and detection culture medium of the clostridium butyricum powder provided by the invention has the following advantages:
(1) the invention can improve the concentration of viable bacteria of clostridium butyricum per unit volume, and the concentration of clostridium butyricum fermented by using commercial peptone as a main nitrogen source is about 13 × 108cfu/mL, the enzymolysis soybean meal is used for replacing commercial peptone, and the concentration of the viable bacteria of the clostridium butyricum in the fermentation liquor reaches about 23 × 108cfu/mL, the sporulation rate is also obviously improved from about 80 percent to nearly 100 percent, the property of the fermentation liquor is stable, and the state of the thalli is stable;
(2) the soybean meal enzymolysis liquid is used for replacing a commercialized fine carbon nitrogen source, so that the cost of a fermentation culture medium is remarkably reduced by about 50-60%, and the total production cost of the bacterial powder is reduced by 20-30%;
(3) according to the invention, the clostridium butyricum is fermented by using the bean pulp enzymolysis liquid, the fermentation liquid is easier to be centrifugally treated than a commercial culture medium, and the viable bacteria yield of the bacterial powder obtained by spray drying is higher than that of the commercial culture medium;
(4) compared with the fermentation culture of a commercial culture medium, the self-made fermentation culture medium is adopted, and the number of liquid fermentation spores and the number of spray-dried bacteria powder are obviously increased.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 preparation method of Clostridium butyricum
The preparation method of the clostridium butyricum comprises the following steps:
s1, taking clostridium butyricum strains, inoculating the clostridium butyricum strains in a primary seed culture medium of a clostridium butyricum triangular flask, and standing in a incubator for anaerobic culture for 16 hours at 37 ℃ to obtain primary seed fermentation liquor; the seed culture medium comprises the following components in percentage by weight: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of ferrous chloride, 0.01 percent of calcium thioglycolate, 97.695 percent of water and 6.5 percent of pH value;
s2, secondary seed culture: inoculating the primary seed fermentation liquor obtained in the step S1 according to the volume of 3% of the secondary culture liquor, and standing in a incubator for anaerobic culture for 16h at 37 ℃ to obtain secondary seed fermentation liquor; the secondary culture solution comprises the following components in percentage by weight: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of chlorite, 0.01 percent of calcium thioglycolate, 97.695 percent of water and pH 6.5;
s3, three-stage seed culture: inoculating the secondary seed fermentation liquor obtained in the step S2 according to 3% of the volume of the tertiary culture liquor, and performing anaerobic culture in a fermentation tank at 37 ℃ for 24 hours to obtain tertiary seed liquor;
s4, fermentation culture: fermenting and inoculating the tertiary seed liquid obtained in the step S3 according to the volume of the fermentation liquid of 2%, carrying out anaerobic culture in a fermentation tank for 24h at 37 ℃, and finishing fermentation by using sodium hydroxide with the pH of 6.5 in the fermentation process until more than 98% of spores are formed to obtain primary thalli;
s5, cell concentration: heating the primary thalli obtained in the step S4 to 60 ℃, adjusting the pH value to 6.0 by using citric acid, naturally settling and concentrating for 4 hours to 60%, removing the supernatant, centrifuging at 8000rpm by using a tubular or disc centrifuge, and continuously collecting thalli to obtain bacterial sludge;
s6, adding a protective agent: and (4) mixing the bacterial sludge obtained in the step S5 according to the ratio of bacterial sludge: protective agent 1:8, mixing to obtain bacterial sludge containing a protective agent;
s7, spray drying to prepare powder: and (5) spray drying the bacterial sludge containing the protective agent obtained in the step (S6) at the air inlet temperature of 180 ℃ to obtain the protective agent.
EXAMPLE 2 method for hydrolyzing carbon and nitrogen sources in culture Medium
The preparation method of the enzymolysis soybean meal and the corn flour comprises the following steps: is prepared by hydrolyzing soybean meal with complex enzyme (alkaline protease and neutral protease in a mass ratio of 1: 1) and spray drying.
In 1 ton enzymolysis tank equipment, 100kg of soybean meal, 150kg of corn flour and 2 ton of water are fully stirred and uniformly mixed, then the mixture is heated to 85 ℃ for curing and fully dissolved, the temperature is reduced to 55 ℃, the pH value is 7.0, 5 ten thousand active unit neutral protease and 1.0 percent of 5 ten thousand active unit fungal alpha-amylase are added for maintaining for 4 hours, the pH value is adjusted to 7.5, 5 ten thousand active unit alkaline protease is added, and the temperature is maintained for 4 hours at 55 ℃.
Example 3 Medium component screening assay
(1) Determination of the optimal carbon Source
After pretreatment, selecting a self-prepared clostridium butyricum sample, respectively culturing by using glucose, starch, sucrose, maltose and lactose as carbon sources, carrying out anaerobic culture at 37 ℃ for 24h, and comparing counting results of culture media of the carbon sources.
The results show that the counting results were highest when glucose was used as the carbon source, so glucose was selected as the best carbon source.
(2) Determination of optimal Nitrogen sources
After pre-treating a self-prepared clostridium butyricum sample, respectively culturing by using self-made peptone, beef extract, urea and ammonium sulfate as nitrogen sources, wherein anaerobic culture is carried out at 37 ℃ for 24 hours, and counting results of each nitrogen source culture medium are compared.
The results show that the counting results are highest when the self-made peptone is used as a nitrogen source, so the self-made peptone is selected as the optimal nitrogen source.
Example 4A method for preparing a self-made peptone
2.0 tons of soybean meal 100kg water are fully stirred and uniformly mixed, then the mixture is heated to 85 ℃ for curing and fully dissolved, the temperature is reduced to 55 ℃, the pH value is 7.0, 5 ten thousand active unit neutral protease is added for maintaining for 4 hours, the pH value is adjusted to 7.5, 5 ten thousand active unit alkaline protease is added, the mixture is maintained for 4 hours at the temperature of 55 ℃, and the enzymatic hydrolysate is injected into a spray dryer for spray drying to obtain the soybean meal.
The invention also provides a preparation method of the culture medium, which comprises the following operation processes: weighing the components according to the formula proportion, mixing, fully dissolving, adjusting the pH of the culture medium to 7.2 by using food-grade NaOH solution with the mass concentration of 1mol/L, and sterilizing at 121 ℃ for 20min to obtain the traditional Chinese medicine.
Example 5 culture medium for rapidly detecting clostridium butyricum and preparation method thereof
The culture medium for rapidly detecting clostridium butyricum comprises the following components in percentage by weight: each liter of culture medium comprises the following components in parts by weight: 15 parts of self-made peptone, 5 parts of yeast powder, 10 parts of glucose, 3 parts of sodium thioglycolate, 0.5 part of L-cysteine salt, 2.5 parts of sodium chloride, 25 parts of agar and 0.001 part of resazurin.
The preparation process of the culture medium is as follows: weighing the components according to the formula proportion, mixing, fully dissolving, adjusting the pH of the culture medium to 7.2 by using food-grade NaOH solution with the mass concentration of 1mol/L, and sterilizing at 121 ℃ for 20min to obtain the traditional Chinese medicine.
Example 6 comparison of self-made Medium to commercial Medium
Different batches of clostridium butyricum samples produced by three biological engineering companies are respectively detected on three culture media. The results are shown in the following table.
The results show that: (1) the rapid detection culture medium disclosed by the invention is convenient to operate, uniform and full in colony morphology, easy to identify and has certain characteristics of clostridium butyricum. The method has the advantages of high detection viable bacteria amount, stable result and low cost. The detection time is short, and the detection of one sample can be completed within 10-15 min.
(2) TSC medium is expensive, especially the price of added serine. The culture medium needs to be covered by an upper layer culture medium, the upper layer culture medium and the lower layer culture medium are different, and colonies on the sandwich culture plate are easy to gather and influence the counting result. The operation is complicated and time-consuming. The dilution liquid has large oiliness and foam, the quantity is influenced when the dilution liquid is measured, and the device is difficult to clean after the experiment.
(3) The TSN culture medium is convenient to operate, but the viable bacteria content is low, and the cost is higher than that of the rapid detection culture medium disclosed by the invention.
Example 7 Rapid detection method of Clostridium butyricum
The clostridium butyricum detection method based on the culture medium comprises the following steps:
(1) sample preparation and dilution: aseptically weighing 10.00g of a sample to be detected, adding the sample into a 250mL triangular flask containing 90mL of sterile water and small glass beads, and fully oscillating the sample on a shaking table for 10min to prepare a product with the weight ratio of 1: 10 diluting liquid; then, using a 1mL pipette, pipette 1: and (3) transferring 1mL of 10 diluent into a test tube filled with 9mL of sterile water, and fully shaking and uniformly mixing to obtain a mixture 1: 100 dilution. According to the bacteria content of the sample, 10 times of gradually increased dilution is made according to the operation sequence.
(2) Inoculation and culture: selecting 2 suitable dilutions, wherein each gradient is 3 parallel, respectively adding 100 mu L of diluted sample dilution liquid to a Clostridium butyricum culture medium in a plate, uniformly coating, placing the coated plate in a biochemical incubator after solidification, inverting the plate, culturing at 37 ℃, and strictly performing anaerobic culture for 24 hours.
(3) And (3) counting bacterial colonies, namely taking out the cultured plates after culture, respectively counting the corresponding bacterial colonies according to morphological analysis, selecting effective data of the bacterial colonies of the plates between 30 and 300 for calculation, wherein the method is calculated according to GB/T13093 plus 2006, and finally measuring that the concentration of the viable bacteria of the clostridium butyricum reaches about 23 × 108cfu/mL。
Example 8 Low cost preparation of Clostridium butyricum powder
Take a 5 ton fermenter as an example:
firstly, a 500mL triangular flask containing 450mL of culture medium with pH 6.5 is taken and sterilized by autoclaving at 118 ℃. Cooling to 37 ℃, and taking the clostridium butyricum inoculated in an aseptic operation table. Standing in an incubator at 37 ℃ for 16h, sampling, and microscopic examining a small amount of fusiform spores with normal shape and no mixed bacteria. The formula of the culture medium is as follows: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of ferrous chloride and 0.01 percent of calcium thioglycolate.
Second-level seed culture: a5000 ml Erlenmeyer flask containing 4000ml of medium pH 6.5. Sterilizing at 118 deg.C under high pressure, cooling to 37 deg.C, adding the first-stage seed fermentation liquid in aseptic operation table, and standing and culturing at 37 deg.C for 16 h. The sampling microscopic examination shows that the sample has normal shape and a small amount of clostridia and has no mixed bacteria. The formula of the culture medium is as follows: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of ferrous chloride, 0.01 percent of calcium thioglycolate and pH 6.5.
③ culturing seeds: 1 ton fermentation tank containing 800L culture medium, sterilizing at 121 deg.C for 30min, controlling pH to 6.5 with alkali, introducing nitrogen, cooling to 37 deg.C, inoculating secondary seed into the fermentation tank by flame, and performing anaerobic culture for 24h with spore rate of more than 80. And introducing air for later use. The formula of the culture medium is as follows: 1.5 percent of soluble starch, 0.5 percent of yeast powder, 0.2 percent of dipotassium phosphate, 0.005 percent of ferrous chloride and 0.01 percent of calcium thioglycolate.
Fourthly, fermentation culture: 5 tons of fermentation tank containing 4 tons of fermentation liquid is sterilized at 121 ℃ for 30min, the pH is controlled by alkali to be 6.5, nitrogen is introduced to cool to 37 ℃, 80L of tertiary seed liquid is inoculated into the fermentation tank, and simultaneously, single-degree high-pressure inorganic salt is inoculated to sterilize at 121 ℃ for 30 min. Maintaining the pressure of nitrogen at 0.05MPa, rotating speed at 100r/min, and culturing for more than 24 h. The number of spores in the fermentation liquor reaches more than 23 hundred million/mL.
The formula of the culture medium is as follows: enzymolysis liquid containing 3 percent of soybean meal and 5 percent of corn flour, 0.05 percent of yeast powder, 0.2 percent of dipotassium hydrogen phosphate, 0.05 percent of magnesium chloride, 0.05 percent of calcium chloride, 0.005 percent of ferrous chloride, 0.01 percent of calcium thioglycolate and 0.01 percent of sodium thioglycolate.
Fifth, thallus concentration: heating to 60 deg.C, adjusting pH to 6.0 with citric acid, and naturally settling and concentrating for 4 hr to obtain 60%. The supernatant was removed and the cell was collected by a tube or disk centrifuge.
Sixthly, adding a protective agent: bacterial sludge according to bacterial sludge weight: the protective agent is added with 5% of maltodextrin, 0.1% of compound multivitamin and 90% of water according to the weight ratio of 1: 8.
And (c) spray drying to prepare powder: and (3) spray-drying the bacterial sludge with the protective agent, wherein the air inlet temperature is 180 ℃.
Detecting the spore number of the bacterial powder: per gram of fungal powder content 300 hundred million/g
Therefore, the content of the clostridium butyricum powder produced by the method is extremely high, and the average value is 364 × 108cfu/mL, which is much higher than the number of Clostridium butyricum produced in the prior art.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (2)
1. The culture medium for detecting clostridium butyricum is characterized by comprising the following components in parts by weight per liter: 15 parts of self-made peptone, 5 parts of yeast powder, 10 parts of glucose, 3 parts of sodium thioglycolate, 0.5 part of L-cysteine salt, 2.5 parts of sodium chloride, 25 parts of agar and 0.001 part of resazurin;
the preparation method of the self-made peptone comprises the following steps: 2.0 tons of soybean meal 100kg water are fully stirred and uniformly mixed, then the mixture is heated to 85 ℃ for curing and fully dissolved, the temperature is reduced to 55 ℃, the pH value is 7.0, 5 ten thousand active unit neutral protease is added for maintaining for 4 hours, the pH value is adjusted to 7.5, 5 ten thousand active unit alkaline protease is added for maintaining for 4 hours at the temperature of 55 ℃, and the enzymatic hydrolysate is injected into a spray dryer for spray drying to obtain the soybean meal.
2. A method for preparing the culture medium according to claim 1, which comprises the following steps: weighing the components according to the formula proportion, mixing, fully dissolving, adjusting the pH of the culture medium to 7.2 by using food-grade NaOH solution with the mass concentration of 1mol/L, and sterilizing at 121 ℃ for 20min to obtain the traditional Chinese medicine.
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| CN114410524A (en) * | 2022-01-20 | 2022-04-29 | 厦门昶科生物工程有限公司 | Industrial production method of clostridium butyricum powder |
| CN114672446B (en) * | 2022-05-31 | 2022-08-12 | 广东海洋大学 | Preparation method and application of clostridium butyricum preparation |
| CN114874949B (en) * | 2022-06-06 | 2023-09-12 | 江苏三仪生物工程有限公司 | Clostridium butyricum, fermentation product thereof, microbial inoculum containing clostridium butyricum and animal feed additive |
| CN115161242B (en) * | 2022-07-26 | 2023-06-20 | 宜宾五粮液股份有限公司 | Method for directional enrichment culture of clostridium microorganisms |
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