CN114774324A - Method for optimizing and separating probiotics - Google Patents
Method for optimizing and separating probiotics Download PDFInfo
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- CN114774324A CN114774324A CN202210504188.8A CN202210504188A CN114774324A CN 114774324 A CN114774324 A CN 114774324A CN 202210504188 A CN202210504188 A CN 202210504188A CN 114774324 A CN114774324 A CN 114774324A
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- 239000006041 probiotic Substances 0.000 title claims abstract description 19
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 36
- 238000000926 separation method Methods 0.000 claims abstract description 25
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- 238000012258 culturing Methods 0.000 claims abstract description 15
- 239000012470 diluted sample Substances 0.000 claims abstract description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 17
- 238000010790 dilution Methods 0.000 claims description 16
- 239000012895 dilution Substances 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 235000014655 lactic acid Nutrition 0.000 claims description 12
- 239000004310 lactic acid Substances 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 10
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 abstract description 9
- 241000186000 Bifidobacterium Species 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 241000193749 Bacillus coagulans Species 0.000 description 9
- 229940054340 bacillus coagulans Drugs 0.000 description 9
- 239000000203 mixture Substances 0.000 description 5
- 230000000529 probiotic effect Effects 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000002068 microbial inoculum Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention discloses an optimized separation method of probiotics, which is characterized by comprising the following steps: s1: preparing a diluted sample solution; s2: separating and culturing; culturing for 40-45 hours under anaerobic condition, then culturing for 6-8 hours under aerobic condition, S3: primary screening of strains; s4: and (4) re-screening the strains. The invention carries out aerobic culture treatment in the separation culture, effectively reduces the separation and purification difficulty of each strain on the form, and improves the viable count and the purification rate of the bifidobacterium. After the separation culture, two procedures of bacterial strain primary screening and bacterial strain secondary screening are carried out, the bacterial colony diameter/transparent ring diameter value is screened out in the primary screening process, then bacterial strain secondary screening work is carried out on the basis, and the secondary screening is carried out by carrying out step-by-step culture through an LB culture medium and a secondary screening culture medium so as to ensure the separation sufficiency and improve the purification effect of the average value.
Description
Technical Field
The invention relates to the technical field of probiotic separation, in particular to an optimal separation method for probiotics.
Background
With the improvement of living standard of people, high-quality health food is the pursuit and requirement of the public, and the food quality safety is concerned by people. The use of antibiotics is totally limited in China, which has great influence on the traditional livestock industry, and researches show that the use of antibiotics can be greatly reduced by using probiotic preparations to adjust the balance of animal intestinal flora. Probiotics are active microorganisms which are beneficial to a host and change the composition of flora at a certain part of the host by colonizing in a human body. The health of the intestinal tract is kept by promoting the absorption of nutrients by regulating the immune function of the host mucous membrane and the system or by regulating the balance of flora in the intestinal tract, so that single microorganisms or mixed microorganisms with definite compositions which are beneficial to the health are generated.
The strains currently used in probiotic preparations for feed are mainly: bacillus, saccharomycetes, lactic acid bacteria, actinomycetes, bifidobacteria and the like are processed into a feed additive for use, which is a mode with high economic benefit. Under the influence of processing techniques such as granulation and powder preparation of the microbial inoculum and storage conditions, the activity of the microbial inoculum is reduced to a certain extent, and the microbial inoculum is digested to a certain extent, so that the problem that probiotics can reach the intestinal tract alive is solved.
Bacillus coagulans (Bacillus coagulans) belongs to the genus Bacillus in taxonomy, can decompose saccharides to generate lactic acid, has the probiotic characteristics of common lactic acid bacteria, has the characteristics of strong stress resistance, gastric acid resistance, high temperature resistance and the like due to the capability of producing spores, and can be applied to the industries of medical treatment, food, livestock raising, aquatic products and the like. Before experimental analysis of bacillus coagulans, the bacillus coagulans needs to be separated and extracted, so that research on high-density fermentation of the bacillus coagulans is carried out, and reference is provided for application of the bacillus coagulans in industrial production of feed microbial agents, and therefore a method for separating and extracting the bacillus coagulans needs to be provided to realize separation and extraction of the bacillus coagulans.
Disclosure of Invention
The invention aims to provide an optimized probiotic separation method to realize separation and extraction of bacillus coagulans and solve the technical problems in the background technology.
The technical scheme of the invention provides an optimized separation method of probiotics, which comprises the following steps:
s1: preparing a diluted sample solution, weighing 20-25g of feed, and homogenizing in 200-250mL of sterile water to prepare a diluted sample solution with the volume ratio of 1: 10, and continuously carrying out tenfold gradient dilution, wherein the dilution gradient is 10-1To 10-9；
S2: separating and culturing, respectively taking a certain amount of dilution gradient as 10-4To 10-9The diluted sample solution is placed on a culture medium flat plate, and 2 to 3 parallel samples are made for the diluted sample solution of each dilution gradient; culturing for 40-45 hours under anaerobic condition, culturing for 6-8 hours under aerobic condition,
s3: primarily screening strains, measuring the diameter of a bacterial colony and the diameter of a transparent ring, and preserving strains of the strains with larger values of the bacterial colony diameter/the diameter of the transparent ring;
s4: re-screening the strains, respectively inoculating the strains to be detected into 50mL of LB culture medium, placing the strains in a shaking table at 45-50 ℃ for shaking culture at the speed of 120-150r/min for 20-22h, and preparing seed liquid; transferring the seed solution to a 100mL rescreening culture medium by an inoculation amount of 5-8%, placing the rescreening culture medium in a shaking table at 45-50 ℃ for shaking culture at 150r/min for 25-30h, and measuring the content of lactic acid in fermentation liquor to screen the strain with strong acid production.
In a preferred embodiment, the LB medium comprises the following parameters: 10-12g/L of peptone, 5-8g/L of yeast powder and 10-12g/L of sodium chloride, and the pH value is 7.0.
In a preferred embodiment, the parameters of the components of the screening plate medium are: 10-12g/L of peptone, 5-8g/L of yeast powder, 10-12g/L of sodium chloride, 20-25g/L of glucose, 20-23g/L of calcium carbonate and 20-25g/L of agar powder, and the pH value is 7.0.
In a preferred embodiment, the composition parameters of the rescreening medium are as follows: 10-12g/L of peptone, 5-8g/L of yeast powder, 10-12g/L of sodium chloride and 50-55g/L of glucose, and the pH value is 7.0.
In a preferred embodiment, the culture temperature of the LB culture medium is 36-38 ℃; the culture temperature of the screening plate culture medium is 35-38 ℃; the culture temperature of the rescreening culture medium is 35-37 ℃.
The technical scheme of the invention has the beneficial effects that:
1. the invention carries out aerobic culture treatment in separation culture, effectively reduces the separation and purification difficulty of each strain on the form, and improves the viable count and the purification rate of the bifidobacterium.
2. After separation and culture, two procedures of primary screening and secondary screening of the strains are carried out, the strains with larger colony diameter/transparent ring diameter value are screened out during primary screening, and then strain secondary screening work is carried out on the basis, wherein the secondary screening is carried out by carrying out step-by-step culture through an LB culture medium and a secondary screening culture medium, so that the separation sufficiency is ensured, and the purification effect of the average value is improved.
Drawings
FIG. 1 is a histogram of the ratio of colony diameter (H)/clearing zone diameter (C);
FIG. 2 is a bar graph of the determination of lactic acid production capacity for 5 generations of subculture and fermentation.
Detailed Description
The present invention will be described in further detail with reference to the drawings and the detailed description. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Example 1
The technical scheme of the invention provides an optimized separation method of probiotics, which comprises the following steps:
s1: preparing a diluted sample solution, weighing 20g of feed, homogenizing in 200mL of sterile water to prepare a mixture of 1: 10 ofSample solution is released, ten times of gradient dilution is carried out continuously, and the dilution gradient is 10-1To 10-9；
S2: separating and culturing, respectively taking a certain amount of dilution gradient as 10-4To 10-9The diluted sample solution is placed on a culture medium flat plate, and 2 to 3 parallel samples are made for the diluted sample solution of each dilution gradient; culturing under anaerobic condition for 40 hr, culturing under aerobic condition for 6 hr,
s3: primarily screening the strains, measuring the diameter of a bacterial colony and the diameter of a transparent ring, and preserving the strains with larger values of the diameter of the bacterial colony/the diameter of the transparent ring;
s4: re-screening the strains, respectively inoculating the strains to be detected into 50mL of LB culture medium, placing the strains in a shaking table at 45 ℃ for shaking culture at 120r/min for 20h, and preparing into seed liquid; transferring the seed solution into 100mL of a re-screening culture medium by using the inoculation amount of 5%, placing the re-screening culture medium in a shaking table at 45 ℃ for shaking culture for 25h at 120r/min, and measuring the lactic acid content in fermentation liquor to screen strains with strong acid yield.
In a preferred embodiment, the parameters of each component of the LB culture medium are as follows: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride, and the pH value is 7.0.
In a preferred embodiment, the parameters of the components of the screening plate medium are: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride, 20g/L of glucose, 20g/L of calcium carbonate and 20g/L of agar powder, and the pH value is 7.0.
In a preferred embodiment, the composition parameters of the rescreening medium are as follows: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 50g/L of glucose, and the pH value is 7.0.
In a preferred embodiment, the culture temperature of the LB medium is 36 ℃; the culture temperature of the screening plate culture medium is 35 ℃; the culture temperature of the rescreened culture medium is 35 ℃.
Example 2
The technical scheme of the invention provides an optimized separation method of probiotics, which comprises the following steps:
s1: preparing a diluted sample solution, weighing 25g of feed, and homogenizing in 250mL of sterile water to prepare a mixed solution of 1: 10, and continuously carrying out ten times of gradient dilution, wherein the dilution gradient is10-1To 10-9；
S2: separating and culturing, respectively taking a certain amount of dilution gradient as 10-4To 10-9The diluted sample solution is placed on a culture medium flat plate, and 2 to 3 parallel samples are made for the diluted sample solution of each dilution gradient; culturing for 45 hours under anaerobic condition, culturing for 8 hours under aerobic condition,
s3: primarily screening the strains, measuring the diameter of a bacterial colony and the diameter of a transparent ring, and preserving the strains with larger values of the diameter of the bacterial colony/the diameter of the transparent ring;
s4: re-screening the strains, respectively inoculating 50mL of LB culture medium to the strains to be detected, placing the strains in a shaking table at 50 ℃ for shaking culture at 150r/min for 22h, and preparing seed liquid; transferring the seed solution into a 100mL rescreening culture medium by 8 percent of inoculation amount, placing the rescreening culture medium in a shaking table at 50 ℃ for shaking culture for 30h at 150r/min, and measuring the content of lactic acid in fermentation liquor to screen the bacterial strain with strong acid production.
In a preferred embodiment, the parameters of each component of the LB culture medium are as follows: 10-12g/L of peptone, 8g/L of yeast powder and 12g/L of sodium chloride, and the pH value is 7.0.
In a preferred embodiment, the parameters of the components of the screening plate medium are: peptone 12g/L, yeast powder 8g/L, sodium chloride 12g/L, glucose 25g/L, calcium carbonate 23g/L, agar powder 25g/L, and pH 7.0.
In a preferred embodiment, the parameters of each component of the rescreening culture medium are as follows: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 50g/L of glucose, and the pH value is 7.0.
In a preferred embodiment, the cultivation temperature of the LB medium is 38 ℃; the culture temperature of the screening plate culture medium is 38 ℃; the culture temperature of the rescreened medium was 37 ℃.
With the embodiment as a detection target, the following detection works are carried out:
1. bacterial strain separation primary screen
The results of screening the strains having a larger colony diameter (H)/clearing circle diameter (C) by primary plate separation are shown in FIG. 1.
As can be seen from FIG. 1, 6 of the 15 strains with the larger H/C values were larger than 3.5, and the H/C values of the strains were less than 3.5, namely, 1 (3.6), 3 (3.8), 6 (3.9), 8 (4.2), 9 (3.7) and 11 (3.8), respectively.
2. Bacterial strain re-screening
In order to obtain a strain with good genetic stability and high lactic acid yield, 6 strains obtained are subjected to 5-generation subculture and fermentation lactic acid production performance measurement, and the result is shown in fig. 2.
As can be seen from FIG. 2, the lactic acid production of strain No. 8 was the highest (25.3g/L), and the lactic acid production of other strains was less than 25 g/L. Thus, strain No. 8 can be identified as the most preferred strain to isolate.
The invention carries out aerobic culture treatment in the separation culture, effectively reduces the separation and purification difficulty of each strain on the form, and improves the viable count and the purification rate of the bifidobacterium. After separation and culture, two procedures of primary screening and secondary screening of the strains are carried out, the strains with larger colony diameter/transparent ring diameter value are screened out during primary screening, and then strain secondary screening work is carried out on the basis, wherein the secondary screening is carried out by carrying out step-by-step culture through an LB culture medium and a secondary screening culture medium, so that the separation sufficiency is ensured, and the purification effect of the average value is improved.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention. Structures, devices, and methods of operation not specifically described or illustrated herein are generally practiced in the art without specific recitation or limitation.
Claims (5)
1. An optimized separation method of probiotics is characterized by comprising the following steps:
s1: preparing a diluted sample solution, weighing 20-25g of feed, and homogenizing in 200-250mL of sterile water to prepare a diluted sample solution with the volume ratio of 1: 10, and continuously carrying out ten times of gradient dilution, wherein the dilution gradient is 10-1To 10-9；
S2: separating and culturing, respectively taking a certain amount of dilution gradientIs 10-4To 10-9The diluted sample solution is placed on a culture medium flat plate, and 2 to 3 parallel samples are made for the diluted sample solution of each dilution gradient; culturing for 40-45 hours under anaerobic condition, culturing for 6-8 hours under aerobic condition,
s3: primarily screening strains, measuring the diameter of a bacterial colony and the diameter of a transparent ring, and preserving strains of the strains with larger values of the bacterial colony diameter/the diameter of the transparent ring;
s4: re-screening the strains, respectively inoculating the strains to be detected into 50mL of LB culture medium, placing the strains in a shaking table at 45-50 ℃ for shaking culture at the speed of 120-150r/min for 20-22h, and preparing seed liquid; transferring the seed solution into 100mL of a rescreening culture medium by using the inoculation amount of 5-8%, placing the rescreening culture medium in a shaker at 45-50 ℃ for shake culture at the speed of 150r/min for 25-30h, and measuring the lactic acid content in the fermentation liquor to screen the strain with strong acid production.
2. The method for optimizing and separating probiotics according to claim 1, wherein the LB medium comprises the following components: 10-12g/L of peptone, 5-8g/L of yeast powder, 10-12g/L of sodium chloride and pH 7.0.
3. The method for optimizing and separating probiotics according to claim 1, wherein the parameters of each component of the screening plate culture medium are as follows: 10-12g/L of peptone, 5-8g/L of yeast powder, 10-12g/L of sodium chloride, 20-25g/L of glucose, 20-23g/L of calcium carbonate and 20-25g/L of agar powder, and the pH value is 7.0.
4. The method for optimizing and separating probiotics according to claim 1, wherein the rescreened culture medium comprises the following parameters: 10-12g/L of peptone, 5-8g/L of yeast powder, 10-12g/L of sodium chloride and 50-55g/L of glucose, and the pH value is 7.0.
5. The method for optimizing and separating probiotics according to claim 1, wherein the culture temperature of an LB culture medium is 36-38 ℃; the culture temperature of the screening plate culture medium is 35-38 ℃; the culture temperature of the rescreening culture medium is 35-37 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242074A (en) * | 2011-04-12 | 2011-11-16 | 叶春 | Screen method for facultative anaerobe, and apparatus thereof |
| CN109136145A (en) * | 2018-09-11 | 2019-01-04 | 安徽万士生物制药有限公司 | One kind separating Bifidobacterium and its purification process from rabbit excrement |
| WO2022057741A1 (en) * | 2020-09-18 | 2022-03-24 | 山东得益乳业股份有限公司 | Bifidobacterium animalis, breeding method therefor and use thereof |
| CN114410472A (en) * | 2021-12-27 | 2022-04-29 | 乌兰察布职业学院 | Screening method of cellulase-producing strain |
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- 2022-05-10 CN CN202210504188.8A patent/CN114774324A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242074A (en) * | 2011-04-12 | 2011-11-16 | 叶春 | Screen method for facultative anaerobe, and apparatus thereof |
| CN109136145A (en) * | 2018-09-11 | 2019-01-04 | 安徽万士生物制药有限公司 | One kind separating Bifidobacterium and its purification process from rabbit excrement |
| WO2022057741A1 (en) * | 2020-09-18 | 2022-03-24 | 山东得益乳业股份有限公司 | Bifidobacterium animalis, breeding method therefor and use thereof |
| CN114410472A (en) * | 2021-12-27 | 2022-04-29 | 乌兰察布职业学院 | Screening method of cellulase-producing strain |
Non-Patent Citations (2)
| Title |
|---|
| TAYE YESHAMBEL ET AL.: ""Isolation and Identification of Lactic Acid Bacteria from Cow Milk and Milk Products"", 《THE SCIENCE WORLD JOURNAL》, vol. 2021, pages 1 - 6 * |
| 郑丽君 等: ""婴儿粪便中益生菌的分离筛选及培养"", 《农产品加工》, vol. 7, no. 4, pages 8 - 12 * |
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