CN110669680A - Culture method of black yeast and process for extracting beta glucan from black yeast - Google Patents
Culture method of black yeast and process for extracting beta glucan from black yeast Download PDFInfo
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- 241001480003 Chaetothyriales Species 0.000 title claims abstract description 91
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 title claims abstract description 36
- 229920002498 Beta-glucan Polymers 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000012136 culture method Methods 0.000 title abstract description 9
- 239000000243 solution Substances 0.000 claims abstract description 41
- 239000000706 filtrate Substances 0.000 claims abstract description 32
- 239000001963 growth medium Substances 0.000 claims abstract description 31
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 27
- 239000012138 yeast extract Substances 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000007787 solid Substances 0.000 claims abstract description 17
- 238000001914 filtration Methods 0.000 claims abstract description 16
- 230000010355 oscillation Effects 0.000 claims abstract description 10
- 230000000415 inactivating effect Effects 0.000 claims abstract description 8
- 239000011259 mixed solution Substances 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims description 23
- 230000004913 activation Effects 0.000 claims description 18
- 108010059892 Cellulase Proteins 0.000 claims description 14
- 108010059820 Polygalacturonase Proteins 0.000 claims description 14
- 229940106157 cellulase Drugs 0.000 claims description 14
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 10
- 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 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 10
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 241000223678 Aureobasidium pullulans Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241000223651 Aureobasidium Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
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- 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
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P19/08—Dextran
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- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Virology (AREA)
- Botany (AREA)
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Abstract
The invention relates to a culture method of black yeast and a process for extracting beta glucan from the black yeast. The method comprises the following steps: inoculating the black yeast to an activated culture medium, inoculating the black yeast to a solid culture medium, and extracting, concentrating and separating to obtain a black yeast extract culture solution after the black yeast extract culture solution is obtained; adding the culture solution of the black yeast extract into water, adjusting pH, performing ultrasonic oscillation treatment, and filtering to obtain primary filter residue and primary filtrate; mixing the primary filter residue with water, performing enzymolysis reaction, and filtering to obtain secondary filter residue and secondary filtrate; and inactivating the secondary filtrate, mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state. The culture method of the black yeast and the process for extracting the beta glucan from the black yeast extract have high extraction rates of the black yeast and the beta glucan, wherein the detection result shows that the effective component content of the black yeast extract is 39.8-43.9%, and the extraction rate of the beta glucan is 4.37-5.05%.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method of black yeast and a process for extracting beta glucan from the black yeast.
Background
Black yeast (Aureobasidium pullulans), also known as Basidiomycetes Aureobasidium and Aureobasidium pullulans, is a yeast-like fungus and exists in various living environments. The periphery of the cell wall of black yeast produces polysaccharide material, the most important of which is beta glucan.
At present, the black yeast and the corresponding beta glucan are directly extracted from the fungus products, the extraction purity is low, the time is long, and the comprehensive cost is high.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a culture method of black yeast and a process for extracting beta glucan from the black yeast. The culture method of the black yeast and the process for extracting the beta glucan from the black yeast extract have high extraction rates of the black yeast and the beta glucan, wherein the detection result shows that the effective component content of the black yeast extract is 39.8-43.9%, and the extraction rate of the beta glucan is 4.37-5.05%.
The scheme of the invention is to provide a method for culturing black yeast, which is characterized by comprising the following steps:
(1) inoculating the black yeast into an activation culture medium, and transferring the strain once every 30-34 h during the period to obtain an activated strain;
(2) inoculating the activated strain obtained in the step (1) into a solid culture medium to obtain a black yeast culture solution;
(3) extracting the culture solution of the black yeast obtained in the step (2), and then concentrating and separating to obtain a culture solution of the black yeast extract.
Preferably, in the step (1), the black yeast accounts for 0.06-0.08% of the weight of the activation medium.
Preferably, in the step (1), the temperature during the culture of the activation medium is 32-35 ℃ and the time is 10-15 d.
Preferably, in step (1), the activation medium comprises the following raw materials in parts by weight: 2.0-2.2 parts of glucose, 0.8-1.0 part of peptone, 0.3-0.5 part of monopotassium phosphate and 92-96 parts of distilled water.
Preferably, in the step (2), the activated strain accounts for 8-12% of the weight of the solid medium.
Preferably, in the step (2), the temperature during the solid medium culture is 35-37 ℃ and the time is 20-30 d.
Based on the same technical concept, the invention also provides a culture solution of the black yeast extract obtained by the culture method.
Also, based on the same technical concept, the invention further provides a process for extracting beta-glucan from the culture solution of the black yeast extract, which comprises the following steps:
(a) adding the culture solution of the black yeast extract into water, adjusting the pH value to be alkaline, carrying out ultrasonic oscillation treatment and then filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, fully reacting, and filtering to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
Preferably, in the step (a), the pH is 7.5-8.5; the power of the ultrasonic oscillation is 600-800W, and the time is 8-12 min.
Preferably, in the step (b), the weight ratio of the primary filter residue to the cellulase to the pectinase is 100: 0.2-0.4: 0.6-0.8.
The invention has the beneficial effects that:
the culture method of the black yeast and the process for extracting the beta glucan from the black yeast extract have high extraction rates of the black yeast and the beta glucan, wherein the detection result shows that the effective component content of the black yeast extract is 39.8-43.9%, and the extraction rate of the beta glucan is 4.37-5.05%.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1
The embodiment provides a method for culturing black yeast, which comprises the following steps:
(1) uniformly mixing 2.0g of glucose, 0.8g of peptone, 0.3g of monopotassium phosphate and 92g of distilled water to obtain an activated culture medium;
(2) inoculating black yeast into an activation culture medium, wherein the weight of the black yeast accounts for 0.06% of the weight of the activation culture medium, culturing for 10d at the temperature of 32 ℃, and transferring the strain once every 30h during the culture period to obtain an activated strain;
(3) inoculating the activated strain obtained in the step (2) into a solid culture medium, wherein the activated strain accounts for 8% of the weight of the solid culture medium, and culturing at 35 ℃ for 20 days to obtain a black yeast culture solution;
(4) and (4) extracting the black yeast culture solution obtained in the step (3), and then concentrating and separating to obtain a black yeast extract culture solution.
The present invention also provides a process for extracting beta-glucan from a culture solution of an extract of saccharomyces cerevisiae, comprising the following steps:
(a) adding the culture solution of the black yeast extract into water, adjusting pH to 7.5, performing ultrasonic oscillation treatment under 600W power for 8min, and filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, wherein the weight ratio of the primary filter residue to the cellulase to the pectinase is 100:0.2:0.6, and filtering after full reaction to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
Example 2
The embodiment provides a method for culturing black yeast, which comprises the following steps:
(1) uniformly mixing 2.2g of glucose, 1.0g of peptone, 0.5g of monopotassium phosphate and 96g of distilled water to obtain an activated culture medium;
(2) inoculating black yeast into an activation culture medium, wherein the weight of the black yeast accounts for 0.08 percent of the weight of the activation culture medium, culturing for 15d at the temperature of 35 ℃, and transferring the strain once every 34h during the culture period to obtain an activated strain;
(3) inoculating the activated strain obtained in the step (2) into a solid culture medium, wherein the activated strain accounts for 12% of the weight of the solid culture medium, and culturing at 37 ℃ for 30d to obtain a black yeast culture solution;
(4) extracting the culture solution of the black yeast obtained in the step (2), and then concentrating and separating to obtain a culture solution of the black yeast extract.
The present invention also provides a process for extracting beta-glucan from a culture solution of an extract of saccharomyces cerevisiae, comprising the following steps:
(a) adding the culture solution of the black yeast extract into water, adjusting pH to 8.5, performing ultrasonic oscillation treatment for 12min under the power condition of 800W, and filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, wherein the weight ratio of the primary filter residue to the cellulase to the pectinase is 100:0.4:0.8, and filtering after full reaction to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
Example 3
The embodiment provides a method for culturing black yeast, which comprises the following steps:
(1) uniformly mixing 2.0g of glucose, 1.0g of peptone, 0.3g of monopotassium phosphate and 96g of distilled water to obtain an activated culture medium;
(2) inoculating black yeast into an activation culture medium, wherein the weight of the black yeast accounts for 0.06% of the weight of the activation culture medium, culturing for 15d at the temperature of 32 ℃, and transferring the strain once every 30h during the culture period to obtain an activated strain;
(3) inoculating the activated strain obtained in the step (2) into a solid culture medium, wherein the activated strain accounts for 8% of the weight of the solid culture medium, and culturing at 37 ℃ for 20 days to obtain a black yeast culture solution;
(4) and (4) extracting the black yeast culture solution obtained in the step (3), and then concentrating and separating to obtain a black yeast extract culture solution.
The present invention also provides a process for extracting beta-glucan from a culture solution of an extract of saccharomyces cerevisiae, comprising the following steps:
(a) adding the culture solution of the black yeast extract into water, adjusting pH to 7.5, performing ultrasonic oscillation treatment for 12min under 600W power, and filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, wherein the weight ratio of the primary filter residue to the cellulase to the pectinase is 100:0.2:0.8, and filtering after full reaction to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
Example 4
The embodiment provides a method for culturing black yeast, which comprises the following steps:
(1) uniformly mixing 2.2g of glucose, 0.8g of peptone, 0.5g of monopotassium phosphate and 92g of distilled water to obtain an activated culture medium;
(2) inoculating black yeast into an activation culture medium, wherein the weight of the black yeast accounts for 0.08 percent of the weight of the activation culture medium, culturing for 10 days at the temperature of 35 ℃, and transferring the strain once every 34 hours during the culture period to obtain an activated strain;
(3) inoculating the activated strain obtained in the step (2) into a solid culture medium, wherein the activated strain accounts for 12% of the weight of the solid culture medium, and culturing at 37 ℃ for 20 days to obtain a black yeast culture solution;
(4) and (4) extracting the black yeast culture solution obtained in the step (3), and then concentrating and separating to obtain a black yeast extract culture solution.
The present invention also provides a process for extracting beta-glucan from a culture solution of an extract of saccharomyces cerevisiae, comprising the following steps:
(a) adding the culture solution of the black yeast extract into water, adjusting pH to 8.5, performing ultrasonic oscillation treatment for 8min under the power condition of 800W, and filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, wherein the weight ratio of the primary filter residue to the cellulase to the pectinase is 100:0.4:0.6, and filtering after full reaction to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
Example 5
The embodiment provides a method for culturing black yeast, which comprises the following steps:
(1) uniformly mixing 2.1g of glucose, 0.9g of peptone, 0.4g of monopotassium phosphate and 94g of distilled water to obtain an activated culture medium;
(2) inoculating black yeast into an activation culture medium, wherein the weight of the black yeast accounts for 0.07 percent of the weight of the activation culture medium, culturing for 12 days at the temperature of 33 ℃, and transferring the strain once every 32 hours during the culture period to obtain an activated strain;
(3) inoculating the activated strain obtained in the step (2) into a solid culture medium, wherein the activated strain accounts for 10% of the weight of the solid culture medium, and culturing at 36 ℃ for 25 days to obtain a black yeast culture solution;
(4) extracting the culture solution of the black yeast obtained in the step (2), and then concentrating and separating to obtain a culture solution of the black yeast extract.
The present invention also provides a process for extracting beta-glucan from a culture solution of an extract of saccharomyces cerevisiae, comprising the following steps:
(a) adding the culture solution of the black yeast extract into water, adjusting pH to 8.0, performing ultrasonic oscillation treatment for 10min under the power of 700W, and filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, wherein the weight ratio of the primary filter residue to the cellulase to the pectinase is 100:0.3:0.7, and filtering after full reaction to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
In order to verify the extraction concentration of the black yeast extract and the extraction concentration of β -glucan according to the present invention, the products obtained in examples 1 to 5 were measured, and the test results are shown in table 1.
TABLE 1 Black yeast and beta glucan concentration test results
Results and conclusions:
in embodiments 1 to 5 of the present invention, the ratio of the effective components in the black yeast extract is 39.8 to 43.9%, and the extraction rate of the β -glucan is 4.37 to 5.05%, both of which reach a high level, and have significant effects.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. The method for culturing the black yeast is characterized by comprising the following steps of:
(1) inoculating the black yeast into an activation culture medium, and transferring the strain once every 30-34 h during the period to obtain an activated strain;
(2) inoculating the activated strain obtained in the step (1) into a solid culture medium to obtain a black yeast culture solution;
(3) extracting the culture solution of the black yeast obtained in the step (2), and then concentrating and separating to obtain a culture solution of the black yeast extract.
2. The method for culturing black yeast according to claim 1, wherein the black yeast accounts for 0.06-0.08% of the weight of the activation medium in the step (1).
3. The method for culturing black yeast according to claim 1, wherein the temperature during the step (1) of culturing in the activation medium is 32 to 35 ℃ for 10 to 15 days.
4. The method for culturing black yeast according to claim 1, wherein in the step (1), the activation medium comprises the following raw materials in parts by weight: 2.0-2.2 parts of glucose, 0.8-1.0 part of peptone, 0.3-0.5 part of monopotassium phosphate and 92-96 parts of distilled water.
5. The method for culturing black yeast according to claim 1, wherein the activated strain accounts for 8-12% of the weight of the solid medium in the step (2).
6. The method for culturing black yeast according to claim 1, wherein the temperature in the solid medium culture period in step (2) is 35-37 ℃ for 20-30 days.
7. The extract of black yeast cultured by the method according to any one of claims 1 to 6.
8. A process for extracting beta-glucan from the culture solution of the extract of black yeast as claimed in claim 7, comprising the steps of:
(a) adding the culture solution of the black yeast extract into water, adjusting the pH value to be alkaline, carrying out ultrasonic oscillation treatment and then filtering to obtain primary filter residue and primary filtrate;
(b) mixing the primary filter residue obtained in the step (a) with water, adding cellulase and pectinase, fully reacting, and filtering to obtain secondary filter residue and secondary filtrate;
(c) inactivating the secondary filtrate obtained in the step (b), mixing with the primary filtrate, and concentrating the mixed solution to obtain the beta glucan in a colloidal state.
9. The process for extracting beta-glucan according to claim 8, wherein in the step (a), the pH is 7.5-8.5; the power of the ultrasonic oscillation is 600-800W, and the time is 8-12 min.
10. The process for extracting beta-glucan according to claim 8, wherein in the step (b), the weight ratio of the primary filter residue to the cellulase to the pectinase is 100: 0.2-0.4: 0.6-0.8.
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| JP2004049013A (en) * | 2002-07-16 | 2004-02-19 | Asahi Denka Kogyo Kk | NEW MICROORGANISM AND METHOD FOR PRODUCING beta-GLUCAN USING THE SAME |
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2019
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