CN115777810A - Method for producing Tibetan tea by using aspergillus extracellular enzyme preparation - Google Patents
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- CN115777810A CN115777810A CN202211565921.3A CN202211565921A CN115777810A CN 115777810 A CN115777810 A CN 115777810A CN 202211565921 A CN202211565921 A CN 202211565921A CN 115777810 A CN115777810 A CN 115777810A
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- enzyme preparation
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 50
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 23
- 241000228212 Aspergillus Species 0.000 title claims abstract description 21
- 241001122767 Theaceae Species 0.000 title claims abstract 21
- 238000000855 fermentation Methods 0.000 claims abstract description 32
- 230000004151 fermentation Effects 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 7
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 5
- 238000012869 ethanol precipitation Methods 0.000 claims abstract description 5
- 229940088598 enzyme Drugs 0.000 claims description 47
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 9
- 102000030523 Catechol oxidase Human genes 0.000 claims description 4
- 108010031396 Catechol oxidase Proteins 0.000 claims description 4
- 108010059892 Cellulase Proteins 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 108010059820 Polygalacturonase Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 229940106157 cellulase Drugs 0.000 claims description 4
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000000643 oven drying Methods 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims 1
- 231100000678 Mycotoxin Toxicity 0.000 abstract description 4
- 239000002636 mycotoxin Substances 0.000 abstract description 4
- 239000000796 flavoring agent Substances 0.000 abstract description 3
- 235000019634 flavors Nutrition 0.000 abstract description 3
- 244000269722 Thea sinensis Species 0.000 description 33
- 235000013616 tea Nutrition 0.000 description 31
- 230000000694 effects Effects 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000007269 microbial metabolism Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IPMYMEWFZKHGAX-UHFFFAOYSA-N Isotheaflavin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C(C1=C2)=CC(O)=C(O)C1=C(O)C(=O)C=C2C1C(O)CC2=C(O)C=C(O)C=C2O1 IPMYMEWFZKHGAX-UHFFFAOYSA-N 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- UXRMWRBWCAGDQB-UHFFFAOYSA-N Theaflavin Natural products C1=CC(C2C(CC3=C(O)C=C(O)C=C3O2)O)=C(O)C(=O)C2=C1C(C1OC3=CC(O)=CC(O)=C3CC1O)=CC(O)=C2O UXRMWRBWCAGDQB-UHFFFAOYSA-N 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 235000020279 black tea Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000009569 green tea Nutrition 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000014620 theaflavin Nutrition 0.000 description 1
- IPMYMEWFZKHGAX-ZKSIBHASSA-N theaflavin Chemical compound C1=C2C([C@H]3OC4=CC(O)=CC(O)=C4C[C@H]3O)=CC(O)=C(O)C2=C(O)C(=O)C=C1[C@@H]1[C@H](O)CC2=C(O)C=C(O)C=C2O1 IPMYMEWFZKHGAX-ZKSIBHASSA-N 0.000 description 1
- 229940026509 theaflavin Drugs 0.000 description 1
- 235000008118 thearubigins Nutrition 0.000 description 1
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Abstract
The invention discloses a method for producing Tibetan tea by using an aspergillus extracellular enzyme preparation, which comprises the following steps: (1) Culturing aspergillus in a PDB culture medium to obtain a fermentation broth; (2) Centrifuging the fermentation liquor obtained in the step (1) to obtain a crude enzyme solution, and then obtaining an enzyme preparation by using an ethanol precipitation method; (3) And (3) adding the enzyme preparation obtained in the step (2) into green raw tea for enzymatic fermentation to obtain the Tibetan tea. The product produced by the aspergillus ectoenzyme preparation has the unique flavor of the Tibetan tea, the fermentation time is shorter than that of the common fermented Tibetan tea, the production efficiency of the Tibetan tea is improved, and the Tibetan tea is prevented from being infected by mycotoxin in the long-time fermentation process.
Description
Technical Field
The invention belongs to the field of Tibetan tea production, and particularly relates to a method for producing Tibetan tea by using an aspergillus extracellular enzyme preparation.
Background
The Tibetan tea is a typical representative of black tea in China and is a living necessity of frontier minority nationality. In recent years, the Tibetan tea is popular with consumers due to the unique health care effect, and the market for internal sale is continuously expanded. Pile fermentation is a key process in the quality forming process of Tibetan tea. In the pile fermentation process, polyphenols, alkaloids, amino acids and other substances in the raw material tea are subjected to a series of complex biochemical reactions such as oxidation, reduction, polymerization, degradation, complexation and the like under the action of the microbial compound exoenzyme, so that the unique pure and thick taste of the ya-an Tibetan tea is formed.
The pile fermentation process of the traditional Tibetan tea is an open microbial fermentation system, a plurality of problems are faced in the Tibetan tea production process, the problems of long pile fermentation period, inconsistent pile and reactor core quality conversion, mycotoxin possibly caused by microbial metabolism and the like become keys for restricting the development of the Yaan Tibetan tea industry, and the development of a clean production new process of the Yaan Tibetan tea by utilizing a modern biological technology becomes the focus of attention of the Yaan Tibetan tea industry.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: solves the problems of long pile fermentation period, inconsistent quality conversion of the pile and the reactor core, possible mycotoxin caused by microbial metabolism and the like of the traditional microbial fermentation tea-storing pile fermentation.
The technical scheme of the invention is as follows: a method for producing Tibetan tea by using an aspergillus extracellular enzyme preparation comprises the following steps:
(1) Culturing aspergillus in a PDB culture medium to obtain a fermentation broth;
(2) Centrifuging the fermentation liquor obtained in the step (1) to obtain a crude enzyme solution, and then obtaining an enzyme preparation by using an ethanol precipitation method;
(3) And (3) adding the enzyme preparation obtained in the step (2) into green raw tea for enzymatic fermentation to obtain the Tibetan tea.
Further, the method for enzymatic fermentation is as follows: and uniformly inoculating the enzyme preparation into green raw tea, culturing for 24h at the temperature of 30-60 ℃, and drying in an oven at the temperature of 80 ℃ to obtain the Tibetan tea product. Preferably at 55 ℃.
Further, the enzyme preparation is added in an amount of 50-100U/30g.
Further, in the step (1), the culture conditions are as follows: culturing at 35 deg.C and 150r/min in PDB culture medium for 7d.
Further, the aspergillus is a strain with high enzyme production capability separated from Tibetan tea products in Yaan area, and the enzyme production capability refers to the capability of producing polyphenol oxidase, cellulase, pectinase and protease.
Further, in the step (2), the centrifugation method is as follows: centrifuging the fermentation liquor in a 12000r/min centrifuge for 20min, and taking supernatant.
Further, the ethanol precipitation method comprises the following steps: adding anhydrous ethanol into the crude enzyme solution until the ethanol content is 75%, precipitating with ethanol for 24h, rinsing the precipitate, and freeze drying to obtain enzyme preparation.
Compared with the prior art, the invention has the following beneficial effects:
the product produced by the aspergillus ectoenzyme preparation has the unique flavor of the Tibetan tea, the fermentation time is shorter than that of the common fermented Tibetan tea, the production efficiency of the Tibetan tea is improved, and the Tibetan tea is prevented from being infected by mycotoxin in the long-time fermentation process.
Drawings
FIG. 1 is a graph for 24h evaluation of fermentation with exogenous enzymes added at different concentrations under different temperature conditions.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
Example 1
(1) Separating and screening high-enzyme-producing strains: the Aspergillus fungus strain is separated from Tibetan tea products produced by different Tibetan tea production enterprises of Yaan by using a PDA culture medium, and the strain with higher enzyme production capability is screened out by selecting the culture medium by using polyphenol oxidase, cellulase, pectinase and protease for later use.
(2) Determination of optimal enzyme production conditions: the optimum enzyme-producing conditions of the high-enzyme-producing strain are explored by single-factor tests (inoculation amount, culture time and culture temperature) by taking the enzyme-producing capability as an index.
(3) Preparing fermentation liquor: scraping the separated and screened high-yield enzyme strains into sterile water, uniformly shaking to prepare spore suspension, and adjusting the concentration of the spore suspension to 10 7 CFU/mL. And (3) sucking a proper amount of spore suspension, inoculating into a PDB culture medium, and culturing for 7d in a constant-temperature shaking incubator at 35 ℃ and 150r/min to obtain fermentation liquor.
(4) Extracting a crude enzyme solution: centrifuging the fermentation liquor obtained after the fermentation liquor is centrifuged for 20min by a 12000r/min centrifuge, and taking supernate to obtain crude enzyme liquid.
(5) Extracellular enzyme preparation: adding 3 times volume of anhydrous ethanol into the supernatant, performing alcohol precipitation for 24h, centrifuging at 6000r/min for 10min after alcohol precipitation, rinsing the precipitate, and freeze-drying to obtain dry powder, i.e. the extracellular enzyme preparation. And respectively detecting the enzyme activities of polyphenol oxidase, cellulase, pectinase and protease of the extracellular enzyme preparation.
(6) The process optimization: uniformly inoculating enzyme preparations (0, 50U and 100U) with different concentrations into green tea (30 g) with water content of 35% for enzymatic reaction, culturing at different temperatures (35 deg.C, 45 deg.C and 55 deg.C) for 24h, and oven drying at 80 deg.C to obtain Tibetan tea product. The contents of theaflavin, thearubigin and theabrownin of the Tibetan tea obtained under different conditions are shown in table 1.
TABLE 1 fermentation of tea pigment component at different temperatures for 24 hr by adding exogenous enzymes with different concentrations
The soup color, the aroma, the taste and the leaf bottom are analyzed item by referring to an evaluation method in the national standard GB/T32719.5-2018. The liquor color of the Tibetan tea product prepared by adding exogenous enzymes with different concentrations and fermenting for 24 hours is compared, so that the fermentation speed of the Tibetan tea can be improved by adding the exogenous enzymes, and the liquor color is positively correlated with the enzyme activity; the unique flavor of the Tibetan tea is gradually developed by adding the enzyme preparation, the product has orange red liquor color, sweet and mellow taste, and the aroma is old and sweet; the leaf bottom is gradually deepened along with the increase of the enzyme activity.
Claims (8)
1. A method for producing Tibetan tea by using an aspergillus extracellular enzyme preparation is characterized by comprising the following steps:
(1) Culturing aspergillus in a PDB culture medium to obtain a fermentation broth;
(2) Centrifuging the fermentation liquor obtained in the step (1) to obtain a crude enzyme solution, and then obtaining an enzyme preparation by using an ethanol precipitation method;
(3) And (3) adding the enzyme preparation obtained in the step (2) into green raw tea for enzymatic fermentation to obtain the Tibetan tea.
2. The method for producing Tibetan tea by using the aspergillus extracellular enzyme preparation is characterized in that the method for enzymatic fermentation is as follows: uniformly inoculating the enzyme preparation into green raw tea, culturing at 30-60 deg.C for 24 hr, and oven drying at 80 deg.C to obtain Tibetan tea product.
3. The method for producing Tibetan tea using the extracellular enzyme preparation of aspergillus according to claim 2, wherein the culture is performed at 55 ℃.
4. The method for producing Tibetan tea by using the aspergillus extracellular enzyme preparation according to claim 2, wherein the adding amount of the enzyme preparation is 50-100U/30g.
5. The method for producing Tibetan tea by using the aspergillus extracellular enzyme preparation according to claim 1, wherein in the step (1), the culture conditions are as follows: culturing at 35 deg.C and 150r/min in PDB medium for 7d.
6. The method for producing Tibetan tea by using the extracellular enzyme preparation of aspergillus according to claim 1, wherein the aspergillus is a strain which is separated from Tibetan tea products in Yaan area and has the capability of producing polyphenol oxidase, cellulase, pectinase and protease.
7. The method for producing Tibetan tea by using the aspergillus extracellular enzyme preparation is characterized in that in the step (2), the centrifugation method comprises the following steps: centrifuging the fermentation liquor in a 12000r/min centrifuge for 20min, and taking the supernatant.
8. The method for producing Tibetan tea by using the aspergillus extracellular enzyme preparation is characterized in that the ethanol precipitation method comprises the following steps: adding anhydrous ethanol into the crude enzyme solution until the ethanol content is 75%, precipitating with ethanol for 24h, rinsing the precipitate, and freeze drying to obtain enzyme preparation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211565921.3A CN115777810A (en) | 2022-12-07 | 2022-12-07 | Method for producing Tibetan tea by using aspergillus extracellular enzyme preparation |
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| CN202211565921.3A CN115777810A (en) | 2022-12-07 | 2022-12-07 | Method for producing Tibetan tea by using aspergillus extracellular enzyme preparation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113632861A (en) * | 2021-08-17 | 2021-11-12 | 四川农业大学 | Instant Tibetan tea powder and preparation method thereof |
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| CN105779307A (en) * | 2016-04-18 | 2016-07-20 | 云南中烟工业有限责任公司 | Trichoderma aureoviride strain and method for preparing Pu'er Tea tobacco flavor |
| CN107616243A (en) * | 2017-09-29 | 2018-01-23 | 四川农业大学 | A kind of preparation technology for hiding tea |
| CN109055273A (en) * | 2018-09-10 | 2018-12-21 | 安徽农业大学 | A kind of green brick tea pile-fermentation strain composition and application |
-
2022
- 2022-12-07 CN CN202211565921.3A patent/CN115777810A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182500A (en) * | 2007-11-01 | 2008-05-21 | 广东省微生物研究所 | Glucomannan enzyme preparation method |
| CN102719367A (en) * | 2012-07-10 | 2012-10-10 | 湖南农业大学 | Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase |
| CN103060286A (en) * | 2012-12-10 | 2013-04-24 | 华南理工大学 | Lipase made of aspergillus niger strains, and producing method and utilization thereof |
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| CN109055273A (en) * | 2018-09-10 | 2018-12-21 | 安徽农业大学 | A kind of green brick tea pile-fermentation strain composition and application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113632861A (en) * | 2021-08-17 | 2021-11-12 | 四川农业大学 | Instant Tibetan tea powder and preparation method thereof |
| CN113632861B (en) * | 2021-08-17 | 2024-02-13 | 四川农业大学 | Instant Tibetan tea powder and preparation method thereof |
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