CN114480151B - Yeast and application thereof in tea tree quality improvement and efficacy enhancement - Google Patents
Yeast and application thereof in tea tree quality improvement and efficacy enhancement Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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Abstract
Yeast strain classified asHanseniaspora uvarum WYCCW10240 with a preservation number of CCTCC NO: M20211275, a preservation time of 2021, 10 months and 14 days, a preservation unit of China center for type culture Collection, and an address of university of Wuhan, wuhan. The invention discloses a saccharomycete strain, which can greatly improve the yield and the content of tea polyphenol, amino acid, caffeine and total flavone of tea leaves in the tea leaf surface spraying process, so as to improve the quality and the quality of the tea leaves, increase the economic benefit, and respectively improve the content of the tea polyphenol, the amino acid, the caffeine and the total flavone of the tea leaves treated by saccharomycete in the application by 72.47 percent, 88.31 percent, 45.18 percent and 82.67 percent relative to a control group, so that the saccharomycete strain can be used as a high-efficiency microbial inoculum for improving the quality and the efficiency of the tea leaves with average yield increasing of 24.4 percent.
Description
Technical Field
The invention belongs to the field of plant production, relates to foliar spraying of tea trees, and particularly relates to saccharomycetes and application thereof in quality improvement and efficiency enhancement of tea trees.
Background
Tea tree, school name:Camellia sinensis(l.) o.ktze, is a camellia family, genus camellia shrub or small arbor, and is hairless in shoots. She Gezhi, oblong or oval. The leaves of the tea tree can be used for preparing tea (different from the tea tree), seeds can squeeze oil, the tea tree is fine in material quality, and the wood can be used for carving. The distribution is mainly concentrated between 16 degrees in south latitude and 30 degrees in north latitude, tea trees like warm and moist climate, buds start to sprout when the average air temperature is above 10 ℃, the growth optimum temperature is 20-25 ℃, and the annual precipitation is above 1000 mm; light-preference and yin-tolerance, and is suitable for growing under diffuse light; a lifetime is divided into a seedling stage, a juvenile stage, a adult stage and an aging stage. The tree age can reach two hundred years, but the economic age is 40-50 years generally. The southwest of China is the origin center of tea trees, and 60 countries in the world introduce tea trees. In tropical areas, arbor-type tea trees are 15-30 m, the base tree is more than 1.5 m, and the tree age can reach hundreds of years to thousands of years.
However, the tea quality and the like of the tea tree are uneven in the tea tree planting process, and the tea tree is large in difference, so that the quality is not stable enough, and the tea tree is poor in soil, poor in water retention and nutrient, and the yield and quality are affected because the environment for growing the tea tree is generally on a hillside. The present inventors have focused on research related to improving tea quality and yield.
Disclosure of Invention
The invention provides saccharomycetes and application thereof in tea tree quality improvement and efficiency improvement, and solves the problems of poor tea quality and low tea yield.
The technical scheme of the invention is realized as follows:
a yeast strain is classified under the name Hanseniaspora uvarum WYCCW10240 (Hansenula polymorpha WYCCW10240 with grape juice), the preservation number is CCTCC NO: M20211275, the preservation time is 2021, 10 months and 14 days, the preservation unit is China center for type culture Collection, and the address is China university of Wuhan.
The saccharomycete is used as a high-efficiency foliar quality-improving and efficiency-improving microbial inoculum for tea trees.
The application of the saccharomycetes comprises the following steps:
(1) Inoculating saccharomycetes into a YEPD liquid culture medium, and carrying out shaking culture at 28 ℃ for 12 hours to obtain saccharomycetes liquid;
(2) Regulating the concentration of the saccharomycete liquid to OD600 = 0.5 by using saline water, and then loading the saccharomycete liquid into a spraying device for standby;
(3) Uniformly spraying tea leaves according to the amount of 30 kg of prepared bacterial liquid used in each mu of tea garden;
(4) After the leaf surfaces are sprayed according to the three steps, the tea sample collection and related data analysis are carried out after waiting for 5-10 days.
The inoculation amount of the saccharomycete strain in the step (1) is 0.1 hundred million bacteria per square meter of tea garden.
The concentration of the brine in the step (2) is 0.8-1.5%.
The invention has the following beneficial effects:
the invention discloses a saccharomycete strain, which can greatly improve the yield and the tea polyphenol, amino acid, caffeine and total flavone content of tea leaves in the tea tree quality improvement and efficiency improvement process, so as to improve the quality and the quality of the tea leaves, increase the economic benefit, and respectively improve the tea polyphenol, amino acid, caffeine and total flavone content of the tea leaves treated by the saccharomycete strain by 72.47 percent, 88.31 percent, 45.18 percent and 82.67 percent relative to a control group, and increase the yield by 24.4 percent on average, thereby being used as a tea tree high-efficiency leaf surface quality improvement and efficiency improvement microbial inoculum.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a phylogenetic tree.
Figure 2 is a sample collection of tea leaves.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
Yeast strain classified asHanseniaspora uvarumWYCCW10240 with a preservation number of CCTCC NO: M20211275, a preservation time of 2021, 10 months and 14 days, a preservation unit of China center for type culture Collection, and an address of university of Wuhan, wuhan.
Experimental materials
1. Test tea sample
The test tea sample is green tea provided by Fujian Chunlun group limited company, the tea tree grows in a Huan Xizhen Chunlun ecological tea garden in Jinan area of Fuzhou, the elevation is 550m, the climate is warm and moist, the rainfall is abundant, the four seasons evergreen is Fuyun No. 6, the picking standard is one bud two leaves to one bud three leaves, the grade is special, and the tea tree is prepared through the technological processes of picking, deactivating enzyme, twisting and drying.
Instrument and reagent
Spectrophotometry (V-1100D type), high performance liquid chromatography, ultraviolet detector and chromatographic workstation, ultrasonic extractor, electrothermal constant temperature water bath (HWS-26 type), electronic balance (accurate to 0.001), buchner funnel together with decompression suction filtration device, analytical water purifier, SHB-III circulating water type multipurpose vacuum pump, electrothermal constant temperature blast drying oven, pulverizer, high speed centrifuge (3500 r/min), separating funnel, triangular flask, pipette (1 mL, 2 mL, 5 mL, 10 mL), volumetric flask (25 mL, 50 mL, 250 mL, 500 mL volumetric flask), etc.
Methanol reagent, 10% Fu Lin Fen reagent, 7.5% sodium carbonate (Na 2 CO 3 ) Solution, gallic acid standard stock solution (1 mg mL) -1 I.e., 1000. Mu.g.mL -1 Prepared at present), gallic acid working solution, ethyl acetate (A, R), 95 percent ethanol,N-butanol is analytically pure, 2.5% sodium bicarbonate solution, saturated oxalic acid solution, distilled water, etc.
Disclosed formula of yeast YEPD solid medium:
every 1000 g medium components: yeast powder 10g, glucose 20 g, peptone 20 g, agar powder 20 g, deionized water 1000 mL, natural pH.
1. Yeast strainHanseniaspora uvarumIsolation and screening of WYCCW10240
Soil samples are collected between rows of the Ending ecological tea garden of the company Limited in Fujian spring and Lun of Fujian, fujian province, and are stored in a sterile sealed bag by adopting a five-point sampling method, recorded and immediately brought back to a laboratory. 10g of soil sample is randomly taken and placed into a triangular flask which is sterilized in advance and contains 90mL of sterile water and glass beads, and the triangular flask is placed into a shaking table for shaking and uniformly mixing for 10 minutes under the conditions of 28 ℃ and the rotating speed of 150 r/min. Then, the flask was allowed to stand for 10 minutes, and the supernatant was subjected to 10-fold gradient dilution (10 -1 ~10 -9 ) Then coating the yeast source on a YEPD culture medium solid plate, coating 200 mu L of each gradient, and placing the plate into a constant temperature incubator for inversion culture at 28 ℃ overnight to initially obtain the yeast source.
Further streaking and separating out the colony with better selected form, and repeatedly streaking, separating and purifying until no impurity colony appears on the flat plate. Then, the picked and purified single bacteria are streaked on a WL identification culture medium plate to obtain single bacterial colonies, 5 strains with consistent bacterial colony morphology and highest abundance are selected, and the inclined plane is reserved for standby. Single colonies were picked up and inoculated into 5 mL YEPD liquid tubes, shake-cultured at 28℃and 180rpm for 12 h, and the OD600 of the bacterial liquid was adjusted to 1.0 according to 1:100 proportion is taken and 0.5 and mL is connected to 50 mL YEPD liquid culture medium, and is cultured in a shaking table at 28 ℃ and 180rpm for 12 h; and then performing bacterial liquid dilution and spraying according to the operation steps, and screening to obtain the optimal strain WYCCWF10240 by measuring the tea yield, relevant quality indexes and the like. 26S rRNA sequence identified as strain WYCCW10240 by 26S rRNA sequencing and phylogenetic analysis is shown in SEQ ID No. 1; the phylogenetic result is shown in figure 1, and the strain is determined to be Hansenula, and is named as HansenulaHanseniaspora uvarum WYCCW10240。
Application example
The method for spraying tea trees by utilizing the saccharomycetes comprises the following steps:
(1) The yeast strain is prepared according to 10 0 -10 9 Inoculating the ratio of the individual bacteria into a YEPD liquid culture medium, and shake culturing overnight at 28 ℃ to obtain a saccharomycete liquid;
(2) Regulating the concentration of the saccharomycete liquid to OD600 = 0.5 by using 0.8-1.5 wt% sodium chloride solution, and then loading the saccharomycete liquid into a sprayer for later use;
(3) Uniformly spraying tea leaves according to the amount of 30-60 kg of prepared bacterial liquid used in each mu of tea garden; tea trees sprayed by equal amount of deionized water without spraying saccharomycete liquid are used as blank control;
(4) The sample collection of tea leaves is carried out for 8 days, the physical diagram is shown in figure 2, new germination heads are collected and are one leaf and one bud, and then the weight, the tea polyphenol, the amino acid, the caffeine, the total flavone content and the like are measured.
The method for measuring the content of tea polyphenol, amino acid, caffeine and total flavonoids comprises the following steps:
1. tea polyphenol content determination
Method for detecting content of tea polyphenol and catechin in tea leaves by using GB/T8313-2018 [1] Determining the content of tea polyphenol in the tea sample; the absorbance values were measured at 765-nm wavelength using a number of 10 mm cuvettes and spectrophotometers, and the results were finally obtained according to the corresponding formulas and are shown in table 1.
Determination of theanine
And (3) determining the content of theanine in the tea sample according to GB/T23193-2017 high performance liquid chromatography for determining theanine in tea leaves, and performing blank operation after the flow rate and the column temperature are stable. Human HPLC was injected with standard use of 10 pL theanine. 10 μl of test solution was injected under the same chromatographic conditions. The test solution was quantified as peak area. The concentration of the corresponding theanine can be determined from the standard curve from the peak area of the chromatographic peak. The response values of theanine in the test solution are all in the linear range measured by the instrument. After the theanine in the tea sample is heated by boiling water, discharged and purified, the RP-18 column for separating the strong polar compounds is adopted to detect the wavelength 210 and nm, and the high performance liquid chromatograph is used for measuring, and the quality and the quantity are compared with the standard series. Finally, the results are obtained according to the corresponding formulas, and the results are shown in table 1.
Determination of caffeine
The caffeine content in the tea samples was determined according to the ultraviolet spectrophotometry of GB/T8312-2013 "tea caffeine determination", and the absorbance (A) was measured using a 10 mm quartz cuvette with a reagent blank solution as reference at wavelength 274 nm, with the results shown in Table 1.
Determination of Total Flavonoids
Accurately sucking the total flavone extract (diluted by proper times) 2.0 and mL into a 10 mL stopper test tube, adding 60% ethanol 3.0 and mL, measuring the absorbance according to the methods of 1, 2, 3 and 4, taking the reagent blank as a reference (extracting solvent replaces the extract), substituting the absorbance into a standard curve to calculate the total flavone content of the sample liquid, and the result is shown in table 1.
Statistical analysis of data
Statistical analysis was performed using Microsoft Excel 2007 software and SPSS 25.0 software. The Relative Standard Deviation (RSD) was calculated following the above experimental procedure, with 2 replicates of each set of data.
Table 1: statistics of tea polyphenol, amino acid, caffeine and total flavone content of sprayed saccharomycetes liquid group and control group
From the above, the tea polyphenol, amino acid, caffeine and total flavone content of the tea tree group treated by the saccharomycetes are obviously improved compared with the control group.
Via yeastHanseniaspora uvarum The bud weight of the WYCCW10240 treated tea tree was 24.4% less than that of the control group to give 1 gram of green tea, see table 2:
table 2: counting the bud number required by 1 g of green tea
As is clear from Table 2, the yeasts of the present application were usedHanseniaspora uvarum After WYCCW10240, the number of buds required for producing 1 gram of green tea is reduced by 22, and the yield increasing effect is remarkable.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
<110> Fujian Chunlun group Co., ltd
Zhengzhou university of light industry
<120> saccharomycete and application thereof in tea tree quality improvement and efficacy enhancement
<141> 2022-02-24
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 564
<212> DNA
<213> Hanseniaspora uvarum
<400> 1
ggcgagtgag cggtaaaagc tcaaatttga aatctggtac tttcagtgcc cgagttgtaa 60
tttgtagaat ttgtctttga ttaggtcctt gtctatgttc cttggaacag gacgtcatag 120
agggtgagaa tcccgtttgg cgaggatacc ttttctctgt aagacttttt cgaagagtcg 180
agttgtttgg gaatgcagct caaagtgggt ggtaaattcc atctaaagct aaatattggc 240
gagagaccga tagcgaacaa gtacagtgat ggaaagatga aaagaacttt gaaaagagag 300
tgaaaaagta cgtgaaattg ttgaaaggga agggcatttg atcagacatg gtgttttttg 360
catgcactcg cctctcgtgg gcttgggcct ctcaaaaatt tcactgggcc aacatcaatt 420
ctggcagcag gataaatcat taagaatgta gctacttcgg tagtgttata gctttttgga 480
atactgttag ccgggattga ggactgcgct tcggcaagga tgttggcata atggttaaat 540
gccgcccgtc ttgaaacacg gacc 564
Claims (5)
1. A yeast, characterized in that: the classification name of the saccharomycete strain isHanseniaspora uvarum WYCCW10240 with a preservation number of CCTCC NO: M20211275, a preservation time of 2021, 10 months and 14 days, a preservation unit of China center for type culture Collection, and an address of university of Wuhan, wuhan.
2. Use of a yeast according to claim 1, characterized in that: the saccharomycete is used as a high-efficiency foliar quality-improving and efficiency-improving microbial inoculum for tea trees.
3. The use of yeasts according to claim 2, characterized by the following steps:
(1) Inoculating saccharomycetes into a YEPD liquid culture medium, and carrying out shaking culture at 28 ℃ for 12 hours to obtain saccharomycetes liquid;
(2) Regulating the concentration of the saccharomycete liquid to OD600 = 0.5 by using saline water, and then loading the saccharomycete liquid into a spraying device for standby;
(3) Uniformly spraying tea leaves according to the amount of 30 kg of prepared bacterial liquid used in each mu of tea garden;
(4) After the leaf surfaces are sprayed according to the three steps, the tea sample collection and related data analysis are carried out after waiting for 5-10 days.
4. Use of a yeast according to claim 3, characterized in that: the inoculation amount of the saccharomycetes in the step (1) is 0.1 hundred million bacteria per square meter of tea garden.
5. Use of a yeast according to claim 3, characterized in that: the concentration of the brine in the step (2) is 0.8-1.5%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996025039A1 (en) * | 1995-02-17 | 1996-08-22 | The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University | Control of post-harvest fungal disease using saprophytic yeast |
| CN106566778A (en) * | 2016-11-03 | 2017-04-19 | 贵州师范学院 | Meyerozyma caribbica and application thereof |
| CN113604369A (en) * | 2021-02-09 | 2021-11-05 | 吉林农业大学 | Hansenula polymorpha and application thereof |
-
2022
- 2022-02-24 CN CN202210170421.3A patent/CN114480151B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996025039A1 (en) * | 1995-02-17 | 1996-08-22 | The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University | Control of post-harvest fungal disease using saprophytic yeast |
| CN106566778A (en) * | 2016-11-03 | 2017-04-19 | 贵州师范学院 | Meyerozyma caribbica and application thereof |
| CN113604369A (en) * | 2021-02-09 | 2021-11-05 | 吉林农业大学 | Hansenula polymorpha and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 红茶菌中醋酸菌和酵母菌的分离鉴定及其相互作用;王洁琛;陈志周;王颖;刘冰;马倩云;;中国酿造(第03期);全文 * |
| 酵母菌菌株对普洱茶主要功能成分的影响;李雪玲;陈华红;张金丽;王波;周红杰;;食品研究与开发(第21期);全文 * |
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