CN114149925B - Application method of paecilomyces hepiali S2 in tea planting - Google Patents
Application method of paecilomyces hepiali S2 in tea planting Download PDFInfo
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- CN114149925B CN114149925B CN202111313956.3A CN202111313956A CN114149925B CN 114149925 B CN114149925 B CN 114149925B CN 202111313956 A CN202111313956 A CN 202111313956A CN 114149925 B CN114149925 B CN 114149925B
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Classifications
-
- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
-
- 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
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
Abstract
The invention provides an application method of paecilomyces hepiali S2 in tea planting, which adopts a preservation number of CCTCC NO separated from cordyceps sinensis: the culture ferment of M2021317 Paecilomyces hepiali S2 is applied to tea planting. The invention has the beneficial effects that: obviously increases the sprouting density of tea, promotes the growth of tea, and improves the yield of tea and the content of tea polyphenol.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application of paecilomyces hepiali S2 in tea planting.
Background
Cordyceps sinensis is a traditional rare Chinese medicine in China, and has sweet taste, mild nature, and kidney and lung meridian tropism. Has effects in invigorating lung yin, tonifying kidney yang, replenishing qi, relieving cough and eliminating phlegm, and can be used for treating cough, hemoptysis, sexual impotence, spermatorrhea, soreness of waist and knees, and asthenia after illness. The pharmacological research of modern medicine shows that the main pharmacological components of Cordyceps sinensis are adenosine, uridine, cordycepin, cordycepic acid, sterol and peptide. Has effects of reducing blood lipid, resisting cancer, preventing arteriosclerosis, enhancing immunity, relieving fatigue, and improving renal insufficiency.
In recent years, a plurality of medicinal fungi such as hirsutella sinensis and paecilomyces hepialid are separated from wild cordyceps sinensis in a plurality of units at home and abroad, and fermented cordyceps sinensis is cultivated by a biological fermentation technology to obtain a large number of cordyceps sinensis cultures, the main medicinal components of the cordyceps sinensis cultures are similar to those of natural cordyceps sinensis, cultures of hirsutella sinensis and paecilomyces hepialid are carried in pharmacopoeia 2007, 2010, 2015 and 2018, and the cultures of hirsutella sinensis and paecilomyces hepialid are used for preparing the medicines of a larch capsule and a Jinshuibao, and the medicines are applied to clinical treatment of cough, hemoptysis, impotence, spermatorrhea, soreness of waist and knees and asthenia after illness. Chinese patent No. CN201210485017.1, a health product of Cordyceps and its preparation method, chinese patent No. CN201310277019.6, a medicated liquor of Cordyceps powder and its preparation method, and Chinese patent No. CN201210257893.4, a health liquor of Cordyceps and its preparation method, CN201510488835.7, a health liquor of Cordyceps and its preparation method, disclose the application of Cordyceps as health product.
Some researches for years relate to the application of cordyceps sinensis as a feed additive, CN1079204C cordyceps fungus feed additive and a production method thereof, CN 1348703A cordyceps fungus powder green feed additive, CN1013381615B is used for producing the cordyceps sinensis feed additive and products thereof, liquid or solid tray fermentation is used for culturing a large amount of cordyceps sinensis to obtain cordyceps fungus mycelia separated from cordyceps sinensis and producing the cordyceps sinensis mycelia, and the feed additive is prepared. However, application of Cordyceps sinensis in tea planting is disclosed.
Disclosure of Invention
According to the defects of the prior art, the invention exploits a new application of the cordyceps sinensis fermentation product, and provides a method for improving the yield and the quality of tea by applying the cordyceps sinensis fermentation product in tea planting, which is realized by the following technical scheme.
An application method of Paecilomyces hepialid S2 in tea planting adopts a culture ferment of Paecilomyces hepialid S2 separated from Cordyceps sinensis, and is applied to tea planting, wherein Paecilomyces hepialid S2 is preserved in China Center for Type Culture Collection (CCTCC) of university of Wuhan, and the preservation number is CCTCC NO: m2021317.
The paecilomyces hepialid S2 of the cordyceps sinensis is cultured and fermented in a common culture medium containing a carbon source, a nitrogen source and inorganic salt, and the culture fermentation product is obtained and applied to the tea planting process.
The preparation of the paecilomyces hepialid S2 culture ferment of the cordyceps sinensis comprises the following steps:
inoculating Paecilomyces hepialid S2 strain into a seed culture medium, and culturing in a shake flask at a temperature of 22-25 ℃ and a rotating speed of 180-200r/min for 38-46 hours to obtain seed liquid, wherein the concentration of thalli is 0.9-1.2g/100ml, and the shake flask capacity is 150ml/500ml;
inoculating seed liquid obtained by shake flask culture into a liquid fermentation medium according to the proportion of 10-15% of liquid volume ratio, and culturing for 32-44 hours at the temperature of 21-25 ℃ and the rotating speed of 180-200r/min to obtain paecilomyces hepiali S2 fermentation liquid;
adding 42-95% ethanol according to volume of Paecilomyces hepiali S2 fermentation liquor to make ethanol content of S2 fermentation liquor be 8-30%, homogenizing under 100Mpa pressure, then ultrasonic treating, finally adding acetic acid into ultrasonic treating liquor, stirring uniformly to make pH value of acid-base of the liquor be 3.6-4.4, so as to obtain the invented Paecilomyces hepiali S2 culture fermentation liquor preparation.
The seed culture medium comprises glucose, dextrin and starch as carbon source, peptone, bean cake powder and pupa Bombycis powder as nitrogen source, and MgSO 4 、KH 2 PO 4 Is a mineral element.
The liquid fermentation medium comprises the following components: glucose 0.8-1.2%, dextrin 1-1.5%, starch 0.6-1.0%, peptone 0.9-1.5%, bean cake powder 0.5-1.0%, pupa Bombycis powder 0.7-1.3%, and MgSO 4 0.05-0.07％、KH 2 PO 4 0.08-0.13％。
The beneficial effects of the invention are as follows:
1. the fermentation product of the paecilomyces hepialid S2 can obviously improve the bud ratio of the tea and the yield of the tea, can also improve the content of tea polyphenol, and exploits the new application of the cordyceps sinensis.
2. The paecilomyces hepialid S2 is prepared by using few raw materials, low in consumption, convenient in source, short in culture and fermentation period and low in cost, can be prepared by using common fermentation equipment, and is easy to realize industrial production.
3. In the existing tea production process, the fertilization method and the dosage of planting are often improved, or a growth and development regulator is adopted, the cordyceps sinensis Paecilomyces hepiali S2 is separated from cordyceps sinensis, and a fermentation product is nontoxic and harmless to human bodies, is a safe and green organic product, and provides a new selection method for planting the green organic tea.
Detailed Description
Embodiments of the invention are described in detail below with reference to the attached drawings, but the invention can be implemented in a number of different ways, which are defined and covered by the claims.
In the embodiment of the invention, the cordyceps sinensis bacteria adopted in the experiment are cordyceps sinensis medicinal fungi S2 obtained by repeated separation and purification after surface sterilization from wild natural fresh cordyceps sinensis in the Jade tree area of Qinghai province, and the cordyceps sinensis bacteria S2 is paecilomyces hepiali Paecilomyces hepialid which is obtained from the Jade tree area of Qinghai province through colony morphology, microscopic characteristics and rRNA gene ITS2-5.8S-1TS2 region sequence determination results and is preserved in China center for type culture collection of microorganisms of university of Wuhan and with the preservation number of CCTCC NO of M20211317.
The preparation of the paecilomyces hepialid S2 culture ferment of the cordyceps sinensis is carried out by the following steps:
inoculating the Paecilomyces hepialid S2 strain into a seed culture medium with glucose, dextrin and starch as carbon sources, peptone, bean cake powder and silkworm chrysalis powder as nitrogen sources and MgSO4 and KH2PO4 as mineral elements, and culturing in a shake flask at the speed of 180-200r/min at 22-25 ℃ for 38-46 hours to obtain seed liquid with the thallus concentration of 0.9-1.2g/100ml and the shake flask capacity of 150ml/500ml.
Inoculating seed liquid obtained by shake flask culture into a liquid fermentation culture medium according to the liquid volume ratio of 10-15%, wherein the culture medium comprises the following components: glucose 0.8-1.2%, dextrin 1-1.5%, starch 0.6-1.0%, peptone 0.9-1.5%, bean cake powder 0.5-1.0%, silkworm chrysalis powder 0.7-1.3%, mgSO40.05-0.07%, KH2PO40.08-0.13%, and culturing at 21-25 deg.C and rotation speed 180-200r/min for 32-44 hr to obtain Paecilomyces hepiali S2 fermentation broth.
Adding appropriate amount of 42-95% ethanol to make ethanol content of S2 fermentation broth 8-30%, homogenizing under 100Mpa pressure, then performing ultrasonic treatment, adding appropriate amount of acetic acid into the ultrasonic treatment solution, and stirring to pH value of 3.6-4.4 to obtain Paecilomyces hepiali S2 fermentation preparation.
The test design scheme of the paecilomyces hepiali S2 applied to the tea leaves is carried out according to the design scheme of a unified test block set formulated in the fertilizer test process:
the block design in the test scheme is totally designed with four treatments, each treatment is repeated 2 times, and the total area of the test plot area is 5 mu. Four treatment groups of a protective row and a test component A, B, C, D are arranged among groups of the test group, wherein the treatment group A is a blank control group, clear water is applied, the test group B is 30 times of diluent of the paecilomyces hepialid S2 fermentation preparation, the test group C is 60 times of diluent of the paecilomyces hepialid S2 fermentation preparation, and the test group D is 2100 times of diluent of the paecilomyces hepialid S.
The Sichuan tea production area is selected in a test field, the soil fertility is uniform, the pH value of the soil is 5.3, the organic matter content is 27, the total nitrogen is 0.19, the alkali nitrogen is 125, the total phosphorus is 0.65, the available phosphorus is 25, the total potassium is 13.6, the quick-acting potassium is 139, good watering facilities are provided, the test tea tree is a local main variety, and the field management of the test field, such as fertilization, irrigation and pest control, is performed according to the conventional management of the field in the test period.
Test data acquisition and processing: according to the conventional test method, 4 pieces of tea leaves in each 1 square ruler are firstly investigated by a random sampling method in a delimited area of a test land, the weight of hundred buds is measured, then new germinated tea leaves in the delimited area are collected according to a bud-by-leaf collection standard, weighing and metering are carried out, the investigation is carried out once a week in the full germination period of the tea leaves, and the obtained investigation data are subjected to data statistical analysis according to a general mathematical method.
Analysis of tea quality: and (3) analyzing tea polyphenol and free amino acid of the tea, wherein the content of the tea polyphenol in the tea is determined by adopting a conventional ferric tartrate colorimetric method. The analysis of free amino acid in tea leaves is determined by adopting a high-pressure liquid chromatography method, wherein the analysis is performed by adopting reverse-phase high-performance liquid chromatography after phenyl isothiocyanate is subjected to pre-column derivatization, and the model of a chromatographic column is Shim-pack vp-odsc, and the mobile phase A is: 0.1mol/L sodium acetate-acetonitrile (97:3) buffer, B: acetonitrile-water (4:1) solution, the column temperature is 40 ℃, the sample injection amount is 10ul, and the linear gradient elution operation is carried out under the chromatographic operation condition of 254nm detection wavelength, and the result shows that the method has good separation effect on 18 amino acids in tea under the above operation condition, and the correlation coefficient is 0.994.
Example 1
The paecilomyces hepiali S2 (Paecilomyes hepialiS 2) separated from Cordyceps sinensis is preserved in China general microbiological culture collection center (CCTCC NO) of university of Wuhan with the preservation number of CCTCC NO: m2021317.
Seed culture medium composition (%): glucose 1.3, dextrin 0.6, starch 0.3, peptone 0.9, bean cake powder 1.2, pupa Bombycis powder 0.6, mgSO 4 0.06、KH 2 PO 4 And 0.12, the pH of the culture medium is natural. The shake flask has a capacity of 500ml, the liquid amount of the seed culture medium is 120ml, the preserved and activated fungus blocks are inoculated into the sterilized shake flask seed culture medium, and the seed liquid is obtained by continuous culture for 42 hours under the conditions of a culture temperature of 23 ℃ and a rotating speed of 180 ℃.
Then, the seed liquid with 15 percent of volume ratio is transplanted into a 500ml triangular flask filled with 140ml of fermentation medium, and the continuous fermentation is finished for 37 hours under the conditions of 24 ℃ of fermentation temperature and 190r/min of rotation speed, so that the paecilomyces hepialid S2 fermentation liquid is measured by adopting a high-pressure liquid chromatography.
The results show that compared with the products of enterprises such as Jiangxi, jiangsu, zhejiang, qinghai and the like in China, the S2 fermentation product contains high content of adenosine, uridine, ergosterol and 18 amino acids. In addition, in the S2 fermentation product, 15 kinds of cyclic dipeptides such as high content of cyclo- (Gly-Pro), cyclo- (Pro-Ala), cyclo- (Ala-Val), cyclo- (Pro-Tyr), cyclo- (Pro-Val), cyclo- (Ala-Ile), cyclo- (PHe-Ala), cyclo- (Pro-Leu), cyclo- (Ile-Val), cyclo- (P He-Val), cyclo- (Leu-Ile), cyclo- (PHe-Ile), and cyclo- (PHe-PHe) were detected. Wherein cyclo- (Pro-Leu) is a cyclic dipeptide of both the alpha and beta configurations.
Adding 42% pure grain to the fermentation broth to brew Chinese liquor to make the alcohol content of the fermentation broth reach 13%, cooling in a 4-7% (v/v) refrigerator, ultrasonically extracting with ultrasonic extractor at rotation speed of 3r/min and power of 35KHZ for 35min, and adding edible vinegar to adjust pH of the S2 fermentation product to 3.8-4.2.
Example two
Promotion of germination by paecilomyces hepiali S2 culture ferment: the test design is carried out by formulating a unified test scheme according to the fertilizer test process, the design of the granule is carried out by designing four treatments in total, each treatment is repeated for 2 times, and the total area of the test plot is 5 mu. The test group A, B, C, D comprises four treatment groups, wherein the treatment group A is a blank control group, clear water is applied, the test group B is 30 times of diluent of the paecilomyces hepialid S2 fermentation preparation, the test group C is 60 times of diluent of the paecilomyces hepialid S2 fermentation preparation, and the test group D is 2100 times of diluent of the paecilomyces hepialid S.
The Sichuan tea production area is selected in a test field, the soil fertility is uniform, the pH value of the soil is 5.3, the organic matter content is 27, the total nitrogen is 0.19, the alkali nitrogen is 125, the total phosphorus is 0.65, the available phosphorus is 25, the total potassium is 13.6, the quick-acting potassium is 139, good watering facilities are provided, the test tea tree is a local main variety, and the field management of the test field, such as fertilization, irrigation and pest control, is performed according to the conventional management of the field in the test period.
Test data acquisition and processing: the method is carried out according to a conventional test method, namely, 4 blocks are firstly investigated by a random sampling method in a delimited block of a test land, each block is 1 square, the bud number of tea leaves is investigated, the weight of hundred buds is measured, the investigation is carried out once a week in the full germination period of the tea leaves, and the investigation data obtained by the investigation are subjected to data statistics analysis according to a general mathematical method, and the results are shown in table 1.
TABLE 1S2 effect of ferment on germination of tea leaves during the full-bloom stage
Example III
Effects of Paecilomyces hepiali S2 culture ferment on tea yield and tea quality: the experimental design and protocol was essentially as described in example 2.
The test design makes a unified test block design scheme according to the fertilizer test process, four treatments are designed in total, each treatment is repeated 2 times, and the total area of the test field cells is 5 mu. The test group A, B, C, D comprises four treatment groups, wherein the treatment group A is a blank control group, clear water is applied, the test group B is 30 times of diluent of the paecilomyces hepialid S2 fermentation preparation, the test group C is 60 times of diluent of the paecilomyces hepialid S2 fermentation preparation, and the test group D is 2100 times of diluent of the paecilomyces hepialid S.
The Sichuan production area is selected in a test field, the soil fertility is uniform, the pH value of the soil is 5.3, the organic matter content is 27, the total nitrogen is 0.19, the alkali nitrogen is 125, the total phosphorus is 0.65, the available phosphorus is 25, the total potassium is 13.6, the quick-acting potassium is 139, good watering facilities are provided, the test tea tree is a local main variety, and the field management such as fertilization, irrigation and pest control of the test field are performed according to the conventional management of the field in the test period.
Test data acquisition and processing: according to a conventional test method, namely, in a delimited area of a test land, 4 pieces are firstly investigated by a random sampling method, each piece is 1 square, newly germinated tea leaves in the delimited area are collected according to a collection standard of one leaf per bud, weighing and metering are carried out, the obtained investigation data are investigated once a week in the full germination period of the tea leaves, and data statistics analysis is carried out according to a general mathematical method, and the results are shown in tables 2-3.
TABLE 2 effect of S2 on tea yield increase
TABLE 3 influence of S2 on tea chemical composition
| Treatment of | Water extract% | Rate of change% | Tea polyphenols% | Rate of change% | Free amino acids% | Rate of change% | Caffeine% | Rate of change% |
| CK | 40.1 | 10.9 | 4.9 | 3.9 | ||||
| 100-fold dilution | 40.7 | +1.4％ | 13.2 | +12.1％ | 5.1 | +4％ | 4.0 | +2.5％ |
| 70-fold diluent | 40.8 | +1.7％ | 14.0 | +28.4％ | 5.3 | 8.2％ | 4.1 | +5％ |
| 30-fold dilution | 42.3 | +5.5％ | 15.7 | +44％ | 5.7 | 16.3％ | 4.2 | +7.7％ |
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. An application method of paecilomyces hepialid S2 for improving the bud ratio and the yield of tea leaves in tea planting is characterized in that a preparation of a culture ferment of paecilomyces hepialid S2 separated from cordyceps sinensis is adopted and applied to tea planting; the paecilomyces hepialid S2 is preserved in China center for type microbiological collection center of Wuhan university, and the preservation number is CCTCC NO: m2021317;
the preparation of the culture ferment of the paecilomyces hepiali S2 comprises the following steps:
inoculating Paecilomyces hepialid S2 strain into a seed culture medium, and culturing in a shake flask at a temperature of 22-25 ℃ and a rotating speed of 180-200r/min for 38-46 hours to obtain seed liquid, wherein the concentration of thalli is 0.9-1.2g/100ml, and the shake flask capacity is 150ml/500ml;
inoculating seed liquid obtained by shake flask culture into a liquid fermentation medium according to the proportion of 10-15% of liquid volume ratio, and culturing for 32-44 hours at the temperature of 21-25 ℃ and the rotating speed of 180-200r/min to obtain paecilomyces hepiali S2 fermentation liquid;
adding 42-95% ethanol according to the volume of the Paecilomyces hepiali S2 fermentation liquor to ensure that the ethanol content of the S2 fermentation liquor is 8-30%, homogenizing under 100Mpa pressure, then carrying out ultrasonic treatment, finally adding acetic acid into the ultrasonic treatment liquor, and stirring uniformly to ensure that the pH value of acid-base of the solution is 3.6-4.4, thus obtaining the preparation of the culture fermentation product of the Paecilomyces hepiali S2.
2. The method according to claim 1, wherein the seed culture medium comprises glucose, dextrin, and starch as carbon source, peptone, bean cake powder, and pupa Bombycis powder as nitrogen source, and MgSO 4 、KH 2 PO 4 Is a mineral element.
3. The method of claim 1, wherein the liquid fermentation medium composition is: glucose 0.8-1.2%, dextrin 1-1.5%, starch 0.6-1.0%, peptone 0.9-1.5%, bean cake powder 0.5-1.0%, pupa Bombycis powder 0.7-1.3%, and MgSO 4 0.05-0.07%、KH 2 PO 4 0.08-0.13%。
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