Preparation method of ginseng pu'er tea

Abstract

The present application relates to a kind of ginseng pu'er tea preparation method, belong to fermentation tea technical field, the present application is extracted fermentation product after ginseng stem leaf powder is fermented by Pediococcus pentosaceus, Isachy orientalis and orange thermophilic sac fungi processing, join in pu'er ripe tea loose tea and be pressed into tight tea after being obtained ginseng pu'er tea.Rg3, CK and Rh2 content higher in ginseng pu'er tea prepared by the method of the present application.

Description

Technical Field

[0001] This invention belongs to the field of fermented tea technology, and specifically relates to a method for preparing ginseng Pu-erh tea. Background Technology

[0002] Most ginseng, whether wild or cultivated, is cultivated with a focus on its roots, while the stems and leaves are generally discarded as waste, resulting in a low recycling rate. Ginseng stems and leaves contain proto-saponins such as Rb1, Rb2, and Rc, but these have large molecular weights and high polarity, making them difficult to be directly absorbed by the intestines after oral administration.

[0003] Currently, most ginseng Pu-erh tea sold on the market is made by mixing ginseng root slices with Pu-erh tea. There are no reports of ginseng Pu-erh tea being made using ginseng stems and leaves as raw materials. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for preparing ginseng Pu-erh tea.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing ginseng Pu-erh tea, comprising the following steps: S1, Take dried ginseng stems and leaves, pulverize them to obtain ginseng stem and leaf powder; S2: Add 12-15 parts glucose, 4-5 parts yeast extract, 0.4-0.7 parts potassium dihydrogen phosphate, 0.15-0.25 parts magnesium sulfate, 0.8-1.2 parts Tween-80, and 1000 parts water to every 100 parts by weight of ginseng stem and leaf powder, adjust the pH to 6.5, and sterilize to obtain a mixed culture medium; S3, inoculate *Pediococcus pentosaceus* at 4%-5% of the mixed culture medium volume. Pediococcus pentosaceus Seed liquid, 32-35℃, 200r / min aerobic fermentation for 24 hours; After fermentation in steps S4 and S3 is complete, add 4%-6% *Issa mesasura* (Oriental Isaac yeast) to the mixed culture medium. Issatchenkia orientalis Seed liquid, adjust the temperature to 28-32℃, and ferment at 200r / min for 36 hours in a micro-aerobic manner; After fermentation in steps S5 and S4 is complete, inoculate with *Ascomycota orangeensis* at 2-3% of the culture medium volume. Thermoascus aurantiacus Spore suspension, adjust the temperature to 42-45℃, and aerobic ferment at 180r / min for 48 hours; S6, after fermentation is complete, filter, collect the filtrate, extract with 70% ethanol under reflux for 2 hours, concentrate under reduced pressure and spray dry to obtain ginseng stem and leaf extract. S7. Take loose Pu-erh ripe tea and ginseng stem and leaf extract in a mass ratio of 45-55:8; dissolve the ginseng stem and leaf extract in 1.5 times its mass of edible ethanol to obtain a mixture; spray the mixture into the loose Pu-erh ripe tea and stir evenly; press into compressed tea. S8, post-fermentation and sterilization: aged for 72 hours at 38-40℃ and 65% humidity, then sterilized by electron beam irradiation, to obtain ginseng Pu'er tea.

[0006] Further, the method for preparing the Pediococcus pentosaceus seed culture is as follows: The Pediococcus pentosaceus seed culture is prepared by inoculating the strain into a liquid culture medium containing: peptone: 10.0g, beef extract powder: 8.0g, yeast powder: 4.0g, glucose: 20.0g, magnesium sulfate: 0.2g, sodium acetate: 5.0g, diammonium hydrogen citrate: 2.0g, dipotassium hydrogen phosphate: 2.0g, manganese sulfate: 0.04g, Tween 80: 1.0g, and 1L of water, and incubating at 37℃ under slightly aerobic conditions (3%-5% O2, 10% CO2) for 48h.

[0007] Furthermore, the method for preparing the *Issa mesasula* seed culture is as follows: The *Issa mesasula* seed culture is prepared by inoculating the strain into a liquid culture medium containing: yeast extract: 3.0g, malt extract: 3.0g, glucose: 10.0g, casein peptone: 5.0g, and water: 1L, and then incubating aerobically at 30°C for 36h.

[0008] Furthermore, the method for preparing the *Ascomycota orangeensis* spore suspension is as follows: the *Ascomycota orangeensis* seed liquid is prepared by culturing on PDA slant medium at 45°C for 72 hours, washing off the spores with sterile water to obtain a concentration of 1×10⁻⁶. 8 CFU / mL spore suspension.

[0009] Furthermore, the specific method for vacuum concentration and spray drying in step S6 is as follows: vacuum concentration is carried out under vacuum conditions of -0.06 to -0.08 MPa and 55-70℃ until the relative density is 1.10-1.20. The concentrate is then spray dried, with the inlet air temperature set at 160-200℃ and the outlet air temperature at 70-90℃.

[0010] Beneficial effects: 1. This invention uses ginseng stems and leaves as raw materials to prepare ginseng Pu'er tea, which can realize waste utilization and increase the economic benefits of ginseng cultivation; 2. The ginseng Pu-erh tea prepared by the method of the present invention has a high content of Rg3, CK and Rh2. Detailed Implementation

[0011] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0012] In this invention, *Pediococcus pentosaceus*, *Issa mesasura*, and *Thermophilus orangeus* were all purchased from Beina Chuanglian Biotechnology Co., Ltd., with strain numbers BNCC192624, BNCC225398, and BNCC391490, respectively. The preparation method of Pediococcus pentosaceus seed culture is as follows: the strain is inoculated into a liquid culture medium containing: peptone: 10.0g, beef extract powder: 8.0g, yeast powder: 4.0g, glucose: 20.0g, magnesium sulfate: 0.2g, sodium acetate: 5.0g, diammonium hydrogen citrate: 2.0g, dipotassium hydrogen phosphate: 2.0g, manganese sulfate: 0.04g, Tween 80: 1.0g, and 1L of water, and cultured at 37℃ in a slightly aerobic (3%-5% O2, 10% CO2) environment for 48h.

[0013] The preparation method of the seed culture of *Issa mesasura* is as follows: the strain is inoculated into a liquid culture medium containing: yeast extract: 3.0g, malt extract: 3.0g, glucose: 10.0g, casein peptone: 5.0g, and water: 1L, and cultured aerobically at 30℃ for 36h.

[0014] The preparation method of the *Ascomycota orangeensis* seed culture is as follows: culture on PDA slant medium at 45℃ for 72 hours, wash away the spores with sterile water, and prepare a solution with a concentration of 1×10⁻⁶. 8 CFU / mL spore suspension.

[0015] Example 1 A method for preparing ginseng Pu-erh tea includes the following steps: S1, Take dried ginseng stems and leaves, pulverize them to obtain ginseng stem and leaf powder; S2: Add 14 parts glucose, 4.5 parts yeast extract, 0.5 parts potassium dihydrogen phosphate, 0.2 parts magnesium sulfate, 1 part Tween-80, and 1000 parts water to every 100 parts by weight of ginseng stem and leaf powder, adjust the pH to 6.5, and sterilize to obtain a mixed culture medium. S3, inoculate *Pediococcus pentosaceus* at 4.5% of the mixed culture medium volume. Pediococcus pentosaceus Seed liquid, 32-35℃, 200r / min micro-aerobic fermentation for 24 hours; After fermentation in steps S4 and S3 is complete, add 5% *Issa mesasura* (Oriental Isaac yeast) per unit volume of the mixed culture medium. Issatchenkia orientalis Seed liquid, adjust the temperature to 28-32℃, and aerobic ferment at 200r / min for 36 hours; After fermentation in steps S5 and S4 is complete, inoculate with *Ascomycota orangeensis* at 2.5% of the culture medium volume. Thermoascus aurantiacus Spore suspension, adjust the temperature to 42-45℃, and aerobic ferment at 180r / min for 48 hours; S6, after fermentation, filter, collect the filtrate, extract with 70% ethanol under reflux for 2 hours, concentrate under reduced pressure and spray dry to obtain ginseng stem and leaf extract; the specific method of concentration under reduced pressure and spray drying is as follows: concentrate under reduced pressure at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 55-70℃ to a relative density of 1.15, spray dry the concentrate, and set the inlet air temperature to 160-200℃ and the outlet air temperature to 70-90℃; S7. Take loose Pu-erh ripe tea and ginseng stem and leaf extract in a mass ratio of 50:8; dissolve the ginseng stem and leaf extract in 1.5 times its mass of edible ethanol to obtain a mixture; spray the mixture into the loose Pu-erh ripe tea and stir evenly; press into compressed tea. S8, post-fermentation and sterilization: aged for 72 hours at 38-40℃ and 65% humidity, then sterilized by electron beam irradiation, to obtain ginseng Pu'er tea.

[0016] Example 2 A method for preparing ginseng Pu-erh tea includes the following steps: S1, Take dried ginseng stems and leaves, pulverize them to obtain ginseng stem and leaf powder; S2, add 12 parts glucose, 4 parts yeast extract, 0.4 parts potassium dihydrogen phosphate, 0.15 parts magnesium sulfate, 0.8 parts Tween-80, and 1000 parts water to every 100 parts ginseng stem and leaf powder, adjust the pH to 6.5, and sterilize to obtain a mixed culture medium; S3, inoculate *Pediococcus pentosaceus* at 4% of the mixed culture medium volume. Pediococcus pentosaceus Seed liquid, 32-35℃, 200r / min aerobic fermentation for 24 hours; After fermentation in steps S4 and S3 is complete, add 4% *Issa mesasura* (Oriental Isaac yeast) to the mixed culture medium. Issatchenkia orientalis Seed liquid, adjust the temperature to 28-32℃, and ferment at 200r / min for 36 hours in a micro-aerobic manner; After fermentation in steps S5 and S4 is complete, inoculate with *Ascomycota orangeensis* at 2% of the culture medium volume. Thermoascus aurantiacus Spore suspension, adjust the temperature to 42-45℃, and aerobic ferment at 180r / min for 48 hours; S6, after fermentation, filter, collect the filtrate, extract with 70% ethanol under reflux for 2 hours, concentrate under reduced pressure and spray dry to obtain ginseng stem and leaf extract; the specific method of concentration under reduced pressure and spray drying is as follows: concentrate under reduced pressure at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 55-70℃ to a relative density of 1.10, spray dry the concentrate, set the inlet air temperature to 160-200℃ and the outlet air temperature to 70-90℃.

[0017] S7. Take loose Pu-erh ripe tea and ginseng stem and leaf extract in a mass ratio of 45:8; dissolve the ginseng stem and leaf extract in 1.5 times its mass of edible ethanol to obtain a mixture; spray the mixture into the loose Pu-erh ripe tea and stir evenly; press into compressed tea. S8, post-fermentation and sterilization: aged for 72 hours at 38-40℃ and 65% humidity, then sterilized by electron beam irradiation, to obtain ginseng Pu'er tea.

[0018] Example 3 A method for preparing ginseng Pu-erh tea includes the following steps: S1, Take dried ginseng stems and leaves, pulverize them to obtain ginseng stem and leaf powder; S2: Add 15 parts glucose, 5 parts yeast extract, 0.7 parts potassium dihydrogen phosphate, 0.25 parts magnesium sulfate, 1.2 parts Tween-80, and 1000 parts water to every 100 parts by weight of ginseng stem and leaf powder, adjust the pH to 6.5, and sterilize to obtain a mixed culture medium. S3, inoculate *Pediococcus pentosaceus* at 5% of the volume of the mixed culture medium. Pediococcus pentosaceus Seed liquid, 32-35℃, 200r / min micro-aerobic fermentation for 24 hours; After fermentation in steps S4 and S3 is complete, add 6% *Issa mesasura* (Oriental Isaac yeast) to the mixed culture medium. Issatchenkia orientalis Seed liquid, adjust the temperature to 28-32℃, and aerobic ferment at 200r / min for 36 hours; After fermentation in steps S5 and S4 is complete, inoculate with *Ascomycota orangeensis* at 3% of the culture medium volume. Thermoascus aurantiacus Spore suspension, adjust the temperature to 42-45℃, and aerobic ferment at 180r / min for 48 hours; S6, after fermentation, filter, collect the filtrate, extract with 70% ethanol under reflux for 2 hours, concentrate under reduced pressure and spray dry to obtain ginseng stem and leaf extract; the specific method of concentration under reduced pressure and spray drying is as follows: concentrate under reduced pressure at a vacuum degree of -0.06 to -0.08 MPa and a temperature of 55-70℃ to a relative density of 1.20, spray dry the concentrate, and set the inlet air temperature to 160-200℃ and the outlet air temperature to 70-90℃.

[0019] S7. Take loose Pu-erh ripe tea and ginseng stem and leaf extract in a mass ratio of 55:8; dissolve the ginseng stem and leaf extract in 1.5 times its mass of edible ethanol to obtain a mixture; spray the mixture into the loose Pu-erh ripe tea and stir evenly; press into compressed tea. S8, post-fermentation and sterilization: aged for 72 hours at 38-40℃ and 65% humidity, then sterilized by electron beam irradiation, to obtain ginseng Pu'er tea.

[0020] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that it does not include steps S4 and S5.

[0021] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that it does not include steps S3 and S5.

[0022] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that it does not include step S5. Experimental Example I. Determination of Ginsenoside Content (HPLC Method) The ginseng Pu-erh tea prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were used. Each treatment was repeated three times. For each repeated treatment, 5g of tea sample was added to 250mL of boiling water and steeped for 5min. After filtration, the samples were tested. The results are shown in Table 1 below.

[0023] Table 1. Ginsenoside content of each treatment As can be seen from Table 1 above, the preparation method of this application can increase the content of Rg3, Rh2 and CK in ginseng Pu-erh tea.

[0024] II. Animal experiments on antioxidant and immune regulation (D-galactose-induced aging mouse model) Take the ginseng Pu-erh tea prepared in Examples 1 to 3 and Comparative Examples 1 to 3, add 5g of the tea to 250mL of boiling water, cover and steep for 5 minutes, shaking once every 1 minute during steeping. After steeping, filter with gauze or filter paper and collect the filtrate. Add another 250mL of boiling water, treat in the same way, combine the two filtrates, and concentrate the filtrate to 5mL to obtain a concentration of 1g crude drug / mL.

[0025] According to the FDA's "Guidance on Estimating the Maximum Safe Starting Dose for Initial Clinical Treatment Trials in Healthy Adult Volunteers" and "Pharmacological Experimental Methodology," the conversion factor for body surface area between humans and mice is approximately 12.3.

[0026] Recommended daily tea intake for humans: 5-10g of tea leaves Based on a 60 kg adult, the human dose is approximately 0.083-0.167 g / kg. The equivalent dose in mice = human dose × 12.3 ≈ 1.02-2.05 g / kg The 10g crude drug / kg used in this invention is approximately 5-10 times the recommended human dose, for the following reasons: Purpose of pharmacodynamic validation: Higher doses make it easier to observe significant differences and demonstrate product effectiveness; Safety window assessment: Simultaneously verify that the product has no toxic effects at high doses; Dosage regimen: The concentrated tea (equivalent to 10 g / kg·d of raw medicinal material) from Examples 1-3 and Comparative Examples 1-3 was administered by gavage at a dose of 10 mL / kg body weight. The blank control group and model group were administered an equal volume of distilled water by gavage. Administration was once daily for 6 consecutive weeks. Each group consisted of 15 mice, with three replicates per group. After gavage, the thymus index, spleen index, serum SOD, serum MDA, and IL-2 were measured. The results are shown in Table 2 below.

[0027] Table 2. Statistical table of thymus index, spleen index, serum SOD, serum MDA and IL-2 in mice. As can be seen from Table 2 above, Examples 1 to 3 are superior to Comparative Examples 1 to 3 in terms of restoring immune organ index and reducing lipid peroxidation product MDA. In particular, the recovery of IL-2 level is close to that of the normal control group. The experimental group has a synergistic enhancing effect on T cell immune function, which is not achieved by single-strain fermentation or simple compound preparation.

[0028] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for preparing ginseng Pu-erh tea, characterized in that: It includes the following steps: S1, Take dried ginseng stems and leaves, pulverize them to obtain ginseng stem and leaf powder; S2: Add 12-15 parts glucose, 4-5 parts yeast extract, 0.4-0.7 parts potassium dihydrogen phosphate, 0.15-0.25 parts magnesium sulfate, 0.8-1.2 parts Tween-80, and 1000 parts water to every 100 parts by weight of ginseng stem and leaf powder, adjust the pH to 6.5, and sterilize to obtain a mixed culture medium; S3, inoculate *Pediococcus pentosaceus* at 4%-5% of the mixed culture medium volume. Pediococcus pentosaceus Seed liquid, 32-35℃, 200r / min aerobic fermentation for 24 hours; After fermentation in steps S4 and S3 is complete, add 4%-6% *Issa mesasura* (Oriental Isaac yeast) to the mixed culture medium. Issatchenkia orientalis Seed liquid, adjust the temperature to 28-32℃, and ferment at 200r / min for 36 hours in a micro-aerobic manner; After fermentation in steps S5 and S4 is complete, inoculate with *Ascomycota orangeensis* at 2-3% of the culture medium volume. Thermoascus aurantiacus Spore suspension, adjust the temperature to 42-45℃, and aerobic ferment at 180r / min for 48 hours; S6, after fermentation is complete, filter, collect the filtrate, extract with 70% ethanol under reflux for 2 hours, concentrate under reduced pressure and spray dry to obtain ginseng stem and leaf extract. S7. Take loose Pu-erh ripe tea and ginseng stem and leaf extract in a mass ratio of 45-55:8; dissolve the ginseng stem and leaf extract in 1.5 times its mass of edible ethanol to obtain a mixture; spray the mixture into the loose Pu-erh ripe tea and stir evenly; press into compressed tea. S8, post-fermentation and sterilization: aged for 72 hours at 38-40℃ and 65% humidity, then sterilized by electron beam irradiation, to obtain ginseng Pu'er tea.

2. The method for preparing ginseng Pu-erh tea according to claim 1, characterized in that: The method for preparing the Pediococcus pentosaceus seed culture is as follows: The Pediococcus pentosaceus seed culture is prepared by inoculating the strain into a liquid culture medium containing: peptone: 10.0g, beef extract powder: 8.0g, yeast powder: 4.0g, glucose: 20.0g, magnesium sulfate: 0.2g, sodium acetate: 5.0g, diammonium hydrogen citrate: 2.0g, dipotassium hydrogen phosphate: 2.0g, manganese sulfate: 0.04g, Tween 80: 1.0g, and water 1L, and incubating at 37℃ for 48h in a slightly aerobic manner.

3. The method for preparing ginseng Pu-erh tea according to claim 1, characterized in that: The method for preparing the seed culture of *Isaccharomyces cerevisiae* is as follows: The strain is inoculated into a liquid culture medium containing: yeast extract: 3.0g, malt extract: 3.0g, glucose: 10.0g, casein peptone: 5.0g, and water: 1L, and cultured aerobically at 30℃ for 36h.

4. The method for preparing ginseng Pu-erh tea according to claim 1, characterized in that: The method for preparing the orange thermophilic ascomycete spore suspension is as follows: The method for preparing the orange thermophilic ascomycete seed liquid is as follows: culturing on PDA slant medium at 45℃ for 72 hours, washing off the spores with sterile water to prepare a solution with a concentration of 1×10⁻⁶. 8 CFU / mL spore suspension.

5. The method for preparing ginseng Pu-erh tea according to claim 1, characterized in that: The specific method for vacuum concentration and spray drying in step S6 is as follows: vacuum concentration is carried out under vacuum conditions of -0.06 to -0.08 MPa and 55-70℃ until the relative density is 1.10-1.

20. The concentrate is then spray dried, with the inlet air temperature set at 160-200℃ and the outlet air temperature at 70-90℃.

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