CN107156346A - Fine and soft ganoderma tea of dance and preparation method thereof - Google Patents
Fine and soft ganoderma tea of dance and preparation method thereof Download PDFInfo
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- CN107156346A CN107156346A CN201710338778.7A CN201710338778A CN107156346A CN 107156346 A CN107156346 A CN 107156346A CN 201710338778 A CN201710338778 A CN 201710338778A CN 107156346 A CN107156346 A CN 107156346A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/14—Tea preparations, e.g. using additives
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention discloses fine and soft ganoderma tea of one kind dance and preparation method thereof.The fine and soft ganoderma tea of dance is made of with the raw material of following weight proportions, 150 parts of black tea, 25 75 parts of Effects of Extracts of Grifola frondosa on Active, 75 25 parts of Ganodenna Lucidum P.E.The black tea powder, Effects of Extracts of Grifola frondosa on Active and Ganodenna Lucidum P.E of sieving were mixed after certain time, ethanol in proper amount is added, is well mixed, sieving granulation；Sieved again by drying, obtain finished product.The present invention has instant, mouthfeel suitable, and effective active composition is easier to be absorbed by human body, can strengthen immunity the characteristics of, provide more multi items for health protection tea, be easy to consumer to select.
Description
Technical field
The present invention relates to a kind of preparation method of health protection tea, particularly a kind of fine and soft ganoderma tea of dance and preparation method thereof.
Background technology
Black tea is one of teas with strongest influence power in Chinese six big teas, China be produce and drink earliest in the world it is red
, just there is the record of production black tea in the country of tea early in China of Ming Dynasty initial stage (1368-1375).Substantial amounts of research report is confirmed
Antitumor, anti-mutation, suppression pathogen, the antiviral effect of black tea colorant, and it is many to cardiovascular and cerebrovascular disease etc.
Preventive and therapeutic action.The catechin and the antioxidation activity of theaflavin contained in black tea is even better than glutathione, Vitamin C
Acid and tocopherol.There are some researches show, the antioxidation activity of contained theaflavin is no less than contained catechin in green tea in black tea, and
And catechin does not significantly change its activity for removing free radical to the conversion of theaflavin during black tea processing and fermentation.Separately there is text
Report is offered, black tea polyphenols have the effect for suppressing aortic endothelial cell propagation, there is antitumor, anti-cancer.With red
Raising of the medical evidence and people of tea healthcare function to health perception, it is with unique qualitative characteristics in addition, and black tea is also got over
Favored to get over by common people.
Ganoderma lucidum is Polyporaceae (Polyporaceae) fungi red sesame (Leyss.exFr.) Karst. or purple sesame
G.sinense Zhao, Xu et Zhang drying fructification.Ganoderma lucidum is the treasure in motherland's pools of traditional Chinese medicine, is have " celestial
The reputation of grass ".The fructification of ganoderma lucidum has strong pharmacological activity, there is analgesic, calmness, removing toxic substances, can be with antitumor, tune
Immunity, reduction blood glucose are saved, there is nourishing and fit keeping function, promote longevity.The pharmaceutical component of ganoderma lucidum enriches very much, wherein effectively
Composition includes GL-B, triterpene compound, amino acid, protein, alkaloid and trace element etc., each active ingredient it
Between have synergy.
Grifola frondosus (Grifola frondosa) is subordinate to Basidiomycotina, Hymenomycetes, Holobasidiomycetidae, non-gill fungus
Mesh, Polyporaceae, tree flower Pseudomonas, also known as polyporus frondosus, chestnut mushroom, lotus flower bacterium, it is fine and soft that Japan is commonly called as dance.Grifola frondosus is subtropical zone
Macro fungi into temperate forests, it is nutritious with unique fragranced, delicious in taste and living containing abundant biology
Property material.In recent years, the research of the fungi polysaccharide as BRM has been carried out in a deep going way both at home and abroad, grifolan shows
The curative effect of work is also gradually recognized by people.Find that grifolan has obvious by substantial amounts of chemistry and pharmacology analysis
Antitumor, anti HIV-1 virus improves function of immune system, regulation blood glucose, blood fat and cholesterol levels, hypotensive activity, to preventing
The generation and transfer of cancer cell, AIDS, hypertension, obesity, the disorder of diabetes and function of immune system have certain
Curative effect, it is and without any side effects.Therefore, grifola frondosus is a kind of precious pharmaceutical fungi and edible fungi of great Development volue.
Health protection tea is that based on tea, equipped with appropriate Chinese medicine, existing tea flavour, there is slight flavour of a drug again, and have health protection and therapeutic action
Beverage.Health protection tea is combined because rich in tealeaves nutritional ingredient, effective component, being matched somebody with somebody assistant raw material special component, and with now
The ideological trend gone back to nature in the world is coincide, thus is welcome by suitable crowd.The development of health protection tea, with the hair of society
Exhibition, scientific and technological progress is improved constantly, and various health protection teas emerge in large numbers like the mushrooms after rain.But current health protection tea manufacture, Bu Shaodou
Rest on and indiscriminately imitate ancient prescription, or using a certain teas as raw material, simple medication is combined into, and lacks scientific experiment, detection and analysis, causes health care
The reputation of tea is affected.Moreover, the health protection tea species of in the market is various, effect emphasizes particularly on different fields, and taste also differs widely.It is full
The demand of sufficient different consumer groups, the kind of more health protection teas can be more convenient the selection of people.
The content of the invention
The purpose of the present invention, is to provide fine and soft ganoderma tea of a kind of dance and preparation method thereof.The present invention has instant, mouthfeel
Properly, effective active composition be easier absorbed by human body, can strengthen immunity the characteristics of, provide more product for health protection tea
Kind, it is easy to consumer to select.
What the present invention was realized in.The fine and soft ganoderma tea of dance, it be made of with the raw material of following weight proportions,
150 parts of black tea,
20-80 parts of Effects of Extracts of Grifola frondosa on Active,
80-20 parts of Ganodenna Lucidum P.E.
The fine and soft ganoderma tea of foregoing dance, it is characterised in that：It is made of with the raw material of following weight proportions,
150 parts of black tea,
25-80 parts of Effects of Extracts of Grifola frondosa on Active,
80-25 parts of Ganodenna Lucidum P.E.
The fine and soft ganoderma tea of foregoing dance, it is characterised in that：It is made of with the raw material of following weight proportions,
150 parts of black tea,
25-75 parts of Effects of Extracts of Grifola frondosa on Active,
75-25 parts of Ganodenna Lucidum P.E.
The preparation method of the foregoing fine and soft ganoderma tea of dance, is prepared according to the following steps；
A, black tea sterilizing, crushing, obtain A product；
B, Effects of Extracts of Grifola frondosa on Active sieving, obtain B product；
C, Ganodenna Lucidum P.E sieving, obtain C product；
D, A product, B product and C product are placed in mixer, after mixing D product；
E, finished product that D product are pelletized to obtain.
In the fine and soft ganoderma lucidum tea preparation method of foregoing dance, described step a is that black tea is used into Co60 irradiation sterilizations, irradiation
Dosage 6Kgy, crushed 80 mesh sieves, obtained A product；Described step d is that A product, B product and C product are placed in mixer, mixing 28~
35 minutes, obtain D product；Described step e is that appropriate 80% ethanol is added into D product, is well mixed, and is pelletized by 14 mesh sieves, then
By 65 DEG C of drying, moisture≤10%, 14 mesh sieve whole grain is controlled, E product are obtained.
In the fine and soft ganoderma lucidum tea preparation method of foregoing dance, the Effects of Extracts of Grifola frondosa on Active in described step b is made in the steps below
It is standby：
B1, grifola frondosus raw material refluxing extraction is multiple, extract solution sieving merging, obtain b1 product；
B2, b1 product are concentrated to give b2 product；
B3, by b2 product drying and screenings, obtain Effects of Extracts of Grifola frondosa on Active.
In the fine and soft ganoderma lucidum tea preparation method of foregoing dance, described step b1 is to be flowed back after grifola frondosus raw material is cleaned with water
Extract 3 times, the 1st 10 times of amounts extract 2h, the 2nd 8 times of amounts extract 1.5h, and the 3rd time 6 times of amounts extract 1h, and extract solution crosses 200 mesh not
Become rusty steel screen cloth, merges 3 filtrates, obtains b1 product；Described step b2 is to be concentrated in vacuo to b1 product relative at 70 DEG C -80 DEG C
Density 1.10-1.15, vacuum -0.06~-0.08Mpa obtains b2 product；Described step b3 is to be spray-dried b2 product, entrance
180 ± 5 DEG C of temperature, 70 ± 5 DEG C of outlet temperature crosses 80 mesh sieves, obtains Effects of Extracts of Grifola frondosa on Active.
In the fine and soft ganoderma lucidum tea preparation method of foregoing dance, the Ganodenna Lucidum P.E in described step c is prepared according to the following steps：
C1, ganoderma lucidum fruitbody crushed, obtain c1 product；
C2, c1 product refluxing extraction is multiple, extract solution sieving merging, obtain c2 product；
C3, c2 product are concentrated, obtain c3 product；
C4, by c3 product drying and screenings, obtain Ganodenna Lucidum P.E.
In the fine and soft ganoderma lucidum tea preparation method of foregoing dance, described step c2 is, by c1 product by water refluxing extraction 2 times, the
10 times of amount 2h, second of 8 times of amount 1.5h, the mesh screen of extract solution 200, merging filtrate obtains merging filtrate c2 product；Described
Step c3 is that c2 product are concentrated under reduced pressure, and vacuum -0.06~-0.08Mpa is concentrated into the relative density at 70 DEG C -80 DEG C
1.05-1.15, obtains concentrate c3 product；Described step c4 is to be spray-dried c3 product, 180 ± 5 DEG C of inlet temperature, outlet temperature
70 ± 5 DEG C of degree, crosses 80 mesh sieves, obtains Ganodenna Lucidum P.E.
Compared with the prior art, the present invention adds the extract of ganoderma lucidum and grifola frondosus in black tea, make ganoderma lucidum, grifola frondosus and
The effective active composition of tealeaves is sufficiently combined together, and makes the effective active composition in product be easier to be inhaled by human body
Receive, more enrich the healthcare function of tea；The ganoderma health preserving black tea of the present invention not only has the advantages that mouthfeel and the health care of black tea, also
The medicinal function of ganoderma lucidum and grifola frondosus is combined, with protect liver, nourishing the stomach, antitoxic heart-soothing and sedative, improving eyesight, lowering blood pressure and blood fat, drop blood
Sugar, the effects such as improving body immunity, anti-aging, health and beauty, millet paste is limpid bright when brewing, and ganoderma lucidum is mingled with tea perfume
Delicate fragrance, is returned sweet after mouthfeel is bitter；In addition, the fine and soft ganoderma tea of dance of the present invention, with ganoderma lucidum and tealeaves delicate fragrance, soup look is red gorgeous, grows
Sweet alcohol, tea residue is scarlet bright, has been obviously improved the quality and added value of black tea, is imitated with significant business efficiency and society
Rate, also provides more multi items for health protection tea, is easy to consumer to select.
Therapeutic efficiency：There is the fine and soft ganoderma tea of the dance of the present invention liver-protecting and stomach-nourishing, improving eyesight of calming the nerves, regulation blood fat, raising human body to exempt from
The effect of epidemic disease power.
Present invention also offers a kind of animal test method for detecting strengthen immunity, using mtt assay detection ConA inductions
Mouse lymphocyte propagation transformation function, ear swelling method detect mouse delayed allergy, Jerne improves slide methods inspection
Mouse antibodies cellulation number is surveyed, Hemagglutination Method detection mice serum hemolysin level, half intracorporal method detection mouse peritoneal macrophage is thin
Endocytosis erythrophage ability, it is interiorIntravenous injection detects carbonic clearance ability, and determination of lactate dehydrogenase method detects NK cytoactives,
Reference《Health food is examined and assessment technique specification》Test method is carried out.
Brief description of the drawings
Fig. 1 is the fine and soft ganoderma tea process diagram of dance；
Fig. 2 is Effects of Extracts of Grifola frondosa on Active process chart；
Fig. 3 is Ganodenna Lucidum P.E process chart.
Embodiment
Embodiment 1：The fine and soft ganoderma tea of dance, is made with the raw material of following weight,
150 parts of black tea,
75 parts of Effects of Extracts of Grifola frondosa on Active,
25 parts of Ganodenna Lucidum P.E.
The specific preparation process of Effects of Extracts of Grifola frondosa on Active is as follows：
B1, grifola frondosus raw material is cleaned after extract 2h with water refluxing extraction 3 times, the 1st 10 times of amounts, the 2nd time 8 times of amounts are extracted
1.5h, the 3rd time 6 times of amounts extract 1h, and extract solution crosses 200 mesh stainless steel mesh, merges 3 filtrates, obtains b1 product；
B2, the relative density 1.10-1.15 being concentrated in vacuo to b1 product at 70 DEG C -80 DEG C, vacuum -0.06~-
0.08Mpa, obtains b2 product；
B3, b2 product are spray-dried, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature, cross 80 mesh sieves, obtain grifola frondosus
Extract.
The specific preparation process of Ganodenna Lucidum P.E is as follows：
C1, ganoderma lucidum fruitbody crushed, obtain c1 product；
C2, by c1 product by water refluxing extraction 2 times, 10 times of amounts 2h, second of 8 times of amount 1.5h, the mesh of extract solution 200 for the first time
Filter is sieved through, merging filtrate obtains merging filtrate c2 product；
C3, c2 product are concentrated under reduced pressure, vacuum -0.06~-0.08Mpa is concentrated into the relative density at 70 DEG C -80 DEG C
1.05-1.15, obtains concentrate c3 product；
C4, c3 product are spray-dried, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature, cross 80 mesh sieves, obtain ganoderma lucidum and carry
Take thing.
The specific preparation process of the fine and soft ganoderma tea of dance is as follows：
A, black tea used into Co60 irradiation sterilizations, irradiation dose 6Kgy crushed 80 mesh sieves, and obtained A product；
B, Effects of Extracts of Grifola frondosa on Active sieving, obtain B product；
C, Ganodenna Lucidum P.E sieving, obtain C product；
D, A product, B product and C product are placed in mixer, mix 28~35 minutes, obtain D product；
E, appropriate 80% ethanol is added into D product, be well mixed, pelletized by 14 mesh sieves, then pass through 65 DEG C of drying, control
Moisture≤10%, 14 mesh sieve whole grain, obtains finished product；Finally finished particle by 2.5g/ bags dispense that fine and soft ganoderma lucidum tea bag must be waved, controlled
Content uniformity processed is ± 5%, then mounted box, inspection, storage.
Embodiment 2：The fine and soft ganoderma tea of dance, is made with the raw material of following weight,
150 parts of black tea,
50 parts of Effects of Extracts of Grifola frondosa on Active,
50 parts of Ganodenna Lucidum P.E.
The specific preparation process of Effects of Extracts of Grifola frondosa on Active is as follows：
B1, grifola frondosus raw material is cleaned after extract 2h with water refluxing extraction 3 times, the 1st 10 times of amounts, the 2nd time 8 times of amounts are extracted
1.5h, the 3rd time 6 times of amounts extract 1h, and extract solution crosses 200 mesh stainless steel mesh, merges 3 filtrates, obtains b1 product；
B2, the relative density 1.10-1.15 being concentrated in vacuo to b1 product at 70 DEG C -80 DEG C, vacuum -0.06~-
0.08Mpa, obtains b2 product；
B3, b2 product are spray-dried, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature, cross 80 mesh sieves, obtain grifola frondosus
Extract.
The specific preparation process of Ganodenna Lucidum P.E is as follows：
C1, ganoderma lucidum fruitbody crushed, obtain c1 product；
C2, by c1 product by water refluxing extraction 2 times, 10 times of amounts 2h, second of 8 times of amount 1.5h, the mesh of extract solution 200 for the first time
Filter is sieved through, merging filtrate obtains merging filtrate c2 product；
C3, c2 product are concentrated under reduced pressure, vacuum -0.06~-0.08Mpa is concentrated into the relative density at 70 DEG C -80 DEG C
1.05-1.15, obtains concentrate c3 product；
C4, c3 product are spray-dried, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature, cross 80 mesh sieves, obtain ganoderma lucidum and carry
Take thing.
The specific preparation process of the fine and soft ganoderma tea of dance is as follows：
A, black tea used into Co60 irradiation sterilizations, irradiation dose 6Kgy crushed 80 mesh sieves, and obtained A product；
B, Effects of Extracts of Grifola frondosa on Active sieving, obtain B product；
C, Ganodenna Lucidum P.E sieving, obtain C product；
D, A product, B product and C product are placed in mixer, mix 28~35 minutes, obtain D product；
E, appropriate 80% ethanol is added into D product, be well mixed, pelletized by 14 mesh sieves, then pass through 65 DEG C of drying, control
Moisture≤10%, 14 mesh sieve whole grain, obtains finished product；Finally finished particle by 2.5g/ bags dispense that fine and soft ganoderma lucidum tea bag must be waved, controlled
Content uniformity processed is ± 5%, then mounted box, inspection, storage.
Embodiment 3：The fine and soft ganoderma tea of dance, is made with the raw material of following weight,
150 parts of black tea,
25 parts of Effects of Extracts of Grifola frondosa on Active,
75 parts of Ganodenna Lucidum P.E.
The specific preparation process of Effects of Extracts of Grifola frondosa on Active is as follows：
B1, grifola frondosus raw material is cleaned after extract 2h with water refluxing extraction 3 times, the 1st 10 times of amounts, the 2nd time 8 times of amounts are extracted
1.5h, the 3rd time 6 times of amounts extract 1h, and extract solution crosses 200 mesh stainless steel mesh, merges 3 filtrates, obtains b1 product；
B2, the relative density 1.10-1.15 being concentrated in vacuo to b1 product at 70 DEG C -80 DEG C, vacuum -0.06~-
0.08Mpa, obtains b2 product；
B3, b2 product are spray-dried, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature, cross 80 mesh sieves, obtain grifola frondosus
Extract.
The specific preparation process of Ganodenna Lucidum P.E is as follows：
C1, ganoderma lucidum fruitbody crushed, obtain c1 product；
C2, by c1 product by water refluxing extraction 2 times, 10 times of amounts 2h, second of 8 times of amount 1.5h, the mesh of extract solution 200 for the first time
Filter is sieved through, merging filtrate obtains merging filtrate c2 product；
C3, c2 product are concentrated under reduced pressure, vacuum -0.06~-0.08Mpa is concentrated into the relative density at 70 DEG C -80 DEG C
1.05-1.15, obtains concentrate c3 product；
C4, c3 product are spray-dried, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature, cross 80 mesh sieves, obtain ganoderma lucidum and carry
Take thing.
The specific preparation process of the fine and soft ganoderma tea of dance is as follows：
A, black tea used into Co60 irradiation sterilizations, irradiation dose 6Kgy crushed 80 mesh sieves, and obtained A product；
B, Effects of Extracts of Grifola frondosa on Active sieving, obtain B product；
C, Ganodenna Lucidum P.E sieving, obtain C product；
D, A product, B product and C product are placed in mixer, mix 28~35 minutes, obtain D product；
E, appropriate 80% ethanol is added into D product, be well mixed, pelletized by 14 mesh sieves, then pass through 65 DEG C of drying, control
Moisture≤10%, 14 mesh sieve whole grain, obtains finished product；Finally finished particle by 2.5g/ bags dispense that fine and soft ganoderma lucidum tea bag must be waved, controlled
Content uniformity processed is ± 5%, then mounted box, inspection, storage.
Embodiment 4：Strengthen immunity zoopery
With inbred strais male mice C57BL/6J, 6~8 week old, 18~22g of body weight.Orally recommended according to the people of tested material
Amount set the basic, normal, high dosage group of sample be respectively 250,500,1000mg/kg BW, while setting 1 negative control group, test every group
15 mouse.The fine and soft ganoderma lucidum tea bag of the dance prepared in embodiment 1 is brewed 10 minutes with boiling water, taken out after the fine and soft ganoderma lucidum tea bag of dance, point
Do not add water and be configured to 1.25,2.50,5.00mg/ml strength solutions (being configured by sample gross weight), give corresponding dosage group animal
Gavage, gavage volume is 0.2ml/10g BW, and negative control group gives isometric pure water, daily gavage 1 time, gives the time 30
My god.
The mouse spleen lymphocyte transformation experiment of 1.MTT methods ConA inductions
1.1 instruments and material
RPMI1640 cell culture fluids, calf serum, 2 mercapto ethanol (2-ME), penicillin, streptomysin, concanavalin A
(ConA), hydrochloric acid, isopropanol, MTT, Hank's liquid, PBS (pH7.2-7.4)
Gauze, 200 eye mesh screens, 24 well culture plates, 96 well culture plates (flat), operating theater instruments, large syringe inner core, two
Carbonoxide incubator, ELIASA, spectrophotometer, superclean bench, autoclave, sterile filters.
1.2 experimental procedure
1.2.1 preparation of reagents
Complete culture solution RPMI1640 nutrient solution filtration sterilizations, with 10% calf serum of preceding addition, 1% glutamine
(200mmol/L), penicillin (100U/mL), streptomysin (100ug/L) and 5 × 10-5mol/L 2 mercapto ethanol, with sterile
1mol/L HCl or 1mol/L NaOH adjust pH to 7.0-7.2, i.e. complete culture solution.
ConA liquid is configured to 100ug/mL solution, filtration sterilization, in (- 20 DEG C) preservations of low temperature refrigerator with distilled water.
Sterile Hank's liquid is with preceding with 3.5% sterile NaHCO3 tune pH7.2-7.4.
5mgMTT is dissolved in 1mL pH7.2 PBS by MTT liquid, now with the current.
4mL 1mol/L HCl, prepared before use are added in acid isopropyl alcoholic solution 96mL isopropanols.
1.2.2 prepared by splenocyte suspension
It is sterile to take spleen, it is placed in and fills in appropriate (3-5ml) sterile Hank's liquid plate, and one block of yarn is placed above in spleen
Cloth, is gently ground spleen with large syringe inner core, and individual cells suspension is made.Through 200 mesh sieve net filtrations, washed with Hank's liquid
2 times, 10min (1000r/min) is centrifuged every time.Then cell is suspended in 2mL complete culture solution, uses full-automatic cell meter
Number instrument meter number splenocytes, or with the blue dyeing counting viable count (should be more than 95%) of platform phenol, adjustment cell concentration is 3 × 106
Individual/mL.
1.2.3 lymphproliferation response
Cell suspension point holes is added in 24 well culture plates, per hole 1mL, a hole adds 75 μ l ConA liquid (equivalent to 7.5 μ
G/mL), another hole is put in 5%CO2,37 DEG C of CO2 incubators as control and cultivates 72h.Culture terminates preceding 4h, is gently sucked per hole
Supernatant 0.7mL, adds the RPMI1640 nutrient solutions that 0.7mL is free of calf serum, while MTT (5mg/mL) 50 μ l/ holes are added,
Continue to cultivate 4h.After culture terminates, 1mL acid isopropyl alcohol is added per hole, ultrasonic vibration (2 seconds) or artificial piping and druming are mixed, and make purple
Color crystallization is completely dissolved.Then it is dispensed into 96 well culture plates, each hole dispenses 3 holes as Duplicate Samples, uses enzyme linked immunosorbent detection
Instrument, OD value is determined with 570nm wavelength.Also lysate can be directly moved into 2mL cuvettes, in wavelength on spectrophotometer
570nm determines OD values.
1.3 data processings and result
ConA induction mouse lymphocyte conversion test result (being shown in Table 1) show, the lymph of 3 dosage group mouse of sample
Cell transformation capacity is above negative control group (F=3.514, P=0.025), and wherein high dose group is compared with negative control group
Difference is statistically significant.
The immune function of mice correlation test result of table 1
2 dinitrofluorobenzene inducing mouse delayed allergies (ear swelling method)
2.1 material
DNFB, acetone, sesame oil, barium sulphide, card punch.
2.2 experimental procedure
2.2.1 preparation of reagents
DNFB solution D NFB solution answers Fresh, weighs DNFB50mg, puts cleaning and dries in bottle, by what is prepared in advance
5mL acetone sesame oil solution (acetone：Sesame oil=1：1) bottle, is poured into, the sealing of bottle stopper blend compounds cloth is covered.After mixing, with 250 μ l
Syringe is taken by bottle cap.
2.2.2 sensitization
Lose hair or feathers per mouse skin of abdomen barium sulphide, scope about 3cm × 3cm, with the μ l of DNFB solution 50 uniformly smear sensitization.
2.2.3 the generation of delayed allergy (DTH) and measure
After 5 days, uniformly it is applied to mouse right ear (two sides) with the μ l of DNFB solution 10 and is attacked.24h cervical vertebras take off after attack
Mortar puts to death mouse, cuts left and right auricular concha.Diameter 8mm auricle is removed with card punch, is weighed.
2.2.4 data processing and result
Delayed allergy result of the test as shown in table 1, shows the left and right auricle weight of three dosage group mouse of sample
Difference is above negative control group, and wherein high dose group difference compared with negative control group is statistically significant.
From the ConA mouse lymphocyte conversion tests induced and delayed allergy result of the test, it is possible to determine that cell
Immunity test result is positive.
3. antibody-producting cell detection
3.1 instruments and material
Hemolysis plaque automatic image analyzer, CO2gas incubator, water bath with thermostatic control, centrifuge, operating theater instruments, 200 mesh
Screen cloth, SRBC, complement (guinea pig serum), Hank's liquid, RPMI1640 nutrient solutions, SA buffer solutions (C4H4N2O30.46g,
MgCl20.1g, CaCl2.2H2O 0.2g, NaCl 8.38g, NaHCO3 0.252g and C8H11N2NaO3 0.3g, plus steam
Distilled water is to 1000mL), sterile saline, agarose.
3.2 experimental procedure：
3.2.1 SRBC
Sheep taking blood from jugular vein, sheep blood is put into the sterilizing conical flask of bead, shaken in one direction, with de- fibre
Dimension, physiological saline 2500rpm/min, 15min are washed 3 times, are prepared hematocrit SRBC, be put into 4 DEG C of refrigerators and save backup.
3.2.2 the preparation of complete medium：Newborn calf serum is through sheep red blood cell (SRBC) (v/v, 5：1) absorb, 4 DEG C, 30-
After 60min, add in incomplete culture medium RPMI 1640, be prepared into the complete medium containing 20% calf serum.
3.2.3 prepared by complement
Femoral artery takes blood, stands 30-60min, and 2500rpm/min, 15min separation serum mixes 5-10 guinea pig serum
After conjunction, with hematocrit SRBC with 5：1 (v/v) ratio is mixed, and 4 DEG C of refrigerators place 30-60min, or concussion, 2500rpm/min,
15min separates serum, and packing, -80 DEG C of refrigerators are preserved.Used time is with complete medium 1：10 (v/v) dilution proportions.
3.2.4 animal is immunized
2% (v/v) cell suspension (about 1 × 108 SRBC), every mouse abdominal cavity note are made with physiological saline by hematocrit SRBC
Penetrate 0.2mL.
3.2.5 prepared by splenocyte suspension
Mouse cervical dislocations of the SRBC after immune 4-5 days is put to death, it is sterile to take spleen, and one block of yarn is placed above in spleen
Cloth, is gently ground spleen with large syringe inner core, is made individual cells suspension, 200 mesh sieve net filtrations, 1000rpm/min,
10min, removes supernatant, and Hank ' liquid is washed 2 times, and 5 × 106~1 × 107 cell/mL are prepared into complete medium RPMI 1640
Splenocyte suspension.
3.2.6 the measure of plaque
3.2.6.1 bottom culture medium prepares 0.5g agaroses and added in 100mL sterile salines, dissolves by heating, treats temperature
Spend when 50 DEG C or so, added to the amount in 1mL/ holes in six well culture plates, it is standby after agar solidification.
3.2.6.2 top layer culture medium prepares 0.5g agaroses and added in 100mL Hank ' s liquid (PH7.2-7.4), heats molten
In solution, the test tube that 46-50 DEG C of constant temperature is added to the amount of 0.5mL/ test tubes.
3.2.6.3 the μ l 20%SRBC (normal saline, v/v) of bed board 50,200 μ l splenocyte suspensions are successively added and contained
It is rapid to mix in the test tube for having 0.5mL top layers, pour into six orifice plates and pave top layer mixed liquor, each sample do two it is parallel
Hole.
3.2.6.4 Plaque assay：Prepare culture plate to be put into 37 DEG C, be incubated 1h in 5%CO2 incubators, then per hole
Add complement (v/v, 1 that 500 μ l are diluted with complete medium：10), continue to be incubated 2h, it is empty that automatic image analyzer reads haemolysis
Spot image analysis software counts hemolysis plaque number.The average value for taking parallel hole plaque number is the hemolysis plaque numerical value of sample, with sky
The splenocyte of spot number/106 is represented.
3.3 data processings and result
As shown in table 1, its result shows that the antibody tormation of each dosage group mouse of sample is thin to antibody-producting cell measurement result
Born of the same parents' number is above negative control group.
4. the measure (Hemagglutination Method) of serum hemolysin
4.1 instruments and material
SRBC, physiological saline, Microhemagglutination brassboard, centrifuge
4.2 experimental procedure
4.2.1 SRBC sheep taking blood from jugular vein, sheep blood is put into the sterilizing conical flask of bead, in one direction
Shake, to take off fiber, be put into 4 DEG C of refrigerators and save backup, can preserve 2 weeks.
4.2.2 animal is immunized and serum separation takes sheep blood, with brine 3 times, every time centrifugation (2000r/min)
10min.Hematocrit SRBC is made into 2% (v/v) cell suspension with physiological saline, every mouse intraperitoneal injection 0.2ml is immunized.
After 4~5d, extract eyeball and take blood in centrifuge tube, place about 1h, solidification blood and tube wall are peeled off, serum is fully separated out,
2000r/min centrifuges 10min, collects serum.
4.2.3 the serum of different dilution factors is respectively placed in micro by agglutinating reaction physiological saline by serum doubling dilution
In blood coagulation tests plate, per the μ l of hole 100,100 μ l 0.5% (v/v) SRBC suspensions are added, are mixed, in the square position for loading moistening
Capping, in 37 DEG C of incubation 3h, observes hemagglutination degree.
4.3 data processings and result
Serum hemolysin measurement result (as shown in table 1), shows that the antibody product of each dosage mouse of sample is above feminine gender
Control group.
5. mouse carbonic clearance is tested
5.1 instruments and reagent
Spectrophotometer, full-automatic ELIASA, timer, hemoglobin pipet, india ink, Na2CO3
5.2 experimental procedure
5.2.1 solution is prepared
Injection prepared Chinese ink is by 3~4 times of normal saline dilution of india ink stoste.
Na2CO3 solution takes 0.1gNa2CO3, plus distilled water is to 100mL.
5.2.2 injection prepared Chinese ink is weighed, and the india ink of dilution is injected from mouse tail vein, based on every 10g body weight 0.1mL
Calculate.Treat that prepared Chinese ink injects, timing immediately.
5.2.3 determine
Prepared Chinese ink is injected after 2 minutes and 10 minutes, is taken the μ l of blood 20 from angular vein clump respectively, is existed side by side and be added into 2mL
0.1%Na2CO3In solution.With spectrophotometer or full-automatic ELIASA at 600nm wavelength densitometric value (OD), with
Na2CO3Solution makees blank control.
Mouse is put to death, liver and spleen is taken, organ surface blood stains are blotted with filter paper, are weighed.
The ability of mouse carbonic clearance is represented with phagocytic index.Phagocytic index a is calculated as follows：
K=(lgOD1-lgOD2)/(t2- t1)
Phagocytic index a=body weight/(liver weight+spleen weight) ×
5.2.4 data processing and result
As shown in table 2, its result shows the mouse carbonic clearance energy of each dosage group mouse of sample to mouse carbonic clearance experimental result
Power is above negative control group.
The immune function of mice correlation test result of table 2
6. Turnover of Mouse Peritoneal Macrophages swallows fluorescent microsphere test method
6.1 instruments and material
Hank's liquid, calf serum, PBS, physiological saline, fluorescent microsphere (2 μm of φ), the white egg of 1% calf serum
In vain (BSA).
Flow cytometer, is cleaned by ultrasonic instrument, and carbon dioxide cell incubator, centrifuge, 6 well culture plates, cell is scraped, and filters
Device (75m), streaming loading pipe, the special sheath fluid of flow cytometer, liquid-transfering gun and suction nozzle, white blood cell count(WBC) plate, operating theater instruments are a set of,
Syringe, dropper, glue head straw.
6.2 experimental procedure
6.2.1 preparation of reagents
6.2.1.1 the Hank's liquid of 5% calf serum is contained：Take 10ml calf serums to add in 200ml Hank's liquid, mix
It is even, match somebody with somebody before use.
6.2.1.2PBS the compound method of buffer solution：KH2PO46.66g, Na2HPO412H2O 6.38g, by above-mentioned examination
Agent, which is dissolved in 1000ml distilled water, adjusts pH value to 7.2.
6.2.1.3 the compound method of 2% sheep red blood cell (SRBC) suspension：Sheep jugular vein blood is taken before experiment, is placed in and fills glass
It is continuous fully to be shaken 5~10 minutes along a direction in the triangular flask of pearl (20 or so), except defibrinating, use physiology salt
Water washing 3 times, each 1500rPm is centrifuged 10 minutes, 4 DEG C of refrigerators are preserved.Supernatant discarding before experiment, by hematocrit physiology
Red cell suspension of the saline into 2%.
6.2.1.4 1%BSA preparation：0.5gBSA is dissolved in 50mlPBS buffer solutions, before use with preferable.
6.2.1.5 fluorescent microsphere preconditioned：37 DEG C of lucifuges of 100 μ l fluorescent microspheres and 10ml 1%BSA are incubated 30min, surpass
Sonication 5min, matches somebody with somebody before use.
6.2.2 peritoneal macrophage swallows fluorescent microsphere process
Experiment is tested for first 4 days to the every sheep erythrocytes of mouse peritoneal injection 0.2ml 2% activation mouse macrophage
The same day puts to death Hank's liquid 3ml/ of mouse, intraperitoneal injection plus calf serum with cervical dislocation, gently rubs belly 20 times,
Fully to wash out peritoneal macrophage, stomach wall is then cut off into an osculum, abdominal cavity washing lotion 2ml is drawn and is filtered with 75 μm of filters
To test tube, adjustment number of macrophages are 4~6 × 105/ml.1ml abdominal cavity washing lotions are drawn in 6 well culture plates with liquid-transfering gun, plus
Enter the fluorescent microsphere (1 × 107/ plate) that preconditioned is crossed, 37 DEG C of carbon dioxide cell incubator (or room temperature) lucifuges are incubated 90
~120 minutes, supernatant (containing non-attached cell and unnecessary fluorescent microsphere) was abandoned in incubation after terminating, 1.0mlPBS buffer solutions are used every time
Gently wash 2 times, go after supernatant to add 4 DEG C of PBS 0.3ml, attached cell is scraped with cell, gently blow and beat uniform
By upper machine analysis after 75 μm of filter filterings.
6.2.3 flow cytomery is analyzed
6.2.3.1 gating：The two-dimentional scatter diagrams of FSC-SSC are first set up, it is thin with macrophage by adjusting FSC and SSC magnitudes of voltage
Born of the same parents' gating defines the macrophage group of analysis, and the interference of other karyocytes, cell fragment etc. is excluded to greatest extent；
6.2.3.2 obtain：The fluorescence channels for launching light in fluorescent microsphere detect the fluorescence intensity of macrophage, every part of sample
5000 macrophages are obtained, data can be shown in two-dimentional scatter diagram and histogram.Gating can be passed through in two-dimentional scatter diagram
Draw a circle to approve the macrophage group of unopsonized fluorescent microsphere and the macrophage group of phagocytosis fluorescent microsphere；Scale can be passed through in histogram
The macrophage group of unopsonized fluorescent microsphere and the macrophage group of phagocytosis fluorescent microsphere are demarcated, total data is through related software point
Analyse the ratio of the macrophage of unopsonized fluorescent microsphere and the macrophage of phagocytosis varying number fluorescent microsphere.
6.3 data processings and result
Number of macrophages × 100 of number of macrophages/counting of phagocytic percentage (%)=phagocytosis fluorescent microsphere
The number of macrophages of the fluorescent microsphere sum/counting for phagocytic index=swallowed
The phagocytic activity of mouse macrophage is represented with phagocytic percentage or phagocytic index.Phagocytic percentage need to carry out data
Conversion, X=Sin-1 P is phagocytic percentage in formula, decimally represents, variance analysis is then carried out again.
Its result as shown in table 2, as a result shows the phagocytic index of the peritoneal macrophage of each dosage group mouse of sample and gulped down
The rate of biting is above negative control group.
7.NK cytoactive detections (determination of lactate dehydrogenase method)
7.1 instruments and material
ELIASA, full-automatic cell calculating instrument, YAC-1 cells, Hank's liquid (pH7.2~7.4), RPMI1640 are trained completely
Nutrient solution, lithium lactate or sodium lactate, nitro tetrazolium chloride (INT), PMS (PMS), NAD, 0.2mol/L
Tris-HCl buffer solutions (pH8.2), 1%NP40 or 2.5%Triton
7.2 experimental procedure
7.2.1 the preparation of LDH matrix liquids
5 × 10-2mol/L of lithium lactate
Nitro tetrazolium chloride (INT) 6.6 × 10-4mol/L
PMS (PMS) 2.8 × 10-4mol/L
Oxidized coenzyme I (NAD) 1.3 × 10-3mol/L
Mentioned reagent is dissolved in 0.2mol/L Tris-HCl buffer solutions (pH8.2)
7.2.2 the passage (YAC-1 cells) of target cell
Target cell is carried out Secondary Culture by 24h before experiment.Washed 3 times, trained completely with RPMI1640 with Hank's liquid using preceding
Nutrient solution adjustment cell concentration is 4 × 105/mL.
7.2.3 the preparation (effector cell) of splenocyte suspension
It is sterile to take spleen, it is placed in the small plate for filling appropriate sterile Hank's liquid, and one piece of gauze is placed above in spleen, use
Large syringe inner core gently grinds spleen, and individual cells suspension is made.Through 200 mesh sieve net filtrations, washed with Hank's liquid 2 times,
10min (1000r/min) is centrifuged every time.Abandon supernatant cytoplasm is upspring, add 0.5mL aqua sterilisas 20 seconds, after splitting erythrocyte
0.5mL2 times of Hank ' s liquid and 8mLHank ' s liquid are added, then cell is suspended in the complete of 2mL by 1000rpm, 10min centrifugations
In full nutrient solution, splenocyte is counted with full-automatic cell calculating instrument；Or trained completely with RPMI1640s of the 1mL containing 10% calf serum
Nutrient solution is resuspended, and (viable count should be more than 95%) is counted after being diluted with 1% glacial acetic acid, with the blue dyeing counting viable count of platform phenol
(should be more than 95%), be finally 2 × 107/mL with RPMI1640 complete culture solutions adjustment cell concentration.
7.3 NK cytoactive detections
Taking target cell and each 100 μ l of effector cell, (effect target compares 50:1), add in U-shaped 96 well culture plate；Target cell is natural
Release aperture adds target cell and each 100 μ l of nutrient solution, and target cell maximum release aperture adds target cell and 1%NP40 or 2.5%Triton
Each 100 μ l；Above-mentioned items are all provided with three multiple holes, and 4h is cultivated in 37 DEG C, 5%CO2 incubators, then by 96 well culture plates with
1500r/min centrifuges 5min, is drawn per hole in the well culture plate of 100 μ l horizontalizations bottom of supernatant 96, while the μ l of LDH matrix liquids 100 are added,
3min is reacted, the 1mol/L μ l of HCl 30 are added per hole, OD value (OD) is determined at ELIASA 490nm.
NK cytoactives are calculated as follows：
NK cytoactives %=(reacting hole OD- Spontaneous releases hole OD)/(maximum release aperture OD- Spontaneous releases hole OD) ×
100%
7.4 data processings and result
NK cytoactives need to carry out data conversion, X=Sin-1 P is NK cytoactives in formula, is decimally represented, so
Carry out variance analysis again afterwards.
Its result as shown in table 2, as a result shows that the NK cytoactives of each dosage group mouse of sample are above negative control group.
8. conclusion
Breed transformation function by the ConA mouse lymphocytes induced to detect, delayed allergy detection, mouse resists
Body cellulation number detects that mice serum hemolysin level detection, Turnover of Mouse Peritoneal Macrophages phagocytosis red blood cell ability is detected, carbon
Ability detection is cleaned up, as a result NK cytoactive detections show that composition of the present invention has the function of strengthen immunity.
Claims (9)
1. the fine and soft ganoderma tea of dance, it is characterised in that：It is made of with the raw material of following weight proportions,
150 parts of black tea,
20-80 parts of Effects of Extracts of Grifola frondosa on Active,
80-20 parts of Ganodenna Lucidum P.E.
2. the fine and soft ganoderma tea of dance as claimed in claim 1, it is characterised in that：It is made of with the raw material of following weight proportions,
150 parts of black tea,
25-80 parts of Effects of Extracts of Grifola frondosa on Active,
80-25 parts of Ganodenna Lucidum P.E.
3. the fine and soft ganoderma tea of dance as claimed in claim 2, it is characterised in that：It is made of with the raw material of following weight proportions,
150 parts of black tea,
25-75 parts of Effects of Extracts of Grifola frondosa on Active,
75-25 parts of Ganodenna Lucidum P.E.
4. the preparation method of fine and soft ganoderma tea is waved as described in claim 1,2 or 3, it is characterised in that：It is prepared according to the following steps；
A, black tea sterilizing, crushing, obtain A product；
B, Effects of Extracts of Grifola frondosa on Active sieving, obtain B product；
C, Ganodenna Lucidum P.E sieving, obtain C product；
D, A product, B product and C product are placed in mixer, after mixing D product；
E, finished product that D product are pelletized to obtain.
5. the fine and soft ganoderma lucidum tea preparation method of dance as claimed in claim 4, it is characterised in that：Described step a is to use black tea
Co60 irradiation sterilizations, irradiation dose 6Kgy crushed 80 mesh sieves, obtained A product；Described step d is to be placed in A product, B product and C product
In mixer, mix 28~35 minutes, obtain D product；Described step e is that appropriate 80% ethanol is added into D product, is well mixed,
Pelletized by 14 mesh sieves, then by 65 DEG C of drying, control moisture≤10%, 14 mesh sieve whole grain, obtain finished product.
6. the fine and soft ganoderma lucidum tea preparation method of dance as claimed in claim 4, it is characterised in that：Grifola frondosus in described step b is carried
Thing is taken, is prepared according to the following steps：
B1, grifola frondosus raw material refluxing extraction is multiple, extract solution sieving merging, obtain b1 product；
B2, b1 product are concentrated to give b2 product；
B3, by b2 product drying and screenings, obtain Effects of Extracts of Grifola frondosa on Active.
7. the fine and soft ganoderma lucidum tea preparation method of dance as claimed in claim 6, it is characterised in that：Described step b1 is, by grifola frondosus
Starting material with water refluxing extraction 3 times, the 1st 10 times of amounts extract 2h, and the 2nd 8 times of amounts extract 1.5h, and the 3rd time 6 times of amounts extract 1h, carry
Take liquid to cross 200 mesh stainless steel mesh, merge 3 filtrates, obtain b1 product；Described step b2 is, by b1 product be concentrated in vacuo to 70 DEG C-
Relative density 1.10-1.15 at 80 DEG C, vacuum -0.06~-0.08Mpa obtains b2 product；Described step b3 is, by b2 product
Spray drying, 180 ± 5 DEG C of inlet temperature, 70 ± 5 DEG C of outlet temperature crosses 80 mesh sieves, obtains Effects of Extracts of Grifola frondosa on Active.
8. the fine and soft ganoderma lucidum tea preparation method of dance as claimed in claim 4, it is characterised in that：Ganoderma lucidum in described step c is extracted
Thing, is prepared according to the following steps：
C1, ganoderma lucidum fruitbody crushed, obtain c1 product；
C2, c1 product refluxing extraction is multiple, extract solution sieving merging, obtain c2 product；
C3, c2 product are concentrated, obtain c3 product；
C4, by c3 product drying and screenings, obtain Ganodenna Lucidum P.E.
9. the fine and soft ganoderma lucidum tea preparation method of dance as claimed in claim 8, it is characterised in that：Described step c2 is to lead to c1 product
Water refluxing extraction 2 times are crossed, for the first time 10 times of amount 2h, second of 8 times of amount 1.5h, the mesh screen of extract solution 200, merging filtrate is obtained
Merging filtrate c2 product；Described step c3 is that c2 product are concentrated under reduced pressure, vacuum -0.06~-0.08Mpa, be concentrated into 70 DEG C -
Relative density 1.05-1.15 at 80 DEG C, obtains concentrate c3 product；Described step c4 is to be spray-dried c3 product, inlet temperature
180 ± 5 DEG C, 70 ± 5 DEG C of outlet temperature crosses 80 mesh sieves, obtains Ganodenna Lucidum P.E.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107865142A (en) * | 2017-12-06 | 2018-04-03 | 四川省茶马古道生物科技有限公司 | A kind of gold chestnut mushroom black tea processing technology |
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| CN1064988A (en) * | 1992-04-04 | 1992-10-07 | 陈喜霖 | Preparation of ganoderma lucidum health tea |
| JP3527625B2 (en) * | 1997-09-09 | 2004-05-17 | アイ・エム・ビー株式会社 | Bourbon stalk composition for beverages |
| CN1899322A (en) * | 2006-07-19 | 2007-01-24 | 北京恩华医药研究院 | Chinese medicine preparation with organism immunity increasing function |
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2017
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|---|---|---|---|---|
| CN1064988A (en) * | 1992-04-04 | 1992-10-07 | 陈喜霖 | Preparation of ganoderma lucidum health tea |
| JP3527625B2 (en) * | 1997-09-09 | 2004-05-17 | アイ・エム・ビー株式会社 | Bourbon stalk composition for beverages |
| CN1899322A (en) * | 2006-07-19 | 2007-01-24 | 北京恩华医药研究院 | Chinese medicine preparation with organism immunity increasing function |
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| CN107865142A (en) * | 2017-12-06 | 2018-04-03 | 四川省茶马古道生物科技有限公司 | A kind of gold chestnut mushroom black tea processing technology |
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