CN115785300A - A Ganoderma water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -dextran, and its preparation method - Google Patents
A Ganoderma water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -dextran, and its preparation method Download PDFInfo
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Abstract
The invention provides a ganoderma aqueous extract ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan, which is prepared by the following method (1) crushing ganoderma fruit body, adding water for boiling extraction, and filtering to obtain ganoderma aqueous extract; (2) Performing ultrafiltration treatment on the ganoderma water extract to obtain ganoderma water extract ultrafiltrate. The ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan provided by the invention has the advantages of low extraction cost, low energy consumption, high extraction rate, suitability for industrial production, fragrant and slightly bitter taste, multiple efficacies of improving immunity, resisting inflammation, regulating intestinal flora and the like, and suitability for various groups to drink.
Description
Technical Field
The invention relates to the field of edible fungus polysaccharide preparation, in particular to ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan and a preparation method thereof.
Background
Ganoderma (Ganoderma lucidum) is a traditional medicinal fungus, and has effects of strengthening body resistance, consolidating constitution, tonifying, and prolonging life. The polysaccharide is one of main active ingredients of the ganoderma, especially ganoderma beta-glucan, and has outstanding immunomodulatory activity. Researches show that beta- (1 → 3,1 → 6) -glucan in the ganoderma lucidum is a high-efficiency biological reaction regulatory factor, plays an important role in activating immune cells such as lymphocytes and macrophages, promoting the generation of cytokines, activating a complement system, promoting the generation of antibodies, specifically binding with receptors such as Dectin-1 on the cell surface, activating the immune system and the like, and has good thickening effect and thermal stability due to unique high-level conformation in an aqueous solution, thereby having important development and application prospects.
At present, many ganoderma lucidum products are available on the market, but most ganoderma lucidum beta- (1 → 3,1 → 6) -glucan has low content, and the problems of time consumption, low yield and difficult large-scale application such as boiling water extraction, concentration and alcohol precipitation, column chromatography separation and the like commonly used in the extraction and preparation process of ganoderma lucidum are solved.
Therefore, there is a need to develop a new method for enriching beta- (1 → 3,1 → 6) -glucan of ganoderma lucidum, and obtain the extract rich in the component for the back-end product development to meet the increasing demand of functional products.
Disclosure of Invention
The invention firstly provides a preparation method of ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan, which comprises the following steps:
(1) Pulverizing Ganoderma fruiting body, adding water, boiling and extracting, and filtering to obtain Ganoderma water extractive solution;
(2) Performing ultrafiltration treatment on the ganoderma water extract to obtain ganoderma water extraction ultrafiltrate;
wherein the ratio of the lucid ganoderma sporocarp to the water is 1g: 20-30 mL of water with the temperature of 80-110 ℃; the time is 1-3 hours; repeatedly extracting for 1-3 times, and mixing extractive solutions;
wherein the ultrafiltration is carried out by an ultrafiltration membrane with the molecular weight cutoff of 50 ten thousand daltons;
the invention also provides ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan, which is prepared by the following method:
(1) Pulverizing Ganoderma encarpium, adding water, boiling for extraction, and filtering to obtain Ganoderma water extractive solution;
(2) Performing ultrafiltration treatment on the ganoderma water extract to obtain ganoderma water extract ultrafiltrate;
the ganoderma aqueous extract ultrafiltrate provided by the invention is used as a functional raw material rich in beta- (1 → 3,1 → 6) -glucan, and can be prepared into a medicament or a preparation for preventing or treating immune diseases, or a health food or a beverage for improving the immunity of the organism. When the ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan is used for preparing medicaments, health-care food or beverages, the ganoderma lucidum water extraction ultrafiltrate can be separately applied with other medicaments, health-care food, water or beverages, and can also be prepared into a single product according to a certain proportion, for example, the ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan is mixed with water or beverages at the ratio of 1:1-4, to make a new beverage, and so on.
The ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan provided by the invention has the following innovation points:
the method adopts a simple boiling water extraction combined ultrafiltration interception refining green process, greatly improves the content of beta- (1 → 3,1 → 6) -glucan in the ganoderma lucidum extract, can obviously improve the immunity of organisms, and utilizes the concentrated solution to prepare products so as to achieve the effects of reducing cost and improving quality.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.
FIG. 1HPSEC-MALLS-RI method for determining beta- (1 → 3,1 → 6) -glucan Standard Curve
FIG. 2 shows polysaccharide molecular weight distribution of Ganoderma lucidum water extractive solution and Ganoderma lucidum water extractive ultrafiltrate
FIG. 3 comparison of immunological activities of water extractive solution and ultrafiltrate of Ganoderma
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
(1) A preparation method of ganoderma lucidum water extraction ultrafiltrate rich in ganoderma lucidum beta-glucan comprises the following steps:
treating raw materials: drying to obtain Ganoderma encarpium (Hunong Ganoderma lucidum No. 4, edible fungus research institute of Shanghai institute of agricultural science), pulverizing into about 1-3 cm to obtain Ganoderma encarpium coarse powder;
preparing a ganoderma lucidum water extracting solution: according to the mass volume ratio of the ganoderma lucidum fruiting body coarse powder to distilled water of 1g: adding distilled water in a proportion of 20mL, extracting at 100 ℃ for 2 hours, filtering, continuously adding distilled water with the same amount as that of the first extraction into filter residues, extracting at 100 ℃ for 1 hour, filtering, and combining the two filtrates to obtain the ganoderma lucidum water extract;
preparing ganoderma lucidum water extraction ultrafiltrate: performing ultrafiltration treatment on the ganoderma lucidum water extract by using a hollow fiber ultrafiltration membrane (GE company, america) with the molecular weight cutoff of 50 ten thousand daltons, wherein the part penetrating through the filter membrane is a filtrate, the part which is not filtered is a cutoff solution, performing ultrafiltration concentration until the mass-volume ratio of ganoderma lucidum fruiting body coarse powder to the cutoff solution is 1g.
And (4) sterilization and preservation: sterilizing the ganoderma lucidum water extraction ultrafiltrate for 10 to 15 minutes at the temperature of between 100 and 121 ℃ and storing for later use.
(2) Detection of polysaccharide content
Freeze drying the ganoderma water extract and ganoderma water extract ultrafiltrate to obtain solid powder, respectively weighing appropriate amount of powder to dissolve, and measuring total sugar content by phenol-sulfuric acid method. The specific method comprises the following steps: 5% phenol solution, concentrated sulfuric acid and 0.1mg/mL glucose standard solution are prepared. Taking glucose standard (Sigma-Aldrich company, USA) solution (with concentration of 0.00, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10 mg/mL) and 1mL sample determination solution, respectively placing in a long test tube, adding 0.5mL5% phenol solution and 2.5mL concentrated sulfuric acid, mixing, placing in 100 deg.C water bath for 15min, taking out, cooling to room temperature, placing 200 μ L in an ELISA plate, and determining absorbance value at 490 nm. And drawing a standard curve according to the concentration of the glucose standard substance and the corresponding absorbance value, and calculating the total sugar content.
The content of reducing sugar in the ganoderma water extract and the water extraction ultrafiltrate is determined according to a DNS method. The specific method comprises the following steps: preparing a DNS reagent, a 1mg/mL glucose standard solution and a sample determination solution. Adding 0, 0.2, 0.4, 0.8, 1.2, 1.6 and 2mL of glucose standard solution into a 25mL volumetric flask, and adding water to make up to 2mL (the concentration of the glucose standard is 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL); 2mL of sample measuring solution is put into a 25mL volumetric flask; respectively adding 5mL of the LDNS reagent, placing in a water bath at 100 ℃ for 5min, cooling, diluting to 25mL, shaking up, taking 200 mu L of the mixture to an enzyme label plate, measuring the absorbance value at 520nm, drawing a standard curve according to the concentration of the glucose standard substance and the corresponding absorbance value, and calculating the content of the reducing sugar.
The calculation formula of the polysaccharide content is as follows:
polysaccharide content (%) = total sugar content (%) -reducing sugar content (%)
The polysaccharide content comparison result (table 1) shows that the polysaccharide content of the ganoderma lucidum water extraction liquid is 20.01%, and the polysaccharide content of the ganoderma lucidum water extraction ultrafiltrate is doubled to reach 45.27%, which indicates that the polysaccharide content of the ganoderma lucidum water extraction ultrafiltrate is effectively increased by ultrafiltration interception.
TABLE 1 comparison of polysaccharide content in aqueous Ganoderma extract and aqueous ultrafiltrates
(3) Content detection of beta- (1 → 3,1 → 6) -glucan in ganoderma lucidum water extract and ganoderma lucidum water extract ultrafiltrate
Analyzing the content of beta- (1 → 3,1 → 6) -glucan in the ganoderma water extract and ganoderma water ultrafiltrate by high performance gel size exclusion chromatography-multi-angle laser disperser-differential refractometer combined analysis method (HPSEC-MALLS-RI).
The specific chromatographic conditions are as follows: with TSK-GEL series G6000PW XL And G4000PW XL The column (7.8 mm. Times.300mm, TOSOH, japan) was used as an analytical column with 0.15mol/L of NaNO 3 And 0.05mol/L NaH 2 PO 4 (pH = 7) as a mobile phase, and elution analysis was performed at 35 ℃ at a flow rate of 0.5 mL/min.
The polysaccharide powder obtained after freeze drying is prepared into the concentration of 5mg/mL, and is centrifuged at 12000 Xg at a high speed for 10min, and then the supernatant is sampled by 100 mu L. Weighing beta- (1 → 3,1 → 6) -glucan standard (scleroglucan, elicityl company, france) to prepare concentrations of 0mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL and 2mg/mL, sampling, making a standard curve (figure 1) according to polysaccharide concentration corresponding to peak area measured by a differential refractometer, and calculating the content of beta- (1 → 3,1 → 6) -glucan in the ganoderma lucidum water extract and ganoderma lucidum water extract ultrafiltrate. Each sample was repeated 3 times and averaged.
Analyzing polysaccharide molecular weight distribution of the Ganoderma water extractive solution and Ganoderma water extractive ultrafiltrate by high performance gel size exclusion chromatography (HPSEC-MALLS-RI) combined analysis method (specific column chromatography and elution conditions are as above). The light source wavelength of the laser light scattering instrument is 623.8nm, and the refractive index increment (dn/dc) of the polysaccharide in the solution is calculated according to 0.146 mL/g. The molecular weight was calculated by collecting and analyzing light scattering data using Astra (version 6.1.1, wyatt technology, santa Barbara, calif.) data analysis software.
The molecular weight analysis result shows (figure 2), the ganoderma lucidum water extracting solution mainly contains a beta- (1 → 3,1 → 6) -glucan component with the molecular weight of 200 ten thousand to 300 ten thousand daltons (the retention time is 22min to 26 min) and a low molecular weight polysaccharide component with the molecular weight of about 1 ten thousand (the retention time is 42min to 47 min), and the proportion of the macromolecular beta- (1 → 3,1 → 6) -glucan component in the ganoderma lucidum water extracting ultrafiltrate obtained by ultrafiltration interception is greatly improved. The content comparison result shows that the content of the beta- (1 → 3,1 → 6) -glucan in the ganoderma lucidum water extract is 4.33%, the content of the ganoderma lucidum water extract ultrafiltrate is obviously improved to 37.18%, which indicates that the beta- (1 → 3,1 → 6) -glucan component in the ganoderma lucidum water extract is efficiently enriched by ultrafiltration interception, and the specific data are shown in the following table 2.
TABLE 2 beta- (1 → 3,1 → 6) -Glucan content results
(4) Determination of enhanced immunity of Ganoderma water extractive solution and Ganoderma water extractive ultrafiltrate by activating NF-kB pathway through Dectin-1 receptor binding
Preparing a reagent: (1) growth medium: DMEM medium (Invitrogen, USA), 10% (v/v) fetal bovine serum (Invitrogen, USA), 100U/mL penicillin (Sigma-Aldrich, USA) and 100 μ g/m streptomycin (Sigma-Aldrich, USA), 100 μ g/mL Normocin (Invivogen, USA). (2) selecting a culture medium: 50mL of the complete medium was added to 200. Mu.L of HEK-Blue CLR Selection (InvivoGen, USA), and the mixture was stored at 4 ℃. (3) detection culture medium: HEK-Blue protection (InvivoGen, USA) is dissolved in 50mL of sterile water and stored at 4 ℃ in the dark.
And (3) cell culture: culturing HEK-Blue hDectin-1a cell strain (Invivogen, USA) in DMEM complete medium for two generations, culturing in selective medium, each culture condition 5% 2 37 ℃ and saturated humidity.
The determination method comprises the following steps: taking HEK-Blue hDectin-1a cells in logarithmic growth phase, and regulating cell density to 3 × 10 with detection culture medium 5 one/mL in 96-well platesIn the kit, 180. Mu.L of cell suspension and 20. Mu.L of different sample solutions were added to each well, sterile water was used as a blank, and the final concentration of the sample was 200. Mu.g/mL, and three replicates were set for each sample. Culturing in incubator for 24h, and measuring absorbance value with microplate reader at 630nm wavelength.
The activity comparison results show that (figure 3), the ganoderma lucidum water extract and the ganoderma lucidum water extract ultrafiltrate both have certain activity of activating NF-kB, but the activity is different in strength. The activity of the ganoderma water extraction ultrafiltrate which is enriched with beta- (1 → 3,1 → 6) -glucan by ultrafiltration is obviously superior to that of the ganoderma water extracting solution, and especially the activity of the ganoderma water extraction ultrafiltrate is equivalent to that of high-purity beta- (1 → 3,1 → 6) -glucan under the condition of low concentration (10 mu g/mL), which shows that the active ingredients are effectively retained after ultrafiltration enrichment.
Claims (2)
1. A method for preparing ganoderma lucidum water extraction ultrafiltrate rich in beta- (1 → 3,1 → 6) -glucan comprises the following steps:
(1) Pulverizing Ganoderma fruiting body, adding water, boiling and extracting, and filtering to obtain Ganoderma water extractive solution;
(2) Performing ultrafiltration treatment on the ganoderma water extract to obtain ganoderma water extract ultrafiltrate;
wherein the ratio of the ganoderma lucidum sporocarp to the water is 1g: 20-30 mL of water with the temperature of 80-110 ℃; the time is 1-3 hours;
wherein the ultrafiltration is carried out by an ultrafiltration membrane with the molecular weight cutoff of 50 ten thousand daltons.
2. An aqueous ultrafiltrate of β - (1 → 3,1 → 6) -glucan enriched ganoderma lucidum prepared by the method of claim 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1618819A (en) * | 2004-10-20 | 2005-05-25 | 上海市农业科学院 | Preparation method of ganoderma polysaccharide |
| JP2006037016A (en) * | 2004-07-29 | 2006-02-09 | Shiriusu:Kk | METHOD FOR EXTRACTION OF beta-GLUCAN FROM DEER HORN AND GANODERMA LUCIDUM |
| CN101935358A (en) * | 2010-09-10 | 2011-01-05 | 上海市农业科学院 | Ganoderma lucidum polysaccharide and preparation method thereof |
| CN102219866A (en) * | 2011-06-15 | 2011-10-19 | 浙江工业大学 | Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006037016A (en) * | 2004-07-29 | 2006-02-09 | Shiriusu:Kk | METHOD FOR EXTRACTION OF beta-GLUCAN FROM DEER HORN AND GANODERMA LUCIDUM |
| CN1618819A (en) * | 2004-10-20 | 2005-05-25 | 上海市农业科学院 | Preparation method of ganoderma polysaccharide |
| CN101935358A (en) * | 2010-09-10 | 2011-01-05 | 上海市农业科学院 | Ganoderma lucidum polysaccharide and preparation method thereof |
| CN102219866A (en) * | 2011-06-15 | 2011-10-19 | 浙江工业大学 | Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp |
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