CN110483653A - A kind of preparation method and product and application with immunocompetent lentinan component - Google Patents

Abstract

The invention discloses a preparation method of a lentinan component with immune activity, which comprises the following steps: (1) coarsely pulverize dried mushrooms, and then superfinely pulverize them; (2) leaching with hot water (1) (3) Use a microfiltration membrane module with a pore size of 50‑100nm to process the lentinan water extract to achieve solid-liquid separation, or use a PPS filter bag to filter out the insoluble matter in the lentinan water extract. Substance, lentinan extract B; (4) The lentinan extract B in (3) was treated by ultrafiltration using three kinds of ultrafiltration membrane modules. The present invention realizes the effective separation of Lentinan components through multi-stage crushing and multi-stage filtration, and also provides convenience for recovering trehalose and mannitol rich in small molecular components. The technical scheme of the present invention is simple It is easy to operate, easy to realize large-scale preparation and production, and the production process is green and safe. The content of protein in the lentinan component obtained through the technical scheme is ≤1%, the content of flavonoids is ≤3.2%, and the content of reducing sugar is ≤2.8%.

Description

Preparation method, product and application of a lentinan component with immune activity

technical field

The invention relates to the field of extraction and purification of natural products, in particular to a preparation method of a lentinan component with immune activity.

Background technique

Lentinan is an effective active ingredient extracted from the fruiting bodies of Lentinus edodes, and is the main active substance of Lentinus edodes. A large number of studies have shown that it has significant anti-virus, anti-tumor, immune function regulation and stimulation of interferon formation. Therefore, lentinan has great application value in the field of medicine and health food. my country is a big producer of shiitake mushrooms, and the trade volume of shiitake mushrooms accounts for more than 95% of the world, which provides a rich source for the preparation of shiitake polysaccharides. In view of this, lentinan has been extensively studied in my country and even in the world, and has become an important functional ingredient in health products, and has broad application prospects in food and biomedicine.

In addition, shiitake mushrooms are also rich in small molecular substances such as trehalose and mannitol, and these two substances have a very broad application market and good development prospects. However, trehalose and mannitol in shiitake mushrooms are often neglected, and the separation and purification of the active lentinan that has been focused on research is mostly at the laboratory level, and it is urgent to improve the industrialization and large-scale preparation of high-purity lentinan components. . At present, the technology that can be used for large-scale separation and purification of lentinan is relatively lacking, and it is more urgent to prepare large-scale and industrialized technology of lentinan with higher purity.

There are mainly three problems in the industrial separation of polysaccharides in the prior art: (1) the purity of polysaccharides obtained by separation and purification is low; (2) the technical solutions used are difficult in the actual industrial scale-up process, including high scale-up costs and some equipment cannot be scaled up , Poor safety after amplification, etc.; (3) The technical solutions used are not green and sustainable enough, including high energy consumption, low efficiency, and low utilization of raw materials. In the following, representative technical solutions in the prior art will be cited as examples to clarify the deficiencies of the prior art in the field of industrialized separation and preparation of polysaccharides. Patent CN201010525618.1 discloses a technical method for large-scale separation and purification of polysaccharides using ultrafiltration concentration, Cellulose DE52 ion exchange column, gel column chromatography and dialysis. Although this technical solution can obtain high-purity polysaccharide components, but In addition to ultrafiltration and concentration, other technical solutions obviously have limitations in industrialization, especially gel column chromatography. Patent CN200610116956.3 discloses a method for purifying lentinan using an improved chromatographic column (DEAE-cellulose as a filler), but this method is eluted with NaOH solution, resulting in subsequent necessary impurity removal steps and in food The limitations in field application, therefore, are not conducive to its industrial scale-up. Patent CN201120045307.5 discloses a device for separating, purifying and concentrating lentinan. The device uses ceramic membrane microfiltration (0.05 μm ~ 0.5 μm) and reverse osmosis. The function of the microfiltration in this method is solid-liquid separation, so , its separation effect on lentinan is poor, and it is also difficult to realize the separation between lentinan fractions. Patent CN201310753452.2 discloses a method for extracting and grading lentinan. The technical solution described in this method involves various organic reagents such as chlorobenzene, calcium chloride, Sevag reagent and acetone; the purification steps of this method are complicated and the efficiency is low , In addition, after being treated by such methods, the recovery and utilization of the remaining components except the polysaccharide component is facing great difficulties, which is not green enough and sustainable.

In summary, the higher physiological and pharmacological activities of lentinan are well known to those skilled in the art. However, the existing technology for the industrial extraction, separation and purification of lentinan has problems of "high purity, low efficiency, and difficult industrialization", " High efficiency, industrialization, low purity" and difficult "green, sustainable development" and other issues. Therefore, in view of the above problems, there is an urgent need in the art to find a solution that can realize industrialization and large-scale preparation while ensuring the high purity of polysaccharides, and the technical solution is as green and sustainable as possible.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of lentinan components with immune activity. The preparation method is easy to realize large-scale and industrialized preparation and production, and it is easy to realize automation. The production process is green and safe. Through this preparation method , can realize the separation of various components in the mushroom extract, and the purity of the mushroom polysaccharide prepared by this method is high.

In order to solve the above-mentioned technical problems, the technical solution of the present invention is a preparation method of an immune-active lentinan component, the steps of which are as follows:

(1) Using dried shiitake mushrooms as raw materials, coarsely pulverize them with a pulverizer, and then use a mechanical vibration ultrafine pulverizer with a power of 3Kw to process dried shiitake mushrooms as fine particles of shiitake mushrooms with a size of 200 to 600 meshes;

(2) Use hot water to extract the fine particles of shiitake mushrooms in (1), the ratio of solid to liquid is 1:5-20, the extraction temperature is 60-120°C, the extraction time is 2-8h, and the material residue is removed by centrifugation at 5000rpm for 10min. Collect the water extract of lentinan, the solid content of which is 3-8%;

(3) Use a microfiltration membrane module with a pore size of 50-100nm to treat the lentinan extract to achieve solid-liquid separation, and the pressure is 0.1-0.8MPa, or use a PPS filter bag to filter out the insoluble substances in the lentinan extract, and the lentinan extract B;

(4) Three kinds of ultrafiltration membrane modules are used for ultrafiltration treatment of the lentinan extract after microfiltration and/or filter bag filtration in (3), and the three kinds of ultrafiltration membrane modules are respectively A1 Membrane, A2 membrane of 5kDa～10kDa and A3 membrane of 2～3kDa, the arrangement order of the membrane is from large to small molecular weight cut off or from small to large, membrane A1 cuts off component B1, membrane A1 filters out-membrane A2 cuts off component B2, membrane A2 filter out-membrane A3 retains component B3 and membrane A3 filters out component B4; in addition, components B1 and B4 can also be obtained from membrane A1 and membrane A3 by correspondingly processing the mushroom extract B;

Further, the B1, B2, and B3 components are concentrated using membrane A4 of a 200-350Da nanofiltration membrane, and freeze-dried or vacuum-dried to obtain dry lentinan components;

Further, the B4 is treated by nanofiltration of the membrane A4, the pressure is 1.6-3.0MPa, the molecular weight of the obtained membrane A4 intercepted liquid B4-1 is concentrated at 360Da, containing 50-70% trehalose, and the membrane A4 filtrate B4 -2 The molecular weight distribution is concentrated at 180Da, containing 45-70% of mannitol.

Further, the solid content of the lentinan extract B is 3-8%.

Further, membrane A1, membrane A2, and membrane A3 are roll-type ultrafiltration membranes, and the membrane A1 is selected from the roll-type ultrafiltration membrane of SEPRO Company of the United States whose model is PE10HR, and membrane A2 is selected from the model of PE5 of SEPRO Company of the United States. Roll type ultrafiltration membrane, membrane A3 is selected from the type H2540F30 roll type ultrafiltration membrane of General Electric Company of the United States.

An application of the lentinan component prepared by the above method in the preparation of immune drugs.

Further, the lentinan component is used to promote the proliferation of T lymphocytes.

Still further, the lentinan fraction is used to stimulate the spleen to secrete cytokines and increase the content of immunoglobulins.

Using the above-mentioned technical scheme, the effective separation of Lentinan components is realized through multi-stage crushing and multi-stage filtration, and it also facilitates the recovery of trehalose and mannitol rich in small molecular components, so as to realize the Comprehensive utilization of the extract, and the technical solution of the present invention is simple and easy to operate, easy to realize large-scale, industrialized production, and easy to realize automation, the production process is green and safe, and the protein content in the lentinan component obtained by the technical solution is ≤1 %, flavonoid content ≤ 3.2%, reducing sugar content ≤ 2.8%.

Description of drawings

Fig. 1 is a technical roadmap of the present invention;

Fig. 2 is the HPAEC-PAD chromatogram of the monosaccharide composition of the mushroom concentrate in Example 1 of the present invention;

Fig. 3 is the monosaccharide composition HPAEC-PAD chromatogram of each molecular component of Lentinan in Example 1 of the present invention (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 respectively are standard monosaccharides Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Fru, GalA and GlcA);

Fig. 4 is a HPSEC elution chromatogram comparison chart of lentinan components B1, B2, and B3 in Example 1 of the present invention;

Figure 5 is a liquid chromatogram of trehalose at different concentrations in Example 1 of the present invention;

Fig. 6 is the trace elution curve of the phenol-sulfuric acid method of the distilled water elution component of the water extract of shiitake mushrooms in comparative example 1 treated by AB-8 resin;

Fig. 7 is the HPGPC analysis comparison chart of graded alcohol precipitation HPGPC in the mushroom concentrate liquid solid content 1.5% in comparative example 2;

Figure 8 is a graph showing the proliferation rate of B lymphocytes in immunosuppressed mice with polysaccharides of different components in Example 1 of the present invention;

Figure 9 is a graph showing the spleen index of different components of polysaccharides acting on immunosuppressed mice in Example 1 of the present invention;

Fig. 10 is a thymus index diagram of polysaccharides of different components in Example 1 of the present invention acting on immunosuppressed mice;

Figure 11 is a diagram of the serum IgG content of different components of polysaccharides in Example 1 of the present invention acting on immunosuppressed mice;

Fig. 12 is a graph showing the serum IgM content of different polysaccharide components in Example 1 of the present invention acting on immunosuppressed mice.

Detailed ways

The specific embodiments of the present invention will be further described below in conjunction with the accompanying drawings. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

Example 1

As shown in Figure 1, dry shiitake mushrooms are taken as raw materials, and after being preliminarily crushed by a pulverizer, the shiitake mushroom powder is processed into fine particles of 200-600 meshes with a mechanical vibration ultrafine pulverizer. Lentinan was extracted with hot water, the ratio of solid to liquid was 1:10, the temperature was set at 100°C, and the extraction time was 4h. After the extraction, centrifuge at 5000rpm for 10min to remove material residue, and collect the lentinan extract. A microfiltration membrane module with a pore size of 100nm is used to treat the extract of Lentinus edodes polysaccharides to remove insoluble substances in the extract of Lentinus edodes, and the operating pressure is 0.4MPa. The concentrated liquid of shiitake mushrooms was hydrolyzed by sulfuric acid, and the monosaccharide composition was mainly determined by HPAEC-PAD to be Glc, Gal, and Man. The chromatogram of HPAEC-PAD is shown in Figure 2.

Add the lentinan extract to the feed tank, and start ultrafiltration to purify the lentinan components: ① Turn on the molecular weight cut-off of 2.5kDa roll-type ultrafiltration membrane to purify the lentinan extract B, control the pressure at 0.4-0.8MPa, and the temperature Control at 10-25°C, collect the filtrate (B4) and retentate; ②Close the 2.5kDa coiled ultrafiltration membrane module, open the 5kDa coiled ultrafiltration membrane module, control the pressure at 0.4-0.8MPa, and control the temperature at 10 -25°C, the collected filtrate is the 2.5kDa and 5kDa ultrafiltration fraction (B3), and the retentate undergoes the next ultrafiltration stage; ③Close the 5kDa membrane module, open the 10kDa ultrafiltration membrane module, and use Continue ultrafiltration, control the pressure at 0.4-0.8MPa, and control the temperature at 10-25°C. The collected filtrate is the 5kDa-10kDa component (B2), and the retentate is the 10kDa retentate (B1). The components B1, B2, and B3 are concentrated by nanofiltration membrane at a pressure of 2.0-2.6 MPa, and freeze-dried to obtain dry components of lentinan.

The obtained 2.5kDa filtrate B4 is continuously subjected to nanofiltration treatment using a 350Da nanofiltration membrane, the temperature is controlled at 10-25°C, the pressure is controlled at 2.2-2.4MPa, the obtained 350Da nanofiltration retentate is B4-1, and the obtained nanofiltration The output liquid is B4-2, and B4-1 and B4-2 are processed by freeze-drying.

The yield and physical and chemical properties of each component were measured and analyzed, and the results are shown in Figure 2-4. Wherein, the yield of the obtained B1 component is 2.71%, and its sugar content measured by the phenol sulfuric acid method is 95.72%. The HPGPC measurement shows that the elution peak shape of the B1 component is uniform and symmetrical, indicating that the molecular weight distribution of the component has good uniformity, and Concentrated distribution at 136kDa; measured by HPSEC-MALS, its polydispersity is 1.795, Rh is 4.96nm, [η] is 9.99mL/g, Mark-Houwink equation parameter K value is 1.881×10-2mL/g, α value is 0.662; B1 sample was hydrolyzed with 2M H2SO4 for 2 hours, and the HPAEC-PAD test results showed that its monosaccharide composition was mainly composed of Glc, Gal and Man in a ratio of 36.93:8.55:5.0, and also contained a small amount of Fuc, Xyl and GlcA.

The yield of the obtained B2 is 1.52%, the sugar content is 60.45%, and the purity is high. The molecular weight is mainly distributed in the range of 35 to 90 kDa as determined by HPGPC, the polydispersity is 1.241 as determined by HPSEC-MALS, the Rh is 1.648nm, and [η] is 6.24mL /g, Mark-Houwink equation parameter K value is 2.476×10-2mL/g, α value is 0.652; monosaccharide composition is Glc:Gal:Man:Xyl:GlcA=28.93:7.72:7.99:0.57:0.94.

The yield of the obtained B3 component is 5.45%, the sugar content is 38.67%, the molecular weight distribution is determined to be concentrated in 2.1～53kDa by HPGPC, and its polydispersity is 1.274 as measured by HPSEC-MALS, Rh is 1.137nm, [η] The K value of the Mark-Houwink equation parameter is 1.367×10-2mL/g, and the α value is 0.413; the monosaccharide composition is Glc:Gal:Man=16.35:2.00:1.16, and contains a small amount of Xyl.

The yield of the obtained B4-1 component was 26.59%, and the molecular weight determined by HPGPC was about 360Da. The sample was hydrolyzed with 2M H2SO4, and the detection result of HPAEC-PAD showed that only Glc was detected; the trehalose content was found quantitatively by liquid chromatography to 63%.

The yield of B4-2 obtained was 63.74%, and its molecular weight was determined to be about 180Da by HPGPC. At the same time, 2M H2SO4 was used to hydrolyze the sample, and the results of HPAEC-PAD showed that it did not contain Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Fru , GalA and GlcA and other monosaccharides, the main substances contained in this supplementary certificate are not monosaccharides, and the quantitative experiment of liquid chromatography shows that the content of mannitol reaches 48%.

Example 2

Dried shiitake mushrooms are taken as raw materials, and after being preliminarily crushed by a pulverizer, the shiitake mushroom powder is processed into fine particles of 200-600 meshes by a mechanical vibrating ultrafine pulverizer. Lentinan was extracted with boiling water, the ratio of solid to liquid was 1:10, and the extraction time was 6 hours. After the extraction is completed, filter the residue with three layers of gauze, and then centrifuge at 5000 rpm for 10 minutes to completely remove the residue of the material to obtain the extract of lentinan. The lentinan extract was manually filtered with a pp filter bag to remove insoluble substances in the extract of the mushroom, and the filtrate was collected.

Add the lentinan extract B to the feed liquid tank of the membrane separation equipment, and start ultrafiltration to purify the lentinan components: ① Turn on the roll-type ultrafiltration membrane with a molecular weight cut-off of 10kDa to purify the lentinan extract B, and control the pressure at 0.4- 0.8MPa, the temperature is controlled at 10-25°C, discharge and collect the ultrafiltration retentate from the discharge port to obtain the 10kDa retentate component B1, and refill the filtrate into the tank; ②Close the 10kDa roll-type ultrafiltration membrane module, Open the 5kDa roll-type ultrafiltration membrane module, control the pressure at 0.4-0.8MPa, and control the temperature at 10-25°C. Release the retentate from the discharge port and collect it to obtain 5kDa-10kDa ultrafiltration component B2. Add it into the tank; ③Close the 5kDa membrane module, open the 2.5kDa ultrafiltration membrane module, continue ultrafiltration of the 5kDa ultrafiltration retentate, control the pressure at 0.4-0.8MPa, and control the temperature at 10-25°C. The collected retentate is 2.5 kDa～5kDa component (B3), the filtrate is the 2.5kDa filtrate (B4).

Concentrate components B1, B2, and B3 using nanofiltration membrane, control the pressure at 2.0-2.6 MPa, concentrate the volume to 1/10 of the original mentioned, and then concentrate under reduced pressure, freeze-dry to obtain the dry component of Lentinan.

The yield and physical and chemical properties of each component were determined and analyzed. The yield of the obtained B1 component was 1.97%, and its sugar content was 86.21% as determined by the phenol-sulfuric acid method. The HPGPC elution peak shape was uniform and symmetrical, showing that the B1 component had a better molecular distribution uniformity and concentrated distribution at 136kDa, indicating that the B1 component The purity is high; the B1 sample was hydrolyzed with 2M H2SO4 for 2 hours, and the HPAEC-PAD test results showed that its monosaccharide composition was Glc:Gal:Man=33.14:10.57:7.50, and it also contained a small amount of Fuc, Xyl and GclA. The quality of the obtained B2 is 1.11%, the sugar content is 64.65%, and its molecular weight is mainly distributed in 35～90kDa as determined by HPGPC; its monosaccharide composition is mainly composed of Glc, Gal and Man in proportions of 26.74, 4.99 and 5.21 as determined by HPAEC-PAD, and the other Contains a small amount of Fuc and Xyl. The yield of the obtained B3 component was 3.96%, the sugar content was 38.67%, and the molecular weight distribution was determined to be concentrated in the range of 2.1 to 53 kDa as determined by HPGPC; after B3 was hydrolyzed with concentrated sulfuric acid, the determination by HPAEC-PAD showed that it was mainly Glc, Gal and Man are composed of 16.35, 2.00 and 1.16 in proportion, and contain a small amount of Xyl.

Comparative example 1 (AB-8 resin purified lentinan aqueous extract)

Dried shiitake mushrooms are taken as raw materials, and after being preliminarily crushed by a pulverizer, the shiitake mushroom powder is processed into fine particles of 200-600 meshes by a mechanical vibrating ultrafine pulverizer. Lentinan was extracted with boiling water, the ratio of solid to liquid was 1:10, and the extraction time was 4 hours. After the extraction is completed, the residue is removed by filtering with gauze, and then the material residue is completely removed by centrifugation at 5000 rpm for 10 minutes to obtain the extract of lentinan.

Use AB-8 resin to treat lentinan extract: (1) Packing pretreatment: first add ethanol or methanol equivalent to 0.5 times the volume of the filled resin into the adsorption column, and then put the new resin into the column; make the liquid level higher than The resin layer is about 0.3mm, and soak for 24 hours; use 2BV ethanol to pass through the resin layer at a flow rate of 2BV/h, and soak for 4 hours; use ethanol or methanol to pass through the resin layer at a flow rate of 2BV/h, wash until the effluent is added with water Not white and turbid. And wash ethanol with water at the same flow rate; use 2BV of 5% HCL solution to pass through the resin layer at a flow rate of 4BV/h, soak for 4 hours, wash with water at the same flow rate until the pH of the effluent is neutral; use 2BV of 2% NaOH solution , pass through the resin layer at a flow rate of 4BV/h, soak for 4 hours, and then wash with water at the same flow rate until the pH of the effluent is neutral; (2) Column packing: Pour the processed filler into a glass chromatograph with a specification of 26mm×600mm In the column, gently tap the column body with the ear washing ball while the filler is settling, and finally balance it with distilled water overnight; (3) Loading: Load the sample at no more than 5% of the column volume, and measure 10mL of mushroom concentrate , use a dropper to quickly add dropwise to the upper end of the column, try to make the sample evenly adsorbed on the same height of the resin, and wait for it to adsorb for 20 minutes; (3) Elution: turn on the constant flow pump, rotate at 45rpm, and elute 4 columns with deionized water Volume, adopt automatic partial collector to collect 129 tubes altogether of the sample of ultrapure water elution; (4) each tube collects the elution curve (as shown in Figure 6) of the phenol sulfuric acid tracer of polysaccharide determination through phenol sulfuric acid method, And collect samples according to this figure, wherein the collected liquids from the 10th to 20th tubes are combined to obtain W10-20 components, thus, W21-33, W34-39, W40-129 components are obtained.

The total sugar content of the obtained components was measured by the phenol sulfuric acid method, and the molecular mass distribution of the components was measured by HPGPC. The results showed that the molecular weight of the ultrapure water eluted component W10-20 was as high as 524kDa; while W21 -33, W34-39, and W40-129 have poor molecular mass distribution uniformity, which is similar to the molecular mass distribution HPGPC diagram of the water extract, so they do not have a separation effect.

Comparative example 2 (separation of lentinan components by fractional alcohol precipitation method)

Dried shiitake mushrooms are taken as raw materials, and after being preliminarily crushed by a pulverizer, the shiitake mushroom powder is processed into fine particles of 200-600 meshes by a mechanical vibrating ultrafine pulverizer. Lentinan was extracted with boiling water, the ratio of solid to liquid was 1:10, and the extraction time was 4 hours. After the extraction is completed, the residue is removed by filtering with gauze, and then the material residue is completely removed by centrifugation at 5000 rpm for 10 minutes to obtain the extract of lentinan.

Adjust the lentinan extract to a solid content of 1.5%, slowly add absolute ethanol to the lentinan extract until the final concentration of ethanol in the system is 20%, and after standing at 4°C for 6 hours, use a refrigerated centrifuge at 8000rpm for 20min. Centrifuge at 4°C to collect 20% concentration of centrifuged precipitate; continue to add ethanol to the supernatant to increase the concentration to 30% Concentration of centrifugal precipitation; supernatant continues to add ethanol to increase the concentration to 40%, after standing at 4 ° C for 6 hours, use a refrigerated centrifuge to centrifuge at 8000 rpm, 20 min, 4 ° C, and collect the centrifugal precipitation of 40% concentration; thus, Until the concentration of ethanol in the system reaches 80%. Dissolve the obtained precipitates of each component with appropriate amount of distilled water, and use a vacuum rotary evaporator to repeatedly add water to concentrate the solution until all ethanol is removed, and then freeze-dry it.

As shown in FIG. 7 , HPGPC was used to measure the molecular mass distribution of the precipitated components at ethanol concentrations of 30%, 40%, 50%, 60%, 70% and 80%. The results showed that except for the 60% ethanol alcohol precipitated fraction, the molecular mass distribution of other alcohol precipitated fractions was not uniform, presenting multiple molecular mass concentrated distribution regions (including 184Da～200kDa). The 60% alcohol precipitation fraction with a better molecular weight distribution in the fractional alcohol precipitation was hydrolyzed with 2M H2SO4 for 2 hours, and the HPAEC-PAD test results showed that its monosaccharide composition was Glc, Gal and Man according to the composition of 37.39:15.47:4.16. Contains a small amount of GlcA.

Compared with the method of Example 1 or 2, the steps used in the separation of components in the lentinan aqueous extract using this method are very cumbersome, and it is necessary to add ethanol step by step to precipitate different lentinan components; secondly, the method takes a long time, Low efficiency; again, ethanol is introduced in the method, and the ethanol needs to be removed by methods such as vacuum evaporation in the later stage, which increases the cost, and there are potential safety hazards in the recovery and storage of ethanol; finally, the obtained component with the best molecular weight distribution Its monosaccharide composition is different from the monosaccharide composition of the membrane separation method, and the Glc content in fraction B1 obtained from membrane separation accounts for a larger proportion.

Comparative example 3 (ammonium sulfate salting out method separates lentinan component)

Dried shiitake mushrooms are taken as raw materials, and after being preliminarily crushed by a pulverizer, the shiitake mushroom powder is processed into fine particles of 200-600 meshes by a mechanical vibrating ultrafine pulverizer. Lentinan was extracted with boiling water, the ratio of solid to liquid was 1:10, and the extraction time was 4 hours. After the extraction is completed, the residue is removed by filtering with gauze, and then the material residue is completely removed by centrifugation at 5000 rpm for 10 minutes to obtain the extract of lentinan.

Adjust the solid content of the lentinan extract to 2%, add ammonium sulfate until the saturation of ammonium sulfate is 20%, let stand at 4°C for 6-12h to settle, and then centrifuge (8000rpm, 20min) to collect the supernatant and precipitate respectively, and Continue to add ammonium sulfate to the supernatant to increase the saturation of ammonium sulfate from 20% to 25%, and collect the supernatant and precipitate respectively under the same conditions; thus, add ammonium sulfate to 90% with a gradient of 5% in turn. Collect each gradient precipitation fraction and the final supernatant fraction; use dialysis to remove ammonium sulfate, concentrate the dialyzed fluid, freeze-dry, and weigh to calculate the yield.

The molecular weight distribution of each component is measured, and the results show that: (1) the recovery rate of the component obtained by ammonium sulfate salting-out is low, and only 30%, 35%, 65%, and 70% gradients obtain precipitation, and the precipitation yield is respectively 0.4%, 3.5%, 4.6% and 3.4%; (2) due to the addition of a large amount of ammonium sulfate in the polysaccharide system, each component needs to be desalted during the recovery process, and the final remaining 90% ammonium sulfate supernatant Due to the high content of ammonium salt in the liquid, it is difficult to recover, and the ammonium salt and the sample liquid are viscous and difficult to dry; (3) 30% of the precipitated component was successfully recovered, and the total sugar content was determined to be 9.98% by the phenol-sulfuric acid method. ±2.14%, indicating that its main component is not polysaccharide; (4) 2M H2SO4 was used to hydrolyze 30% of the precipitated fraction for 2 hours, and the HPAEC-PAD test results showed that its monosaccharide composition was Glc, and its content was only 4.79%.

In summary, the ammonium sulfate salt-out method is compared with the three-stage ultrafiltration membrane separation method of embodiment 1 or 2, and its separation steps are loaded down with trivial details, time-consuming, has introduced ammonium salt, and separation effect is not good; Therefore, the ammonium sulfate salt-out method It is not suitable for separating the lentinan components from the lentinan extract.

Physical and chemical properties and immune activity experiments were carried out for the product obtained in Example 1

1. Materials and methods

Levamisole hydrochloride (Shandong Renhetang Pharmaceutical Co., Ltd.), cyclophosphamide (Jiangsu Shengdi Pharmaceutical Co., Ltd.), 15% sheep red blood cells (SRBC), 1640 medium, red blood cell lysate (Beijing Suolaibao), fetal bovine serum (BiologicalIndustries), ConA, LPS, MTT (Sigma), CCK8 (Japan Collaborative Research Institute), IgG, IgM, IgA, IgE Kit (Wuhan Uersen), IL-6, TNF-α, IL-1β (Wuhan Boster), ferric chloride, mannitol, and trehalose are all analytically pure in China.

2. Main instruments and equipment

3K15-high-speed refrigerated centrifuge (Sigma Company), METTLER TOLEDO AL104 electronic balance (Mettler-Toledo Instrument Co., Ltd.), -80 ℃ ultra-low temperature refrigerator (Thermo, USA), CO2 incubator (Thermo, USA), multi-functional enzyme label Instrument Varioskan Flash (Thermo, USA), Bruker 400M NMR (Bruker, Germany), LC-MS 1290-6400 (Agilent, USA), Waters High Performance Liquid Chromatography (Suzhou Curtis White Industrial Equipment Co., Ltd.)

3. Experimental method

3.1 Analysis of physical and chemical properties

(1) Determination of neutral sugar content: with glucose as a standard product, the content of neutral sugar in the sample was determined by the phenol-sulfuric acid method, and the sample was measured in parallel three times, and the average value was taken.

(2) Determination of uronic acid content: with glucuronic acid as a standard, the sulfuric acid-carbazole method was used to determine the content of uronic acid in the sample, and the samples were measured in parallel three times, and the average value was taken.

(3) Determination of protein content: with bovine serum albumin (BSA) as a standard, the protein content was determined by the Coomassie brilliant blue method, the samples were measured in parallel three times, and the average value was taken.

(4) Determination of total phenolic content: using gallic acid as the conversion equivalent, the modified Folin–Ciocalteu method was used to determine the total phenolic content. Add 25 μL of gallic acid standard or polysaccharide sample solution to the 96-well plate, and then add 125 μL of Folin-Ciocalteu reagent correspondingly, mix and react for 10 min, add 125 μL of 7.5% Na2CO3 solution (mass fraction), and place at 25 ° C for 30 min, Measured with a microbore ultraviolet spectrophotometer (EL340, Bio-TekInstruments Inc., Winooski, VT, USA) at 765nm, the results are expressed as the percentage of gallic acid equivalent per gram of sample dry weight, and all standards and samples are paralleled three times.

(5) Determination of flavonoid content: rutin solutions of different concentrations were used as standard solutions for the determination of ketone content, and a standard curve of flavonoid content was drawn according to the absorbance value of the standard, and all standards and samples were parallelized three times.

3.2 Monosaccharide composition analysis

The composition of monosaccharides was analyzed by an ICS-5000 ion chromatograph. Accurately weigh 5.0 mg of sample, add 0.5 mL of ultrapure water to dissolve it. Stir 0.5mL of 12M H2SO4 solution in ice bath for 0.5h, then add 2mL of ultrapure water, and place the sample in 105°C oil bath for 2h. The hydrolyzate was diluted and analyzed by ion chromatography after passing through a 0.22 μm filter membrane.

Chromatographic column: CarboPacPA20 analytical column (3mm×150mm), CarboPacPA20 analytical column (3mm×30mm); eluent: 0.25M NaOH solution, 1M CH3COONa; elution flow rate: 0.5mL/min; injection volume: 10μL; column temperature 35°C.

3.3 Determination of trehalose and mannitol in B4-1 and B4-2

Liquid phase analysis: Prepare trehalose and mannose standard products at 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL and 5.0mg/mL respectively, and establish corresponding standard curves. Quantitative analysis of trehalose in B4-1 and mannitol in B4-2 was carried out by high performance gel permeation chromatography.

3.4 Immunological activity experiment

The immune activity of B1, B2 and B3 components of lentinan was evaluated by the immune activity test. (1) The clean female BALB/c mice were modeled by the current method of multiple intraperitoneal injections of cyclophosphamide; (2) Randomly grouped , model group, polysaccharide group, protoplasm group and positive group mice intraperitoneal injection of the cyclophosphamide 100mg/kg bw model, intragastric administration of high, medium and low doses were 15mg/kg, 30mg/kg, 60mg/kg; ( 3) Another 14 mice were taken as the normal control group, and the same volume of normal saline was administered to the stomach; (4) Delayed hypersensitivity of the model group, the polysaccharide group, the homogenate group and the positive control group were measured—tissue swelling degree , T lymphocyte proliferation rate, B lymphocyte proliferation rate, the impact of NK cell activity in immunosuppressed mice, IL-1β, IL-6, IgA, IgE indicators in the supernatant of mouse spleen homogenate, and the effect on the lentinan group The immune activity of the points was evaluated.

Indicator measurement:

(1) After the mice were killed, the organs were weighed, and the organ index was calculated;

(2) Delayed hypersensitivity—tissue swelling;

(3) T lymphocyte proliferation test;

(4) B lymphocyte proliferation experiment;

(5) Determine the content of immunoglobulin (IgG, IgM) in the serum according to the instructions of the ELISA kit;

(6) NK cytotoxicity (%)=(OD target cell control group absorbance value-(OD sample group absorbance value-OD blank control group absorbance value)/OD effector cell control group absorbance value)×100%;

(7) Spleen cytokines and immunoglobulins of immunosuppressed mice: Accurately weigh the weight of the mouse spleen, add pre-cooled PBS according to the ratio of 1:9 (w:v), and homogenize with a homogenizer, 10000r/ Centrifuge for min and collect the supernatant. The content of IL-1β, IL-6, IgA, and IgE in the supernatant of mouse spleen homogenate was determined according to the operating instructions of the ELISA kit.

4. Experimental results

The results show that the preparation of the present invention can obtain lentinan components with higher purity, wherein the purity of B1 is the highest and can reach 95%, followed by the purity of B2 can reach more than 60%, and the purity of B3 can also reach about 40%. Comprehensive separation of polysaccharide fractions. In addition, the experimental results show that B4-1 mainly contains trehalose, which is as high as more than 60%, and the mannitol content in B4-2 is also close to 50%, and they are discarded as waste materials in the production of lentinan. Therefore, the present invention It can also provide a comprehensive and comprehensive extraction technology for the existing lentinan production process, and provide a new green and sustainable development technology route for the existing relevant personnel and units of polysaccharide production. As shown in Figure 8-12, the immune activity experiment shows that B1, B2 and B3 can increase the spleen index and thymus index of immunosuppressed mice, promote the proliferation of T cells, enhance the allergic delayed reaction of immunosuppressed mice, stimulate The spleen secretes cytokines and immunoglobulins, among which B3 has the strongest immune activity, followed by B2, and finally B1.

In summary, the present invention provides a method for preparing lentinan components with different molecular weights with immunological activity, through ultrafine pulverization, hot water extraction, three-stage ultrafiltration separation, and nanofiltration concentration, so that the separation of polysaccharides in this process is simple. It consumes less energy, has high purity polysaccharides, is green, pollution-free, and sustainable, and has a stable separation effect and good reproducibility. Extraction technology brings higher social production value.

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (10)

1. a kind of preparation method with immunocompetent lentinan component, it is characterised in that: steps are as follows,

(1) dried thin mushroom first carries out coarse crushing with pulverizer, then with mechanical oscillation micronizer, obtains the mushroom of 200~600 mesh Fine particle；

(2) using mushroom fine particle in hot water extraction (1), solid-liquid ratio 1:5-20, Extracting temperature is 60-120 DEG C, when extraction Between be 2-8h, by 5000rpm, material residue is removed in 10min centrifugation, collects lentinan Aqueous extracts；

(3) it uses aperture to handle lentinan Aqueous extracts for 50-100nm micro-filtration membrane module and realizes separation of solid and liquid, pressure 0.1- 0.8MPa, or the insoluble substance in lentinan Aqueous extracts, lentinan extracting solution B are removed using PPS sock filtration；

(4) using the lentinan extracting solution B in three kinds of hyperfiltration membrane assembly hyperfiltration treatments (3), three kinds of hyperfiltration membrane assemblies are respectively Molecular cut off is A1 film, the A2 film of 5kDa~10kDa and the A3 film of 2~3kDa of 10kDa~300kDa, and film puts in order It is descending or ascending for molecular cut off, film A1 retain component B1, film A2 retain component B2, film A3 retain component B3 with And film A3 filters out component B4.

2. the preparation method of lentinan component according to claim 1, it is characterised in that: the lentinan Aqueous extracts Solid content be 3-8%.

3. the preparation method of lentinan component according to claim 1, it is characterised in that: use 200-350Da nanofiltration B1, B2, B3 component is concentrated in the film A4 of film, obtains lentinan dry product component after freeze-drying or vacuum drying.

4. the preparation method of lentinan component according to claim 1, it is characterised in that: the B4 uses the film A4 Nanofiltration processing, pressure 1.6-3.0MPa, gained film A4 trapped fluid B4-1, film A4 filter liquor B4-2.

5. the preparation method of lentinan component according to claim 1, it is characterised in that: the film A1, film A2, film A3 For rolling ultrafiltration membrane.

6. the preparation method of lentinan component according to claim 5, it is characterised in that: the film A1 is selected from beauty The rolling ultrafiltration membrane of the model PE10HR of SEPRO company, state, film A2 are selected from the rolling of the model PE5 of U.S. SEPRO company Ultrafiltration membrane, film A3 are selected from the rolling ultrafiltration membrane of the model H2540F30 of General Electric Company.

7. a kind of lentinan component as prepared by the preparation method of any one of claims 1 to 6.

8. a kind of lentinan component by claim 7 is in the application prepared on immunogenic pharmaceutical.

9. lentinan component according to claim 8 is preparing the application on immunogenic pharmaceutical, it is characterised in that: described Lentinan component B1, B2 and B3 are used to promote the proliferation of T lymphocyte.

10. lentinan component according to claim 8 is preparing the application on immunogenic pharmaceutical, it is characterised in that: institute Lentinan component B1, B2 and B3 are stated for stimulating spleen secrete cytokines and improving immunoglobulin content.