CN104059161A - Pleurotus eryngii leftover crude polysaccharide and preparation method thereof - Google Patents

Abstract

The invention discloses a pleurotus eryngii leftover crude polysaccharide which is prepared by the following steps: crude polysaccharide extract, ethanol precipitation, dialysis and drying. The pleurotus eryngii leftover crude polysaccharide has the advantages of high yield, low protein content and greatly higher content of beta-glucan.

Description

A kind of crude polysaccharide of Pleurotus eryngii leftovers and preparation method thereof

technical field

The invention mainly relates to the field of edible fungus extraction, in particular to a crude polysaccharide from Pleurotus eryngii leftovers and a preparation method thereof.

Background technique

Pleurotus eryngii (Pleurotus Eryngii) is a new species of rare edible fungi that has been successfully developed and cultivated in recent years, integrating edible and medicinal uses. Modern pharmacological research shows that Pleurotus eryngii polysaccharide is rich in content and is one of the main active ingredients. It has significant pharmacological effects in antithrombosis, blood lipid regulation, immune function regulation, antitumor, and antiradiation.

Pleurotus eryngii leftovers are the by-products of Pleurotus eryngii processing, such as mushroom feet, mushroom slices, and stipe bases, which are discarded during processing because they are difficult to eat or have low commodity value.

Conventional crude polysaccharide extraction is only to crush the leftovers of Pleurotus eryngii into coarse powder, and the obtained crude polysaccharide has a low yield and low polysaccharide content. Sugar content is low. At the same time, ordinary pulverization extracts polysaccharides with high protein content, but the existing deproteinization technology is relatively complicated and will cause loss of polysaccharides, which is not conducive to large-scale production.

Contents of the invention

The technical problem to be solved by the present invention is to provide a kind of crude polysaccharide of Pleurotus eryngii leftovers and its preparation method, to overcome the low yield of crude polysaccharide in the prior art, the low content of β-glucan in crude polysaccharide, and the difficulty of industrial production Shortcomings.

The present invention firstly provides a kind of crude polysaccharide of Pleurotus eryngii leftovers, which is prepared by the following method:

Extraction of crude polysaccharides: crush the leftovers of Pleurotus eryngii into nano-scale powders, extract in boiling water for 1-2 hours to obtain crude polysaccharide extracts;

Ethanol precipitation: add absolute ethanol to the crude polysaccharide extract until the ethanol content reaches 70%-80%, let it stand at room temperature for 12-15h, and collect the precipitate;

Dialysis and drying: dissolve the precipitate with water, dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da, and freeze-dry the solution after dialysis to obtain the crude polysaccharide of Pleurotus eryngii leftovers.

Specifically, the Pleurotus eryngii leftovers crude polysaccharide of the present invention is prepared by the following method:

Extraction of crude polysaccharides: use Pleurotus eryngii leftovers as raw materials, crush them into nano-scale powder, add 15-20 times the weight of water, soak at room temperature for 30-60min, heat to boiling, keep boiling slightly for 60-120min, centrifuge at 9700g for 10- 15min; the supernatant was concentrated under reduced pressure until the ratio of solid to liquid (mass/volume) was 1:6-1:7;

Ethanol precipitation: Slowly add absolute ethanol to the concentrated solution, stir while adding, until the ethanol content reaches 70%-80%, stand at room temperature for 12-15h, centrifuge to remove the supernatant of alcohol precipitation, and collect the precipitate.

Dialysis and drying: dissolve the precipitate with water, dialyze the crude polysaccharide solution at 4°C for 3-4 days with a dialysis bag with a molecular weight cut-off of 3500Da, freeze-dry the solution after dialysis, and obtain the crude polysaccharide of Pleurotus eryngii.

The present invention also provides a method for preparing the crude polysaccharide of Pleurotus eryngii leftovers, which comprises the steps of:

Extraction of crude polysaccharides: crush the leftovers of Pleurotus eryngii into nano-scale powders, extract in boiling water for 1-2 hours to obtain crude polysaccharide extracts;

Ethanol precipitation: add absolute ethanol to the crude polysaccharide extract until the ethanol content reaches 70%-80%, let it stand at room temperature for 12-15h, and collect the precipitate;

Dialysis and drying: dissolve the precipitate with water, dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da, and freeze-dry the solution after dialysis to obtain the crude polysaccharide of Pleurotus eryngii.

The Pleurotus eryngii leftover crude polysaccharide prepared by the invention has a high yield of more than 10%, a polysaccharide content of more than 60%, a low protein content, and a greatly increased content of beta-glucan in the crude polysaccharide, which exceeds 30%. HPSEC-MALLS-RI analysis showed that the crude polysaccharide mainly had three sugar peaks, and the monosaccharide analysis showed that the crude polysaccharide was mainly composed of glucose, and the crude polysaccharide had a significant effect on the release of NO from the RAW264.7 macrophage cell line. Therefore, the preparation method is convenient, efficient, low in cost, high in yield and content of crude polysaccharide, and comprehensive in waste utilization, suitable for large-scale production.

In the invention, the nanoscale crushing of the leftovers of Pleurotus eryngii releases a large amount of polysaccharides on the cell wall, thereby improving the yield of crude sugar, and greatly increasing the content of polysaccharides and β-glucan. At the same time, crude polysaccharides showed good cellular immune activity.

Therefore, the use of Pleurotus eryngii leftovers to extract Pleurotus eryngii crude polysaccharides not only has rich sources of raw materials and low production costs, but also saves resources and reduces environmental pollution, so as to achieve the purpose of comprehensive utilization of Pleurotus eryngii resources.

Description of drawings

Figure 1 Molecular weight distribution of crude polysaccharides

Figure 2 Monosaccharide composition of crude polysaccharides

Figure 3 Crude polysaccharides promote the amount of NO released by RAW264.7 macrophage cell line

Detailed ways:

Example 1:

1. Extraction of Pleurotus eryngii crude polysaccharide: take the leftovers of Pleurotus eryngii (Xinghan, Shanghai Guosen Biotechnology Co., Ltd.) B, crushing time 6h, particle size 10-1000nm), weigh 10g, add 200mL distilled water, soak at room temperature for 60min, heat to boiling, keep boiling slightly for 120min, centrifuge at 9700g for 12min. Repeat the above steps for the precipitate, extract once more, and combine the supernatant.

2. Concentration of the crude polysaccharide extract: the supernatant was concentrated to 60 mL under reduced pressure in a rotary evaporator.

3. Ethanol precipitation: Slowly add absolute ethanol to the concentrated solution, stir while adding, until the ethanol content reaches 70%, let it stand at room temperature for 12 hours, remove the supernatant of alcohol precipitation by centrifugation, and collect the precipitate.

4. Dialysis and drying: Dissolve the precipitate in 70 mL of water, and use a dialysis bag with a molecular weight cut-off of 3500 Da (Sinopharm Chemical Reagent Co., Ltd.) to dialyze the crude polysaccharide solution at 4°C for 3 days to remove small molecular substances. The dialyzed crude polysaccharide solution was frozen in a -20°C refrigerator for 2 hours, then frozen in a -80°C refrigerator for 3 hours, and finally put into a freeze dryer to freeze-dry to obtain a dry crude polysaccharide.

The yield of the prepared crude polysaccharide is 12.30%, the polysaccharide content is 61.55%, and the β-glucan content is 31.63%.

Analytical balance weighs the weight of crude polysaccharide dry product. The crude polysaccharide yield was calculated according to the following formula.

Detection of polysaccharide and β-glucan content:

Determination of polysaccharide content: Determination by phenol-sulfuric acid method, accurately weigh 5mg of this product, put it in a 2ml centrifuge tube, add 1ml of distilled water, heat to dissolve, cool to room temperature, and centrifuge. The supernatant was diluted 100 times, 1.0m1 of the sample was accurately drawn, put into a 15ml test tube, and phenol and sulfuric acid reagents were added respectively. After a full reaction, the absorbance was measured at 490nm, and the sugar content of the sample was calculated using dextran as a standard.

Determination of β-glucan content: The content of β-glucan in the polysaccharide sample was determined according to the method in the Yeast and Mushroom β-glucan Detection Kit of Megazyme Company.

Example 2:

1. Extraction of Pleurotus eryngii crude polysaccharide: take the leftovers of Pleurotus eryngii (Xinghan, Shanghai Guosen Biotechnology Co., Ltd.) B, crushing time 6h, particle size 10-1000nm), weigh 10g, add 150mL distilled water, soak at room temperature for 30min, heat to boiling, keep boiling slightly for 120min, centrifuge at 9700g for 12min. Repeat the above steps for the precipitation, and then extract once more for 60 minutes, and combine the supernatants.

2. Concentration of the crude polysaccharide extract: the supernatant was concentrated to 70 mL under reduced pressure in a rotary evaporator.

3. Ethanol precipitation: Slowly add absolute ethanol to the concentrated solution, stir while adding, until the ethanol content reaches 75%, let it stand at room temperature for 15 hours, remove the supernatant of alcohol precipitation by centrifugation, and collect the precipitate.

4. Dialysis and drying: Dissolve the precipitate in 50 mL of water, and use a dialysis bag with a molecular weight cut-off of 3500 Da (Sinopharm Chemical Reagent Co., Ltd.) to dialyze the crude polysaccharide solution at 4°C for 3 days to remove small molecular substances. The dialyzed crude polysaccharide solution was frozen in a -20°C refrigerator for 2 hours, then frozen in a -80°C refrigerator for 3 hours, and finally put into a freeze dryer to freeze-dry to obtain a dry crude polysaccharide.

The yield of the prepared crude polysaccharide was 10.97%, the polysaccharide content was 67.33%, and the β-glucan content was 35.60%.

Example 3:

1. Sample preparation: Weigh 5 mg of the crude polysaccharide prepared in Example 1, add 1 mL of HPLC mobile phase, fully dissolve, centrifuge at 13200 r/min for 10 min, and filter the supernatant through a 0.25 μm water phase microporous membrane before performing HPSEC-MALLS- RI analysis.

2. Chromatographic analysis conditions: TSK PWXL6000 and TSK PWXL3000 gel chromatographic columns are connected in series for analysis, and the mobile phase is a solution containing 0.05 mol/L NaH ₂ PO ₄ and 0.15 mol/L NaNO ₃ (pH=7, 0.02 % sodium azide), the flow rate is 0.5mL/min, the temperature of the chromatographic column is kept constant at 35°C with a column thermostat; the wavelength of the light source of the laser detector is 623.8nm. The refractive index increment (dn/dc) of the polysaccharide in the solution is calculated as 0.146mL/g.

3. Molecular weight calculation: use Astra (version6.1.1, Wyatt Technology, Santa Barbara, CA) data analysis software to collect and analyze light scattering data, and calculate molecular weight and other polysaccharide molecular properties.

The analysis spectrum of HPSEC-MALLS-RI is shown in Figure 1. The crude polysaccharide component obtained after the Pleurotus eryngii leftovers were pulverized by nano-powder mainly contained three peaks, and the molecular weight distribution range was relatively wide, ranging from 250,000 to 5 million Daltons. Example 4:

1. Sample preparation: Weigh 2 mg of the crude polysaccharide prepared in Example 2 into a glass bottle with a cover, add 2 mL of 2 mol/L trifluoroacetic acid, take an oil bath at 110°C for 4 hours, cool to room temperature, and blow dry with a nitrogen blower at 50°C , add methanol to dry, repeat twice until there is no sour smell, rinse the glass bottle with 2mL ultrapure water in a centrifuge tube, centrifuge at 13200r/min for 10min, and analyze the supernatant by high performance anion chromatography (HPAEC).

2. Chromatographic conditions: chromatographic column CarboPac ^TM PA20 (3×150nm); mobile phase 0.8% NaOH solution (pH=12); time 30min; column temperature 30°C; flow rate 0.45mL/min; injection volume 25μL.

The monosaccharide composition detected by high performance anion chromatography is shown in Figure 2. The crude polysaccharide component is composed of five monosaccharides, arabinose, galactose, glucose, xylose and mannose after hydrolysis, among which glucose accounts for the highest proportion, reaching more than 70%, followed by mannose and galactose, which are about 10%. %, while the ratio of xylose and arabinose is very low.

Example 5:

Determination of the activity of the crude polysaccharides from Pleurotus eryngii leftovers to stimulate macrophages to release NO in vitro:

1. Sample preparation: Accurately weigh the Pleurotus eryngii offal crude polysaccharide samples prepared in Example 1 and Example 2 respectively in a sterilized eppendorf tube, and prepare a sample solution with a concentration of 5 mg/mL with sterile PBS . After fully dissolving, centrifuge at 15,000 r/min for 40 min, transfer the supernatant to a new sterile eppendorf tube under aseptic conditions, and dilute the sample to 2 mg/ml and 0.5 mg/ml for use.

2. Cell culture: Take the RAW264.7 macrophage cell line in the logarithmic growth phase (purchased from the Institute of Cells, Chinese Academy of Sciences), and use DMEM complete medium (Gibco Company) to subculture at 37 ° C under the condition of 5% CO ₂ . 0.05% trypsin (Gibco Company) was digested, and the suspension was centrifuged at 1000 rpm/min for 3 minutes to collect cells and counted for later use.

3. Reagent preparation and standard curve drawing

Griess reagent: add 6.25ml H ₃ PO ₃ in the beaker, add distilled water 250ml, add 2.5g sulfanilamide (sulfanilamide, Sigma company) and 0.25g naphthylethylenediamine dihydrochloride (naphthylethylenediaminedihydrochloride, Sigma company) respectively, use Magnetic stirrer until it is completely dissolved, and the brown reagent bottle is stored in a refrigerator at 4°C.

Standard curve drawing: prepare sodium nitrite solutions with different concentrations, the concentration gradient is 0, 5, 10, 15, 20, 25, 30, 35, 40μM, a total of nine; take 100μL in a 96-well plate hole, add 50μL Griess reagent , Determination of absorbance at 543nm, standard curve for each concentration 3 replicates, draw a standard curve according to the absorbance.

4. Determination of the amount of NO released by sample-stimulated macrophages: collect RAW264.7 cells, and dilute the cells to 5×10 ⁵ /mL with colorless RPMI1640 (10% fetal bovine serum + 1% antibiotic liquid, Gibco company) medium , added to a 96-well plate, 180 μL was added to each well, and then 20 μL of the sample to be tested was added. The positive control was LPS (1 μg/mL, Sigma Company), and incubated at 37° C. for 48 hours. Take 100 μL of the supernatant in a 96-well plate, add 50 μl of Griess reagent, incubate at room temperature for 10 min, and measure the absorbance at 543 nm. Cellular NO release was calculated according to the standard curve.

The results are shown in Figure 3. From the results, it can be seen that the crude polysaccharides of Pleurotus eryngii leftovers prepared in Examples 1 and 2 can significantly promote the release of NO from the RAW264.7 macrophage cell line, and can significantly enhance the macrophage The phagocytosis and killing ability of phagocytes, thereby enhancing the anti-tumor ability of the body.

Claims (3)

1. a kind of Pleurotus eryngii leftovers crude polysaccharide, is characterized in that it is prepared by following method: Extraction of crude polysaccharides: crush the leftovers of Pleurotus eryngii into nano-scale powders, extract in boiling water for 1-2 hours to obtain crude polysaccharide extracts; Ethanol precipitation: add absolute ethanol to the crude polysaccharide extract until the ethanol content reaches 70%-80%, let it stand at room temperature for 12-15h, and collect the precipitate; Dialysis and drying: dissolve the precipitate with water, dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da, and freeze-dry the solution after dialysis to obtain the crude polysaccharide of Pleurotus eryngii leftovers. 2. Pleurotus eryngii leftovers crude polysaccharide according to claim 1 is characterized in that it is prepared by the following method: Extraction of crude polysaccharides: use Pleurotus eryngii leftovers as raw materials, crush them into nano-scale powder, add 15-20 times the weight of water, soak at room temperature for 30-60min, heat to boiling, keep boiling slightly for 60-120min, centrifuge at 9700g for 10- 15min;; The supernatant was concentrated under reduced pressure until the ratio of solid to liquid (mass/volume) was 1:6-1:7; Ethanol precipitation: Slowly add absolute ethanol to the concentrated solution, stir while adding, until the ethanol content reaches 70%-80%, stand at room temperature for 12-15h, centrifuge to remove the supernatant of alcohol precipitation, and collect the precipitate; Dialysis and drying: dissolve the precipitate with water, dialyze the crude polysaccharide solution at 4°C for 3-4 days with a dialysis bag with a molecular weight cut-off of 3500Da, freeze-dry the solution after dialysis, and obtain the crude polysaccharide of Pleurotus eryngii. 3. a method for preparing the crude polysaccharide of Pleurotus eryngii leftovers described in claim 1 or 2, is characterized in that it comprises the steps: Extraction of crude polysaccharides: crush the leftovers of Pleurotus eryngii into nano-scale powders, extract in boiling water for 1-2 hours to obtain crude polysaccharide extracts; Ethanol precipitation: add absolute ethanol to the crude polysaccharide extract until the ethanol content reaches 70%-80%, let it stand at room temperature for 12-15h, and collect the precipitate; Dialysis and drying: dissolve the precipitate with water, dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da, and freeze-dry the solution after dialysis to obtain the crude polysaccharide of Pleurotus eryngii.