CN102702378B - Method for extracting polysaccharides with cell immunocompetence from needle mushroom root waste materials - Google Patents
Method for extracting polysaccharides with cell immunocompetence from needle mushroom root waste materials Download PDFInfo
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- CN102702378B CN102702378B CN201210219695.3A CN201210219695A CN102702378B CN 102702378 B CN102702378 B CN 102702378B CN 201210219695 A CN201210219695 A CN 201210219695A CN 102702378 B CN102702378 B CN 102702378B
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Abstract
The invention relates to a method for extracting polysaccharides with cell immunocompetence from needle mushroom root waste materials and belongs to the field of biomedicine. The method comprises the steps of: drying and crushing needle mushroom roots to prepare ultrafine needle mushroom root powder, wherein the needle mushroom roots are the cut root parts as waste materials in a needle mushroom processing course; adding a proper amount of 85% ethanol to the crushed ultrafine powder to soak for 2 hours at room temperature; centrifuging to collect a precipitate; adding deionized water according to a ratio of water to material namely 28mL/g based on the raw material as a reference; carrying out ultrasonic treatment for 19.8 minutes at the power of 680W; centrifuging to collect a supernate; carrying out rotary evaporation on the supernate to obtain a proper amount of at room temperature; adding anhydrous ethanol the volume of which is 4 times that of a concentrated solution in the concentrated solution; precipitating for 12 hours at 4 DEG C; centrifuging to collect a precipitate; and placing the precipitate in an oven of 60 DEG C to bake so as to obtain crude needle mushroom root polysaccharide. An ultrasonic wave extraction technology is adopted, and the polysaccharide yield is 16.20%. Compared with the traditional hot water extraction, by using the method disclosed by the invention, the yield is improved by 70%, and the time is shortened by 92%.
Description
One, technical field
The present invention relates to a kind of method of extracting the polysaccharide with Cell-mediated Immunity from needle mushroom root waste material, belong to biomedicine field, product can be widely used in the fields such as functional food, biological medicine.Wherein needle mushroom root is in the needle mushroom course of processing, and the root the base of a fruit cut as waste material divides.
Two, background technology
Needle mushroom (Flammulina velutipes) has another name called plain mushroom, dried mushroom, be subordinate to Mycophyta, Hymenomycetes, Agaricales, Tricholomataceae, gold thread Pseudomonas, contain higher protein, carbohydrate and robust fibre, and lipid content is lower, also containing the multivitamins such as VB1, VB2, VC, and be rich in the several mineral materials such as calcium, phosphorus, iron, is a kind of rare high nutritious prod.
Along with the mankind deepen gradually to the understanding of protective foods and needle mushroom nutritive value, needle mushroom will have boundless DEVELOPMENT PROSPECT, meanwhile, also the waste material that has increasing needle mushroom root is produced.According to China edible mushrooms association, add up, cut-off to 2007 year China's needle mushroom ultimate production is about 144.1 ten thousand tons, wherein needle mushroom root accounts for 15% left and right of needle mushroom weight, be about 21.6 ten thousand tons, yet in the production process of needle mushroom, mushroom root goes out of use mostly, and even the feed of continuous cropping livestock is all not satisfactory, is not fully utilized.Discarded in production intermediate links of needle mushroom root, has increased the cost of processing waste to enterprise like this.Therefore, the rational exploitation and utilization of needle mushroom root is extremely urgent, so need to open up an approach for the utilization of needle mushroom root.
In the exploitation of polysaccharide, separation and Extraction is one of crucial step, and it not only affects the yield of product, is also worth closely related with the activity of product.Flammulina velutipes can be water-soluble, be insoluble to the organic solvents such as alcohol, ether, ketone, thereby polysaccharide traditional extraction process mainly contains hot water extraction, soda acid extraction, enzyme process etc.Traditional hot water extraction and soda acid extraction, required extraction time is long, extraction temperature is high, energy consumption is large, and for a long time high temperature or acid-alkali medium likely cause polysaccharide to degrade, and make the extraction of polysaccharide be difficult to reach ideal effect, these have all limited the exploitation of polysaccharide greatly.Along with scientific and technological progress, the more method of employing of extracting about polysaccharide is at present ultrasonic extraction, utilize strong cavitation effect that Ultrasonic Radiation produces, mechanical vibration, disturbance effect, high acceleration, emulsification, spread, smash and the multiple effect such as stirring, increase frequency and the speed of material molecule, thereby accelerate target component and enter solvent, realize and efficiently extract rapidly entocyte, thereby improved efficiency, shortened the time; Leaching process does not relate to chemical reagent simultaneously, and this has reduced to a certain extent on the bioactive impact of lixiviate material, so ultrasound assisted extraction technique is used widely in Effective Component of Chinese Medicine extraction process.Utilizing ultrasonic extraction needle mushroom root polysaccharide, study its suitable extraction conditions, improve polysaccharide yield, is the important channel of improving needle mushroom root waste utilization rate, is also the basis of follow-up bioactivity research.
Three, summary of the invention
Technical problem
The object of this invention is to provide and a kind ofly can from needle mushroom root waste material, extract the method with Cell-mediated Immunity polysaccharide, the method has been avoided traditional extraction technology, and extraction time is long, the drawback that extraction yield is low, energy consumption is high.This method is simple to operate simultaneously, and the polysaccharide yield of extraction is higher, can farthest improve the utilization ratio of waste material, reduces the pollution to environment.
Technical scheme
From needle mushroom root waste material, extract a method with Cell-mediated Immunity polysaccharide, make by the following method:
1) preparation of needle mushroom root super-fine powder: the airing of fresh needle mushroom root is after 1 thousand, being put in 60 ℃ of air blast in baking oven dries, then through 30B pulverizer, pulverize, double roll crusher is broken, 80 mesh sieves divide, and micronizer mill is pulverized, and 300 mesh sieves divide, make needle mushroom root super-fine powder, be placed in-20 ℃ standby.
2) take needle mushroom root super-fine powder, add under appropriate 85% ethanol room temperature standing 2h with remove portion pigment and impurity, according to ratio of water to material 10～50mL/g, add deionized water again, utilize DCTZ-2000 thermostatic ultrasonic extractor, with 200～680W supersound extraction, 5～25min, centrifuging and taking supernatant liquor; Supernatant liquor employing Sevag reagent (propyl carbinol: trichloromethane=1: 4) method Deproteinization, under 8000r/min condition, centrifugal 10min gets supernatant;
3) use Rotary Evaporators by supernatant concentration to appropriate, add 4 times of volume dehydrated alcohols, alcohol precipitation 12h at 4 ℃, 5000r/min is centrifugal, and 15min gets precipitation, in 60 ℃ of oven for drying, obtains needle mushroom root Crude polysaccharides powder;
4) needle mushroom root Crude polysaccharides powder is spent to ionized water and be made into 10mg/mL solution, add DEAE-Mierocrystalline cellulose-52 exchange column, then use successively deionized water, 0.1,0.3mol/L NaCl solution gradient wash-out, separation obtains 3 components;
5) calculation formula of Crude polysaccharides yield:
Crude polysaccharides yield (%)=Crude polysaccharides quality (g)/ultra micro grain weight (g) * 100%
Beneficial effect
The present invention compares with traditional hot water extraction, and extraction efficiency is high, and needle mushroom root polysaccharide yield is high.
In the Application and Development of mushroom root waste material, it is one of crucial step that activeconstituents is wherein extracted, and it not only merely affects the yield of product, and direct relation economic benefit also has close relationship with the biological activity of product.Due to traditional hot water extraction, extraction time is long, polysaccharide yield is low, be difficult to farthest improve the utilization ratio of mushroom root waste material, in order to improve this situation, the present invention has adopted ultrasonic wave extraction, studied ultrasonic time, ratio of water to material, this 3 single factors impact on polysaccharide yield of ultrasonic power, screen the central point of each factor, on this basis, adopt the BBD method of Design experts V6.0 software, carry out the extracting parameter optimization Test of 3 factor 3 levels, use Design experts V6.0 software to carry out statistical study simultaneously, determine the optimum combination of 3 factor different levelss, the processing parameter that last optimization needle mushroom root polysaccharide extracts is ultrasonic power 680W, ultrasonic time 19.8min, ratio of water to material is 28mL/g, result of study shows according to processing parameter of the present invention, the yield of needle mushroom root polysaccharide can reach 16.20%, compare with traditional hot water lixiviate, yield has improved 70%, time shorten 92%.
The present invention adopts ultrasonic wave extraction, utilize the multiple effects such as strong cavitation effect that Ultrasonic Radiation produces, mechanical vibration, disturbance effect, the penetration power of the frequency of increase material molecule and speed, solvent, thereby the stripping of effective constituent in promotion cell, significantly improves polysaccharide yield and extraction efficiency, it is simple that while ultrasonic wave extraction has equipment, processing ease, the advantages such as applied widely, production efficiency is high, and energy waste is little.Therefore, this technology is the making full use of of waste material of needle mushroom root, has opened up new approach.
Four, accompanying drawing explanation
Fig. 1 is the present invention and contrast extraction conditions comparison.
Fig. 2 is needle mushroom root polysaccharide DEAE-Mierocrystalline cellulose-52 elution curves.
Fig. 3 is needle mushroom root polysaccharide and the impact of purified components on macrophage proliferation rate thereof.
Five, embodiment
(1) test design
1, hot water extraction
With reference to accounting for the report of building the people such as ripple, take the optimal conditions of needle mushroom traditional hot water lixiviate polysaccharide as raw material adopts and slightly make an amendment, extract 80 ℃ of temperature, extraction time 4h, during ratio of water to material 28ml/g, the yield of polysaccharide is 9.51%.
2, ultrasonic extraction
Select ultrasonic power, ultrasonic time and ratio of water to material to do single factor experiment, by single factor experiment, tentatively determine the optimum range of 3 variablees, carry out Three factors-levels Box-Behnken test design, test level and the coding of each factor are listed in table 1, and Box-Behnken test design and response value thereof are in Table 2.
3, elution process:
Needle mushroom root Crude polysaccharides powder is spent to ionized water and be made into 10mg/mL solution, add DEAE-Mierocrystalline cellulose-52 exchange columns (2.6cm * 30cm), each applied sample amount is 10mL, then use successively deionized water, 0.1, 0.3, 0.5, 0.7mol/L NaCl solution gradient wash-out, using DHL-2B computer digital display constant flow pump coutroi velocity is 1mL/min, fraction collection (10mL/ pipe), collect phenolsulfuric acid method color developing detection for liquid pipe, at wavelength, be to measure absorbancy under 490nm, the pipe number of collecting of take is X-coordinate, light absorption value (490nm) is ordinate zou drafting DEAE-Mierocrystalline cellulose-52 chromatographic column elution curve, select the interval Fractional Collections sample in optimal absorption peak.Elution curve is shown in Fig. 2.
4, activity
Adopt CCK-8 method.Its principle that detects cytoactive is that WST-8 can be reduced to by the desaturase in the cell mitochondrial in viable cell the orange-yellow formazan dyestuff with high water soluble under the effect of electron carrier 1-Methoxy PMS, the quantity of formazan and the quantity of viable cell that generate are directly proportional; and the quantity of formazan can be weighed with the light absorption value at 450nm place, thereby the quantity that indirectly reflects viable cell, so within the scope of certain cell quantity, can reflect by OD450 value the quantity of survivaling cell.
(2) embodiment
Needle mushroom root, by Nanjing, Gao Gu edible mushrooms scientific & trading Co., Ltd. provides.
Needle mushroom root super-fine powder: after the airing of fresh needle mushroom root is extremely half-dried, is put in 60 ℃ of air blast in baking oven and dries, then pulverize through 30B pulverizer, double roll crusher is broken, and 80 mesh sieves divide, and micronizer mill is pulverized, 300 mesh sieves divide, and make needle mushroom root super-fine powder, be placed in-20 ℃ standby.
Raw materials pretreatment: take 15g needle mushroom root super-fine powder, add appropriate 85% dehydrated alcohol, under room temperature, standing 2h is with remove portion pigment and impurity, and 5000r/min is centrifugal, and 10min gets precipitation, standby.
1, hot water lixiviate needle mushroom root Crude polysaccharides (contrast)
(1) the learnt from else's experience throw out of alcohol pre-treatment, take raw material as benchmark, adds 420mL deionized water fully to mix, lixiviate 4h in 80 ℃ of water-baths (can in good time stir during lixiviate) by ratio of water to material 28mL/g;
(2) the centrifugal 15min of vat liquor 5000r/min gets supernatant, and supernatant liquor employing Sevag reagent (propyl carbinol: trichloromethane=1: 4) method Deproteinization, under 8000r/min condition, centrifugal 10min gets supernatant;
(3) supernatant liquor is put 50 ℃ of Rotary Evaporators (N-1100EYELA type Rotary Evaporators, Tokyo physics and chemistry apparatus Co., Ltd.) and is evaporated in right amount, and concentrated solution adds 4 times of volume dehydrated alcohols in 4 ℃ of refrigerators precipitation 12h;
(4) the centrifugal 15min taking precipitate of 5000r/min, throw out is put 60 ℃ of oven for drying, can obtain the needle mushroom root Crude polysaccharides of hot water lixiviate.Crude polysaccharides yield is 9.51%, and extraction time is 240min.
2, supersound extraction needle mushroom root Crude polysaccharides (the present invention)
(1) the learnt from else's experience throw out of alcohol pre-treatment, by ratio of water to material 28mL/g, adding 420mL deionized water fully to mix, is ultrasonic 19.8min under 680W with DCTZ-2000 multipurpose thermostatic ultrasonic extractor (Beijing Hongxianglong Biotechnology Development Co., Ltd) at power;
(2) the centrifugal 15min of vat liquor 5000r/min gets supernatant, and supernatant liquor employing Sevag reagent (propyl carbinol: trichloromethane=1: 4) method Deproteinization, under 8000r/min condition, centrifugal 10min gets supernatant;
(3) supernatant liquor is put 50 ℃ of Rotary Evaporators (N-1100EYELA type Rotary Evaporators, Tokyo physics and chemistry apparatus Co., Ltd.) and is evaporated in right amount, and concentrated solution adds 4 times of volume dehydrated alcohols in 4 ℃ of refrigerators precipitation 12h;
(4) the centrifugal 15min taking precipitate of 5000r/min, throw out is put 60 ℃ of oven for drying, can obtain the needle mushroom root Crude polysaccharides of ultrasonic extraction.Crude polysaccharides yield is 16.20%, and the time used is 19.8min.
As shown in Figure 1, adopt the present invention to compare with traditional hot water extraction, needle mushroom root Crude polysaccharides yield has improved 70%, time shorten 92%.
3. determination of activity
(1) in 96 orifice plates, every hole adds the scavenger cell RAW264.7 cell suspension of 100 μ L logarithmic phases, and final concentration is 2 * 10
5individual/mL, is placed in 5%CO
2, 37 ℃ of incubators (HEPA Class100 cell culture incubator) cultivate 4h.
(2) discard nutrient solution, every hole adds Crude polysaccharides, FSP-1, FSP-2, the FSP-3 solution of 100 μ L DMEM substratum preparation different concns.Every kind of sample is divided into 4 groups, and Group I adds DMEM substratum, is Normal group; Group II adds respectively the polysaccharide soln of 5 μ g/mL, 25 μ g/mL and 50 μ g/mL to Group IV, every group establish 4 parallel.
(3) 96 orifice plates are placed in after 5%CO2,37 ℃ of incubators cultivation 20h, every hole adds 10 μ LCCK-8 solution, and room temperature jolts 10min, continues to cultivate 4h, is determined at the absorbancy at 450nm place by microplate reader.
Macrophage proliferation rate calculation formula is as follows:
Macrophage proliferation rate=A
1/ A
0, A wherein
1for the light absorption value of sample sets parallel test, A
0light absorption value for Normal group parallel test.
Needle mushroom root polysaccharide and purified components thereof are on the impact of macrophage proliferation rate as shown in Figure 3, except FSP-3, Crude polysaccharides, FSP-1 and FSP-2 are greater than 1 to the proliferation rate of scavenger cell, illustrate that Crude polysaccharides, FSP-1 and FSP-2 can promote macrophage proliferation.
Test level and the coding of each factor of table 1 Box-Behnken test design
Table 2 Box-Behnken test design and response value thereof
Claims (1)
1. from needle mushroom root waste material, extract a method with Cell-mediated Immunity polysaccharide, wherein needle mushroom root is in the needle mushroom course of processing, and the root the base of a fruit cut as waste material divides, and comprising:
1) take needle mushroom root super-fine powder, add under 85% ethanol room temperature standing 2h with remove portion pigment and impurity, centrifuging and taking precipitation, add again deionized water, the consumption of deionized water is 10～50mL/g, utilize thermostatic ultrasonic extractor, with 200～680W supersound extraction, 5～25min, centrifuging and taking supernatant liquor; Supernatant liquor adopts Sevag method Deproteinization, and under 8000r/min condition, centrifugal 10min gets supernatant;
2) use Rotary Evaporators that supernatant is concentrated, in concentrated solution, add 4 times of volume dehydrated alcohols, alcohol precipitation 12h at 4 ℃, 5000r/min is centrifugal, and 15min gets precipitation, in 60 ℃ of oven for drying, obtains needle mushroom root Crude polysaccharides powder;
3) the Crude polysaccharides powder making is spent to ionized water and be made into 10mg/mL solution, use DEAE-Mierocrystalline cellulose-52 chromatography column, use successively deionized water, 0.1,0.3mol/L NaCl solution gradient wash-out, obtain successively 3 components; And by mouse macrophage RAW264.7 culture experiment, find needle mushroom root Crude polysaccharides and there is immunoregulatory activity with deionized water and the resulting purified product of 0.1mol/LNaCl eluant solution.
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| CN103141816B (en) * | 2013-03-19 | 2015-04-22 | 中华全国供销合作总社济南果品研究院 | Processing method and application of enoki mushroom roots |
| CN103788223B (en) * | 2014-01-16 | 2016-05-04 | 山东省农业科学院农产品研究所 | A kind of extracting method of flammulina velutipes |
| CN103788229B (en) * | 2014-02-19 | 2016-02-17 | 浙江省农业科学院 | A kind of method simultaneously extracting polysaccharide and polyphenol from needle mushroom |
| CN104277135A (en) * | 2014-09-23 | 2015-01-14 | 华南农业大学 | Pleurotus tuber-regium polysaccharide and extraction method thereof |
| CN104387485A (en) * | 2014-11-12 | 2015-03-04 | 绥化学院 | Method for extracting polysaccharides in flammulina velutipes by synergism of complex enzymes and high-pressure hot water extraction process |
| CN105111324A (en) * | 2015-08-28 | 2015-12-02 | 武汉工程大学 | Flash extraction method of polysaccharide from Flammulina velutipes leftovers |
| CN109021138A (en) * | 2018-08-09 | 2018-12-18 | 合肥福泉现代农业科技有限公司 | A method of polyferose is prepared from the polyoses extract of enoki mushroom roots waste |
| CN109988249B (en) * | 2018-11-13 | 2020-12-15 | 时代生物科技（深圳）有限公司 | Preparation method and application of flammulina velutipes polysaccharide FVP |
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