CN103524636B - A kind of preparation method of portobello mushroom polysaccharide - Google Patents
A kind of preparation method of portobello mushroom polysaccharide Download PDFInfo
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Abstract
The invention discloses a kind of preparation method with the portobello mushroom polysaccharide of immunoregulatory activity, with applicable modern large-scale production.Brown mushroom powder is carried out microwave extraction; Be cooled to room temperature; Adopt macroporous adsorbent resin to carry out removal of impurities, afterwards, adjust ph scope is 3.0-10.0, adopts the process of anion-cation exchange resin series connection resin column deproteinated; Ultra-filtration technique is adopted to be further purified the brown mushroom Crude polysaccharides after removal of impurities, deproteinated; The molecular weight ranges retained in purge process is 100000-800000D; By above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze, make the moisture fully charge in material, vacuum-drying afterwards obtains portobello mushroom polysaccharide.The series connection removal of impurities of the method integrated application microwave extraction technology, resin and deproteinated technology, ultrafiltration technology purifying and Freeze Drying Technique, gained portobello mushroom polysaccharide purity can reach more than 90%, and shows obvious immunoregulation effect.
Description
Technical field
The present invention relates to health food processing technique field, particularly relate to a kind of preparation method with the portobello mushroom polysaccharide of immunoregulatory activity.
Background technology
Brown mushroom Agaricuscrocopeluspeck, it is tawny that its mushroom cement layer cell contains brown pigments, the fibrous scale of the many lifes of mushroom lid, therefore named brown mushroom or brown squama mushroom, belongs to load Jun Gang ﹑ agaric Mu ﹑ Agaricus edibilis, Agaricus.The large handle of brown mushroom cap is thick, bacterial context is plump, aromatic flavour is agreeable to the taste, be rich in the multiple nutrients materials such as polysaccharide, protein, robust fibre, amino acid and mineral substance, there is high nutritive health-care medical value, therefore become the higher-grade edible mushrooms in fashionable America and Europe and World Developed Countries or area.
Numerous research shows, brown mushroom has significantly antitumor, reducing blood-fat, hypoglycemic, anti-ageing, hypotensive and improve the nourishing functions such as immunity of organisms.Wherein, polysaccharide, as the main activeconstituents of brown mushroom, has become the focus of Recent study, has had comparatively wide DEVELOPMENT PROSPECT.
At present; also rare on domestic and international market have portobello mushroom polysaccharide extract and related products; preparation method's report for portobello mushroom polysaccharide in document is also only limited to the techniques such as traditional hot water return (ultrasonic, microwave) extraction, water extract-alcohol precipitation; polysaccharide yield and purity are all very restricted; be not suitable for the requirement of modern large-scale production, greatly limit the application of portobello mushroom polysaccharide in protective foods and pharmaceutical prod.
Therefore, research and development portobello mushroom polysaccharide extracts preparation method, forms portobello mushroom polysaccharide product preparation process, for raising portobello mushroom polysaccharide added value of product, meets the need of market, has great economic benefit and social benefit.
Summary of the invention
The object of the invention is the technological deficiency for existing in prior art, a kind of preparation method of high purity portobello mushroom polysaccharide of applicable large-scale production is provided.
The technical scheme adopted for realizing object of the present invention is:
A preparation method for portobello mushroom polysaccharide, comprises the steps:
(1) brown mushroom powder is carried out microwave extraction;
(2) brown for above-mentioned gained mushroom Aqueous extracts is cooled to room temperature, obtains brown mushroom Crude polysaccharides solution;
(3) adopt macroporous adsorbent resin to carry out removal of impurities to step (2) gained brown mushroom Crude polysaccharides solution, afterwards, adjust ph scope is 3.0-10.0, adopts the process of anion-cation exchange resin series connection resin column deproteinated;
(4) ultra-filtration technique is adopted to be further purified the brown mushroom Crude polysaccharides after removal of impurities, deproteinated; The molecular weight ranges retained in purge process is 100000-800000D;
(5) by above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze, make the moisture fully charge in material, vacuum-drying afterwards obtains portobello mushroom polysaccharide.
The step that described employing macroporous adsorbent resin carries out removal of impurities process to brown mushroom Crude polysaccharides solution is as follows: with glucose meter, getting concentration is that 1.0-50mg/mL brown mushroom Crude polysaccharides solution joins and processedly good is equipped with in the adsorption column of macroporous adsorbent resin, adsorb with the flow velocity of 0.1-3BV/h, wash-out is carried out with 3-10 times of cylinder ponding, elution flow rate is 0.5-5BV/h, obtains deimpurity elutriant.
The step that described employing anion-cation exchange resin carries out deproteinated process is as follows: macroporous adsorbent resin process of learning from else's experience remove deimpurity elutriant, concentrated, adjustment concentration is to 1.0-50mg/mL, adjust ph 3.0-10.0, successively by processed good anionite-exchange resin and cationic exchange numerical value series connection resin column, adsorb with the flow velocity of 0.1-3BV/h, carry out wash-out with 3-10 times of cylinder ponding, elution flow rate is 0.5-5BV/h, obtains Deproteinated portobello mushroom polysaccharide solution; Wherein, the mass ratio of anionite-exchange resin and resin cation (R.C.) is 1:0.5-1:10.
Step (4) described ultra-filtration technique comprises the steps: to the further grading purification of the portobello mushroom polysaccharide after deproteinated the portobello mushroom polysaccharide solution getting deproteinated process gained, carry out ultrafiltration, molecular weight cut-off scope is 100000-800000D, operating pressure is 0.2-0.8Mpa, temperature is 20-50 DEG C, and the rotating speed of rotor is 200-800r/min; Collection retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution, dry, obtains portobello mushroom polysaccharide.
Described macroporous adsorbent resin is nonpolar macroporous adsorption resin.
Described anionite-exchange resin is 201x7, D201 or D301, or approximate model resin; Described Zeo-karb is D113 or 001x7, or approximate model resin.
Described property macroporous adsorbent resin is D101, AB-8 or D4020, or approximate model resin.
In step (3), Crude polysaccharides solution is by before macroporous resin column, and with glucose meter, adjustment concentration is to 1.0-50mg/mL; After crossing macroporous resin column in step (4), polysaccharide soln is before deproteinated process, should concentrate, and with glucose meter, adjustment concentration is to 1.0-50mg/mL, and pH value should be adjusted to 3.0-10.0.
In step (5), pre-freeze is to-60 to-80 DEG C, maintains at least 4h; Vacuum drying temperature is-45 DEG C.
During microwave extraction in step (1), microwave power is 200-800W, and solid-liquid ratio is 1:10-1:100.
Compared with prior art, the invention has the beneficial effects as follows:
1, the preparation method of portobello mushroom polysaccharide of the present invention, combine the multiple technologies such as microwave extraction, macroporous adsorbent resin, ion exchange resin series connection removal of impurities, deproteinated technology, ultrafiltration and lyophilize, portobello mushroom polysaccharide (molecular weight ranges 100000-800000D) purity of preparation is high, content is greater than 90%, and productive rate can reach more than 3.0%.Present method is efficient, easy, economical, and, without organic solvent, meet environmental requirement, be applicable to industrial scale and produce
2, the preparation method of portobello mushroom polysaccharide of the present invention adopts macroporous resin, ion exchange resin column tandem process to carry out removal of impurities, deproteinated process to brown mushroom Crude polysaccharides; albumen decreasing ratio can reach more than 87%; relative to traditional Sevag method, trichloroacetic acid method etc.; there is the advantages such as environmental protection, efficient, safety, meet the requirement of large-scale production.
3, the preparation method of portobello mushroom polysaccharide of the present invention adopts ultrafiltration technology purification technique, carries out grading purification to polysaccharide, and molecular weight cut-off scope is 100000-800000D, purification effect is good, operating process is simple, and whole technique can be carried out continuously, is beneficial to scale operation.
4, the present invention adopts Freeze Drying Technique to carry out cryodrying to portobello mushroom polysaccharide, ensure that the original structure of portobello mushroom polysaccharide to greatest extent, unaffectedly provides guarantee for it is active.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
1, brown mushroom raw material being crushed to 60 orders, taking 100 grams, be placed in Microwave Extraction Apparatus, is that Extraction solvent extracts with water, and microwave frequency is 500W, and extraction time is 5 minutes, solid-liquid ratio 1:40.
2, above-mentioned brown mushroom Aqueous extracts is cooled to room temperature, centrifugal, get supernatant liquor, and add water regulate concentration to 1.0mg/mL(with glucose meter).
3, macroporous adsorbent resin is adopted, removal of impurities process is carried out to brown mushroom Crude polysaccharides solution: joined by above-mentioned portobello mushroom polysaccharide solution and processedly good be equipped with in the adsorption column of D4020 macroporous adsorbent resin, with 0.1BV/h(column volume/hour) flow velocity adsorb, wash-out is carried out with 3 times of cylinder ponding, flow velocity is 0.5BV/h, collects water elution liquid.
4, ion exchange resin is adopted to carry out deproteinated process to brown mushroom crude polysaccharide extract solution: to get the above-mentioned elutriant through removal of impurities process, suitably concentrated, adjust concentration to 1.0mg/mL(with glucose meter), and add ammoniacal liquor adjust ph to 10.0, with 1BV/h(column volume/hour) good D201 ion exchange resin column and 001x7 ion exchange resin column (the mass ratio 1:0.5 of D201 resin and 001 × 7 resin) are housed by processed successively, wash-out is carried out with 6 times of cylinder ponding, flow velocity is 5BV/h, collects water elution liquid.
5, the ultra-filtration and separation of portobello mushroom polysaccharide: suitably concentrated by above-mentioned elutriant, carries out ultrafiltration.Molecular weight cut-off scope is 100000-800000D, and operating pressure is 0.4Mpa, and temperature is 35 DEG C, and the rotating speed of rotor is carry out loop ultrafiltration under the condition of 800r/min.Collect the concentrated solution retained, and with distilled water cleaning, merge washing lotion and concentrated solution.
6, the lyophilize of portobello mushroom polysaccharide: by above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze to-60 DEG C, after temperature of charge reaches pre-freezing temperature, then maintain at least 4h, to ensure the moisture fully charge in material.At-45 DEG C, carry out vacuum-drying afterwards, time of drying 20h.Finally prepare portobello mushroom polysaccharide product 3.0 grams, in white sponge, adopt sulfuric acid-phynol method to measure its content, result shows that polysaccharide content is that 92.28%(is with glucose meter).
Embodiment 2
1, brown mushroom raw material is crushed to 80 orders, takes 100 grams, being placed in Microwave Extraction Apparatus is that Extraction solvent extracts with water, and microwave frequency is 800W, and extraction time is 4 minutes, solid-liquid ratio 1:100.
2, above-mentioned brown mushroom Aqueous extracts is cooled to room temperature, centrifugal, get supernatant liquor, and add water regulate concentration to 20mg/mL(with glucose meter).
3, macroporous adsorbent resin is adopted, removal of impurities process is carried out to brown mushroom Crude polysaccharides extracting solution: joined by above-mentioned portobello mushroom polysaccharide solution and processedly good be equipped with in the adsorption column of D101 macroporous adsorbent resin, with 1.0BV/h(column volume/hour) flow velocity adsorb, wash-out is carried out with 10 times of cylinder ponding, flow velocity is 2.0BV/h, collects water elution liquid.
4, ion exchange resin is adopted to carry out deproteinated process to brown mushroom crude polysaccharide extract solution: to get the above-mentioned Crude polysaccharides solution through removal of impurities process, suitably concentrated, adjust concentration to 20mg/mL(with glucose meter), and add ammoniacal liquor adjust ph to 8.0, with 0.1BV/h(column volume/hour) good D301 ion exchange resin column and D113 ion exchange resin column (the mass ratio 1:5 of D301 resin and D113 resin) are housed by processed successively, wash-out is carried out with 10 times of cylinder ponding, flow velocity is 1.2BV/h, collects water elution liquid.
5, the ultra-filtration and separation of portobello mushroom polysaccharide: suitably concentrated by above-mentioned elutriant, carries out ultrafiltration.Molecular weight cut-off scope is 100000-800000D, and operating pressure is 0.2Mpa, and temperature is 50 DEG C, and the rotating speed of rotor is carry out loop ultrafiltration under the condition of 600r/min.Collection retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution.
6, the lyophilize of portobello mushroom polysaccharide: by above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze to-60 DEG C, after temperature of charge reaches pre-freezing temperature, then maintain at least 4h, to ensure the moisture fully charge in material.At-45 DEG C, carry out vacuum-drying afterwards, time of drying 20h.Finally prepare portobello mushroom polysaccharide product 3.3 grams, in white sponge, adopt sulfuric acid-phynol method to measure its content, result shows that polysaccharide content is that 90.15%(is with glucose meter).
Embodiment 3
1, brown mushroom raw material is crushed to 80 orders, takes 100 grams, being placed in Microwave Extraction Apparatus is that Extraction solvent extracts with water, and microwave frequency is 200W, and extraction time is 5 minutes, solid-liquid ratio 1:10.
2, above-mentioned brown mushroom Aqueous extracts is cooled to room temperature, centrifugal, get supernatant liquor, and add water regulate concentration to 50mg/mL(with glucose meter).
3, macroporous adsorbent resin is adopted, removal of impurities process is carried out to brown mushroom Crude polysaccharides extracting solution: joined by above-mentioned portobello mushroom polysaccharide extracting solution and processedly good be equipped with in the adsorption column of AB-8 macroporous adsorbent resin, with 3BV/h(column volume/hour) flow velocity adsorb, wash-out is carried out with 5 times of cylinder ponding, flow velocity is 5BV/h, collects water elution liquid
4, ion exchange resin is adopted to carry out deproteinated process to brown mushroom crude polysaccharide extract solution: to get the above-mentioned Crude polysaccharides extract through removal of impurities process, suitably concentrated, adjust concentration to 50mg/mL(with glucose meter), and add dilute hydrochloric acid adjust ph to 3.0, with 3BV/h(column volume/hour) good 201x7 ion exchange resin column and D113 ion exchange resin column (the mass ratio 1:10 of 201x7 resin and D113 resin) are housed by processed, wash-out is carried out with 3 times of cylinder ponding, flow velocity is 0.5BV/h, collects elutriant.
5, the ultra-filtration and separation of portobello mushroom polysaccharide: suitably concentrated by above-mentioned elutriant, carries out ultrafiltration.Molecular weight cut-off scope is 100000-800000D, and operating pressure is 0.8Mpa, and temperature is 20 DEG C, and the rotating speed of rotor is carry out loop ultrafiltration under the condition of 200r/min.Collection retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution.
6, the lyophilize of portobello mushroom polysaccharide: by above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze to-60 DEG C, after temperature of charge reaches pre-freezing temperature, then maintain at least 4h, to ensure the moisture fully charge in material.At-45 DEG C, carry out vacuum-drying afterwards, time of drying 20h.Finally prepare portobello mushroom polysaccharide product 3.0 grams, in white sponge, adopt sulfuric acid-phynol method to measure its content, result shows that polysaccharide content is that 93.50%(is with glucose meter).
Experimental analysis
The immunity moderation of portobello mushroom polysaccharide and anti-cerebral anoxia active function are evaluated:
1, portobello mushroom polysaccharide is on the impact of mouse immune organ thymus gland, index and spleen index
Mouse random packet is also weighed on an empty stomach, and male and female dual-purpose, physiological saline group physiological saline, uses the portobello mushroom polysaccharide aqueous solution for tested group, continuous gavage 7d, every day 1 time, 20mLkg
-1.And record the food consumption of mouse every day.After administration 3d, claim a Mouse Weight and note down, according to body weight adjustment dosage.Again weigh on an empty stomach when 8d, and after administration 1h, pluck eyeball sacrificed by exsanguination mouse, extract Thymus and spleen, claim its quality with analytical balance, calculate organ index.
Test-results is as shown in table 1:
Table 1: portobello mushroom polysaccharide is on the impact of young mice thymus gland, index and spleen index
Note: compared with physiological saline group, * p<0.10, * * p<0.05
Result shows, compare with physiological saline group, the high, medium and low dosage group of portobello mushroom polysaccharide all can make the thymus index of young mice, index and spleen index increases, and high dose group shows significant difference (p<0.05) to thymus index, index and spleen index, middle dosage group has shown different (p<0.10) to thymus index, index and spleen index.Illustrate that portobello mushroom polysaccharide can strengthen cellular immunization and humoral immunization process, thus enhancing body non-specific immune function, improve Abwehrkraft des Koepers.
2, to dehisce after brown spore mushroom Polysaccharides on Mice broken end the impact of breathing time
Mouse random packet, male and female dual-purpose, physiological saline group physiological saline, uses the portobello mushroom polysaccharide aqueous solution for tested group, continuous gavage 7d, every day 1 time, 20mLkg
-1.And record the food consumption of mouse every day.After administration 3d, claim a Mouse Weight and note down, according to body weight adjustment dosage.After last administration 1h, successively mouse basal part of the ear rear portion sacrificed by decapitation mouse, dehisce after calculating mouse broken end with stopwatch immediately breathing time and rate elongation.Test-results is as shown in table 2:
Table 2: portobello mushroom polysaccharide is on the impact of breathing time of dehiscing after young mice broken end
Note: compared with physiological saline group, * p<0.10, * * p<0.05
Result shows: compare with physiological saline group, the high, medium and low dosage group of portobello mushroom polysaccharide all can extend the mouth breathing time of young mice, and high dose group shows significant difference (p<0.05), middle dosage group has shown different (p<0.10).Show that portobello mushroom polysaccharide has reduction and organizes oxygen consumption, improve substance metabolism and energy metabolism, show certain anti-cerebral anoxia effect.
Method synthesis of the present invention utilizes the multiple technologies such as microwave extraction technology, macroporous adsorbent resin, ion exchange resin column series connection removal of impurities, deproteinated technology, ultrafiltration and lyophilize, portobello mushroom polysaccharide (molecular weight ranges 100000-800000D) purity of preparation is high, content reaches more than 90%, productive rate can reach the quality of total mass that 3.0%(productive rate is more than 90% portobello mushroom polysaccharide finally obtained/the obtain brown mushroom raw material of corresponding product), gained polysaccharide shows obvious immunoregulation effect.Present method is efficient, environmental protection, economy, be applicable to industrial scale and produce.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. a preparation method for portobello mushroom polysaccharide, is characterized in that, comprises the steps:
(1) brown mushroom powder is carried out microwave extraction; Microwave power is 200-800W, and solid-liquid ratio is 1:10-1:100;
(2) brown for above-mentioned gained mushroom Aqueous extracts is cooled to room temperature, obtains brown mushroom Crude polysaccharides solution;
(3) adopt macroporous adsorbent resin to carry out removal of impurities to step (2) gained brown mushroom Crude polysaccharides solution, afterwards, adjust ph scope is 3.0-10.0, adopts the process of anion-cation exchange resin series connection resin column deproteinated;
(4) ultra-filtration technique is adopted to be further purified the brown mushroom Crude polysaccharides after removal of impurities, deproteinated; The molecular weight ranges retained in purge process is 100000-800000D;
(5) by above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze, make the moisture fully charge in material, vacuum-drying afterwards obtains portobello mushroom polysaccharide;
The step that described employing macroporous adsorbent resin carries out removal of impurities process to brown mushroom Crude polysaccharides solution is as follows: with glucose meter, getting concentration is that 1.0-50mg/mL brown mushroom Crude polysaccharides solution joins and processedly good is equipped with in the adsorption column of macroporous adsorbent resin, adsorb with the flow velocity of 0.1-3BV/h, wash-out is carried out with 3-10 times of cylinder ponding, elution flow rate is 0.5-5BV/h, obtains deimpurity elutriant;
The step that described employing anion-cation exchange resin carries out deproteinated process is as follows: macroporous adsorbent resin process of learning from else's experience remove deimpurity elutriant, concentrated, adjustment concentration is to 1.0-50mg/mL, adjust ph 3.0-10.0, successively by processed good anionite-exchange resin and cationic exchange numerical value series connection resin column, adsorb with the flow velocity of 0.1-3BV/h, carry out wash-out with 3-10 times of cylinder ponding, elution flow rate is 0.5-5BV/h, obtains Deproteinated portobello mushroom polysaccharide solution; Wherein, the mass ratio of anionite-exchange resin and resin cation (R.C.) is 1:0.5-1:10;
Step (4) described ultra-filtration technique comprises the steps: to the further grading purification of the portobello mushroom polysaccharide after deproteinated the portobello mushroom polysaccharide solution getting deproteinated process gained, carry out ultrafiltration, molecular weight cut-off scope is 100000-800000D, operating pressure is 0.2-0.8Mpa, temperature is 20-50 DEG C, and the rotating speed of rotor is 200-800r/min; Collection retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution, dry, obtains portobello mushroom polysaccharide.
2. the method preparing portobello mushroom polysaccharide according to claim 1, is characterized in that, described macroporous adsorbent resin is nonpolar macroporous adsorption resin.
3. the method preparing portobello mushroom polysaccharide according to claim 1, is characterized in that, described anionite-exchange resin is 201x7, D201 or D301; Described Zeo-karb is D113 or 001x7.
4. the method preparing portobello mushroom polysaccharide according to claim 1, is characterized in that, described property macroporous adsorbent resin is D101, AB-8 or D4020.
5. the method preparing portobello mushroom polysaccharide according to claim 1, is characterized in that, in step (3), Crude polysaccharides solution is by before macroporous resin column, and with glucose meter, adjustment concentration is to 1.0-50mg/mL; After crossing macroporous resin column in step (4), polysaccharide soln is before deproteinated process, should concentrate, and with glucose meter, adjustment concentration is to 1.0-50mg/mL, and pH value should be adjusted to 3.0-10.0.
6. the method preparing portobello mushroom polysaccharide according to claim 1, is characterized in that, in step (5), pre-freeze is to-60 to-80 DEG C, maintains at least 4h; Vacuum drying temperature is-45 DEG C.
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| KR20030072417A (en) * | 2002-03-04 | 2003-09-15 | 박홍제 | Manufacture method of a functional tea and food, using extract of mushroom mycelium by new extraction technique. |
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