CN103524636A - Preparation method for portobello mushroom polysaccharide - Google Patents
Preparation method for portobello mushroom polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method for portobello mushroom polysaccharide with immunoregulatory activity in order to be suitable for modern large-scale production. The preparation method comprises the following steps: performing microwave extraction on portobello mushroom powder; cooling the portobello mushroom powder to room temperature; removing impurities through macroporous adsorption resin, then adjusting the pH value to be 3.0-10.0, and performing deproteinization by connecting anion and cation exchange resin in series with a resin column; performing further purification on the portobello mushroom polysaccharide subjected to impurity removal and deproteinization by an ultrafiltration technology, wherein the retained molecular weight in a purification process is 100,000-800,000 D; pre-freezing the portobello mushroom polysaccharide ultrafiltrate until water in the material is completely frozen, and performing vacuum drying to obtain the portobello mushroom polysaccharide. According to the method, the microwave extraction technology, the resin series connection impurity removal and deproteinization technology and the ultrafiltration technical purification and freeze-drying technology are comprehensively applied, so that the purity of the obtained portobello mushroom polysaccharide can be up to over 90 percent, and an obvious immunoregulation effect can be achieved.
Description
Technical field
The present invention relates to health food processing technique field, particularly relate to a kind of preparation method with the brown lentinan of immunoregulatory activity.
Background technology
Brown mushroom Agaricus crocopelus peck, its mushroom cement layer cell contains brown pigments and is tawny, the many raw fibrous scales of mushroom lid, therefore named brown mushroom or brown squama mushroom, belong 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 comparatively wide DEVELOPMENT PROSPECT.
At present; also rare on domestic and international market have brown lentinan extract and a related products; preparation method's report for brown lentinan 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 limited the application of brown lentinan in protective foods and pharmaceutical prod.
Therefore, research and develop brown lentinan and extract preparation method, form brown lentinan product preparation process, for improving brown lentinan added value of product, meet the need of market, there is great economic benefit and social benefit.
Summary of the invention
The object of the invention is for the technological deficiency existing in prior art, a kind of preparation method of the brown lentinan of high purity of applicable large-scale production is provided.
For realizing the technical scheme that object of the present invention adopts, be:
A preparation method for brown lentinan, comprises the steps:
(1) brown mushroom powder is carried out to microwave extraction;
(2) the brown mushroom water extraction of above-mentioned gained liquid is cooled to room temperature, obtains brown mushroom Crude polysaccharides solution;
(3) adopt macroporous adsorbent resin to carry out removal of impurities to the brown mushroom Crude polysaccharides of step (2) gained solution, afterwards, regulating pH value scope is 3.0-10.0, adopts anion-cation exchange resin series connection resin column deproteinated to process;
(4) the brown mushroom Crude polysaccharides after adopting ultra-filtration technique to removal of impurities, deproteinated is further purified; The molecular weight ranges of holding back 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 brown lentinan.
The step that described employing macroporous adsorbent resin carries out removal of impurities processing to brown mushroom Crude polysaccharides solution is as follows: with glucose meter, getting concentration is that the brown mushroom Crude polysaccharides of 1.0-50mg/mL solution joins in the processed good adsorption column that macroporous adsorbent resin is housed, flow velocity with 0.1-3BV/h adsorbs, with 3-10 times of cylinder ponding, carry out wash-out, elution flow rate is 0.5-5BV/h, obtains deimpurity elutriant.
The step that described employing anion-cation exchange resin carries out deproteinated processing is as follows: the macroporous adsorbent resin of learning from else's experience is processed removes deimpurity elutriant, concentrated, adjust concentration to 1.0-50mg/mL, regulate pH value 3.0-10.0, successively by processed good anionite-exchange resin and cationic exchange numerical value series connection resin column, with the flow velocity of 0.1-3BV/h, adsorb, with 3-10 times of cylinder ponding, carry out wash-out, elution flow rate is 0.5-5BV/h, obtains Deproteinated brown lentinan solution; Wherein, the mass ratio of anionite-exchange resin and resin cation (R.C.) is 1:0.5-1:10.
The brown lentinan further grading purification of the described ultra-filtration technique of step (4) after to deproteinated comprises the steps: to get the brown lentinan solution that deproteinated is processed gained, carry out ultrafiltration, molecular weight cut-off scope is 100000-800000D, operating pressure is 0.2-0.8Mpa, temperature is 20-50 ℃, and the rotating speed of rotor is 200-800r/min; Concentrated solution is held back in collection, and cleans with distilled water, merges washing lotion and concentrated solution, dry, obtains brown lentinan.
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, by before macroporous resin column, with glucose meter, is adjusted concentration to 1.0-50mg/mL; After crossing macroporous resin column in step (4), polysaccharide soln, before deproteinated is processed, should concentrate, and with glucose meter, adjusts concentration to 1.0-50mg/mL, and pH value should be adjusted to 3.0-10.0.
In step (5), pre-freeze, to-60 to-80 ℃, maintains at least 4h; Vacuum drying temperature is-45 ℃.
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 brown lentinan 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, brown lentinan (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 brown lentinan of the present invention adopts macroporous resin, ion exchange resin column tandem process to carry out removal of impurities, deproteinated processing to brown mushroom Crude polysaccharides; albumen decreasing ratio can reach more than 87%; with respect to traditional Sevag method, Tricholroacetic 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 brown lentinan of the present invention adopts ultrafiltration technology purification technique, and polysaccharide is carried out to grading purification, 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 brown lentinan, has guaranteed to greatest extent the original structure of brown lentinan, is its active unaffected assurance that provides.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
1, brown mushroom raw material is crushed to 60 orders, takes 100 grams, be placed in Microwave Extraction Apparatus, the water of take extracts as extracting solvent, and microwave frequency is 500W, and extraction time is 5 minutes, solid-liquid ratio 1:40.
2, above-mentioned brown mushroom water extraction liquid is cooled to room temperature, centrifugal, get supernatant liquor, and add water regulate concentration to 1.0mg/mL(with glucose meter).
3, adopt macroporous adsorbent resin, brown mushroom Crude polysaccharides solution is carried out to removal of impurities processing: above-mentioned brown lentinan solution is joined in the processed good adsorption column that D4020 macroporous adsorbent resin is housed, with 0.1BV/h(column volume/hour) flow velocity adsorb, with 3 times of cylinder ponding, carry out wash-out, flow velocity is 0.5BV/h, collects water elution liquid.
4, adopt ion exchange resin to carry out deproteinated processing to brown mushroom Crude polysaccharides extract solution: to get the above-mentioned elutriant of processing through removal of impurities, suitably concentrated, adjust concentration to 1.0mg/mL(with glucose meter), and add ammoniacal liquor and regulate pH value to 10.0, with 1BV/h(column volume/hour) successively by processed good D201 ion exchange resin column and the 001x7 ion exchange resin column (the mass ratio 1:0.5 of D201 resin and 001 * 7 resin) of being equipped with, with 6 times of cylinder ponding, carry out wash-out, flow velocity is 5BV/h, collects water elution liquid.
5, the ultra-filtration and separation of brown lentinan: above-mentioned elutriant is suitably concentrated, carry out ultrafiltration.Molecular weight cut-off scope is 100000-800000D, and operating pressure is 0.4Mpa, and temperature is 35 ℃, carries out loop ultrafiltration under the condition that the rotating speed of rotor is 800r/min.The concentrated solution that collection is held back, and clean with distilled water, washing lotion and concentrated solution merged.
6, the lyophilize of brown lentinan: above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze, to-60 ℃, when temperature of charge reaches after pre-freeze temperature, then is maintained at least 4h, to guarantee the moisture fully charge in material.At-45 ℃, carry out afterwards vacuum-drying, time of drying 20h.Finally prepare 3.0 grams of brown lentinan products, be white in color spongy, 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, be placed in Microwave Extraction Apparatus and take water and extract as extracting solvent, microwave frequency is 800W, and extraction time is 4 minutes, solid-liquid ratio 1:100.
2, above-mentioned brown mushroom water extraction liquid is cooled to room temperature, centrifugal, get supernatant liquor, and add water regulate concentration to 20mg/mL(with glucose meter).
3, adopt macroporous adsorbent resin, brown mushroom Crude polysaccharides extracting solution is carried out to removal of impurities processing: above-mentioned brown lentinan solution is joined in the processed good adsorption column that D101 macroporous adsorbent resin is housed, with 1.0BV/h(column volume/hour) flow velocity adsorb, with 10 times of cylinder ponding, carry out wash-out, flow velocity is 2.0BV/h, collects water elution liquid.
4, adopt ion exchange resin to carry out deproteinated processing to brown mushroom Crude polysaccharides extract solution: to get the above-mentioned Crude polysaccharides solution of processing through removal of impurities, suitably concentrated, adjust concentration to 20mg/mL(with glucose meter), and add ammoniacal liquor and regulate pH value to 8.0, with 0.1BV/h(column volume/hour) successively by processed good D301 ion exchange resin column and the D113 ion exchange resin column (the mass ratio 1:5 of D301 resin and D113 resin) of being equipped with, with 10 times of cylinder ponding, carry out wash-out, flow velocity is 1.2BV/h, collects water elution liquid.
5, the ultra-filtration and separation of brown lentinan: above-mentioned elutriant is suitably concentrated, carry out ultrafiltration.Molecular weight cut-off scope is 100000-800000D, and operating pressure is 0.2Mpa, and temperature is 50 ℃, carries out loop ultrafiltration under the condition that the rotating speed of rotor is 600r/min.Concentrated solution is held back in collection, and cleans with distilled water, merges washing lotion and concentrated solution.
6, the lyophilize of brown lentinan: above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze, to-60 ℃, when temperature of charge reaches after pre-freeze temperature, then is maintained at least 4h, to guarantee the moisture fully charge in material.At-45 ℃, carry out afterwards vacuum-drying, time of drying 20h.Finally prepare 3.3 grams of brown lentinan products, be white in color spongy, 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, be placed in Microwave Extraction Apparatus and take water and extract as extracting solvent, microwave frequency is 200W, and extraction time is 5 minutes, solid-liquid ratio 1:10.
2, above-mentioned brown mushroom water extraction liquid is cooled to room temperature, centrifugal, get supernatant liquor, and add water regulate concentration to 50mg/mL(with glucose meter).
3, adopt macroporous adsorbent resin, brown mushroom Crude polysaccharides extracting solution is carried out to removal of impurities processing: above-mentioned brown lentinan extracting solution is joined in the processed good adsorption column that AB-8 macroporous adsorbent resin is housed, with 3BV/h(column volume/hour) flow velocity adsorb, with 5 times of cylinder ponding, carry out wash-out, flow velocity is 5BV/h, collects water elution liquid
4, adopt ion exchange resin to carry out deproteinated processing to brown mushroom Crude polysaccharides extract solution: to get the above-mentioned Crude polysaccharides extract of processing through removal of impurities, suitably concentrated, adjust concentration to 50mg/mL(with glucose meter), and add dilute hydrochloric acid and regulate pH value to 3.0, with 3BV/h(column volume/hour) by processed good 201x7 ion exchange resin column and the D113 ion exchange resin column (the mass ratio 1:10 of 201x7 resin and D113 resin) of being equipped with, with 3 times of cylinder ponding, carry out wash-out, flow velocity is 0.5BV/h, collects elutriant.
5, the ultra-filtration and separation of brown lentinan: above-mentioned elutriant is suitably concentrated, carry out ultrafiltration.Molecular weight cut-off scope is 100000-800000D, and operating pressure is 0.8Mpa, and temperature is 20 ℃, carries out loop ultrafiltration under the condition that the rotating speed of rotor is 200r/min.Concentrated solution is held back in collection, and cleans with distilled water, merges washing lotion and concentrated solution.
6, the lyophilize of brown lentinan: above-mentioned brown mushroom Crude polysaccharides ultrafiltrated pre-freeze, to-60 ℃, when temperature of charge reaches after pre-freeze temperature, then is maintained at least 4h, to guarantee the moisture fully charge in material.At-45 ℃, carry out afterwards vacuum-drying, time of drying 20h.Finally prepare 3.0 grams of brown lentinan products, be white in color spongy, adopt sulfuric acid-phynol method to measure its content, result shows that polysaccharide content is that 93.50%(is with glucose meter).
Experimental analysis
Adjusting immunity and the anti-cerebral anoxia active function of brown lentinan are evaluated:
1, the impact of brown lentinan on mouse immune organ thymus gland, index and spleen index
Mouse random packet is also weighed on an empty stomach, male and female dual-purpose, and physiological saline group physiological saline, tested group with the brown lentinan aqueous solution, continuous gavage 7d, every day 1 time, 20mLkg
-1.And record the mouse food consumption of every day.After administration 3d, claim a Mouse Weight and note down, according to body weight, adjusting dosage.When 8d, again weigh on an empty stomach, and after administration 1h, pluck eyeball sacrificed by exsanguination mouse, extract Thymus and spleen, with analytical balance, claim its quality, calculate organ index.
Test-results is as shown in table 1:
Table 1: the impact of brown lentinan on childhood mouse thymus, index and spleen index
Note: compare * p < 0.10, * * p < 0.05 with physiological saline group
Result shows, with the comparison of physiological saline group, the high, medium and low dosage group of brown lentinan all can make the thymus index of mouse childhood, index and spleen index increase, 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 brown lentinan can strengthen cellular immunization and humoral immunization process, thereby enhancing body non-specific immune function improves Abwehrkraft des Koepers.
2, dehisce after the brown spore mushroom Polysaccharides on Mice broken end impact of breathing time
Mouse random packet, male and female dual-purpose, physiological saline group physiological saline, tested group with the brown lentinan aqueous solution, gavage 7d continuously, every day 1 time, 20mLkg
-1.And record the mouse food consumption of every day.After administration 3d, claim a Mouse Weight and note down, according to body weight, adjusting dosage.After last administration 1h, successively mouse basal part of the ear rear portion sacrificed by decapitation mouse, immediately by dehisce after stopwatch calculating mouse broken end breathing time and rate elongation.Test-results is as shown in table 2:
Table 2: brown lentinan dehisced after the mouse broken end childhood impact of breathing time
Note: compare * p < 0.10, * * p < 0.05 with physiological saline group
Result shows: with the comparison of physiological saline group, the high, medium and low dosage group of brown lentinan all can extend the mouth breathing time of mouse childhood, and high dose group shows significant difference (p < 0.05), and middle dosage group has shown different (p < 0.10).Show that brown lentinan 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, brown lentinan (molecular weight ranges 100000-800000D) purity of preparation is high, content reaches more than 90%, productive rate can reach more than 90% total mass/the obtain quality of the brown mushroom raw material of corresponding product of brown lentinan of 3.0%(productive rate for finally obtaining), gained polysaccharide shows obvious immunoregulation effect.Present method is efficient, environmental protection, economy, is applicable to industrial scale and produces.
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 (10)
1. a preparation method for brown lentinan, is characterized in that, comprises the steps:
(1) brown mushroom powder is carried out to microwave extraction;
(2) the brown mushroom water extraction of above-mentioned gained liquid is cooled to room temperature, obtains brown mushroom Crude polysaccharides solution;
(3) adopt macroporous adsorbent resin to carry out removal of impurities to the brown mushroom Crude polysaccharides of step (2) gained solution, afterwards, regulating pH value scope is 3.0-10.0, adopts anion-cation exchange resin series connection resin column deproteinated to process;
(4) the brown mushroom Crude polysaccharides after adopting ultra-filtration technique to removal of impurities, deproteinated is further purified; The molecular weight ranges of holding back 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 brown lentinan.
2. the method for the brown lentinan of preparation according to claim 1, it is characterized in that, the step that described employing macroporous adsorbent resin carries out removal of impurities processing to brown mushroom Crude polysaccharides solution is as follows: with glucose meter, getting concentration is that the brown mushroom Crude polysaccharides of 1.0-50mg/mL solution joins in the processed good adsorption column that macroporous adsorbent resin is housed, flow velocity with 0.1-3BV/h adsorbs, with 3-10 times of cylinder ponding, carry out wash-out, elution flow rate is 0.5-5BV/h, obtains deimpurity elutriant.
3. the method for the brown lentinan of preparation according to claim 2, it is characterized in that, the step that described employing anion-cation exchange resin carries out deproteinated processing is as follows: the macroporous adsorbent resin of learning from else's experience is processed removes deimpurity elutriant, concentrated, adjust concentration to 1.0-50mg/mL, regulate pH value 3.0-10.0, successively by processed good anionite-exchange resin and cationic exchange numerical value series connection resin column, flow velocity with 0.1-3BV/h adsorbs, with 3-10 times of cylinder ponding, carry out wash-out, elution flow rate is 0.5-5BV/h, obtain Deproteinated brown lentinan solution, wherein, the mass ratio of anionite-exchange resin and resin cation (R.C.) is 1:0.5-1:10.
4. the method for the brown lentinan of preparation according to claim 3, it is characterized in that, the brown lentinan further grading purification of the described ultra-filtration technique of step (4) after to deproteinated comprises the steps: to get the brown lentinan solution that deproteinated is processed gained, carry out ultrafiltration, molecular weight cut-off scope is 100000-800000D, operating pressure is 0.2-0.8Mpa, and temperature is 20-50 ℃, and the rotating speed of rotor is 200-800r/min; Concentrated solution is held back in collection, and cleans with distilled water, merges washing lotion and concentrated solution, dry, obtains brown lentinan.
5. the method for the brown lentinan of preparation according to claim 2, is characterized in that, described macroporous adsorbent resin is nonpolar macroporous adsorption resin.
6. the method for the brown lentinan of preparation according to claim 3, is characterized in that, described anionite-exchange resin is 201x7, D201 or D301; Described Zeo-karb is D113 or 001x7.
7. the method for the brown lentinan of preparation according to claim 1, is characterized in that, described property macroporous adsorbent resin is D101, AB-8 or D4020.
8. the method for the brown lentinan of preparation according to claim 1, is characterized in that, in step (3), Crude polysaccharides solution, by before macroporous resin column, with glucose meter, is adjusted concentration to 1.0-50mg/mL; After crossing macroporous resin column in step (4), polysaccharide soln, before deproteinated is processed, should concentrate, and with glucose meter, adjusts concentration to 1.0-50mg/mL, and pH value should be adjusted to 3.0-10.0.
9. the method for the brown lentinan of preparation according to claim 1, is characterized in that, in step (5), pre-freeze, to-60 to-80 ℃, maintains at least 4h; Vacuum drying temperature is-45 ℃.
10. the method for the brown lentinan of preparation according to claim 1, is characterized in that, during microwave extraction in step (1), microwave power is 200-800W, and solid-liquid ratio is 1:10-1:100.
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| CN106046193A (en) * | 2016-07-26 | 2016-10-26 | 东北师范大学 | Seaweed polysaccharide P155 and preparation process thereof |
| CN106349400A (en) * | 2016-08-29 | 2017-01-25 | 高其品 | Novel method for preparing hericium purified polysaccharides by combined application of macroporous resin |
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| CN106046193B (en) * | 2016-07-26 | 2019-02-19 | 东北师范大学 | A kind of algal polysaccharides P155 and its preparation process |
| CN106349400A (en) * | 2016-08-29 | 2017-01-25 | 高其品 | Novel method for preparing hericium purified polysaccharides by combined application of macroporous resin |
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