CN102690368A - Preparation method of yeast beta-1,3-D-glucan derivative - Google Patents

Preparation method of yeast beta-1,3-D-glucan derivative Download PDF

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CN102690368A
CN102690368A CN2012101994994A CN201210199499A CN102690368A CN 102690368 A CN102690368 A CN 102690368A CN 2012101994994 A CN2012101994994 A CN 2012101994994A CN 201210199499 A CN201210199499 A CN 201210199499A CN 102690368 A CN102690368 A CN 102690368A
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visose
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郭宏昌
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Abstract

The invention discloses a preparation method of a yeast beta-1,3-D-glucan derivative. Puffing technology and acid-base solution treatment are combined in the method to prepare the yeast beta-1,3-D-glucan derivative, so that the time for preparing the yeast beta-1,3-D-glucan can be greatly saved, and the extraction cost can be reduced.

Description

A kind of preparation yeast β-1, the preparation method of 3-D-glucan derivative
Technical field
The invention belongs to biochemical preparation field.Relate to utilize puffing technique and acid-base solution to handle and combine and prepare yeast β-1, the technology that 3-D-VISOSE is biological.
Background technology
Yeast is a kind of important foodstuffs industry mikrobe, and its cell walls divides three layers from outside to inside, is respectively mannosans, protein and VISOSE.The VISOSE of innermost layer has three types: the caustic solubility VISOSE, and alkali is insoluble, the acid-solubility VISOSE, the soda acid insoluble glucan; Wherein the soda acid insoluble glucan is active polysaccharide, removes to contain outside a spot of β-1,6 key; Nearly all be β-1; Therefore 3 keys also claim β-(1,3)-VISOSE.Yeast β-1,3-D-VISOSE can strengthen mammiferous immunizing power, anticancer, antibacterium, antiviral, antimycotic, parasiticide, reducing cholesterol and blood fat, promotion wound healing etc., are a kind of good biological response modifierses.Yeast β-1; The 3-D-VISOSE is restricted in the clinical application of antitumor grade because of its insoluble; Advantages such as viscosity, retentiveness and the thermostability of its height and preparation technology are simple, widespread use in industries such as food, medicine, makeup, agricultural, papermaking and material of construction.From yeast cell, extract β-1,3-D-VISOSE is exactly from cell walls, to remove mannosans and protein.
Yeast cells wall is β-1; A main source of 3-D-VISOSE; Just propose cereuisiae fermentum as far back as the nineteen thirty-nine people and have the immunostimulant characteristic; But discover just that up to nineteen sixty the principal element that immunological characteristic is provided is the VISOSE composition that is present in yeast cells wall, accounts for 15% of cereuisiae fermentum dry weight.
The VISOSE conformation is to its active influence and non-staple, and β-1,3-glycosidic link are only its key with anti-tumor activity.
The low water solubility of alkali-insoluble glucan has limited its application clinically.Though orally can not produce toxic side effect usually, intravenous injection just possibly produce some inestimable toxic side effect, hepatosplenomegaly for example, microthrombusis, recycle system collapse, the formation of inflammatory granuloma etc.Therefore need be to β-1, the 3-D-VISOSE carries out the derivatize modification, obtains water-soluble well and have an immunocompetent Sensor Chip CM 5.
β-1, the derivatize of 3-D-VISOSE replace certain hydrophilic radical at molecular surface, increase water-soluble and maintenance or strengthen its immunocompetence.Present applied derivatize means are a lot, mainly contain carboxymethylation, and carboxyethylation ethylizes, and sulfo groupization is carboxylated etc.Its method is to select different substrates, and in certain medium, control certain reaction condition makes the hydroxyl on the glucone replaced by the substrate group.Difficult point is that control obtains certain substitution value VISOSE, makes this verivate can water-soluble and not destroy its original spirane structure, and this just configuration makes it have special activity.More various derivatization methods, carboxymethylation condition ratio are easier to control.Carboxyl methylation derivant demonstrates the highest activity, as DS when substitution value DS=0.75>1.0 or molecular weight when low, then there is not activity.
Sensor Chip CM 5 is compared with VISOSE has stronger solvability and immune activation effect; Can strengthen the nonspecific defense function such as the macrophage activation function of body; Promote TNF-α synthetic; Simultaneously animal body has been shown that also potential is antiviral, the ability of radioprotective and mutation can protect rat hepatocytes to avoid oxidative damage.The VISOSE soluble derivative has notable antitumor activity simultaneously.Sensor Chip CM 5 can make endotoxemia animal tumor necrosin level significantly reduce, and can suppress the transfer of murine sarcoma cell.
The process for extracting of taking both at home and abroad at present mainly contains acid system, alkaline process, acid-base integration method etc.; The extraction conditions of these methods generally all compares acutely; Acid-base reagent corrodes plant and instrument easily, environmental pollution is serious, and the VISOSE finished product purity that obtains is not high, need be further purified.The VISOSE that alkali broken wall method constitutes yeast cells wall has two-layer: one deck can be by basic hydrolysis, and another layer then is insoluble to alkali.When utilizing alkaline solution that yeast is carried out broken wall, alkali can dissolve the alkali-soluble VISOSE layer in the yeast cells wall, dissolves part lipid simultaneously, thereby makes the permeability of yeast cells wall become big, and intracellular organic matter is separated out easily.The yeast residue after the self-dissolving is adopted in this experiment; Add expanded separating and remove amounts of protein and other impurity; Reduce concentration of lye and consumption in the follow-up alkaline purification operation; So that under relatively mild condition, keep the molecular structure of VISOSE complete as far as possible, improve the yield and the purity of alkali-insoluble glucan finished product, for the comprehensive utilization of beer waste yeast provides theoretical foundation.
In at home and abroad reporting, about β-1, the preparation of 3-D-VISOSE, the methods that adopt acid, alkali or combine with organic solvent more.Once there was the people to compare, test with Sevage method, acid system and alkaline process respectively, and found to be difficult to from cereuisiae fermentum, extract β-1,3-D-VISOSE with the Sevage method; The Crude polysaccharides yield of acid system lixiviate gained is high, but contains a large amount of mannosanss and protein, and purity is not high, and technology is too complicated, and the acid system etching apparatus, the harm workers ' health; The alkaline process extracting technology is simple, and product is the β-1 of higher degree, and the 3-D-VISOSE is effective β-1 from waste yeast, the desirable approach of 3-D-VISOSE.
Summary of the invention
The object of the invention is just in order to provide a kind of preparation yeast β-1; The method of 3-D-VISOSE; Method of the present invention is utilized puffing technique and acid-base solution to handle and is combined and prepares yeast β-1, and the 3-D-VISOSE is biological, not only can practice thrift preparation β-1 greatly; The time of 3-D-VISOSE, and reduce extraction cost.
The object of the invention can be realized through following technique measures:
Method of the present invention realizes through following steps:
A, adopt following (a) or (b) method carry out alkaline purification and expanded;
A, alkaline purification: get the 1M sodium hydroxide that cereuisiae fermentum 100g adds 200-300ml, under temperature 80-100 ℃ condition, do, behind the 2h, centrifugal 15min under the speed of 3000r/min washes 2 times, filter centrifugal, the collecting precipitation thing; Expanded: it is expanded that said throw out is put into microwave oven, and the microwave treatment firepower is high fire, and microwave treatment time is 10 minutes;
B, twin-screw Bulking Machine are expanded: with the cereuisiae fermentum is raw material, adds 1-1.5M sodium hydroxide 15-20ml by every 100g cereuisiae fermentum, under the condition of mixing thoroughly, slowly adds bulking machine, screw speed 30HZ, swelling temperature 120-130 ℃; Said bulking machine is a twin-screw Bulking Machine;
B, neutralizing treatment: with the expanded processing of lowering the temperature of step 1 gained, when its temperature is lower than 50 ℃, slowly adds volume(tric)fraction and be 20% acetate and neutralize, the acetate add-on be the pH value of extract was reduced to 7.0 o'clock till.
C, separation: step 2 gained neutralizer is carried out spinning under rotating speed 4 000 r/min, collect, merge centrifugate; Heating, vacuum is concentrated into the half the of original volume, and the volume(tric)fraction that adds 2 times of volumes then is 95% ethanol;
D, carboxymethylation preparation: with the extracting solution that step 3 obtains, the sodium hydroxide that adds 15g by every 100g cereuisiae fermentum mixes, and 35 ℃ are stirred 0.5 h down; Add Monochloro Acetic Acid 20g by every 100g cereuisiae fermentum then, stirring reaction 3 h under 50 ℃ of conditions, reaction product is used 95% washing with alcohol, precipitates 24 h, obtains the Sensor Chip CM 5 resultant, and resultant is dry;
E, by quality: quality: volume=1:1.25:1.25 gets VISOSE, sodium hydroxide, Monochloro Acetic Acid; The volume(tric)fraction of putting into 2 times of volumes is 95% ethanolic soln, under 50 ℃ of etherification temperatures, etherificate time 3h, 35 ℃ of alkalization temperatures, alkalization time 0.5 h condition, reacts; Under these processing condition, the substitution value of gained Sensor Chip CM 5 is 0.79.
Beneficial effect of the present invention:
Method of the present invention adopts expanded assisted extraction to shorten extraction time significantly, and has improved the extraction yield of polysaccharide, is superior to other process for extracting; It is high than other technologies to adopt auxiliary law to extract polysaccharide yield, and it is feasible to optimize craft science, has accelerated the preparation speed of Sensor Chip CM 5 like this, for the drug development of beer waste yeast polysaccharide and the application of healthcare products are offered help; Not only can practice thrift preparation β-1 greatly, the time of 3-D-VISOSE, and can reduce extraction cost.
Embodiment
To combine embodiment to further describe below the present invention:
Technology of the present invention is following:
Beer waste yeast alkali dissolution → broken microwave (or the expanded fragmentation of twin-screw Bulking Machine) → centrifugal → supernatant concentration → add alcohol deposition → carboxymethylation → vacuum-drying → Sensor Chip CM 5.
Embodiment 1:
1, alkaline purification: get the 100g yeast and add 1M sodium hydroxide 200-300ml, under the temperature 80-100 ℃ condition effect 2h after, centrifugal 15min under the speed of 3000r/min washes 2 times, filter centrifugal, collecting precipitation;
2, microwave bulking: it is expanded that the yeast that will precipitate the alkaline purification of collecting is put into microwave oven, and the microwave treatment firepower be high fiery, and microwave treatment time is 8 minutes; Notice that controlled microwave handles firepower and time, expanded during, stir yeast for special-purpose spoonful with microwave, it is heated evenly;
3, neutralizing treatment: with the expanded processing of lowering the temperature of step 2 gained, when its temperature is lower than 50 ℃, slowly adds volume(tric)fraction and be 20% acetate and neutralize, the acetate add-on is that the pH value of extract was reduced to 7.0 o'clock;
4, separate: neutralizer is carried out spinning under rotating speed 4 000 r/min, collect, merge centrifugate, heating, vacuum is concentrated into the half the of original volume, and the volume(tric)fraction that adds its 2 times of volumes then is 95% ethanol;
5, carboxymethylation preparation: with the extracting solution that step 3 obtains, the sodium hydroxide that adds 15g by every 100g cereuisiae fermentum mixes, and 35 ℃ are stirred 0.5 h down; Add Monochloro Acetic Acid 20g by every 100g cereuisiae fermentum then, stirring reaction 3 h under 50 ℃ of conditions, reaction product is used 95% washing with alcohol, precipitates 24 h, obtains the Sensor Chip CM 5 resultant, and resultant is dry;
6, getting VISOSE, sodium hydroxide, Monochloro Acetic Acid feed ratio is 1:1.25:1.25 (mass/mass/volume); The volume(tric)fraction of putting into 2 times of volumes is 95% ethanolic soln, under 50 ℃ of etherification temperatures, etherificate times 3 h, 35 ℃ of alkalization temperatures, alkalization time 0.5 h condition, reacts; Under these processing condition, the substitution value of gained Sensor Chip CM 5 is 0.79.
Embodiment 2:
1, alkaline purification: the 100g yeast adds 1M sodium hydroxide 200-300ml, temperature 80-100 ℃, the effect 2h after the centrifugal 15min of 3000r/min, wash 2 times, filter centrifugal, collecting precipitation;
2, twin-screw Bulking Machine is expanded: the yeast that deposition is collected, it is expanded slowly to add bulking machine, screw speed 30HZ, swelling temperature 120-130 ℃.Bulking machine is a twin-screw Bulking Machine, screw speed 30HZ, and swelling temperature is controlled;
3, neutralizing treatment: with the processing of lowering the temperature of 1,2 said extracted thing, when its temperature is lower than 50 ℃, slowly adds volume(tric)fraction and be 20% acetate and neutralize, the acetate add-on be the pH value of extract was reduced to 7.0 o'clock till;
4, separate: neutralizer is carried out spinning under rotating speed 4 000 r/min, collect, merge centrifugate, heating, vacuum is concentrated into the half the of original volume, and the volume(tric)fraction that adds its 2 times of volumes then is 95% ethanol;
5, carboxymethylation preparation: with the extracting solution that step 3 obtains, the sodium hydroxide that adds 15g by every 100g cereuisiae fermentum mixes, and 35 ℃ are stirred 0.5 h down; Add Monochloro Acetic Acid 20g by every 100g cereuisiae fermentum then, stirring reaction 3 h under 50 ℃ of conditions, reaction product is used 95% washing with alcohol, precipitates 24 h, obtains the Sensor Chip CM 5 resultant, and resultant is dry;
6, getting VISOSE, sodium hydroxide, Monochloro Acetic Acid feed ratio is 1:1.25:1.25 (mass/mass/volume); The volume(tric)fraction of putting into 2 times of volumes is 95% ethanolic soln, under 50 ℃ of etherification temperatures, etherificate time 3h, 35 ℃ of alkalization temperatures, alkalization time 0.5 h condition, reacts; Under these processing condition, the substitution value of gained Sensor Chip CM 5 is 0.79.

Claims (1)

1. one kind prepares yeast β-1, and the preparation method of 3-D-glucan derivative is characterized in that: said method realizes through following steps:
A, adopt following (a) or (b) method carry out alkaline purification and expanded;
A, alkaline purification: get the 1M sodium hydroxide that cereuisiae fermentum 100g adds 200-300ml, behind effect 2h under the temperature 80-100 ℃ condition, centrifugal 15min under the speed of 3000r/min washes 2 times, filter centrifugal, the collecting precipitation thing; Expanded: it is expanded that said throw out is put into microwave oven, and the microwave treatment firepower is high fire, and microwave treatment time is 10 minutes;
B, twin-screw Bulking Machine are expanded: with the cereuisiae fermentum is raw material, adds 1-1.5M sodium hydroxide 15-20ml by every 100g cereuisiae fermentum, under the condition of mixing thoroughly, slowly adds bulking machine, screw speed 30HZ, swelling temperature 120-130 ℃;
B, neutralizing treatment: with the expanded processing of lowering the temperature of step 1 gained, when its temperature is lower than 50 ℃, slowly adds volume(tric)fraction and be 20% acetate and neutralize, the acetate add-on be the pH value of extract was reduced to 7.0 o'clock till;
C, separation: step 2 gained neutralizer is carried out spinning under rotating speed 4 000 r/min, collect, merge centrifugate; Heating, vacuum is concentrated into the half the of original volume, and the volume(tric)fraction that adds 2 times of volumes then is 95% ethanol;
D, carboxymethylation preparation: with the extracting solution that step 3 obtains, the sodium hydroxide that adds 15g by every 100g cereuisiae fermentum mixes, and 35 ℃ are stirred 0.5 h down; Add Monochloro Acetic Acid 20g by every 100g cereuisiae fermentum then, stirring reaction 3 h under 50 ℃ of conditions, reaction product is used 95% washing with alcohol, precipitates 24 h, obtains the Sensor Chip CM 5 resultant, and resultant is dry;
E, by quality: quality: volume=1:1.25:1.25 gets VISOSE, sodium hydroxide, Monochloro Acetic Acid; The volume(tric)fraction of putting into 2 times of volumes is 95% ethanolic soln, under 50 ℃ of etherification temperatures, etherificate time 3h, 35 ℃ of alkalization temperatures, alkalization time 0.5 h condition, reacts; Under these processing condition, the substitution value of gained Sensor Chip CM 5 is 0.79.
CN2012101994994A 2012-06-18 2012-06-18 Preparation method of yeast beta-1,3-D-glucan derivative Pending CN102690368A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103804511A (en) * 2012-11-13 2014-05-21 安琪酵母股份有限公司 Method for preparing carboxymethyl yeast glucan product and product obtained by using same
CN103804512A (en) * 2014-01-16 2014-05-21 昆山京昆油田化学科技开发公司 Method for preparing galactosyl dextran derivates
CN103804511B (en) * 2012-11-13 2016-11-30 安琪酵母股份有限公司 The product that the preparation method of carboxymethyl yeast dextran product and the method prepare
CN109879983A (en) * 2019-03-21 2019-06-14 石雨 The glucan of a kind of anti-curing cancers and for oral yeast cell wall extract
CN115386135A (en) * 2022-08-18 2022-11-25 广东省科学院生物与医学工程研究所 Carboxymethylated zymosan sponge and preparation method and application thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103804511A (en) * 2012-11-13 2014-05-21 安琪酵母股份有限公司 Method for preparing carboxymethyl yeast glucan product and product obtained by using same
CN103804511B (en) * 2012-11-13 2016-11-30 安琪酵母股份有限公司 The product that the preparation method of carboxymethyl yeast dextran product and the method prepare
CN103804512A (en) * 2014-01-16 2014-05-21 昆山京昆油田化学科技开发公司 Method for preparing galactosyl dextran derivates
CN109879983A (en) * 2019-03-21 2019-06-14 石雨 The glucan of a kind of anti-curing cancers and for oral yeast cell wall extract
CN115386135A (en) * 2022-08-18 2022-11-25 广东省科学院生物与医学工程研究所 Carboxymethylated zymosan sponge and preparation method and application thereof
CN115386135B (en) * 2022-08-18 2023-11-07 广东省科学院生物与医学工程研究所 Carboxymethylated zymosan sponge and preparation method and application thereof

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Application publication date: 20120926