CN102850410A - Method for extracting seaweed polysaccharide from waste beer yeast - Google Patents
Method for extracting seaweed polysaccharide from waste beer yeast Download PDFInfo
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- CN102850410A CN102850410A CN 201210361477 CN201210361477A CN102850410A CN 102850410 A CN102850410 A CN 102850410A CN 201210361477 CN201210361477 CN 201210361477 CN 201210361477 A CN201210361477 A CN 201210361477A CN 102850410 A CN102850410 A CN 102850410A
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Abstract
The invention discloses a method for extracting seaweed polysaccharide from waste beer yeast, belonging to the field of biotechnology. The method mainly uses waste beer yeast as a raw material to prepare seaweed polysaccharide, wherein the main process is that waste beer yeast is pretreated for removing impurities and then is dried for deactivating enzyme, an extract solution of seaweed polysaccharide is obtained by Soxhlet extraction, and seaweed polysaccharide is obtained by decolorizing with activated carbon, precipitating with alkali metal salt for removing protein, removing impurities by ion exchange, concentrating, crystallizing, and drying, wherein the obtained seaweed polysaccharide has the purity of more than 98.5% and can be applied to the fields of medicine, cosmetic, food and so on. The method has the characteristics of high utilization rate of raw materials, low cost, simple process, excellent feasibility, safe operation, and high purity of products; and the industrial production is easy to implement.
Description
One, technical field
The invention belongs to technological field of biochemistry, specially refer to the method that a kind of Sargassum polysaccharides extracts preparation.
Two, background technology
Trehalose is to pass through a-1 by two D-Glucose molecules, and 1 glycosidic link forms, and its molecular formula is C
12H
22O
11, relative molecular mass is 378.33, often exists with two hydrates.Trehalose is white crystal, contains two molecular crystal water, and fusing point is 97 ℃, when being heated to 130 ℃, loses crystal water, and the anhydrous trehalose fusing point reaches 214-216 ℃.The trehalose sweet taste a little less than, be equivalent to 45% of sucrose sweetness, nontoxicity can be water-soluble, Glacial acetic acid and hot ethanol, is insoluble to ether, acetone.Trehalose character is very stable, is the most stable in the natural disaccharide, and without reductibility, Maillard reaction does not occur in heating in food, not by general enzymic hydrolysis, can be had specific trehalase and be hydrolyzed to two molecule glucoses.Trehalose is the non-specific natural protective agent of biological tissue, belongs to the impermeability cryoprotective agent, can make the organisms such as animals and plants and microorganism still can keep vitality in the environment such as freezing, high temperature, dehydration, high osmotic pressure and toxic reagent.Along with progressively going deep into of research, it has been widely used in medical science and field of biology at present, has the scholar to use it for and preserves thrombocyte, cell, tissue, organ, and obtained significant achievement.
Because trehalose has unique biological activity, the various countries scientist has carried out a large amount of research to its production technology.The preparation method of trehalose comprises chemical synthesis, microorganism extraction method, microbe fermentation method, enzymic synthesis method, gene engineering research.
The chemical synthesis of trehalose is at the tetra-acetylated glucose of 2,3,4,6-and 3,4,6-triacetyl-1, produces ethyleneoxide addition between the 2-dehydration-D-glucose and generates.The shortcoming of the standby trehalose of this legal system is low, the separation difficulty of productive rate, also is at present conceptual phase.
Nineteen fifty Laura at first extracts trehalose from yeast.The microorganism extraction method is take yeast, milk-acid bacteria, mould and other microorganism that contains trehalose as extraction source, at first by changing the microbial growth condition, makes the more trehalose of its cylinder accumulation, then adopts appropriate means that trehalose is extracted.The microorganism extraction method is the traditional method of producing trehalose, and through constantly improving, this method is quite ripe, remains so far the common method of producing trehalose.It is raw material that the microorganism extraction method often adopts yeast, and the Sargassum polysaccharides purity for preparing is high, can be used for medicine, molecular biology reagent and field of food.Yet the microorganism extraction method is produced trehalose and is existed the production cycle long, and extraction yield is low, and cost is high, has limited greatly its large-scale production.
The Production by Microorganism Fermentation trehalose is to produce trehalose by microbial fermentation, extracts purifying from fermented liquid again.Its key is the bacterial strain by method breeding high-yield trehaloses such as mutagenesis, cytogamy and gene recombination.As utilize the nutrient solution of the microorganisms such as genus arthrobacter, short stem bacterium, Corynebacterium, nocardia, Rhizoctonia, micrococcus sp to prepare.Yet, this method low conversion rate, by product is many.
It is take glucose, maltose or starch as substrate that the enzymic synthesis method is produced Sargassum polysaccharides, converts trehalose to by corresponding enzyme effect.The enzymic synthesis method is produced Sargassum polysaccharides, and cost is relatively low, but exists product purity low, the application in the fields such as improper medicine, biotechnological formulation.
Under this background, the present invention finishes the preparation of Sargassum polysaccharides take the cereuisiae fermentum waste as raw material, for the Industry Promotion of the microorganism extraction process of Sargassum polysaccharides provides certain technical support.
Three, summary of the invention
The objective of the invention is to propose the method that a kind of Sargassum polysaccharides extracts preparation, method can the Effective Raise Sargassum polysaccharides according to this output and product purity improve productive rate, reduce production costs.
1, invention technical scheme
A kind of Sargassum polysaccharides extracts the method for preparation, mainly is achieved through the following technical solutions:
(1) beer waste yeast pre-treatment
Add an amount of sterilized water in beer waste yeast, cross 100 mesh sieve and sieve, add 5% tartrate, centrifugation obtains pure cereuisiae fermentum after stirring, and is for subsequent use.The pure cereuisiae fermentum 98-105 ℃ of lower drying and processing 40-48h that pre-treatment obtains.
(2) Soxhlet is extracted
Oven dry obtains to put into apparatus,Soxhlet's behind cereuisiae fermentum and the sterilized water certain proportion mixing, and 80-100 ℃, to finish Soxhlet under the 0.09-0.10MPa and extract, after extraction finished, 4 ℃ of extracting solutions were lower centrifugal, get supernatant and obtain the trehalose crude extract.
(3) activated carbon decolorizing
The pH value of regulating extracting solution is 8.5-9.0, and the control temperature is 45-50 ℃, according to the per-cent adding gac of 2%-4%, and lixiviate 0.5-2h, the decolouring number of times is 2-3 time, and is colourless to liquid, the centrifugal supernatant that stays is for subsequent use.
(4) an alkali metal salt precipitation is except albumen
Add an amount of solution of zinc sulfate in the destainer, with adding Ba (OH)
2Regulate pH value to 8.0, stir, leave standstill the rear centrifugal supernatant that stays, for subsequent use.
(5) ion-exchange removal of impurities
Treated macropore strong acid cation resin and macroporous strong basic resin anion(R.A) post are mixed the dress post according to the consumption volume ratio at 2: 1, regulate an alkali metal salt precipitation after the balance and obtain that the flow velocity with 2.0mL/min carries out the cation and anion exchange removal of impurities behind the pH.
(6) crystallizing and drying
To after the effluent liquid of ion-exchange removal of impurities is concentrated, add dehydrated alcohol, the rear input crystal seed that stirs, 4 ℃ leave standstill 12h, obtain the trehalose crystal.Filter and obtain the trehalose crystal, behind a small amount of alcohol flushing, obtain the trehalose sterling in 40 ℃ of lower vacuum-drying 12h.
2, invention technical characterstic
(1) traditional microorganism extraction method production trehalose exists the production cycle long, extraction yield is low, and cost is high, has limited greatly its large-scale production, and the present invention carries out the preparation of trehalose take the beer waste yeast as raw material, the recycling that realizes waste can be arranged, also have simultaneously the raw material availability height, cost is low, technique is simple, easy to implement the method, operational safety, and product purity is high, is easy to Industry Promotion;
(2) the present invention adopts soxhlet extraction to carry out the extraction of Sargassum polysaccharides in the cereuisiae fermentum, compares with traditional reflux method, and extraction yield can improve more than 3 times;
(3) the present invention has adopted an alkali metal salt depositing technology except albumen in the trehalose process for refining, Effective Raise the purity of trehalose, the product purity that obtains reaches more than 98.5%, can be used for the fields such as medicine, makeup and food.
Four, description of drawings
Fig. 1: beer waste yeast prepares the Technology of Trehalose schema
Five, embodiment
The present invention is further described below in conjunction with specific embodiment.
Embodiment one
Take by weighing beer waste yeast 1kg, after the dilution of adding 10L sterilized water is even, crosses 100 mesh sieves and sieve 2 times, add the tartrate final concentration and reach 5%, behind the stirring 20min, the 3000r/min centrifugation obtains pure cereuisiae fermentum, and is for subsequent use.
The pure cereuisiae fermentum that pre-treatment obtains is put into baking oven, 100 ℃ of oven dry 42h.The beer waste yeast that drying and processing is good wraps with bundle puts into apparatus,Soxhlet's, uses the 25L deionized water, at 80 ℃, vacuum tightness is to carry out Soxhlet under the condition of 0.09MPa to extract, after extracting end, extracting solution, the centrifugal 15min of 4000r/min gets supernatant and obtains the trehalose crude extract.
The pH value of regulating the trehalose crude extract is 8.5, adds the gac final concentration and reaches 3%, regulates the crude extract temperature and is 45 ℃ and carries out activated carbon decolorizing, and each treatment time is 1h, and the number of times that decolours is 2 times, and is colourless to liquid, and the centrifugal supernatant that stays is for subsequent use.Adding massfraction in the decolouring treatment solution according to 1: 100 volume ratio is 10% solution of zinc sulfate, and it is rear with Ba (OH) to stir
2Regulator solution pH value to 8.0 leaves standstill the rear centrifugal supernatant that stays.Treated macropore strong acid cation resin D001 and macroporous strong basic resin anion(R.A) post D280 are mixed the dress post according to the consumption volume ratio at 2: 1, regulate the rear flow velocity with 2.0mL/min of an alkali metal salt precipitation acquisition pH to 6.0 after the balance and carry out the cation and anion exchange removal of impurities, after effluent liquid is concentrated into 40%, the dehydrated alcohol that adds 4 times of volumes, rear input crystal seed stirs, 4 ℃ leave standstill 12h, obtain the trehalose crystal.Filter and obtain the trehalose crystal, behind a small amount of alcohol flushing, obtain the trehalose sterling in 40 ℃ of lower vacuum-drying 12h, HPLC testing product purity reaches 98.6%.
Embodiment two
Take by weighing beer waste yeast 1kg, after the dilution of adding 8L sterilized water is even, crosses 100 mesh sieves and sieve 2 times, add the tartrate final concentration and reach 5%, behind the stirring 20min, the 3000r/min centrifugation obtains pure cereuisiae fermentum, and is for subsequent use.
The pure cereuisiae fermentum that pre-treatment obtains is put into baking oven, 105 ℃ of oven dry 42h.The beer waste yeast that drying and processing is good wraps with bundle puts into apparatus,Soxhlet's, uses the 25L deionized water, at 80 ℃, vacuum tightness is to carry out Soxhlet under the condition of 0.09MPa to extract, after extracting end, extracting solution, the centrifugal 15min of 4000r/min gets supernatant and obtains the trehalose crude extract.
The pH value of regulating the trehalose crude extract is 9.0, adds the gac final concentration and reaches 2%, regulates the crude extract temperature and is 45 ℃ and carries out activated carbon decolorizing, and each treatment time is 0.5h, and the number of times that decolours is 2 times, and is colourless to liquid, and the centrifugal supernatant that stays is for subsequent use.Adding massfraction in the decolouring treatment solution according to 1.5: 100 volume ratios is 12% solution of zinc sulfate, and it is rear with Ba (OH) to stir
2Regulator solution pH value to 8.0 leaves standstill the rear centrifugal supernatant that stays.Treated macropore strong acid cation resin D72 and macroporous strong basic resin anion(R.A) post D296 are mixed the dress post according to the consumption volume ratio at 2: 1, regulate the rear flow velocity with 2.0mL/min of an alkali metal salt precipitation acquisition pH to 6.0 after the balance and carry out the cation and anion exchange removal of impurities, after effluent liquid is concentrated into 50%, the dehydrated alcohol that adds 5 times of volumes, rear input crystal seed stirs, 4 ℃ leave standstill 12h, obtain the trehalose crystal.Filter and obtain the trehalose crystal, behind a small amount of alcohol flushing, obtain the trehalose sterling in 40 ℃ of lower vacuum-drying 12h, HPLC testing product purity reaches 98.7%.
Embodiment three
Take by weighing beer waste yeast 1kg, after the dilution of adding 10L sterilized water is even, crosses 100 mesh sieves and sieve 1 time, add the tartrate final concentration and reach 5%, behind the stirring 20min, the 3000r/min centrifugation obtains pure cereuisiae fermentum, and is for subsequent use.
The pure cereuisiae fermentum that pre-treatment obtains is put into baking oven, 100 ℃ of oven dry 45h.The beer waste yeast that drying and processing is good wraps with bundle puts into apparatus,Soxhlet's, uses the 25L deionized water, at 80 ℃, vacuum tightness is to carry out Soxhlet under the condition of 0.09MPa to extract, after extracting end, extracting solution, the centrifugal 15min of 4000r/min gets supernatant and obtains the trehalose crude extract.
The pH value of regulating the trehalose crude extract is 8.5, adds the gac final concentration and reaches 4%, regulates the crude extract temperature and is 45 ℃ and carries out activated carbon decolorizing, and each treatment time is 1h, and the number of times that decolours is 2 times, and is colourless to liquid, and the centrifugal supernatant that stays is for subsequent use.Adding massfraction in the decolouring treatment solution according to 1: 100 volume ratio is 11% solution of zinc sulfate, and it is rear with Ba (OH) to stir
2Regulator solution pH value to 8.0 leaves standstill the rear centrifugal supernatant that stays.Treated macropore strong acid cation resin D72 and macroporous strong basic resin anion(R.A) post D280 are mixed the dress post according to the consumption volume ratio at 2: 1, regulate the rear flow velocity with 2.0mL/min of an alkali metal salt precipitation acquisition pH to 6.0 after the balance and carry out the cation and anion exchange removal of impurities, after effluent liquid is concentrated into 45%, the dehydrated alcohol that adds 4 times of volumes, rear input crystal seed stirs, 4 ℃ leave standstill 12h, obtain the trehalose crystal.Filter and obtain the trehalose crystal, behind a small amount of alcohol flushing, obtain the trehalose sterling in 40 ℃ of lower vacuum-drying 12h, HPLC testing product purity reaches 98.6%.
Embodiment four
Take by weighing beer waste yeast 1kg, after the dilution of adding 9L sterilized water is even, crosses 100 mesh sieves and sieve 2 times, add the tartrate final concentration and reach 5%, behind the stirring 20min, the 3000r/min centrifugation obtains pure cereuisiae fermentum, and is for subsequent use.
The pure cereuisiae fermentum that pre-treatment obtains is put into baking oven, 98 ℃ of oven dry 48h.The beer waste yeast that drying and processing is good wraps with bundle puts into apparatus,Soxhlet's, uses the 30L deionized water, at 80 ℃, vacuum tightness is to carry out Soxhlet under the condition of 0.09MPa to extract, after extracting end, extracting solution, the centrifugal 15min of 4000r/min gets supernatant and obtains the trehalose crude extract.
The pH value of regulating the trehalose crude extract is 9.0, adds the gac final concentration and reaches 2%, regulates the crude extract temperature and is 45 ℃ and carries out activated carbon decolorizing, and each treatment time is 1h, and the number of times that decolours is 2 times, and is colourless to liquid, and the centrifugal supernatant that stays is for subsequent use.Adding massfraction in the decolouring treatment solution according to 1.2: 100 volume ratios is 11% solution of zinc sulfate, and it is rear with Ba (OH) to stir
2Regulator solution pH value to 8.0 leaves standstill the rear centrifugal supernatant that stays.Treated macropore strong acid cation resin D001SC and macroporous strong basic resin anion(R.A) post D380 are mixed the dress post according to the consumption volume ratio at 2: 1, regulate the rear flow velocity with 2.0mL/min of an alkali metal salt precipitation acquisition pH to 6.0 after the balance and carry out the cation and anion exchange removal of impurities, after effluent liquid is concentrated into 40%, the dehydrated alcohol that adds 4 times of volumes, rear input crystal seed stirs, 4 ℃ leave standstill 12h, obtain the trehalose crystal.Filter and obtain the trehalose crystal, behind a small amount of alcohol flushing, obtain the trehalose sterling in 40 ℃ of lower vacuum-drying 12h, HPLC testing product purity reaches 98.8%.
Claims (9)
1. method of from beer waste yeast, extracting Sargassum polysaccharides, it is characterized in that take the beer waste yeast as raw material, after after enzyme, ultrasonic disruption are gone out in pre-treatment removal of impurities, oven dry, extracting acquisition Sargassum polysaccharides crude extract, adopt ultrasonic disruption, Soxhlet to extract the extracting solution that obtains Sargassum polysaccharides, further behind activated carbon decolorizing, an alkali metal salt precipitation deproteinated, ion-exchange removal of impurities, concentrated, crystallizing and drying, obtain the Sargassum polysaccharides sterling.
2. as described in claim 1 beer waste yeast pre-treatment removal of impurities, it is characterized in that: in beer waste yeast, add sterilized water, regulating sterilised yeast suspension concentration is 8%-10%, crossing 100 mesh sieve sieves 1-2 time, the tartrate of adding 5%, stir 15-30min, the 3000r/min centrifugation obtains pure cereuisiae fermentum, and is for subsequent use.
3. drying and processing as described in claim 1, it is characterized in that: in the pure cereuisiae fermentum drying and processing process that pre-treatment obtains, bake out temperature is 98-105 ℃, and the treatment time is 40-48h.
4. Soxhlet extraction process as described in claim 1, it is characterized in that: oven dry obtains the solid-liquid ratio 1 of cereuisiae fermentum and sterilized water: 25-1: 30, extracting temperature is 80-100 ℃, vacuum tightness 0.09-0.10MPa, after extracting end, extracting solution, the centrifugal 15min of 4000r/min gets supernatant and obtains the trehalose crude extract.
5. in the activated carbon decolorizing technique as claimed in claim 1, it is characterized in that: the pH value of extracting solution is 8.5-9.0, and temperature is 45-50 ℃, the addition of gac is 2%-4%, and extraction time is 0.5-2h, and the decolouring number of times is 2-3 time, colourless to liquid, the centrifugal supernatant that stays, for subsequent use.
6. an alkali metal salt depositing technology as claimed in claim 1, it is characterized in that: the massfraction of the solution of zinc sulfate of adding is 10-12%, adding proportion is 1: 100-1.5: 100.
7. ion-exchange impurity removal process as claimed in claim 1, it is characterized in that: the strongly acidic macroporous cation exchange resin of employing can be selected D001,001X7, D001SC, D72, strong basicity punching anionite-exchange resin can be selected D280, D296, D201, a kind of among the D380, yin and yang resin consumption volume ratio is 2: 1, mixes the dress post, and flow velocity is 2.0mL/min.
8. crystallizing and drying technique as claimed in claim 1 is characterized in that: after will being concentrated into 40%-50% through the effluent liquid of ion-exchange removal of impurities, add the dehydrated alcohol of 4-5 times of volume, and the rear input crystal seed that stirs, 4 ℃ leave standstill 12h, obtain the trehalose crystal.Filter and obtain the trehalose crystal, behind a small amount of alcohol flushing, obtain the trehalose sterling in 40 ℃ of lower vacuum-drying 12h.
9. a kind of Sargassum polysaccharides as claimed in claim 1 extracts the method for preparation, and it is characterized in that: the trehalose purity for preparing reaches more than 98.5%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923131A (en) * | 2014-04-23 | 2014-07-16 | 菏泽学院 | Method for preparing trehalose and food fresher by using waste beer yeast |
| CN103980327A (en) * | 2014-05-07 | 2014-08-13 | 国家海洋局第三海洋研究所 | Preparation method of highly pure mycose used for medicinal injections |
| CN103992363A (en) * | 2014-05-09 | 2014-08-20 | 菏泽学院 | Method for preparing trehalose and yeast extract by utilization of waste beer yeast |
| CN104177449A (en) * | 2014-07-24 | 2014-12-03 | 菏泽学院 | Environment-friendly production method for enhancing trehalose yield of beer waste yeast under stress conditions |
| CN104592316A (en) * | 2015-01-31 | 2015-05-06 | 湖南尔康制药股份有限公司 | Preparation method for trehalose for injection |
| CN106399423A (en) * | 2016-09-14 | 2017-02-15 | 天津替代医学科技股份有限公司 | Method for preparing trehalose by using waste beer yeast under adverse stress conditions |
| CN108822166A (en) * | 2018-07-13 | 2018-11-16 | 安徽民祯生物工程有限公司 | A method of trehalose is produced using beer waste yeast |
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- 2012-09-26 CN CN 201210361477 patent/CN102850410A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923131A (en) * | 2014-04-23 | 2014-07-16 | 菏泽学院 | Method for preparing trehalose and food fresher by using waste beer yeast |
| CN103980327A (en) * | 2014-05-07 | 2014-08-13 | 国家海洋局第三海洋研究所 | Preparation method of highly pure mycose used for medicinal injections |
| CN103980327B (en) * | 2014-05-07 | 2016-09-14 | 国家海洋局第三海洋研究所 | A kind of high-purity trehalose preparation method that can be used for pharmaceutical injection agent |
| CN103992363A (en) * | 2014-05-09 | 2014-08-20 | 菏泽学院 | Method for preparing trehalose and yeast extract by utilization of waste beer yeast |
| CN103992363B (en) * | 2014-05-09 | 2016-08-17 | 菏泽学院 | Utilize the method that beer waste yeast prepares trehalose and yeast extract |
| CN104177449A (en) * | 2014-07-24 | 2014-12-03 | 菏泽学院 | Environment-friendly production method for enhancing trehalose yield of beer waste yeast under stress conditions |
| CN104177449B (en) * | 2014-07-24 | 2016-08-17 | 菏泽学院 | Stress conditions is utilized to improve the environment-protection production method of beer waste yeast trehalose yield |
| CN104592316A (en) * | 2015-01-31 | 2015-05-06 | 湖南尔康制药股份有限公司 | Preparation method for trehalose for injection |
| CN106399423A (en) * | 2016-09-14 | 2017-02-15 | 天津替代医学科技股份有限公司 | Method for preparing trehalose by using waste beer yeast under adverse stress conditions |
| CN106399423B (en) * | 2016-09-14 | 2019-11-29 | 中美食品有限公司 | A method of trehalose is prepared under the conditions of environment stress using beer waste yeast |
| CN108822166A (en) * | 2018-07-13 | 2018-11-16 | 安徽民祯生物工程有限公司 | A method of trehalose is produced using beer waste yeast |
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Application publication date: 20130102 |