CN101230106B - Preparation method of banana polysaccharide - Google Patents

Preparation method of banana polysaccharide Download PDF

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Publication number
CN101230106B
CN101230106B CN200810009334XA CN200810009334A CN101230106B CN 101230106 B CN101230106 B CN 101230106B CN 200810009334X A CN200810009334X A CN 200810009334XA CN 200810009334 A CN200810009334 A CN 200810009334A CN 101230106 B CN101230106 B CN 101230106B
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banana
preparation
banana polysaccharide
precipitation
polysaccharide
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CN101230106A (en
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韩金光
伍曾利
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Hainan standard biological Polytron Technologies Inc
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HAINAN STAND BIO-TECHNOLOGY Co Ltd
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

The invention discloses a preparation method of banana polysaccharide, and the preparation method comprises the following processes in sequence: the banana is washed to be clean, cut to pieces and soaked and processed with acid solution, and then blanched with hot water for a few minutes, cooled, and beaten for stand-by, and then water is added to be extracted. The hydrolysis is performed with double enzyme, and then the enzyme inactivation is performed; the liquid is filtered, and the clear liquid is alcoholized and settled statically over night. The liquid is centrifuged and filtered, the deposit is taken and then washed for two times respectively with 95 percent of ethyl alcohol and acetone, and then dried with vacuum, and the banana polysaccharide crude product is obtained. The banana polysaccharide crude product is taken to be dissolved, centrifuged and filtered with water, and then adsorbed, separated and purified through a DEAE chromatography column on the filtrate, the object is collected and then ultrafilted with ultra filtration membrane, and trapped fluid is collected, alcoholized and settled statically over night. The fluid is centrifuged and filtered, the deposit is washed for two times respectively with 95 percent of ethyl alcohol and acetone, and then dried with vacuum, and the high-purity banana polysaccharide is obtained. The extraction method is simple and safe, no pollution is produced, the yield is high, the production cost is low, the purity of the obtained banana polysaccharide is high, and the preparation method is suitable for the large-scale industrialization production.

Description

The preparation method of banana polysaccharide
Technical field
The present invention relates to the production technique of food, medication chemistry, specifically the preparation method of banana polysaccharide.
Background technology
In the life science field, glycobiology is the research forward position and the focus in this field always.Polysaccharide has many-sided complex biological activity and function, and the sugar chain of polysaccharide can be controlled the division and the differentiation of cell, and the growth of regulating cell is with old and feeble.
Sugar degree is nearly 20% in the ripe banana, and banana polysaccharide descends to the murine interleukin that is caused by endoxan certain restraining effect, and certain immunocompetence is promptly arranged, and this Application and Development for banana polysaccharide provides scientific basis.
Yet banana great majority now just eats as a kind of fruit, and its pharmaceutical use often is left in the basket.Banana polysaccharide has the immunocompetence effect.The harm of AIDS (AIDS) makes people to the serious consequence of immunodeficiency more deep understanding arranged, and therefore seeking the medicine with good immunoregulation effect has become one of developing direction of contemporary new drug development.Therefore, research and development utilizes banana polysaccharide to have vast market prospect.
Contain a lot of organized enzymes in the banana, brown stain takes place easily, it is very big unfavorable that the extraction of banana polysaccharide is had.Because the banana polysaccharide in the banana is very easily oxidized,, make the banana polysaccharide blackout that obtains at last so in the extraction purge process of banana polysaccharide, banana polysaccharide is easy to blackout.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of banana polysaccharide.
The present invention adopts following technical scheme:
(1) with banana wash clean, segment, be 3.0~4.5 acid solution immersion treatment with pH, use 90 ℃~100 ℃ hot water blanching numbers minute again, cooling, making beating are standby;
(2) get standby slurries, adding water is heated to more than 90 ℃ and extracts twice, each 3~4 times of water that add banana meat weight extract, collect extracting solution, cool to room temperature, with sour adjust pH to 3.0~4.0, add aspartic proteases such as papain or stomach en-, after stirring evenly, keep the pH value, 45 ℃~50 ℃ condition hydrolysis 3.0~4.0;
(3), add that pancreatin or trypsinase etc. are neutral, Sumizyme MP, after stirring evenly with extracting solution adjust pH to 9.0~9.5 of alkali after with hydrolysis, keep the pH value 9.0~9.5,45 ℃~50 ℃ condition hydrolysis, enzymolysis finish the post-heating intensification and carry out enzyme-deactivating, are cooled to room temperature;
(4) enzymolysis solution transfers pH to 6.0~6.5 centrifugal with acid, filters, and collects filtrate; Filtrate is carried out alcohol precipitation, standing over night;
(5) solution behind the alcohol precipitation in the step (4) is filtered, get precipitation, precipitation is respectively washed 2 times with 95% ethanol and acetone, and vacuum-drying promptly gets the banana polysaccharide crude product;
(6) get the banana polysaccharide crude product, add the water stirring and dissolve, centrifugal, filter, get filtered solution;
(7) filtered solution is carried out chromatographic separation with the DEAE chromatography column;
(8) target compound after the collection chromatographic separation carries out ultrafiltration with ultra-filtration membrane, collects trapped fluid;
(9) trapped fluid carries out alcohol precipitation, standing over night once more;
(10) solution behind the alcohol precipitation in the step (9) is carried out centrifugal, filter, get precipitation, precipitation is respectively washed 2 times with 95% ethanol and acetone, carries out vacuum-drying, promptly gets the banana polysaccharide elaboration.
The time of enzyme-deactivating and temperature are determined according to the kind of enzyme, generally are heated to 100 ℃, are incubated 5~15 minutes.
The alkali that described adjust pH is used can be sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium bicarbonate or ammoniacal liquor.
The acid that described adjust pH is used can be hydrochloric acid, phosphoric acid, citric acid or sulfuric acid.
With the process that enzyme is hydrolyzed, can grasp the hydrolysis process by the control hydrolysis time, hydrolysis time is short, and the insufficient yield that causes of hydrolysis is low, can extend manufacture cycle again but the hydrolysis time process is long, increases cost and increases contaminated chance.Hydrolysis time can be 450~500 minutes in the general step (2).Equally, hydrolysis time is 200~250 minutes in the described step (3).
The phosphate buffered saline buffer of using during described chromatography is 10~30mmol/L pH5.8~6.8 phosphate buffered saline buffers.
It is the ultra-filtration membrane of 5~10KD that molecular retention is adopted in described ultrafiltration.
The present invention is by carrying out double-enzyme hydrolysis to banana, method through the ethanol alcohol precipitation, other impurity is removed in the first step separation and purification, use DEAE chromatography column absorption and purification then, with molecular retention is that the ultra-filtration membrane of 5~10KD carries out ultrafiltration, collect trapped fluid, pass through the alcohol alcohol precipitation again, other impurity is removed in the second step separation and purification.The advantage of this method is as follows:
1, banana directly extracts banana polysaccharide through production technique such as two enzyme enzymolysis, has advantages such as simple, safety, environmental protection, low cost.
2, the prepared banana polysaccharide purity height of method for preparing banana polysaccharide of the present invention, the yield height, of good quality, but advantages such as mass production.
Advantages such as 3, the used raw materials cost of the preparation method of banana polysaccharide of the present invention is low, and the source is wide, and pericarp, pulp all can extract polysaccharide, and medicinal effect is obvious.
4, preparation is simple, and raw material is easy to get, the reaction conditions gentleness, and the yield height, good stability, also very low to preparation equipment and place requirement, be suitable for large-scale industrial production.
Embodiment
Below by specific embodiment the present invention is described in further detail.
Embodiment:
Get 200 ripe bananas, with the banana wash clean, weigh 6.0kg, segment, be 3.0~4.5 hydrochloric acid soln immersion treatment with pH, use 100 ℃ of hot water blanching 7min again, cooling, making beating are standby.Add 20.0l water and be heated to 100 ℃ of extractions 1.5 hours, reduce to room temperature, filter with filter cloth.The banana slag adds 20.0l water again and is heated to 100 ℃ of extractions 1.5 hours, reduces to room temperature, filters with filter cloth.Collect extracted twice liquid, centrifugal, filter, treat that filtered solution is cooled to 45 ℃~50 ℃, transfer pH to 3.0~4.0 with 30% (w%) citric acid, add the ratio of 10 million international units (than vigor) papain, add 50 million international units (than vigor) papain in every kg slurries, after stirring evenly, keep the condition hydrolysis 8 hours of pH3.0~4.0,45 ℃~50 ℃.With 18% (w%) Na0H papain hydrolysis slurries are transferred pH to 9.0~9.5, the ratio that adds 4 million international units (than vigor) pancreatin in every kg slurries, add 20 million international units (than vigor) pancreatin, after stirring evenly, maintain 9.0~9.5,45 ℃~50 ℃ hydrolysis of pH 3.5 hours, enzymolysis finishes post-heating and is warming up to 100 ℃, is incubated 15 minutes and carries out enzyme-deactivating, is cooled to below 30 ℃.Transfer pH6.0~6.5 with 30% (w%) citric acid, centrifugal, suction filtration gets supernatant liquor 36.0kg, and alcohol to the determining alcohol that supernatant liquor adds 95% (v/v) is 70%, and standing over night is carried out alcohol precipitation.Centrifugal, filter, get precipitation, precipitation uses 95% (v/v) ethanol and acetone to wash respectively 2 times, drains, and puts drying in the vacuum drying oven, promptly gets banana polysaccharide crude product 268.34g.The banana polysaccharide crude product adds 6.0L water stirring and dissolving, and is centrifugal, filters, and collects filtrate and gets 5.7kg; Transfer pH to 6.8~7.0 with 18%NaOH, standby.With DEAE furnishing pasty state, adopt slurry type method dress post with water for injection, dress column pressure 0.5Mpa.Clean with 400mmol/L pH6.8 phosphate buffered saline buffer earlier behind the dress post, use 20mmol/L pH6.8 phosphate buffered saline buffer balance again, above-mentioned filtrate is gone up the DEAE chromatography column adsorb, with 20mmol/L pH6.8 phosphate buffered saline buffer wash-out.Collect target compound and get 8.9kg, using molecular retention is that the filter membrane of 100K carries out ultrafiltration, collects trapped fluid and gets 7.0kg, centrifugal, filters, and adding 95% alcohol to determining alcohol is 70%, and standing over night is carried out alcohol precipitation.Centrifugal, filter, get precipitation, precipitation is washed 2 times with 95% ethanol and acetone respectively, drains, and puts drying in the vacuum drying oven, promptly gets banana polysaccharide elaboration 20.21g.Assay is carried out in sampling, and detections such as molecular weight determination are standby.
Assay:
1. polysaccharide is differentiated: phenolsulfuric acid reagent react (positive)
2. polysaccharide content: 99.26% (measuring) with the phenolsulfuric acid method
3. polysaccharide molecule flow measurement: 43109 dalton (with reference to two appendix VH of pharmacopeia in 2005).

Claims (11)

1. the preparation method of a banana polysaccharide, this method is made up of following process successively:
(1) with banana wash clean, segment, be 3.0~4.5 acid solution immersion treatment with pH, use 90 ℃~100 ℃ hot water blanching numbers minute again, cooling, making beating are standby;
(2) get standby slurries, adding water is heated to more than 90 ℃ and extracts twice, each 3~4 times of water that add banana meat weight extract, collect extracting solution, cool to room temperature, with sour adjust pH to 3.0~4.0, add papain, after stirring evenly, keep the pH value, 45 ℃~50 ℃ condition hydrolysis 3.0~4.0;
(3) with extracting solution adjust pH to 9.0~9.5 of alkali after, add pancreatin, after stirring evenly, keep pH value 9.0~9.5 with hydrolysis, 45 ℃~50 ℃ condition hydrolysis, enzymolysis end post-heating carries out enzyme-deactivating, is cooled to room temperature;
(4) transfer pH to 6.0~6.5 with acid, filter, collect filtrate; Filtrate is carried out alcohol precipitation, standing over night;
(5) solution behind the alcohol precipitation in the step (4) is filtered, get precipitation, precipitation is respectively washed with 95% ethanol and acetone, and vacuum-drying promptly gets the banana polysaccharide crude product;
(6) get the banana polysaccharide crude product, add the water stirring and dissolve, centrifugal, filter, get filtered solution;
(7) filtered solution is carried out chromatographic separation with the DEAE chromatography column;
(8) target compound after the collection chromatographic separation carries out ultrafiltration with ultra-filtration membrane, collects trapped fluid;
(9) trapped fluid carries out alcohol precipitation, standing over night once more;
(10) solution behind the alcohol precipitation in the step (9) is carried out centrifugal, filter, get precipitation, each washs precipitation with 95% ethanol and acetone, carries out vacuum-drying, promptly gets the banana polysaccharide elaboration.
2. the preparation method of banana polysaccharide according to claim 1, it is characterized in that: described banana is whole banana, comprises pulp and pericarp.
3. the preparation method of banana polysaccharide according to claim 1, it is characterized in that: the alkali that described adjust pH is used is sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium bicarbonate or ammoniacal liquor.
4. the preparation method of banana polysaccharide according to claim 1, it is characterized in that: the acid that described adjust pH is used is hydrochloric acid, phosphoric acid, citric acid or sulfuric acid.
5. the preparation method of banana polysaccharide according to claim 1 is characterized in that: hydrolysis time is 450~500 minutes in the described step (2).
6. the preparation method of banana polysaccharide according to claim 1 is characterized in that: hydrolysis time is 200~250 minutes in the described step (3).
7. the preparation method of banana polysaccharide according to claim 1 is characterized in that: filtration procedure comprises in the described step (4): carry out coarse filtration with filter cloth earlier, carry out centrifugally again with high speed freezing centrifuge, carry out suction filtration again.
8. the preparation method of banana polysaccharide according to claim 1, it is characterized in that: the filtration procedure in the described step (5) is: centrifugal with high speed freezing centrifuge, pour out supernatant liquor, and collecting precipitation is to container, use 95% ethanol and washing with acetone respectively, drain with suction filter.
9. the preparation method of banana polysaccharide according to claim 1 is characterized in that: the alcohol concn that alcohol precipitation is used in described step (4), (9) is 40%~95%.
10. the preparation method of banana polysaccharide according to claim 1 is characterized in that: the chromatography phosphate buffered saline buffer is 10~30mmol/L in the described step (7), and pH5.8~6.8 phosphate buffered saline buffers carry out wash-out.
11. the preparation method of banana polysaccharide according to claim 1 is characterized in that: it is the ultra-filtration membrane of 5~100KD that molecular retention is adopted in described ultrafiltration.
CN200810009334XA 2008-02-21 2008-02-21 Preparation method of banana polysaccharide Expired - Fee Related CN101230106B (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103360506B (en) * 2013-07-18 2015-10-28 华南理工大学 A kind of extracting method of banana oligosaccharide
JP6523647B2 (en) * 2013-11-14 2019-06-05 大谷 勝 Method for producing banana-derived composition
CN103947968A (en) * 2014-04-30 2014-07-30 桂林军供生化技术开发有限公司 Banana extract, preparation method and application of banana extract
CN105924541B (en) * 2016-05-04 2018-04-17 中国科学院华南植物园 The method that one kind prepares 1,6 glucans of α
CN108142825A (en) * 2017-12-22 2018-06-12 广西兴业百谷米业有限公司 A kind of dietary fiber rice of high-content and preparation method thereof
CN110464872B (en) * 2019-09-26 2020-09-04 海南医学院 Medical hydrogel and preparation method and application thereof
CN114230682B (en) * 2022-02-08 2022-08-19 中国热带农业科学院分析测试中心 Functional banana polysaccharide BPF2 and preparation and application thereof

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CN1337410A (en) * 2001-09-03 2002-02-27 王维新 Production process of extracting fruit polysaccharide from fruits
CN101015336A (en) * 2007-03-09 2007-08-15 伍曾利 Prepn. of coconut extractive

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN1337410A (en) * 2001-09-03 2002-02-27 王维新 Production process of extracting fruit polysaccharide from fruits
CN101015336A (en) * 2007-03-09 2007-08-15 伍曾利 Prepn. of coconut extractive

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