CN103804503A - Method of extracting polysaccharide of enteromorpha by enzymolysis in steps - Google Patents
Method of extracting polysaccharide of enteromorpha by enzymolysis in steps Download PDFInfo
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- CN103804503A CN103804503A CN201210486512.4A CN201210486512A CN103804503A CN 103804503 A CN103804503 A CN 103804503A CN 201210486512 A CN201210486512 A CN 201210486512A CN 103804503 A CN103804503 A CN 103804503A
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Abstract
The invention provides a method of extracting polysaccharide of enteromorpha by enzymolysis in steps. The method comprises the following steps: by taking enteromorpha as a raw material, soaking the enteromorpha powder by water after impurity removal and crushing; then, respectively carrying out enzymolysis in steps on the aqueous extract of the enteromorpha by using pectinase, cellulase and papain; then, inactivating the enzyme, concentrating by centrifuging and precipitating by ethanol; and finally, vacuum-drying to obtain the polysaccharide of the enteromorpha. The method provided by the invention is high in yield, high in product purity, low in energy consumption, free from pollution, high in biological activity, suitable for production of polysaccharide of enteromorpha on a large scale, beneficial to development and utilization of resources polysaccharide of enteromorpha, and remarkable in economic and social values.
Description
Technical field
The present invention relates to a kind of extracting method of sea grass polysaccharide, especially relate to a kind of method of substep enzymolysis and extraction sea grass polysaccharide.
Background technology
Enteromorpha (Enteromorpha) is a kind of large-scale green alga, is commonly called as tongue bar, green laver etc., is the phycophyta of Chlorophyta Ulvales Ulvaceae Enteromorpha, is grown in tideland.In China, Enteromorpha output is very high, especially East Coastal.In recent years, the Fast-propagation of Enteromorpha pollutes China coast, and the pollution causing for administering Enteromorpha excessive multiplication, need to expend a large amount of manpower and materials, therefore if Enteromorpha is developed and utilized, not only can administer Enteromorpha and pollute, and can also turn waste into wealth simultaneously.
Enteromorpha is edible and medicinal algae since ancient times, records Enteromorpha and can " burn last nasal insufflation and stop bleeding from five sense organs or subcutaneous tissue in Compendium of Material Medica; Soup soak smash apply the back of the hand swell and ache "; " stealthy occupy diet spectrum " recorded: " clearing gallbladder, the scrofula that disappears goiter and tumor, rush down swollen, reduce phlegm, control not acclimatized.Enteromorpha is rich in carbohydrate, protein, robust fibre and mineral substance, also contains fat and VITAMIN simultaneously.Studies confirm that in recent years, the main component water-soluble polysaccharide of Enteromorpha is the composition that pharmacologically active is very strong, have the various biological activity such as significant reducing blood-fat, antifatigue, antibacterial, antitumor and strengthening immunity (Lin Wenting. brief talk the development and utilization of Enteromorpha. Chinese food and nutrition, 2007,9:23～25)
What at present the extraction of sea grass polysaccharide was the most frequently used is hot water extraction (Lv Lingling. the optimization research of Enteromorpha water-soluble polysaccharide extraction process. Agriculture of Anhui science, 2009,37 (24): 717,756.), as described in the application number patent application that is 201010224578.7.But adopt hot water extraction from Enteromorpha, to extract that the yield of polysaccharide is lower, foreign matter content is high, consumes energy high, active low.
Summary of the invention
The present invention is exactly for the problems referred to above, provides that a kind of productive rate is high, product purity is high, it is low, pollution-free to consume energy and has retained the extracting method of the sea grass polysaccharide of higher biological activity.
---substep enzymolysis---the deactivation of enzyme---centrifugal, concentrated, ethanol precipitation---vacuum-drying that the production process that technical solution of the present invention adopts is: the removal of impurities of Enteromorpha frond, dry, pulverizing.
Concrete steps are as follows:
Step 1: Enteromorpha is cleaned up to pulverize, sieve after 60～80 ℃ of oven dry (20～60 order).Take gained Enteromorpha powder, add distilled water by solid-liquid ratio 1: 20～60 (w/v, g/ml), fully stir, soak 10～15h, obtain Enteromorpha the first vat liquor.
Step 2: adding acid for adjusting pH value to described Enteromorpha the first vat liquor is 3.5～4.5, and temperature is heated to 40～50 ℃, adds polygalacturonase, fully stirs, and enzymolysis 1～2h, obtains Enteromorpha the second vat liquor.
Step 3: add alkali to regulate pH value to described Enteromorpha the second vat liquor and be 4.5～5.5, temperature is heated to 50～60 ℃, add cellulase, fully stir, enzymolysis 2～4h, obtains Enteromorpha the 3rd vat liquor.
Step 4: adding papoid in described Enteromorpha the 3rd vat liquor, is enzymolysis 1～2h under 5.0～6.0 conditions in 50～60 ℃ of temperature, pH value, obtains Enteromorpha the 4th vat liquor.
Step 5: the above-mentioned product obtaining after substep enzymolysis that completes is heated to 90～100 ℃, residual enzyme is carried out to deactivation, fully stir and soak, process 20～50min, obtain Enteromorpha the 5th vat liquor.
Step 6: at the centrifugal 10～15min of 10000rpm, supernatant liquid filtering final vacuum is concentrated by Enteromorpha the 5th vat liquor; In enriched material, add ethanol, centrifugation, throw out vacuum-drying, obtains sea grass polysaccharide.
Beneficial effect of the present invention:
The present invention adopts substep enzymolysis process to extract sea grass polysaccharide, and in polysaccharide leaching process, substep adds polygalacturonase, cellulase and papoid, makes it under the operating environment that enzyme activity is the highest separately, process respectively Enteromorpha vat liquor.Polygalacturonase, cellulase hydrolyzable pectin substance and Mierocrystalline cellulose, effectively dissolved cell wall more easily discharges polysaccharide in cell; Papoid can not only destroy free protein, and can make the protein degradation of polysaccharide combination, thereby polysaccharide is more fully dissolved in water, and therefore the extraction efficiency of polysaccharide and purity improve greatly, and have retained higher biological activity.
Utilize extracting method gained finished product color and luster of the present invention greyish white, polysaccharide yield 15.92 ± 1.46%, polysaccharide content is 68.35 ± 4.46%.Method is stable, reliable.Antioxidation in vitro experiment confirmation, prepared sea grass polysaccharide is 73.1%～83.7% to the removing ability of ultra-oxygen anion free radical, removing ability and V
cquite, and there is dosage effect, illustrate that it possesses stronger antioxidation activity in vitro.
Non-pollutant discharge in whole technological process of the present invention, is suitable for scale operation, is of value to the exploitation of Enteromorpha resource.
Embodiment
Embodiment 1
Step 1: Enteromorpha is cleaned up, pulverize, cross 40 mesh sieves after 60 ℃ of oven dry.Take 1kg Enteromorpha powder, add 20L distilled water, fully stir, soak 15h.Obtain Enteromorpha the first vat liquor.
Step 2: adding lemon acid for adjusting pH value to described Enteromorpha the first vat liquor is 4.0, and temperature is heated to 45 ℃, adds 15g polygalacturonase, fully stirs, and enzymolysis 1h, obtains Enteromorpha the second vat liquor.
Step 3: add 0.1M sodium hydroxide to regulate pH value to described Enteromorpha the second vat liquor and be 5.0, temperature is heated to 55 ℃, add 10g cellulase, fully stir, enzymolysis 2h, obtains Enteromorpha the 3rd vat liquor.
Step 4: add 30g papoid in described Enteromorpha the 3rd vat liquor, regulating pH value with 0.1M sodium hydroxide is 5.5, at 55 ℃ of enzymolysis 2h of temperature, obtains Enteromorpha the 4th vat liquor.
Step 5: the above-mentioned product obtaining after substep enzymolysis that completes is heated to 100 ℃, residual enzyme is carried out to deactivation, fully stir and soak, process 30min.Obtain Enteromorpha the 5th vat liquor
Step 6: at the centrifugal 10min of 10000rpm, supernatant liquid filtering final vacuum is concentrated by Enteromorpha the 5th vat liquor; To the ethanol that adds 3 times of volumes of enriched material in enriched material, the centrifugal 10min of 4000rpm, throw out vacuum-drying, obtains sea grass polysaccharide 156.8g; Polysaccharide content is 68.1% (adopting phenolsulfuric acid method to measure); Antioxidation in vitro experimental result shows, in the time that sea grass polysaccharide concentration is 0.25mg/mL, the removing ability of ultra-oxygen anion free radical is reached to 76.4%.
Embodiment 2
Step 1: Enteromorpha is cleaned up, pulverize, cross 40 mesh sieves after 60 ℃ of oven dry.Take 1kg Enteromorpha powder, add 20L distilled water, fully stir, soak 10h.Obtain Enteromorpha the first vat liquor.
Step 2: adding lemon acid for adjusting pH value to described Enteromorpha the first vat liquor is 4.0, and temperature is heated to 40 ℃, adds 10g polygalacturonase, fully stirs, and enzymolysis 1h, obtains Enteromorpha the second vat liquor.
Step 3: add 0.1M sodium hydroxide to regulate pH value to described Enteromorpha the second vat liquor and be 5.0, temperature is heated to 50 ℃, add 8g cellulase, fully stir, enzymolysis 2h, obtains Enteromorpha the 3rd vat liquor.
Step 4: add 30g papoid in described Enteromorpha the 3rd vat liquor, regulating pH value with 0.1M sodium hydroxide is 5.5, at 55 ℃ of enzymolysis 2h of temperature, obtains Enteromorpha the 4th vat liquor.
Step 5: the above-mentioned product obtaining after substep enzymolysis that completes is heated to 100 ℃, residual enzyme is carried out to deactivation, fully stir and soak, process 30min.Obtain Enteromorpha the 5th vat liquor.
Step 6: at the centrifugal 10min of 10000rpm, supernatant liquid filtering final vacuum is concentrated by Enteromorpha the 5th vat liquor; To the ethanol that adds 3 times of volumes of enriched material in enriched material, the centrifugal 10min of 4000rpm, throw out vacuum-drying, obtains sea grass polysaccharide 150.2g; Polysaccharide content is 70.6% (adopting phenolsulfuric acid method to measure); Antioxidation in vitro experimental result shows, in the time that sea grass polysaccharide concentration is 0.25mg/mL, the removing ability of ultra-oxygen anion free radical is reached to 80.7%.
Claims (7)
1. a method for substep enzymolysis and extraction sea grass polysaccharide, is characterized in that, comprising:
Step 1: Enteromorpha is cleaned up to pulverize, sieve after 60～80 ℃ of oven dry (20～60 order).Take gained Enteromorpha powder, add distilled water by solid-liquid ratio 1: 20～60 (w/v, g/ml), fully stir, soak 10～15h, obtain Enteromorpha the first vat liquor.
Step 2: adding acid for adjusting pH value to described Enteromorpha the first vat liquor is 3.5～4.5, and temperature is heated to 40～50 ℃, adds polygalacturonase, fully stirs, and enzymolysis 1～2h, obtains Enteromorpha the second vat liquor.
Step 3: add alkali to regulate pH value to described Enteromorpha the second vat liquor and be 4.5～5.5, temperature is heated to 50～60 ℃, add cellulase, fully stir, enzymolysis 2～4h, obtains Enteromorpha the 3rd vat liquor.
Step 4: add papoid in described Enteromorpha the 3rd vat liquor, 50～60 ℃ of temperature, enzymolysis 1～2h under the condition of pH=5.0～6.0, obtains Enteromorpha the 4th vat liquor.
Step 5: the above-mentioned product obtaining after substep enzymolysis that completes is heated to 90～100 ℃, residual enzyme is carried out to deactivation, fully stir and soak, process 20～50min.Obtain Enteromorpha the 5th vat liquor
Step 6: at the centrifugal 10～15min of 10000rpm, supernatant liquid filtering final vacuum is concentrated by Enteromorpha the 5th vat liquor; In enriched material, add ethanol, centrifugation, throw out vacuum-drying.
2. extracting method according to claim 1, is characterized in that, described acid is the one in citric acid, hydrochloric acid or phosphoric acid.
3. extracting method according to claim 1, is characterized in that, adds the amount of polygalacturonase to be equivalent to 1%～3% of Enteromorpha powder weight volume.
4. extracting method according to claim 1, is characterized in that, described alkali is the one in sodium hydroxide, potassium hydroxide or ammoniacal liquor.
5. extracting method according to claim 1, is characterized in that, adds the amount of cellulase to be equivalent to 0.5%～2% of Enteromorpha powder weight volume.
6. extracting method according to claim 1, is characterized in that, adds the amount of papoid to be equivalent to 3%～5% of Enteromorpha powder weight volume.
7. extracting method according to claim 1, is characterized in that, adds the ethanol of 2～3 times of volumes of concentrated solution in step 6, centrifugal after 4 ℃ of standing 3～4h.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567762A (en) * | 2016-02-29 | 2016-05-11 | 福建农林大学 | Enteromorpha prolifera oligosaccharide with auxiliary blood sugar decreasing function and preparation method thereof |
| CN105754005A (en) * | 2016-03-04 | 2016-07-13 | 江苏江大源生态生物科技股份有限公司 | Method for extracting grifola frondosa polysaccharase with high yield |
| CN106509706A (en) * | 2016-11-04 | 2017-03-22 | 中国海洋大学 | Method for removing prometryn and heavy metal in porphyra |
| CN106883309A (en) * | 2017-04-13 | 2017-06-23 | 烟台大学 | The multiplex-enzyme extraction method of laminarin and the laminarin for obtaining |
| CN107232202A (en) * | 2017-07-23 | 2017-10-10 | 青岛海大生物集团有限公司 | A kind of preparation method of sea grass polysaccharide network ammonia copper fungicide |
| CN107641160A (en) * | 2017-10-31 | 2018-01-30 | 青岛绿浒苔农业科技有限公司 | Utilize the method for multi-stage countercurrent enzymolysis coupling technology extraction sea grass polysaccharide |
| CN108003199A (en) * | 2017-07-12 | 2018-05-08 | 中国科学院过程工程研究所 | A kind of Enteromorpha oligosaccharide with function of blood sugar reduction and its preparation method and application |
| CN108007753A (en) * | 2018-01-22 | 2018-05-08 | 上海海洋大学 | A kind of method of chromosome preparation of Enteromorpha sporinite |
| CN108239613A (en) * | 2016-12-27 | 2018-07-03 | 中国海洋大学 | A kind of feed series bacillus, its culture medium and the application in sea grass polysaccharide degrading enzyme is prepared |
| CN108409877A (en) * | 2017-07-12 | 2018-08-17 | 中国科学院过程工程研究所 | Sea grass polysaccharide, enteromorpha oligosaccharide, medical composition and its use |
| CN112608394A (en) * | 2020-12-31 | 2021-04-06 | 青岛海大生物集团有限公司 | Preparation method and application of chlorella polysaccharide derivative |
| CN114672235A (en) * | 2022-04-25 | 2022-06-28 | 山东中胜涂料有限公司 | High-wear-resistance quick-drying water-based acrylic polyurethane finish paint and preparation method thereof |
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Cited By (15)
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| CN105567762B (en) * | 2016-02-29 | 2019-03-12 | 福建农林大学 | A kind of enteromorpha oligosaccharide and preparation method thereof with auxiliary hyperglycemic effect |
| CN105567762A (en) * | 2016-02-29 | 2016-05-11 | 福建农林大学 | Enteromorpha prolifera oligosaccharide with auxiliary blood sugar decreasing function and preparation method thereof |
| CN105754005A (en) * | 2016-03-04 | 2016-07-13 | 江苏江大源生态生物科技股份有限公司 | Method for extracting grifola frondosa polysaccharase with high yield |
| CN106509706A (en) * | 2016-11-04 | 2017-03-22 | 中国海洋大学 | Method for removing prometryn and heavy metal in porphyra |
| CN108239613A (en) * | 2016-12-27 | 2018-07-03 | 中国海洋大学 | A kind of feed series bacillus, its culture medium and the application in sea grass polysaccharide degrading enzyme is prepared |
| CN108239613B (en) * | 2016-12-27 | 2021-07-06 | 中国海洋大学 | Paenibacillus feed, culture medium thereof and application of Paenibacillus feed in preparation of enteromorpha polysaccharide degrading enzyme |
| CN106883309A (en) * | 2017-04-13 | 2017-06-23 | 烟台大学 | The multiplex-enzyme extraction method of laminarin and the laminarin for obtaining |
| CN108003199A (en) * | 2017-07-12 | 2018-05-08 | 中国科学院过程工程研究所 | A kind of Enteromorpha oligosaccharide with function of blood sugar reduction and its preparation method and application |
| CN108409877A (en) * | 2017-07-12 | 2018-08-17 | 中国科学院过程工程研究所 | Sea grass polysaccharide, enteromorpha oligosaccharide, medical composition and its use |
| CN108409877B (en) * | 2017-07-12 | 2020-02-18 | 中国科学院过程工程研究所 | Enteromorpha polysaccharide, enteromorpha oligosaccharide, pharmaceutical composition and application thereof |
| CN107232202A (en) * | 2017-07-23 | 2017-10-10 | 青岛海大生物集团有限公司 | A kind of preparation method of sea grass polysaccharide network ammonia copper fungicide |
| CN107641160A (en) * | 2017-10-31 | 2018-01-30 | 青岛绿浒苔农业科技有限公司 | Utilize the method for multi-stage countercurrent enzymolysis coupling technology extraction sea grass polysaccharide |
| CN108007753A (en) * | 2018-01-22 | 2018-05-08 | 上海海洋大学 | A kind of method of chromosome preparation of Enteromorpha sporinite |
| CN112608394A (en) * | 2020-12-31 | 2021-04-06 | 青岛海大生物集团有限公司 | Preparation method and application of chlorella polysaccharide derivative |
| CN114672235A (en) * | 2022-04-25 | 2022-06-28 | 山东中胜涂料有限公司 | High-wear-resistance quick-drying water-based acrylic polyurethane finish paint and preparation method thereof |
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Application publication date: 20140521 |