CN117757872A - Method for efficiently preparing arrowhead non-starch polysaccharide by utilizing compound enzyme preparation and application - Google Patents
Method for efficiently preparing arrowhead non-starch polysaccharide by utilizing compound enzyme preparation and application Download PDFInfo
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- 229920002472 Starch Polymers 0.000 title claims abstract description 130
- 239000008107 starch Substances 0.000 title claims abstract description 130
- 235000019698 starch Nutrition 0.000 title claims abstract description 130
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 127
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- 238000002360 preparation method Methods 0.000 title claims abstract description 28
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- 238000001816 cooling Methods 0.000 claims description 5
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- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
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- 235000019658 bitter taste Nutrition 0.000 description 1
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
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- 235000003891 ferrous sulphate Nutrition 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the field of natural product preparation and application, and particularly relates to a method for efficiently preparing arrowhead non-starch polysaccharide by utilizing a compound enzyme preparation and application thereof. The complex enzyme preparation provided by the invention can be used for efficiently preparing arrowhead non-starch polysaccharide, and has high polysaccharide yield (76.5+/-2.90%) and low starch residual quantity (4.91+/-0.07%). The arrowhead non-starch polysaccharide prepared by the compound enzyme preparation has strong antioxidant activity, and the clearance rate of the arrowhead non-starch polysaccharide to hydroxyl radicals reaches 96.1+/-2.06% at the concentration of 5 mg/mL; at a concentration of 1mg/mL, the clearance rate of DPPH free radical reaches 95.2+/-4.26 percent. The polysaccharide can be used for preparing antioxidant products such as oral liquid, capsule, tablet, etc.
Description
Technical Field
The invention belongs to the field of natural product preparation, and particularly relates to a method for efficiently preparing arrowhead non-starch polysaccharide by utilizing a compound enzyme preparation and application thereof.
Background
Rhizoma Sagittariae Sagittifoliae (Sagittaria trifolia L.) is tuber of rhizoma Sagittariae Sagittifoliae, and is usually used as vegetable. As a representative variety of agricultural products of' Narcissus, the arrowhead has long planting history in the Jiang Zhe region of China and long medicinal history, and is recorded in Ming Yi Bie Ji, wei jin in the period of Wei jin, and the arrowhead has bitter taste, sweet, slightly cold and no toxicity. It is used for treating diabetes, arthralgia, heat, middle energizer, and qi invigorating. Modern pharmacological research shows that the non-starch polysaccharide is an important functional component of arrowhead, and has the functions of resisting tumor, protecting liver, enhancing organism immunity, resisting oxidation and the like (Journal of the Science of Food and Agriculture,2021,101 (8): 3085-3098).
Due to the interference of coexisting starch, there is a great challenge in efficiently extracting non-starch polysaccharides from arrowhead, and the traditional hot water extraction method causes excessive swelling and even gelatinization of starch, and severely interferes with release of non-starch polysaccharides, so that the yield of arrowhead non-starch polysaccharides is only 8.54+ -1.07% (Chemistry & Biodiversity 2022,19, e 202200219). Patent CN 113801248B discloses a method for extracting arrowhead non-starch polysaccharide by complex enzyme assisted microwave. The complex enzyme consists of alpha-amylase (50U/mg) and cellulase (10U/mg) in a mass ratio of 1:1; the microwave time is 8min under 506W power, and the non-starch polysaccharide yield reaches 36.33 +/-2.57%. Although the method obviously improves the extraction rate of arrowhead non-starch polysaccharide, the influence of the mass ratio of alpha-amylase to cellulase and the enzyme activity on the polysaccharide yield and the polysaccharide components is not considered, and the key problems of starch degradation, conversion and the like in the extraction process are not disclosed.
The invention provides a method for efficiently preparing arrowhead non-starch polysaccharide by utilizing a compound enzyme preparation and application thereof by exploring the influence of the mass ratio of alpha-amylase to cellulase and the enzyme activity on the polysaccharide yield and the starch residue.
Disclosure of Invention
The invention aims to provide a method for efficiently preparing arrowhead non-starch polysaccharide by utilizing a complex enzyme preparation. Compared with the traditional extraction method and the technical scheme disclosed by patent CN 113801248B, the arrowhead non-starch polysaccharide prepared by the compound enzyme preparation provided by the invention has the advantages of high yield and low starch residue; and arrowhead non-starch polysaccharide prepared by the compound enzyme preparation has stronger antioxidant activity.
According to a first aspect of the invention, the invention provides a method for preparing arrowhead non-starch polysaccharide by utilizing a compound enzyme preparation, which takes arrowhead fat-removed powder as a raw material, firstly adopts the compound enzyme preparation for enzymolysis, and places the arrowhead fat-removed powder in a multifunctional microwave synthesis extraction instrument after enzyme deactivation for microwave extraction treatment; centrifuging, concentrating supernatant, precipitating with ethanol, and lyophilizing to obtain rhizoma Sagittariae Sagittifoliae non-starch polysaccharide; the complex enzyme preparation consists of alpha-amylase and cellulase, wherein the mass ratio of the alpha-amylase is as follows: cellulase = 3:2-7:3.
Preferably, the addition amount of the complex enzyme preparation is 2wt% of arrowhead fat-removed powder.
The preparation method of the arrowhead non-starch polysaccharide specifically comprises the following steps:
cleaning fresh rhizoma Sagittariae Sagittifoliae, slicing, oven drying at 60deg.C to constant weight, pulverizing, sieving with 60 mesh sieve to obtain rhizoma Sagittariae Sagittifoliae powder; adding petroleum ether, degreasing for 12h under dark condition, and vacuum filtering to obtain arrowhead defatted powder; soaking arrowhead fat-removed powder in water for 4 hours according to a feed liquid ratio of 1:43g/mL, then regulating the pH value to about 5.0, adding 2% of compound enzyme (composed of alpha-amylase and cellulase with different mass ratios and different enzyme activities), placing in a shaking table, incubating for 2 hours at 50-55 ℃, and placing the enzymolysis liquid in a boiling water bath for enzyme deactivation for 10-15 minutes after finishing; cooling, adjusting pH to 7.0, placing the enzymolysis liquid in a multifunctional microwave synthesis extractor, and treating for 8min under 506W power; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethanol of the volume of the concentrated residual liquid, precipitating for 12 hours at 4 ℃, and freeze-drying the precipitate to obtain arrowhead non-starch polysaccharide.
Polysaccharide yield (%) =m/m×100, wherein: m is the mass (g) of the arrowhead non-starch crude polysaccharide after freeze drying; m is the mass (g) of arrowhead defatted powder;
preferably, the enzyme activity of the alpha-amylase is 4U/mg to 100U/mg, and more preferably 40U/mg;
preferably, the enzyme activity of the cellulase is 10U/mg to 400U/mg, and more preferably 10U/mg;
preferably, the mass ratio of the alpha-amylase to the cellulase is 7:3, the enzyme activity of the alpha-amylase is 40U/mg, and the enzyme activity of the cellulase is 10U/mg. Under the condition, the arrowhead non-starch polysaccharide yield is 76.5+/-2.90%, the starch residual quantity is 4.91+/-0.07%, and the method is remarkably superior to the traditional hot water extraction method (polysaccharide yield: 10.7+/-0.26%, starch residual quantity: 36.6+/-0.65%) and the technical scheme disclosed in patent CN 113801248B (polysaccharide yield: 68.1+/-4.22% and starch residual quantity: 6.02+/-0.25%).
According to a second aspect of the present invention, there is provided arrowhead non-starch polysaccharide, wherein the total sugar content in the arrowhead non-starch polysaccharide is 71.2+/-2.05%, the protein content is 15.6+/-1.87%, the uronic acid content is 2.18+/-0.27%, and the sulfate group content is 5.67+/-0.84%; the particle size of the arrowhead non-starch polysaccharide is 1138+/-43.6 nm, and the potential is-23.8+/-4.37 mV.
According to a third aspect of the present invention, the present invention provides the use of the arrowhead non-starch polysaccharide in the preparation of an antioxidant functional product; can be used for preparing oral liquid, capsule, tablet, etc.
Preferably, the oral liquid contains arrowhead non-starch polysaccharide, erythritol, potassium sorbate and water, wherein the arrowhead non-starch polysaccharide is obtained by the method.
Preferably, the capsules comprise arrowhead non-starch polysaccharide and starch obtained by the method of the invention.
Preferably, the tablets comprise arrowhead non-starch polysaccharide, polyvinylpyrrolidone, calcium hydrophosphate, povidone K30, magnesium stearate and aerosil, which are obtained by the method of the invention.
The invention has the following beneficial effects:
(1) the invention provides a complex enzyme preparation complex enzyme [ alpha-amylase (40U/mg): cellulase (10U/mg) =7:3 ] has remarkable advantages in the aspects of degrading converted starch and breaking wall and promoting release, and can greatly improve the yield of arrowhead non-starch polysaccharide and reduce the starch residue in polysaccharide.
(2) The arrowhead non-starch polysaccharide provided by the invention has remarkable antioxidant activity, is superior to arrowhead polysaccharide prepared by using a hot water extraction method and the technical scheme disclosed by patent CN 113801248B, and can be used for preparing products for treating antioxidant related diseases.
Drawings
FIG. 1. Influence of the mass ratio of alpha-amylase to cellulase on the yield of arrowhead polysaccharide and the starch residue;
FIG. 2. Influence of alpha-amylase enzyme activity on arrowhead polysaccharide yield and starch residue;
FIG. 3 is the effect of cellulase enzyme activity on arrowhead polysaccharide yield and starch residue;
FIG. 4 is an infrared spectrum of arrowhead non-starch polysaccharide;
FIG. 5 is a scanning electron microscope image of arrowhead non-starch polysaccharide;
FIG. 6. Sagittaria sagittifolia non-starch polysaccharide has a scavenging effect on hydroxyl radicals (control 1: sagittaria sagittifolia non-starch polysaccharide prepared by hot water extraction in comparative example scheme A; control 2: sagittaria sagittifolia non-starch polysaccharide prepared by the technical scheme disclosed in patent CN 113801248B in comparative example scheme B);
FIG. 7. Sagittaria Sagittifolia non-starch polysaccharide has a DPPH radical scavenging effect (control 1: sagittaria Sagittifolia non-starch polysaccharide prepared by hot water extraction in comparative example scheme A; control 2: sagittaria Sagittifolia non-starch polysaccharide prepared by the technical scheme disclosed in patent CN 113801248B in comparative example scheme B).
Detailed Description
The arrowhead is supplied by vegetable planting bases in areas of the vehicle workshops in Wu district of Suzhou, jiangsu province, and the variety is Suzhou Huang Cigu; alpha-amylase and cellulase with different enzyme activities are purchased from Shanghai Michlin Biochemical technology Co., ltd; the multifunctional microwave synthesis extractor is provided by Shanghai New instrument microwave chemical technology Co., ltd, and the model is Uwave-2000.
Example 1 Complex enzyme-assisted microwave extraction of Sagittaria Sagittifolia non-starch polysaccharide
The optimal parameters of the feed liquid ratio, the enzymolysis time, the microwave power, the microwave time and the like are taken by the method disclosed by the reference patent CN 113801248B so as to explore the influence of the mass ratio of alpha-amylase to cellulase and the enzyme activity on the yield of arrowhead non-starch polysaccharide and the starch residue. Cleaning fresh rhizoma Sagittariae Sagittifoliae, slicing, oven drying at 60deg.C to constant weight, pulverizing, sieving with 60 mesh sieve to obtain rhizoma Sagittariae Sagittifoliae powder; adding petroleum ether, degreasing for 12h under dark condition, and vacuum filtering to obtain arrowhead defatted powder; soaking arrowhead fat-removed powder in water for 4 hours according to the feed liquid ratio of 1:43g/mL, then regulating the pH value to about 5.0, adding 2wt% of complex enzyme (calculated by taking arrowhead fat-removed powder as a base number and composed of alpha-amylase and cellulase with different enzyme activities and different mass ratios), placing in a shaking table, incubating for 2 hours at 50-55 ℃, and placing the enzymolysis liquid in a boiling water bath to inactivate enzyme for 10-15 minutes after the completion; cooling, adjusting pH to 7.0, placing the enzymolysis liquid in a multifunctional microwave synthesis extractor, and treating for 8min under 506W power; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethyl alcohol of the concentrated solution, precipitating for 12 hours at 4 ℃, and freeze-drying the precipitate to obtain arrowhead non-starch polysaccharide.
Polysaccharide yield (%) =m/m×100, wherein: m is the mass (g) of the arrowhead non-starch crude polysaccharide after freeze drying; m is the mass (g) of the arrowhead defatted powder.
The polysaccharide content is determined by adopting a phenol-sulfuric acid method, and a glucose standard equation is as follows: y=12.479x+0.0082, r 2 =0.9984; the residual starch content is determined by adopting an iodine color development method, and the standard equation of the soluble starch is as follows: y=0.1774x+0.0114, r 2 ＝0.9992。
1.1 influence of the mass ratio of alpha-amylase to cellulase on the yield of arrowhead polysaccharide and the starch residue
The complex enzyme consists of alpha-amylase and cellulase, the enzyme activity of immobilized alpha-amylase is 40U/mg, the enzyme activity of cellulase is 10U/mg, and the influence of different mass ratios (1:4, 3:7, 2:3, 1:1, 3:2, 7:3 and 4:1) on polysaccharide yield and starch residue is examined, and the result is shown in figure 1.
As can be seen from fig. 1, in the case of α -amylase: the mass ratio of the cellulase is between 1:4 and 4:1, and the yield of the arrowhead non-starch polysaccharide is improved and reduced. The highest yield reaches 76.5+/-2.90% at 7:3; but when the mass ratio is continuously increased to 4:1, the yield is reduced to 62.5+/-3.98%. The starch residue showed a trend opposite to the polysaccharide yield, and was 4.91.+ -. 0.07% when the complex enzyme mass ratio (α -amylase: cellulase) reached 7:3.
In combination, in alpha-amylase: when the mass ratio of the cellulase is 7:3, the polysaccharide yield is highest, and the residual starch amount is lowest, so that the mass ratio of the cellulase to the starch is 7:3.
1.2 influence of alpha-amylase enzyme Activity on Sagittaria Sagittifolia polysaccharide yield and starch residue
Immobilization of alpha-amylase: the mass ratio of the cellulase is 7:3, the enzyme activity of the cellulose is 10U/mg, the influence of different alpha-amylase enzyme activities (4U/mg, 10U/mg, 40U/mg, 50U/mg and 100U/mg) on the polysaccharide yield and the starch residue is examined, and the result is shown in figure 2.
As shown in FIG. 2, in the enzyme activity range of 4U/mg to 40U/mg of alpha-amylase, the polysaccharide yield increased with the increase of the enzyme activity; the residual starch content decreases with increasing enzyme activity. Further improves the enzyme activity to 100U/mg, and the polysaccharide yield is 75.8+/-1.06 percent, which is close to 40U/mg (76.5+/-2.90 percent) and 50U/mg (75.8+/-2.34 percent). Therefore, from the viewpoint of cost reduction, 40U/mg of the enzyme activity was selected as the optimum enzyme activity of alpha-amylase.
1.3 influence of cellulase enzyme Activity on Sagittaria Sagittifolia polysaccharide yield and starch residue
Immobilization of alpha-amylase: the mass ratio of the cellulase is 7:3, the enzyme activity of the alpha-amylase is 40U/mg, the influence of different cellulase activities (10U/mg, 30U/mg, 50U/mg and 400U/mg) on the polysaccharide yield and the starch residue is examined, and the result is shown in figure 3.
As shown in FIG. 3, the enzyme activity of cellulase has little influence on the yield of arrowhead non-starch polysaccharide and the starch residue. At 10U/mg, the polysaccharide yield is 76.5+/-2.90%, and the starch residue is 4.91+/-0.07%; at 400U/mg, the polysaccharide yield was 75.8.+ -. 3.49% and the starch residue was 4.88.+ -. 0.21%. Therefore, from the viewpoint of cost reduction, 10U/mg of the enzyme activity was selected as the optimum enzyme activity of the cellulase.
In summary, the most technological conditions of the invention are: the complex enzyme preparation consisting of 40U/mg of alpha-amylase and 10U/mg of cellulase in a mass ratio of 7:3 can be used for efficiently extracting arrowhead non-starch polysaccharide, and the polysaccharide yield is highest (76.5+/-2.90%) and the starch residual quantity is lowest (4.91+/-0.07%) under the condition of the mass ratio and the enzyme activity, so that the economic cost is lowest.
Comparative example
Based on the example 1, in order to further prove that the complex enzyme preparation provided by the invention can efficiently extract arrowhead non-starch polysaccharide, the invention implements the following comparative test:
scheme a: hot water extraction method
Soaking arrowhead fat-removed powder in water for 4 hours according to a feed liquid ratio of 1:43g/mL, and then extracting for 2 hours at 80 ℃; cooling, centrifuging to obtain supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethanol, precipitating at 4deg.C for 12 hr, and lyophilizing the precipitate to obtain arrowhead non-starch polysaccharide; and calculating the yield of the polysaccharide and the starch residue in the polysaccharide.
The results show that: under the conditions of no enzyme and no microwave treatment, the yield of the arrowhead non-starch polysaccharide is 10.7+/-0.26 percent (which is 7.1 times lower than the technical scheme of the invention), and the starch residue is 36.6+/-0.65 percent (which is 7.5 times higher than the technical scheme of the invention).
Scheme B: technical proposal disclosed in patent CN 113801248B
Soaking arrowhead fat-removed powder in water for 4 hours according to the liquid-to-material ratio of 1:43g/mL, then adjusting the pH value to about 5.0, and adding complex enzyme [ alpha-amylase (50U/mg) accounting for 2.0wt% of the weight of the arrowhead fat-removed powder: cellulase (10U/mg) =1:1 ], placing in a shaking table, incubating for 2 hours at 50-55 ℃, and placing the enzymolysis liquid in a boiling water bath to inactivate enzymes for 10-15 min after finishing; cooling, adjusting pH to 7.0, placing the enzymolysis liquid in a multifunctional microwave synthesis extractor, and treating for 8min under 506W power; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethyl alcohol, precipitating at 4 ℃ for 12 hours, and freeze-drying the precipitate to obtain arrowhead non-starch polysaccharide; and calculating the yield of the polysaccharide and the starch residue in the polysaccharide.
The results show that: by utilizing the technical scheme disclosed by the patent CN 113801248B, the yield of the arrowhead non-starch polysaccharide is 68.1+/-4.22 percent (which is 1.1 times lower than the technical scheme of the invention), and the starch residue is 6.02+/-0.25 percent (which is 1.2 times higher than the technical scheme of the invention); the polysaccharide yield is higher than 36.33 +/-2.57 percent disclosed in the patent CN 113801248B, which is probably caused by different production places and varieties of arrowhead raw materials used by the invention.
Example 2 analysis and characterization of the Components of arrowhead non-starch polysaccharide
The total sugar content in the arrowhead non-starch polysaccharide prepared by the corresponding technical scheme of the optimal technological condition of the invention is 71.2+/-2.05% measured by a phenol-sulfuric acid method; the content of the protein is 15.6+/-1.87% measured by a Coomassie brilliant blue method; the content of uronic acid is 2.18+/-0.27% measured by m-hydroxybiphenyl method; the content of sulfuric acid radical is 5.67 plus or minus 0.84 percent by the turbidimetry of barium chloride gelatin.
Particle size and potential measurement: the arrowhead non-starch polysaccharide is prepared into a pure water solution with the concentration of 1mg/mL, and then the pure water solution is injected into a ZS90 Markov nanometer particle size potential analyzer to measure that the average particle size is 1138+/-43.6 nm and the average potential is-23.8+/-4.37 mV.
Infrared spectrometry (as shown in fig. 4): mixing 2mg of dried arrowhead non-starch polysaccharide with 100mg of dried potassium bromide, tabletting, and measuring in FTIR-650 infrared spectrometer with scanning range of 4000cm -1 ～400cm -1 . As shown in FIG. 4, 3400cm -1 The strong absorption peak at the position is caused by the stretching vibration of-OH; 2926cm -1 The absorption peak at the position is caused by C-H stretching or bending vibration; 1631cm -1 And 1403cm -1 Is a typical carbonyl absorption peak; 1033cm -1 The absorption peak at the site is a characteristic absorption peak of the pyran ring; 577cm -1 The absorption peak at this point confirms that the pyran ring is in the alpha-configuration.
The infrared spectrum analysis further proves that the arrowhead non-starch polysaccharide prepared by the technical scheme of the invention accords with the structural characteristics of the polysaccharide.
Scanning electron microscopy (as shown in fig. 5): analyzing the morphology of arrowhead non-starch polysaccharide by adopting a Hitachi S-4700 field emission scanning electron microscope, wherein the morphology is specifically as follows: the arrowhead non-starch polysaccharide is taken and stuck on an objective table of the double-sided conductive adhesive by using a cotton swab, and the non-stuck polysaccharide powder is blown off by using an ear washing ball. And (3) placing the sample in an ion sputtering instrument for foil spraying treatment, sequentially placing the treated sample under a scanning electron microscope, and observing the appearance of the sample after the instrument is set stable. As shown in fig. 5, under the magnification of x 2k, the surface of the arrowhead non-starch polysaccharide prepared by the technical scheme of the invention presents an irregular lamellar and fold morphology, and the surface is uneven and rough.
Example 3 antioxidant Activity of arrowhead non-starch polysaccharide
Scavenging hydroxyl radicals: respectively transferring 1mL (1 mg/mL-5 mg/mL) of arrowhead non-starch polysaccharide solution into three test tubes with a stopper, carrying out three parallel experiments, adding 1mL of each of a ferrous sulfate solution, a salicylic acid-ethanol solution and a hydrogen peroxide solution with the concentration of 6mmol/L into each tube, vibrating and fully and uniformly mixing, and reacting for 30min in a water bath kettle with the temperature of 37 ℃; after the reaction was completed, the absorbance was measured at a wavelength of 510nm by zeroing with distilled water. The arrowhead non-starch polysaccharide prepared by the hot water extraction method in the comparative example scheme A is used as a control 1; the arrowhead non-starch polysaccharide prepared in comparative example B using the technical scheme disclosed in patent CN 113801248B was control 2. The clearance was calculated according to the following formula:
hydroxyl radical scavenging rate= [1- (a) 1 -A 2 )/A 0 ]×100％
Wherein A is 1 Refers to the absorbance of the sample, A 2 Refers to the absorbance value of a control group using distilled water to replace hydrogen peroxide solution, A 0 Refers to the absorbance of a blank group in which distilled water was used instead of the sample.
As shown in FIG. 6, in the concentration range of 1 mg/mL-5 mg/mL, the hydroxyl radical scavenging activity of all samples increased with increasing concentration, and at 5mg/mL, the arrowhead non-starch polysaccharide reached 96.1.+ -. 2.06%, control 1 was 62.8.+ -. 3.48%, and control 2 was 75.2.+ -. 2.85%.
Scavenging DPPH free radicals: respectively transferring 2mL (0.2 mg/mL-1.0 mg/mL) of arrowhead non-starch polysaccharide solution into three test tubes with a support plug, carrying out three parallel experiments, adding 0.1mmol/L DPPH solution into each tube, fully and uniformly mixing, and carrying out light-shielding reaction for 30min; after the reaction, the absorbance at 517nm was measured by zeroing with absolute ethanol. The arrowhead non-starch polysaccharide prepared by the hot water extraction method in the comparative example scheme A is used as a control 1; the arrowhead non-starch polysaccharide prepared in comparative example B using the technical scheme disclosed in patent CN 113801248B was control 2. The clearance was calculated according to the following formula:
DPPH clearance= [1- (a) 1 -A 2 )/A 0 ]×100％
Wherein A is 1 Refers to the absorbance of the sample, A 2 Refers to the absorbance value of a control group with absolute ethyl alcohol instead of DPPH solution, A 0 Refers to the absorbance of a blank group in which distilled water was used instead of the sample.
As shown in FIG. 7, DPPH radical scavenging activity of all samples increased with increasing concentration in the range of 0.2mg/mL to 1mg/mL, and at 1mg/mL, arrowhead non-starch polysaccharide reached 95.2.+ -. 4.26%, control 1 was 61.9.+ -. 4.38%, and control 2 was 74.9.+ -. 3.97%.
In conclusion, the arrowhead non-starch polysaccharide prepared by the technical scheme of the invention has high polysaccharide yield, low starch residue and higher antioxidant activity.
Example 4 preparation of arrowhead non-starch polysaccharide oral liquid
Adding 2.0g of arrowhead non-starch polysaccharide into 100mL of purified water, stirring at room temperature until the arrowhead non-starch polysaccharide is dissolved, adding 0.5g of erythritol and 0.2g of potassium sorbate, uniformly stirring, canning, performing instant sterilization, bottling, and sealing to obtain the arrowhead non-starch polysaccharide oral liquid.
Example 5 preparation of arrowhead non-starch polysaccharide Capsule
Taking 5.0g of arrowhead non-starch polysaccharide which is sieved by a 40-mesh sieve, spraying 90% ethanol according to the proportion of 1:1, uniformly mixing, adding 10% starch to prepare a soft material, sieving by a 20-mesh sieve, granulating, drying in a 60-DEG C oven for 1h, sieving by a 20-mesh sieve, granulating again, and filling in a No. 3 empty capsule under the environment of the relative humidity of less than 65%, thus obtaining the arrowhead non-starch polysaccharide capsule.
Example 6 preparation of arrowhead non-starch polysaccharide tablets
Mixing 5.0g of arrowhead non-starch polysaccharide with 300mg of polyvinylpyrrolidone and 4.0mg of calcium hydrophosphate, grinding and sieving with a 100-mesh sieve; adding 95% ethanol solution of 5% povidone K30 while stirring to prepare soft material, and wet granulating (sieving with 16 mesh sieve); drying at 60deg.C, sieving, granulating, adding 1% magnesium stearate and 2% silica gel micropowder, mixing, and tabletting.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A method for preparing arrowhead non-starch polysaccharide by using compound enzyme preparation takes arrowhead fat-removed powder as raw material, firstly adopts compound enzyme preparation for enzymolysis, and after enzyme deactivation, the arrowhead fat-removed powder is placed in a multifunctional microwave synthesis extraction instrument for microwave extraction treatment; centrifuging, concentrating supernatant, precipitating with ethanol, and lyophilizing to obtain rhizoma Sagittariae Sagittifoliae non-starch polysaccharide; the complex enzyme preparation consists of alpha-amylase and cellulase, wherein the mass ratio of the alpha-amylase is as follows: cellulase = 3:2-7:3.
2. The method of claim 1, wherein the complex enzyme preparation is added in an amount of 2wt% of arrowhead fat-removed powder.
3. The method of claim 1, wherein the complex enzyme formulation comprises an alpha-amylase: cellulase = 7:3.
4. The method according to claim 1, wherein the alpha-amylase enzyme activity is 4U/mg to 100U/mg, preferably 40U/mg.
5. The method according to claim 1, wherein the cellulase enzyme activity is 10U/mg to 400U/mg, preferably 10U/mg.
6. The method according to any one of claims 1-5, comprising in particular the steps of:
cleaning fresh rhizoma Sagittariae Sagittifoliae, slicing, oven drying at 60deg.C to constant weight, pulverizing, sieving with 60 mesh sieve to obtain rhizoma Sagittariae Sagittifoliae powder; adding petroleum ether, degreasing for 12h under dark condition, and vacuum filtering to obtain arrowhead defatted powder; soaking arrowhead degreasing powder in water for 4 hours according to a feed liquid ratio of 1:43g/mL, then adjusting the pH value to 5.0, adding compound enzyme accounting for 2% of the weight of the arrowhead degreasing powder, placing the compound enzyme into a shaking table, incubating for 2 hours at 50-55 ℃, and placing the enzymolysis liquid into a boiling water bath to inactivate enzyme for 10-15 minutes after the completion; cooling, adjusting pH to 7.0, placing the enzymolysis liquid in a multifunctional microwave synthesis extractor, and treating for 8min under 506W power; centrifuging after the completion of the process, collecting supernatant, concentrating the supernatant to 1/4 of the original volume, adding 3 times of absolute ethanol of the volume of the concentrated residual liquid, precipitating for 12 hours at 4 ℃, and freeze-drying the precipitate to obtain arrowhead non-starch polysaccharide.
7. The method of claim 6, wherein the mass ratio of alpha-amylase to cellulase is 7:3, the alpha-amylase enzyme activity is 40U/mg, and the cellulase enzyme activity is 10U/mg; the total sugar content in the obtained arrowhead non-starch polysaccharide is 71.2+/-2.05%, the protein content is 15.6+/-1.87%, the uronic acid content is 2.18+/-0.27%, and the sulfate group content is 5.67+/-0.84%; the particle size of the arrowhead non-starch polysaccharide is 1138+/-43.6 nm, and the potential is-23.8+/-4.37 mV.
8. Use of arrowhead non-starch polysaccharide prepared by the method of claim 7 for preparing an antioxidant functional product.
9. The use according to claim 8, wherein the arrowhead non-starch polysaccharide can be used for preparing oral liquid, capsules or tablets.
10. The method according to claim 9, wherein the oral liquid comprises arrowhead non-starch polysaccharide, erythritol, potassium sorbate and water;
the capsule comprises arrowhead non-starch polysaccharide and starch;
the tablet comprises arrowhead non-starch polysaccharide, polyvinylpyrrolidone, calcium hydrophosphate, povidone K30, magnesium stearate and micro-powder silica gel.
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