CN114150034A - Method for preparing asparagus active polypeptide by compound enzymolysis - Google Patents
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Abstract
The invention relates to the technical field of food preparation, in particular to a method for preparing asparagus active polypeptide by compound enzymolysis, which comprises the steps of raw material treatment, sieving, homogenization, enzymolysis, centrifugation, secondary enzymolysis, ultrafiltration, freeze-drying and the like. The method fully releases the protein wrapped in the asparagus by the technologies of enzymolysis-assisted mechanical pulping, high-speed dispersion, high-pressure homogenization, complex enzyme enzymolysis, ultrafiltration, freeze drying and the like, can be well combined with the protease to hydrolyze the protein, does not generate high temperature in the whole process, does not destroy the activity of the protein and the polypeptide, obtains components with different molecular weights, particularly low molecular weights, has better activity, enables the protein to be dissolved out as much as possible to react with the protease, and obtains an active polypeptide product with better solubility.
Description
Technical Field
The invention relates to the technical field of food preparation, in particular to a method for preparing asparagus active polypeptide by compound enzymolysis.
Background
Asparagus also called asparagus and asparagus is a perennial herb which is a vegetable with both food and medicine. The asparagus has a history of cultivation for more than two thousand years, is native to the coast of the Mediterranean sea and the Xiao-ya-Min-Yi, is transferred from Europe to China in the 20 th century, and is cultivated in vegetable production areas all over the country. The asparagus is a health-care vegetable with excellent quality and rich nutrition, wherein the content of various active ingredients and trace elements is higher than that of common vegetables, and the content of active ingredients such as high-quality protein rutin, quercetin, mannan, steroid saponin, polysaccharide, folic acid and the like is also higher. Researches show that asparagus has the effects of resisting tumors, reducing blood fat, resisting aging, resisting fatigue, resisting mutation, resisting bacteria and preventing and resisting cancers.
In recent years, the asparagus planting and processing are rapidly developed, and with the improvement of living standard and the enhancement of health consciousness of people, the development of functional products by using asparagus raw materials becomes a good direction. Therefore, the method for preparing the asparagus active polypeptide by compound enzymolysis has a good application prospect.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for preparing asparagus active polypeptide by composite enzymolysis.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing asparagus active polypeptide by compound enzymolysis comprises the following steps:
(1) raw material treatment: cleaning fresh Germinatus Phragmitis, adding pectase 0.05-0.15 ‰ of weight of Germinatus Phragmitis, and pulping with wall breaking machine; the asparagus contains a large amount of pectin, and a small amount of compound pectinase is added during pulping, so that the viscosity of materials can be obviously reduced, the pulping is facilitated, the nutritional ingredients and the like wrapped by the pectin are dissolved out, and the subsequent operation is facilitated;
(2) sieving: filtering the asparagus pulp by using a screen to remove large blocks and residues; the material is more uniform, a small amount of fibrous materials can be removed, and equipment damage caused by subsequent dispersing and homogenizing processes is avoided;
(3) homogenizing: firstly, a high-speed disperser is used, the high-speed dispersion is carried out for 10-30min at the set rotating speed of 3000-; homogenizing at 20-40MPa with a high-pressure homogenizer to further reduce and disperse the material particles; the high-speed disperser can play a certain role in crushing, dispersing and homogenizing; homogenizing and emulsifying by using a high-pressure homogenizer to refine particles of the material, so that insoluble component particles are smaller, more components can contact with water molecules, dissolution of nutrient substances is promoted, and subsequent enzymolysis treatment is facilitated;
(4) enzymolysis: slowly heating the materials to 35-45 deg.C, and adding pectase, cellulase and hemicellulase with a volume of 0.2-0.6 ‰ of the feed liquid respectively; placing in an ultrasonic cleaning machine, and performing heat preservation treatment at 40 ℃ for 2h under 20-40W; wherein, the machine is stopped for 10min every 30 min; ultrasonic-assisted enzymolysis is used, so that under the ultrasonic condition, the acting sites of materials and enzyme molecules are fully contacted, the exposure of components such as cellulose, hemicellulose and the like is promoted, and protein molecules are better dissolved out and act;
(5) centrifuging: rapidly cooling the material after enzymolysis to 8-12 ℃, and centrifuging for 10min at 4 ℃ and 3000-; the low temperature condition can protect active components from loss, and centrifugation is used to remove insoluble macromolecular components and retain soluble substances, especially soluble protein mass energy components.
(6) Secondary enzymolysis: taking supernatant, adjusting pH to 7.5-8.5 with sodium bicarbonate, adding appropriate amount of papain and alkaline protease according to the volume of supernatant, stirring at 35-45 deg.C, and performing enzymolysis for 1.5-3 hr; the pH value is adjusted to better exert the activity of the protease, and the low-activity protein macromolecules in the asparagus are degraded into high-activity polypeptide micromolecules under the action of a plurality of composite proteases, so that the action of active ingredients can be better exerted.
(7) And (3) ultrafiltration: respectively centrifuging the clear liquid after enzymolysis at 4000r/min by using 30kDa, 10kDa and 3000Da ultrafiltration centrifugal tubes at 4 ℃ for 10-30min, and respectively obtaining polypeptide components below 3000Da, 3000Da-10kDa, 10kDa-30kDa and above 30kDa according to molecular weight; the polypeptide small molecule component has better activity, and the polypeptide small molecules are separated by a physical method, so that the polypeptides with different molecular weights can be separated, and the activity of the polypeptides can be better tested.
(8) Freeze-drying: respectively placing the collected components in a freeze dryer, freeze-drying until the moisture content is below 5%, and taking out to obtain asparagus active polypeptide; the freeze drying is adopted, the drying temperature is below the freezing point of the material, the drying purpose is achieved through sublimation under the vacuum condition, and the activity of active ingredients such as polypeptide in the asparagus is ensured.
Further, the method for preparing the asparagus active polypeptide by the compound enzymolysis also comprises a finished product preparation process, namely, the asparagus active polypeptide obtained in the step (8) is crushed, screened and packaged to obtain an asparagus active polypeptide finished product.
Further, in the step (1), 0.05-0.15 per mill of pectinase is added in the enzymolysis process, pulping is carried out for 2-5min, standing is carried out for 10-15min at normal temperature, and then pulping is carried out for 1-3min again.
Further, after the pH value is adjusted to 8.0 by using baking soda in the step (6), 0.05-0.30 per mill of papain and 0.01-0.10 per mill of alkaline protease are added according to the volume of the supernatant, the temperature is kept at 35-45 ℃, and the enzymolysis is carried out for 1.5-3 h.
Further, the water used in the method is purified water, and the enzyme preparation is food-grade.
The invention has the technical effects that:
compared with the prior art, the method for preparing the asparagus active polypeptide by compound enzymolysis has the following advantages:
(1) the active peptides are mostly small molecular peptides, and consist of 2-20 amino acids, and some active peptides are sensitive to temperature and are not suitable for heat treatment of intermediate links;
(2) the invention obtains higher protein hydrolysis rate and polypeptide yield by combining the plant source papain and the microorganism source alkaline protease; the polypeptides with different molecular weights can be separated by an ultrafiltration membrane to obtain polypeptide components with different molecular weights, in particular active peptides with the molecular weight lower than 3000 kDa;
(3) the method fully releases the protein wrapped in the asparagus by the technologies of enzymolysis-assisted mechanical pulping, high-speed dispersion, high-pressure homogenization, complex enzyme enzymolysis, ultrafiltration, freeze drying and the like, can be well combined with the protease to hydrolyze the protein, does not generate high temperature in the whole process, does not destroy the activity of the protein and the polypeptide, obtains components with different molecular weights, particularly low molecular weights, has better activity, enables the protein to be dissolved out as much as possible to react with the protease, and obtains an active polypeptide product with better solubility.
The invention uses an Oxford cup method to verify the bacteriostatic ability of the active peptide, and the result proves that the components with the molecular weight of less than 3000kDa have more obvious bacteriostatic effect on the euspora bisporus and have no inhibitory effect on the probiotic streptococcus thermophilus.
Drawings
FIG. 1 is a graph showing the effect of the asparagus active polypeptide of the invention on the alternaria bisporus;
FIG. 2 is a graph showing the effect of asparagus active polypeptides of the present invention on Streptococcus thermophilus.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings of the specification.
Example 1:
the method for preparing the asparagus active polypeptide by compound enzymolysis comprises the following steps:
(1) raw material treatment: cleaning fresh asparagus, adding pectinase with weight of 0.05 per mill of the weight of the asparagus, and pulping by using a wall breaking machine;
(2) sieving: filtering the asparagus pulp by using a 20-mesh screen, and removing large-block residues;
(3) homogenizing: a high-speed disperser is used, the rotation speed is set to be 4000rpm, and high-speed dispersion is carried out for 15min, and a certain homogenization effect is achieved; homogenizing at 30MPa with a high-pressure homogenizer to further reduce and disperse the material particles;
(4) enzymolysis: slowly heating the materials to 40 ℃, and respectively adding 0.5 per mill of pectinase, 0.3 per mill of cellulase and 0.3 per mill of hemicellulase into the materials; placing the mixture in an ultrasonic cleaning machine, and carrying out heat preservation treatment for 2 hours at the temperature of 40 ℃ under the condition of 30W; wherein, the machine is stopped for 10min every 30 min;
(5) centrifuging: rapidly cooling the material after enzymolysis to 10 ℃, centrifuging for 10min at 4 ℃ at 4000r/min by using a centrifuge, and removing residues;
(6) secondary enzymolysis: taking supernatant, adjusting pH to 8.0 with sodium bicarbonate, adding papain 0.15 ‰ and alkaline protease 0.08 ‰ according to volume, stirring at 40 deg.C, and performing enzymolysis for 2 hr;
(7) and (3) ultrafiltration: centrifuging the clear liquid after enzymolysis at 4000r/min respectively by using 30kDa, 10kDa and 3000Da ultrafiltration centrifugal tubes at 4 ℃ for 10-30min to obtain polypeptide components below 3000Da, 3000Da-10kDa, 10kDa-30kDa and above 30kDa respectively;
(8) freeze-drying: respectively placing the collected components in a freeze dryer, freeze-drying until the moisture content is below 5%, and taking out to obtain asparagus active polypeptide; pulverizing, sieving, and packaging to obtain final product of Germinatus Phragmitis active polypeptide.
The invention uses an Oxford cup method to verify the bacteriostatic ability of asparagus active peptide on the two-spore soil-shaped yeast and the streptococcus thermophilus, and the result is shown in figure 1 and figure 2, wherein in figure 1, 1 represents a component of 10kDa-30 kDa; 2 represents a 3000Da-10kDa fraction; 3 represents a fraction below 3000 Da. In FIG. 2, 1 represents a 10kDa-30kDa component; 2 represents a 3000Da-10kDa fraction; 3 represents a fraction below 3000 Da. As shown in the figure, the components with the molecular weight of less than 3000kDa have more obvious bacteriostatic effect on the euspora bisporus and have no inhibitory effect on the probiotic streptococcus thermophilus.
According to the invention, through technologies such as enzymolysis-assisted mechanical pulping, high-speed dispersion, high-pressure homogenization, complex enzyme enzymolysis, ultrafiltration, freeze drying and the like, the protein wrapped in asparagus is fully released, can be better combined with protease to carry out hydrolysis of the protein, the activity of the protein and polypeptide is not destroyed without high temperature in the whole process, the protein is dissolved out as much as possible to react with the protease, and an active polypeptide product with harmful bacteria inhibition and good solubility is obtained.
The above embodiments are only specific examples of the present invention, and the scope of the present invention includes but is not limited to the above embodiments, and any suitable changes or modifications by those of ordinary skill in the art, which are consistent with the claims of the present invention, shall fall within the scope of the present invention.
Claims (7)
1. A method for preparing asparagus active polypeptide by compound enzymolysis is characterized in that: the method comprises the following steps:
(1) raw material treatment: cleaning fresh Germinatus Phragmitis, adding pectase 0.05-0.15 ‰ of weight of Germinatus Phragmitis, and pulping with wall breaking machine;
(2) sieving: filtering the asparagus pulp using a screen;
(3) homogenizing: a high-speed disperser is used for dispersing at high speed and has a homogenizing effect; homogenizing once by using a high-pressure homogenizer to further reduce and disperse the material particles;
(4) enzymolysis: slowly heating the materials to 35-45 deg.C, and adding pectase, cellulase and hemicellulase with a volume of 0.2-0.6 ‰ of the feed liquid respectively; placing in an ultrasonic cleaning machine for heat preservation treatment, wherein stopping the machine for 10min every 30 min;
(5) centrifuging: rapidly cooling the material after enzymolysis to 8-12 ℃, and centrifuging for 10min at 4 ℃ and 3000-;
(6) secondary enzymolysis: taking supernatant, adjusting pH to 7.5-8.5 with sodium bicarbonate, adding papain and alkaline protease according to the volume of supernatant, stirring at 35-45 deg.C, and performing enzymolysis for 1.5-3 hr;
(7) and (3) ultrafiltration: centrifuging the clear solution after enzymolysis by an ultrafiltration centrifugal tube to obtain polypeptide components with different molecular weights;
(8) freeze-drying: and respectively placing the collected components in a freeze dryer, freeze-drying until the moisture content is below 5%, and taking out to obtain the asparagus active polypeptide.
2. The method for preparing asparagus active polypeptide by compound enzymolysis according to claim 1, which is characterized in that: and (3) a finished product preparation process, namely, the asparagus active polypeptide obtained in the step (8) is crushed, screened and packaged to obtain an asparagus active polypeptide finished product.
3. The method for preparing asparagus active polypeptide by compound enzymolysis according to claim 1, which is characterized in that: in the step (1), 0.05-0.15 per mill of pectinase is added in the enzymolysis process, pulping is carried out for 2-5min, standing is carried out for 10-15min at normal temperature, and then pulping is carried out for 1-3min again.
4. The method for preparing asparagus active polypeptide by compound enzymolysis according to claim 1, which is characterized in that: in the step (3), a high-speed disperser is used, the set rotation speed of 3000-.
5. The method for preparing asparagus active polypeptide by compound enzymolysis according to claim 1, which is characterized in that: in the step (4), the ultrasonic cleaning machine is subjected to heat preservation treatment: ultrasonic power is 20-40W for 2 h.
6. The method for preparing asparagus active polypeptide by compound enzymolysis according to claim 1, which is characterized in that: in the step (6), 0.05-0.30 per mill of papain and 0.01-0.10 per mill of alkaline protease are added according to the volume of the supernatant.
7. The method for preparing asparagus active polypeptide by compound enzymolysis according to claim 1, which is characterized in that: in the step (7), the clear liquid after enzymolysis is respectively centrifuged for 10-30min at 4000r/min by using 30kDa, 10kDa and 3000Da ultrafiltration centrifugal tubes respectively at 4 ℃; the four polypeptide components with molecular weight below 3000Da, 3000Da-10kDa, 10kDa-30kDa and above 30kDa are obtained respectively.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115104730A (en) * | 2022-06-28 | 2022-09-27 | 北京姿美堂生物技术股份有限公司 | Composition for improving sleep, sparrow leucomelas-asparagus sleep improvement liquid and tea powder preparation method and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115104730A (en) * | 2022-06-28 | 2022-09-27 | 北京姿美堂生物技术股份有限公司 | Composition for improving sleep, sparrow leucomelas-asparagus sleep improvement liquid and tea powder preparation method and application |
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