CN113999885A - Preparation method of peony stamen protein powder - Google Patents
Preparation method of peony stamen protein powder Download PDFInfo
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- CN113999885A CN113999885A CN202111459730.4A CN202111459730A CN113999885A CN 113999885 A CN113999885 A CN 113999885A CN 202111459730 A CN202111459730 A CN 202111459730A CN 113999885 A CN113999885 A CN 113999885A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
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- General Chemical & Material Sciences (AREA)
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- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
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Abstract
The invention relates to a preparation method of peony stamen protein powder, and belongs to the technical field of food processing. The preparation method of the peony stamen protein powder comprises the following steps: and (3) performing protease enzymolysis treatment on the extracted peony stamen protein, performing glycosylation treatment on an enzymolysis product, and drying to obtain the peony stamen protein. According to the preparation method of the peony stamen protein powder, the peony stamen protein is modified by adopting a method combining enzymolysis treatment and glycosylation reaction, so that the solubility and the emulsibility of the peony stamen protein powder can be improved, no chemical reagent is added, and the preparation method is green, safe, simple to operate and easy for large-scale food processing production.
Description
Technical Field
The invention relates to a preparation method of peony stamen protein powder, and particularly belongs to the technical field of food processing.
Background
The fresh peony flower is bright in color and luster, is quite reputable as 'king in flower', has a habit of eating the peony since ancient times in China, has formed various local characteristic names to eat, and is listed as a new resource food raw material by the national ministry of health in 2013 and developed and utilized by numerous researchers. The peony pistil is the essence of peony, has high carbohydrate and protein content and relatively healthy fatty acid composition, can reduce the blood pressure of a human body, has a relatively good antioxidant effect, and has an important effect on the regulation of the physiological metabolism and the dietary structure of the human body. The peony pistil is used as a plant seed raw material with homology of medicine and food, the protein content accounts for 23.74 percent of dry weight, the amino acid composition is reasonable, the essential amino acid accounts for 38.26 percent, and the peony pistil is a high-quality protein resource. The solubility and the emulsibility of the protein are very important functional properties, and can be used as an emulsifier in food processing, such as a quality improver in bread and cake foods, so as to prevent dough from aging and retrogradation, promote the emulsification and dispersion of the shortening, and improve the texture and the mouthfeel; the added grease is emulsified and dispersed in foods such as minced fish meat, sausages and the like, so that the homogeneity of tissues is improved, and the commodity property and the storage property are improved; the emulsifier is added into milk beverage, so that the grease in the beverage can be emulsified to form a stable emulsifying system, the beverage is prevented from being layered, and the good taste and sensory properties of the beverage are ensured.
Solubility is an important index affecting the functionality of proteins, and the emulsifying properties of proteins need to be dissolved to be expressed, so the solubility and the emulsifying properties of proteins are closely related. The protein emulsifier is a surfactant containing hydrophilic and lipophilic groups, and can make the two phases of water and oil be tightly fused together. In the food industry processing, the protein emulsifier can improve the sensory properties of food, prolong the shelf life of the food, improve the product quality, facilitate the processing and the preservation of the food and be beneficial to developing novel food. However, in the prior art, a method for simultaneously improving the solubility and the emulsibility of the peony stamen protein does not exist, and the application range of the peony stamen cannot be expanded.
Disclosure of Invention
The invention aims to provide a preparation method of peony stamen protein powder, which can improve the solubility and emulsibility of peony stamen protein at the same time.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of peony stamen protein powder comprises the following steps: and (3) performing protease enzymolysis treatment on the extracted peony stamen protein, performing glycosylation treatment on an enzymolysis product, and drying to obtain the peony stamen protein.
According to the preparation method of the peony stamen protein powder, the peony stamen protein is modified by adopting a method combining enzymolysis treatment and glycosylation reaction, so that the solubility and the emulsibility of the peony stamen protein powder can be improved, and the method is high in safety, simple to operate and easy for large-scale food processing production.
Preferably, the enzymatic treatment is carried out under ultrasonic conditions in order to promote the enzymatic reaction. The power of the ultrasonic wave is 100W-300W. The temperature of ultrasonic treatment is 30-50 ℃. The ultrasonic treatment time is 20-40 min. The ultrasonic wave can improve the functional characteristics of the protein, the cavitation generated by the ultrasonic wave can change the molecular structure of the protein, destroy the internal bonds of the protein, expose the hydrophobic groups in the protein, accelerate the processes of proteolysis and glycosylation reaction, and effectively improve the solubility and the emulsibility of the protein.
Further, the enzymatic treatment comprises the following steps: mixing the water dispersion of the peony stamen protein with protease to carry out enzymolysis reaction. The preparation method of the peony stamen protein water dispersion comprises the following steps: mixing the peony stamen protein and water according to the proportion of 1 (6-10) (g/mL).
Preferably, to reduce costs, the protease is papain. The papain is a protease capable of decomposing proteins in acidic, neutral and alkaline environments, is a sulfhydryl (-SH) -containing endopeptidase, has the activities of protease and esterase, has wider specificity, and has stronger hydrolytic capability on animal and plant proteins, polypeptides, esters, amides and the like.
Preferably, the temperature of the enzymolysis reaction of the enzymolysis treatment is 50-65 ℃. The pH value of the enzymolysis reaction is controlled within the range of 6.0-7.0. The time of the enzymolysis reaction of the enzymolysis treatment is 60 min-120 min. The dosage of the papain is 5-15 wt% of the peony stamen protein. By reasonably adjusting and optimizing the concentration, temperature, pH and time conditions of enzymolysis, the enzymolysis efficiency is improved, and the dissolution of protein is further promoted. In order to prevent the extracted protein from being degraded, the enzymatic treatment further comprises the steps of: and (4) carrying out enzyme deactivation treatment on the system after enzymolysis. The temperature of enzyme deactivation treatment is 90-100 ℃. The time of enzyme deactivation is 15 min-25 min.
Preferably, the enzymatic treatment further comprises the steps of: and (4) carrying out centrifugal treatment on the enzyme-inactivated system, and collecting supernatant to obtain an enzymolysis product. The rotation speed of the centrifugation after enzyme deactivation is 4000r/min to 8000r/min, and the centrifugation time is 10min to 20 min.
The glycosylation method is a chemical reaction for combining carbohydrate with amino or carboxyl on protein molecules by covalent bonds, is a process for generating glycoprotein by Maillard reaction without adding any chemical reagent, and is a natural and high-safety protein modification method.
Preferably, the glycosylation reagent for glycosylation treatment is mannooligosaccharides. The mannanoligosaccharide is a novel antigen active substance extracted from the cultured cell wall of yeast. The polysaccharide has good physicochemical properties of low calorie, stability, safety, no toxicity and the like, has the effects of protecting intestinal tracts, improving immunity and the like, and can improve the solubility and the emulsibility of glycosylation products. The manna oligosaccharide is used as a multiplication factor of bifidobacterium, can effectively lead beneficial bacteria in vivo to naturally multiply, improve the structure of intestinal flora and regulate the functions of the intestinal tract, so the manna oligosaccharide has the physiological activities of protecting the liver, resisting tumors, enhancing the immunity, strengthening the intestinal tract peristalsis, reducing cholesterol, resisting aging and the like, is suitable for various intestinal diseases, is also suitable for the daily health care of healthy people, particularly the old and children, has sweet taste and can be used as a substitute of cane sugar. Moreover, the price is low, and the method is suitable for mass production in factories.
The glycosylation process comprises the following steps: mixing an enzymolysis product obtained after enzymolysis with the mannooligosaccharide, performing glycosylation reaction, centrifuging the system after the reaction is finished, and collecting supernatant. The rotating speed of the centrifugation after glycosylation treatment is 4000r/min to 8000r/min, and the centrifugation time is 10min to 20 min.
Preferably, the temperature of the glycosylation reaction of the glycosylation treatment is 60 ℃ to 90 ℃. The pH value of the glycosylation reaction is controlled within the range of 6.0-7.0.
Preferably, the glycosylation process is performed under closed conditions in order to prevent the entry of dust and foreign substances.
Preferably, the glycosylation reaction time of the glycosylation treatment is 90min to 180 min. The dosage of the manna oligosaccharide is 20-30 wt% of the peony stamen protein.
The peony stamen protein is prepared by an alkali extraction and acid precipitation method. The alkali extraction and acid precipitation method has the advantages of high protein extraction rate and purity, easy operation, low cost and the like.
In order to further promote the smooth proceeding of the enzymolysis, the alkali extraction and acid precipitation method comprises the following steps: mixing the degreased peony pistil powder with water, adjusting the pH value to 9.0-9.5, carrying out alkali extraction treatment to obtain an extracting solution, and adjusting the pH value of the extracting solution to 3.5-4.0 by using acid to carry out acid precipitation treatment. Too high pH of the extract can degrade and denature the protein, which is not conducive to protein extraction.
The alkali extraction treatment is carried out by adjusting the pH by adding an alkali as is conventional in the art. The alkali used for the alkali extraction treatment is preferably sodium hydroxide solution. Further preferably, the concentration of the sodium hydroxide solution is 0.1mol/L to 0.5 mol/L.
Preferably, the temperature of the alkali extraction treatment is 30-50 ℃, and the time of the alkali extraction treatment is 1-3 h.
Further preferably, the alkali extraction and acid precipitation method further comprises the following steps: and (4) centrifuging the extracting solution after the alkali extraction treatment, and collecting supernatant. The rotation speed of the centrifugation after the alkali extraction treatment is 4000r/min to 8000r/min, and the centrifugation time is 10min to 20 min.
The acid precipitation treatment employs acid addition to adjust the pH as is conventional in the art. In order to improve the nutritional value of the peony stamen protein, the acid adopted in the acid precipitation treatment is preferably citric acid. Citric acid is edible acid, can enhance normal metabolism in vivo, and has no harm to human body at proper dosage; citric acid is added to certain foods to provide a good mouthfeel and appetite stimulation, and in china, citric acid is used in jams, beverages, cans and candies; citric acid is used as a pH regulator, so that the seasoning effect can be achieved, and the quality of the seasoning can be maintained; the citric acid has chelating effect and pH value regulating effect, and can increase antioxidant, inhibit enzyme activity and prolong food shelf life. Preferably, the acid precipitation treatment adjusts the pH using methods of adding acid solutions that are conventional in the art. Further preferably, the concentration of the citric acid solution is 0.5mol/L to 1 mol/L.
Preferably, the time of the acid precipitation treatment is 60min to 90 min.
Further preferably, the acid precipitation treatment further comprises the following steps: and centrifuging the extracting solution after the acid precipitation treatment, wherein the rotating speed of the centrifugation after the acid precipitation treatment is 4000 r/min-8000 r/min, and the centrifugation time is 10 min-20 min.
In order to further promote the dissolution of the peony stamen protein, the degreased peony stamen powder is mixed with water according to the proportion of 1 (20-30) (g/mL).
Preferably, the preparation method of the defatted peony pistil powder comprises the following steps: mixing the peony pistil powder and petroleum ether according to the proportion of 1 (5-8) (g/mL), and drying to obtain the peony pistil powder. The petroleum ether can remove lipid substances in the peony pistils and promote the extraction of peony pistil proteins by subsequent operations. Preferably, the drying manner is natural air drying.
Preferably, the fineness of the peony pistil powder is 80-100 meshes. Further, the preparation method of the peony pistil powder comprises the following steps: pulverizing peony pistil and sieving to obtain the final product. When the peony pistil powder particles are 80-100 meshes fine, nutrient components can be basically dissolved out, the peony pistil powder particles are crushed too coarse and are not used for dissolving out protein, so that the extraction rate is reduced, and the peony pistil powder particles are crushed too fine, so that the impurity content in an extracting solution is increased. Preferably, the pulverization manner is mechanical pulverization.
Further preferably, the peony pistil for preparing the peony pistil powder is fresh and retains pollen substances on the peony pistil.
Preferably, the drying mode after the glycosylation treatment is vacuum freeze drying. Because the vacuum freeze drying is carried out in a low-temperature and low-oxygen environment, most biological reactions are stagnated, no liquid water exists in the treatment process, and the water is directly sublimated in a solid state, so that the original structure and shape of the peony ophicalcitum are protected to the greatest extent, and finally, a high-quality dried product with both appearance and internal quality is obtained.
Further preferably, the vacuum freeze-drying is specifically: pre-freezing for 4-6 h at-80 to-60 ℃, and then treating for 18-20 h under the vacuum degree of less than or equal to 40 Pa.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
The preparation method of the peony stamen protein powder comprises the following steps:
(1) selecting fresh peony pistils with retained pollen substances on the peony pistils as raw materials, mechanically crushing and sieving with a 80-mesh sieve to obtain peony pistil powder, mixing the peony pistil powder and petroleum ether according to a ratio of 1:5(g/mL) at room temperature to remove lipid substances, naturally drying to obtain degreased powder, sealing, and storing at 4 ℃ for later use.
(2) Mixing the degreased powder in the step (1) with deionized water according to a ratio of 1:20(g/mL) to obtain a mixed solution, adding 0.1mol/L sodium hydroxide, adjusting the pH of the mixed solution to 9.0, then heating the mixed solution in a water bath at 30 ℃ for 3 hours, centrifuging the mixed solution at a rotating speed of 4000r/min for 20 minutes after the reaction is finished, and collecting a supernatant (namely an alkali extraction solution) for later use.
(3) And (3) adjusting the pH of the alkali extraction solution obtained in the step (2) to 3.5 by using 0.5mol/L citric acid solution to precipitate the peony stamen protein, wherein the precipitation time is 60min, centrifuging at the rotating speed of 4000r/min for 20min after the precipitation reaction is finished, and collecting the peony stamen precipitated protein for later use.
(4) Mixing the peony pistil precipitated protein and deionized water according to the proportion of 1:6(g/mL) to obtain a dispersion liquid, carrying out ultrasonic treatment on the dispersion liquid, setting the ultrasonic power to be 100W, the ultrasonic temperature to be 30 ℃, the ultrasonic time to be 40min, then adding papain into the dispersion liquid, wherein the adding amount of the papain is 5 wt% of the peony pistil precipitated protein in the step (3), adjusting the pH value of the dispersion liquid to be 6.0, and then carrying out enzymolysis for 120min at 50 ℃.
(5) After the enzymolysis reaction is finished, heating the mixture in water bath for 25min at the temperature of 90 ℃ for enzyme deactivation reaction treatment, then centrifuging the mixture for 10min at the rotating speed of 8000r/min, and taking supernatant.
(6) And (3) adding mannan-oligosaccharide into the supernatant obtained in the step (5) under a closed condition, wherein the addition amount of the mannan-oligosaccharide is 20 wt% of the peony stamen precipitated protein obtained in the step (3), firstly adjusting the pH value of the solution to 6.0, then heating the solution in a water bath at the temperature of 60 ℃ for 180min, then centrifuging the solution at the rotating speed of 8000r/min for 10min, and taking the supernatant.
(7) Pre-freezing the supernatant obtained in the step (6) at-80 ℃ for 4h, and then drying under the vacuum degree of 40Pa for 20h to obtain the product.
Example 2
The preparation method of the peony stamen protein powder comprises the following steps:
(1) selecting fresh peony pistils with retained pollen substances on the peony pistils as raw materials, mechanically crushing and sieving with a 90-mesh sieve to obtain peony pistil powder, mixing the peony pistil powder and petroleum ether at room temperature according to a ratio of 1:6.5(g/mL) to remove lipid substances, naturally drying to obtain degreased powder, sealing, and storing at 4 ℃ for later use.
(2) Mixing the degreased powder in the step (1) with deionized water according to a ratio of 1:25(g/mL) to obtain a mixed solution, adding 0.25mol/L sodium hydroxide, adjusting the pH of the mixed solution to 9.3, then heating the mixed solution in a water bath at 40 ℃ for 2 hours, centrifuging the mixed solution at a rotating speed of 6000r/min for 15 minutes after the reaction is finished, and collecting a supernatant (namely an alkali extraction solution) for later use.
(3) And (3) adjusting the pH of the alkali extraction solution obtained in the step (2) to 3.7 by using 0.8mol/L citric acid solution to precipitate the peony stamen protein, wherein the precipitation time is 75min, centrifuging at the rotating speed of 6000r/min for 15min after the precipitation reaction is finished, and collecting the peony stamen precipitation protein for later use.
(4) Mixing the peony pistil precipitated protein and deionized water according to a ratio of 1:8(g/mL) to obtain a dispersion liquid, carrying out ultrasonic treatment on the dispersion liquid, setting the ultrasonic power to be 200W, the ultrasonic temperature to be 40 ℃, the ultrasonic time to be 30min, then adding papain into the dispersion liquid, wherein the adding amount of the papain is 10 wt% of the peony pistil precipitated protein in the step (3), adjusting the pH value of the dispersion liquid to be 6.5, and then carrying out enzymolysis for 90min at 58 ℃.
(5) After the enzymolysis reaction is finished, heating the mixture in water bath at 95 ℃ for 20min for enzyme deactivation reaction treatment, then centrifuging the mixture for 10min at the rotating speed of 8000r/min, and taking supernatant.
(6) And (3) adding mannan-oligosaccharide into the supernatant obtained in the step (5) in a closed condition, wherein the addition amount of the mannan-oligosaccharide is 25 wt% of the peony stamen precipitated protein obtained in the step (3), adjusting the pH value of the solution to 6.5, then heating the solution in a water bath at the temperature of 75 ℃ for 135min, then centrifuging the solution at the rotating speed of 8000r/min for 10min, and taking the supernatant.
(7) Pre-freezing the supernatant obtained in the step (6) at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the product.
Example 3
The preparation method of the peony stamen protein powder comprises the following steps:
(1) selecting fresh peony pistils with retained pollen substances on the peony pistils as raw materials, mechanically crushing and sieving with a 100-mesh sieve to obtain peony pistil powder, mixing the peony pistil powder and petroleum ether according to a ratio of 1:8(g/mL) at room temperature to remove lipid substances, naturally drying to obtain degreased powder, sealing, and storing at 4 ℃ for later use.
(2) Mixing the degreased powder in the step (1) with deionized water according to a ratio of 1:30(g/mL) to obtain a mixed solution, adding 0.5mol/L sodium hydroxide, adjusting the pH of the mixed solution to 9.5, heating the mixed solution in a water bath at 50 ℃ for 1h, centrifuging the mixed solution at a rotation speed of 8000r/min for 10min after the reaction is finished, and collecting a supernatant (namely an alkali extraction solution) for later use.
(3) And (3) adjusting the pH of the alkali extraction solution obtained in the step (2) to 4.0 by using 1mol/L citric acid solution to precipitate the peony stamen protein, wherein the precipitation time is 90min, centrifuging for 10min at the rotating speed of 8000r/min after the precipitation reaction is finished, and collecting the peony stamen precipitation protein for later use.
(4) Mixing the peony pistil precipitated protein and deionized water according to a ratio of 1:10(g/mL) to obtain a dispersion liquid, carrying out ultrasonic treatment on the dispersion liquid, setting the ultrasonic power to be 300W, the ultrasonic temperature to be 50 ℃, and the ultrasonic time to be 20min, then adding papain into the dispersion liquid, wherein the adding amount of the papain is 15 wt% of the peony pistil precipitated protein in the step (3), adjusting the pH value of the dispersion liquid to be 7.0, and then carrying out enzymolysis for 60min at 65 ℃.
(5) After the enzymolysis reaction is finished, heating in water bath for 15min at 100 ℃ for enzyme deactivation reaction treatment, then centrifuging for 10min at the rotating speed of 8000r/min, and taking supernatant.
(6) And (3) adding mannan-oligosaccharide into the supernatant obtained in the step (5) in a closed condition, wherein the addition amount of the mannan-oligosaccharide is 30 wt% of the peony stamen precipitated protein obtained in the step (3), adjusting the pH value of the solution to 7.0, heating the solution in a water bath at the temperature of 90 ℃ for 90min, centrifuging the solution at the rotating speed of 8000r/min for 10min, and taking the supernatant.
(7) Pre-freezing the supernatant obtained in the step (6) at-60 ℃ for 6h, and then drying under the vacuum degree of 20Pa for 18h to obtain the product.
Comparative example 1
The preparation method of the peony stamen protein powder of the comparative example comprises the following steps:
prefreezing the peony stamen precipitated protein obtained in the step (3) in the embodiment 2 at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the peony stamen precipitated protein.
Comparative example 2
The preparation method of the peony stamen protein powder of the comparative example comprises the following steps:
(1) mixing the peony stamen precipitated protein obtained in the step (3) in the embodiment 2 with deionized water according to the proportion of 1:8(g/mL) to obtain a dispersion liquid, and carrying out ultrasonic treatment on the dispersion liquid, wherein the ultrasonic power is set to be 200W, the ultrasonic temperature is 40 ℃, and the ultrasonic time is 30 min.
(2) Pre-freezing the dispersion liquid subjected to ultrasonic treatment in the step (1) at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the product.
Comparative example 3
The preparation method of the peony stamen protein powder of the comparative example comprises the following steps:
(1) mixing the peony pistil precipitated protein obtained in the step (3) in the example 2 with deionized water according to the proportion of 1:8(g/mL) to obtain a dispersion, adding papain into the dispersion, wherein the adding amount of the papain is 10 wt% of the peony pistil precipitated protein in the step (3), adjusting the pH of the dispersion to 6.5, performing enzymolysis at 58 ℃ for 90min, performing ultrasonic treatment, setting the ultrasonic power to be 200W, and setting the ultrasonic temperature to be 40 ℃ and the ultrasonic time to be 30 min.
(2) After the enzymolysis reaction is finished, heating the mixture in water bath at 95 ℃ for 20min for enzyme deactivation reaction treatment, then centrifuging the mixture for 10min at the rotating speed of 8000r/min, and taking supernatant.
(3) Pre-freezing the supernatant obtained in the step (2) at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the product.
Comparative example 4
The preparation method of the peony stamen protein powder of the comparative example comprises the following steps:
(1) mixing the peony stamen precipitated protein obtained in the step (3) in the embodiment 2 with deionized water according to the proportion of 1:8(g/mL) to obtain a dispersion liquid, adding mannan-oligosaccharide into the dispersion liquid under a closed condition, wherein the addition amount of the mannan-oligosaccharide is 25 wt% of the peony stamen precipitated protein obtained in the step (3), adjusting the pH value of the solution to 6.5, then carrying out water bath heating at the temperature of 75 ℃ for 135min, then carrying out ultrasonic treatment, setting the ultrasonic power to be 300W, the ultrasonic temperature to be 40 ℃, the ultrasonic time to be 30min, centrifuging at the rotating speed of 8000r/min for 10min after the reaction is finished, and taking a supernatant.
(2) Pre-freezing the supernatant obtained in the step (1) at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the product.
Comparative example 5
The preparation method of the peony stamen protein powder of the comparative example comprises the following steps:
(1) the peony stamen precipitated protein obtained in the step (3) of the example 2 and deionized water are mixed according to the proportion of 1:8(g/mL) to obtain a dispersion liquid, carrying out ultrasonic treatment on the dispersion liquid, setting the ultrasonic power at 200W, the ultrasonic temperature at 40 ℃ and the ultrasonic time at 30min, then adding neutral protease into the dispersion, wherein the neutral protease is obtained by fermenting and extracting bacillus subtilis and belongs to an endonuclease, and the neutral protease is subjected to enzymatic reaction, the macromolecular protein of animals and plants can be hydrolyzed into micromolecular peptide or amino acid which can be used for hydrolyzing various proteins, the optimal temperature of neutral protease is 45-50 ℃, the optimal pH is 6.8-7.0, the addition amount of the neutral protease is 10 wt% of the peony pistil precipitated protein in the step (3), the pH of the dispersion liquid is adjusted to 6.8, and then the enzymolysis is carried out for 90min at 50 ℃.
(2) After the enzymolysis reaction is finished, heating the mixture in water bath at 95 ℃ for 20min for enzyme deactivation reaction treatment, then centrifuging the mixture for 10min at the rotating speed of 8000r/min, and taking supernatant.
(3) And (3) adding mannan-oligosaccharide into the supernatant obtained in the step (2) under a closed condition, wherein the addition amount of the mannan-oligosaccharide is 25 wt% of the peony stamen precipitated protein obtained in the step (3), adjusting the pH value of the solution to 6.5, then heating the solution in a water bath at the temperature of 75 ℃ for 135min, then centrifuging the solution at the rotating speed of 8000r/min for 10min, and taking the supernatant.
(4) Pre-freezing the supernatant obtained in the step (3) at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the product.
Comparative example 6
The preparation method of the peony stamen protein powder of the comparative example comprises the following steps:
(1) adding chitosan into the supernatant obtained in the step (5) in the embodiment 2 in a closed condition, wherein the chitosan is a product obtained by removing partial acetyl of natural polysaccharide chitin, has multiple physiological functions of biodegradability, biocompatibility, nontoxicity, bacteriostasis, cancer resistance, lipid reduction, immunity enhancement and the like, is widely applied to food additives, the optimal pH of the chitosan is 6.5-8.5, the adding amount of the chitosan is 25 wt% of the peony pistil precipitation protein obtained in the step (3), the pH of the solution is adjusted to 6.5, then the solution is heated for 135min in a water bath at the temperature of 75 ℃, and then the supernatant is centrifuged for 10min at the rotating speed of 8000r/min, and the supernatant is obtained.
(2) Pre-freezing the supernatant obtained in the step (1) at-70 ℃ for 5h, and then drying under the vacuum degree of 30Pa for 19h to obtain the product.
Examples of the experiments
The solubility and emulsibility tests of the peony stamen protein powder prepared in the examples 1-3 and the comparative examples 1-6 are shown in the table 1, and the specific test and calculation methods are as follows:
the method for determining the solubility of the peony stamen protein powder comprises the following steps: taking 0.25g of ophicalcitum powder, diluting to 25mL with distilled water, in order to mix the samples uniformly, vortexing at room temperature for 20min, centrifuging at 12000r/min for 10min, collecting the supernatant, diluting appropriately, measuring the protein content by adopting a Coomassie brilliant blue method, and drawing a standard curve by taking bovine serum albumin as a standard substance. The solubility of the protein is the percentage of the protein amount concentration of the supernatant to the total protein mass concentration.
The method for determining the emulsibility of the peony stamen protein powder comprises the following steps: preparing 18mL of 1% peony stamen protein powder water dispersion, adding 6mL of soybean oil, mixing, homogenizing at 10000r/min for 2min, immediately sucking 100 microliters of emulsion from the bottom of a beaker into a glass test tube, adding 4.9mL of 0.1% Sodium Dodecyl Sulfate (SDS) solution, diluting, mixing, and measuring absorbance at 500nm with an ultraviolet-visible spectrophotometer (A)0)。
In the formula: n is the dilution multiple; c is the mass concentration of protein, and the unit is g/mL; phi is the volume ratio of oil added into the solution; a. the0Absorbance at 0.
TABLE 1 solubility and emulsifiability of peony pistil protein powder prepared in examples 1 to 3 of the present invention and comparative examples 1 to 6
| Sample (I) | Solubility/%) | Emulsifiability/(m)2/g) |
| Example 1 | 82.92 | 68.41 |
| Example 2 | 87.12 | 70.75 |
| Example 3 | 79.71 | 63.39 |
| Comparative example 1 | 35.72 | 15.28 |
| Comparative example 2 | 46.29 | 27.56 |
| Comparative example 3 | 77.31 | 41.02 |
| Comparative example 4 | 68.58 | 45.72 |
| Comparative example 5 | 78.96 | 60.37 |
| Comparative example 6 | 75.21 | 59.52 |
As can be seen from the data in Table 1, after the peony stamen protein is subjected to enzymolysis, ultrasonic treatment and ultrasonic treatment, the solubility and the emulsibility of the peony stamen protein can be greatly improved.
Claims (10)
1. The preparation method of peony stamen protein powder is characterized by comprising the following steps: and (3) performing protease enzymolysis treatment on the extracted peony stamen protein, performing glycosylation treatment on an enzymolysis product, and drying to obtain the peony stamen protein.
2. The method for preparing peony stamen protein powder according to claim 1, wherein the enzymatic hydrolysis treatment is performed under ultrasonic conditions.
3. The preparation method of peony stamen protein powder as claimed in claim 2, wherein the power of the ultrasound is 100W-300W, the temperature of the ultrasound is 30-50 ℃, and the time of the ultrasound is 20-40 min.
4. The method for preparing peony stamen protein powder according to claim 1, wherein the protease is papain; the dosage of the papain is 5-15 wt% of the peony stamen protein.
5. The method for preparing peony stamen protein powder according to claim 4, wherein the temperature of the enzymolysis reaction of the enzymolysis treatment is 50-65 ℃; the pH value of the enzymolysis reaction is controlled to be 6.0-7.0; the time of the enzymolysis reaction of the enzymolysis treatment is 60 min-120 min.
6. The method for preparing peony stamen protein powder according to claim 1, wherein the glycosylation reagent for glycosylation treatment is mannooligosaccharide; the dosage of the manna oligosaccharide is 20-30 wt% of the peony stamen protein.
7. The method for preparing peony stamen protein powder according to claim 6, wherein the glycosylation reaction time of the glycosylation treatment is 90min to 180 min; the temperature of the glycosylation reaction of the glycosylation treatment is 60-90 ℃; the pH of the glycosylation reaction is controlled within the range of 6.0-7.0.
8. The method for preparing peony pistil protein powder according to any one of claims 1 to 7, wherein the peony pistil protein is prepared by alkali extraction and acid precipitation.
9. The method for preparing peony stamen protein powder according to claim 8, wherein the alkali extraction and acid precipitation method comprises the following steps: mixing the degreased peony pistil powder with water, adjusting the pH value to 9.0-9.5, carrying out alkali extraction treatment to obtain an extracting solution, and adjusting the pH value of the extracting solution to 3.5-4.0 by using acid to carry out acid precipitation treatment.
10. The method for preparing peony stamen protein powder according to claim 9, wherein the temperature of the alkali extraction treatment is 30-50 ℃ and the time of the alkali extraction treatment is 1-3 hours.
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