CN104152521A - Paeonia suffruticosa pollen protein polypeptide and preparation method and application thereof - Google Patents
Paeonia suffruticosa pollen protein polypeptide and preparation method and application thereof Download PDFInfo
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- CN104152521A CN104152521A CN201410399776.5A CN201410399776A CN104152521A CN 104152521 A CN104152521 A CN 104152521A CN 201410399776 A CN201410399776 A CN 201410399776A CN 104152521 A CN104152521 A CN 104152521A
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Abstract
The invention relates to a method for preparing paeonia suffruticosa pollen protein polypeptide by using paeonia suffruticosa pollen as the raw material, the paeonia suffruticosa pollen protein polypeptide prepared by the method, and application of the protein polypeptide. The paeonia suffruticosa pollen protein polypeptide is paeonia suffruticosa pollen protein or paeonia suffruticosa pollen polypeptide. The method is as below: conducting wall-breaking, crude protein extraction and vacuum drying on the raw material of dried paeonia suffruticosa pollen to obtain the paeonia suffruticosa pollen protein; subjecting the paeonia suffruticosa pollen protein to enzymatic hydrolysis, debitterizing, decolorization and centrifugation or filtration to obtain the paeonia suffruticosa pollen protein polypeptide. The product can be in the forms of powder, granules, tablets, capsules and oral liquid. The paeonia suffruticosa pollen protein polypeptide has efficacy superiorities of lowering cholesterol, lowering blood pressure, promoting fat metabolism, resisting fatigue, enhancing the body immunity and regulating the physiological function of human body, and has broad development and application prospects in the fields of food industry, health care and pharmacy.
Description
Technical field
The invention belongs to peony pollen product technique field, be specifically related to a kind of peony pollen protein polypeptide, preparation method and application thereof.
Background technology
Tree peony (Paeonia suffruticosa Andr.) belongs to Ranunculaceae Paeonia, and machaka, has very high ornamental value and pharmaceutical value.For a long time, the exploitation of tree peony deep processed product but seem serious and lag behind, and production added value is very low, does not form industrial chain.Peony pollen is not only nutritious but also contain various bioactivators.Aminoacids content, more than 25.0%, has been rich in 17 seed amino acids, and vitamins B, Vitamin C content enrich, and also contain the biologically active substance such as tree peony polysaccharide, tree peony flavones.Protein content 39.3%, far above the protein content of general pollen, is called the hat of protein compression body, natural protein by academia.
Peony pollen polypeptide, refers to that protein in peony pollen is through protease hydrolysis, separation, the refining small molecules oligopeptide mixture obtaining.Natural pollen has tough and tensile cell walls, and that the sporopollenin in its structure has is acidproof, alkaline-resisting, heatproof, withstand voltage and to hydrochloric acid in gastric juice and the highly stable physicochemical property of other Digestive tract enzymes, has hindered human body absorbing pollen protein.Pollen is carried out to broken wall, can improve the absorption of protein.Therefore, in order to make full use of peony pollen resource, adopt suitable production technique by these vegetable-protein utilizations, be converted into polypeptide in addition separation and purification, make albumen or health-care polypeptide product, significant to added value and the market competitiveness of raising tree peony industry.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of peony pollen protein polypeptide, peony pollen protein polypeptide preparation and related application prepared by the method.
The invention provides a kind of peony pollen protein polypeptide, be prepared from taking the peony pollen that is dried as raw material.
Further, described peony pollen derives from the red tree peony of phoenix or Paeonia papaveracea.
Further, described peony pollen protein polypeptide is peony pollen albumen, or peony pollen polypeptide.
The present invention also provides a kind of preparation method of peony pollen protein polypeptide, it is characterized in that, described peony pollen protein polypeptide is prepared from taking the peony pollen that is dried as raw material.Further, described peony pollen derives from the red tree peony of phoenix or Paeonia papaveracea.
Further, described peony pollen protein polypeptide is taking the peony pollen that is dried as raw material, through pollen broken wall, crude protein extract, vacuum-drying, obtain peony pollen albumen.Or further, peony pollen albumen, through proteolysis, debitterizing and decoloring, centrifugal or filtration, is obtained to peony pollen polypeptide.
Further, described method also comprises the step of ultra-filtration membrane refining after centrifugal or filtration.
Further, described method also comprises step dry, sterilizing after refining.
Further, described pollen broken wall is ultramicronising broken wall or whirlwind comminuting method broken wall.
Preferably, ultramicronising broken wall, is peony pollen planetary ball mill ultramicronising broken wall, rotating speed 400-600r/min, and grinding time is 0.5-1.5h, ratio of grinding media to material is 6.8-7.Whirlwind comminuting method broken wall, is to adopt whirlwind comminuting method, peony pollen is put into cyclone type high speed sample pulverizer and pulverize 1-5min.
Further, described crude protein is extracted as aqueous soluble protein extraction, salting-in-protein extraction, prolamine extracts or alkali-soluble protein extracts.
Preferably, aqueous soluble protein extracts, and is the pollen after broken wall, and by material-water ratio 1: 10-20 adds distilled water, 4-60 DEG C is stirred extraction 1-8h, and collects supernatant liquor after the centrifugal 10-20min of 4000-8000r/min, and residue is for subsequent use.In supernatant liquor, add step by step a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach successively 40%, 60%, 80%, each 1h that fully stirs, after the centrifugal 10-20min of 4000-8000r/min, collect precipitations at different levels, with the phosphate buffered saline buffer dissolving of pH5.0-7.0, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains water-soluble crude protein solution.
Preferably, further comprise that salting-in-protein extracts, be the residue after above-mentioned extraction with aqueous solution, add 0.15-0.50mol/L sodium chloride solution by solid-liquid ratio 1: 10-20,4-60 DEG C is stirred extraction 1-8h, and collect supernatant liquor after the centrifugal 10-20min of 4000-8000r/min, residue is for subsequent use.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, with the sodium chloride solution dissolving of above-mentioned equal concentration, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of salt.
Preferably, further comprise that prolamine extracts, and is the residue after above-mentioned sodium chloride solution extraction, add 75% ethanolic soln by solid-liquid ratio 1: 10-20,4-60 DEG C is stirred extraction 1-8h, and collects supernatant liquor after the centrifugal 10-20min of 4000-8000r/min, and residue is for subsequent use.In supernatant liquor by material-water ratio 1: 20-30 adds distilled water, fully stirs 1h, and after the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, uses 75% dissolve with ethanol,, with after distill water dialysis 24h, obtain the molten crude protein precipitation of alcohol at 4 DEG C.
Preferably, further comprise that alkali-soluble protein extracts, be the residue after above-mentioned ethanolic soln is extracted, add 0.10-0.75mol/L NaOH solution by solid-liquid ratio 1: 10-20,4-60 DEG C is stirred extraction 1-8h, and collect supernatant liquor after the centrifugal 10-20min of 4000-8000r/min.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, with the NaOH dissolving of above-mentioned equal concentration, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of alkali.
Further, described vacuum lyophilization, be above-mentioned water-soluble crude protein solution, the molten crude protein solution of salt, the molten crude protein precipitation of alcohol, the molten crude protein solution of alkali, vacuum lyophilization respectively, at-55 DEG C～-40 DEG C temperature, obtain peony pollen aqueous soluble protein, salting-in-protein, prolamine, alkali-soluble protein.
The present invention also provides a kind of peony pollen albumen, is one or several in peony pollen aqueous soluble protein, peony pollen salting-in-protein, peony pollen prolamine, peony pollen alkali-soluble protein.
Further, described proteolysis, is above-mentioned peony pollen albumen by material-water ratio 1: 10-20 adds distilled water, adds the proteolytic enzyme of peony pollen albumen butt 3-10%, 35-55 DEG C of insulation enzymolysis 2-12h, then be warming up to the 80-95 DEG C of high temperature enzyme 8-15min that goes out.
Preferably, described proteolytic enzyme is selected from one or several in papoid, flavor protease, Sumizyme MP, neutral protease etc.
Preferred, described proteolytic enzyme is Sumizyme MP, or the combination of flavor protease and neutral protease, or the combination of Sumizyme MP and neutral protease, or the combination of Sumizyme MP and papoid.
Further, described debitterizing and decoloring, is to add stir process 30-40min at 40-65 DEG C of 0.1-0.5% gac.
Further, described centrifugal or filtration, is the centrifugal 20-25min of 4000-10000r/min or Plate Filtration.
Further, described ultrafiltration refining, is peony pollen polypeptide ultra-filtration membrane centrifugal or that filtration obtains to carry out fractional separation by molecular weight, obtains different molecular weight peony pollen polypeptide solution; Molecular weight cut-off is below 10Kda, preferably, below 5Kda, more preferably, below 3Kda, most preferably is below 1Kda.
Further, described peony pollen polypeptide solution is dried as dry or the lyophilize of spraying.
Preferably, spraying is dry to be peony pollen polypeptide solution, and under 0.08-0.2 normal atmosphere, 70-130 DEG C of spraying is dried, and obtains peony pollen polypeptide.Lyophilize is at-55 DEG C～-40 DEG C temperature, adopts vacuum lyophilization, obtains peony pollen polypeptide.
The present invention also provides a kind of peony pollen polypeptide, is prepared from by above-mentioned arbitrary method.
The present invention also provides a kind of peony pollen protein polypeptide preparation, it is characterized in that, be directly prepared from by above-mentioned protein polypeptide, or, it is characterized in that, described protein polypeptide is powder or is further processed into the forms such as particle, tablet, capsule, oral liquid.
The present invention also provides the application of a kind of above-mentioned peony pollen protein polypeptide or preparation, and described application comprises as nutritive food, protective foods, arsenic or protein drinks.
Preferably, described nutritive food is the nutritive food providing for patient or baby; Described protective foods is the protective foods providing for the elderly or sub-health population, and described protein polypeptide beverage refers to the independent Instant Drinks of peony pollen protein polypeptide or the companion's Instant Drinks as existing beverage.
The peony pollen protein polypeptide that the inventive method prepares has good nutritive property, absorption easy to digest, especially some low molecular peptide class, can not only provide rapidly human body energy, and there is unique processing characteristics, can be used as nutritive food, arsenic and protein drinks.Simultaneously, peony pollen protein polypeptide has the cholesterol of reduction, hypotensive and promote metabolism of fat, antifatigue, enhancing body immunity, regulate effect advantages such as human physiological functions to have wide development prospect in foodstuffs industry, protective foods and medicine and other fields.
Embodiment
Further illustrate the present invention below in conjunction with embodiment, should be appreciated that these examples can not serve as restriction of the present invention, without departing from the spirit and substance of the case in the present invention, the amendment of doing or replace and all belong to scope of the present invention.If do not specialize, the means in following embodiment are conventional means known in the art.
Embodiment 1 peony pollen broken wall
With planetary ball mill to pollen ultramicronising broken wall, rotating speed 600r/min, grinding time is 0.5h, ratio of grinding media to material is 7.After measured, the rate of breaking pollen wall of the present embodiment is 100%.
Embodiment 2 peony pollen broken walls
With planetary ball mill to pollen ultramicronising broken wall, rotating speed 400r/min, grinding time is 1.5h, ratio of grinding media to material is 6.8.After measured, the rate of breaking pollen wall of the present embodiment is 96%.
Embodiment 3 peony pollen broken walls
Pollen is put into cyclone type high speed sample pulverizer and pulverize 5min broken wall.After measured, the rate of breaking pollen wall of the present embodiment is 100%.
Embodiment 4 peony pollen broken walls
Pollen is put into cyclone type high speed sample pulverizer and pulverize 1min broken wall.After measured, the rate of breaking pollen wall of the present embodiment is 94%.
Embodiment 5 peony pollen aqueous soluble protein preparations
Step 1, by peony pollen arbitrary embodiment 1-4, adds distilled water by material-water ratio at 1: 10, and 4 DEG C are stirred extraction 8h, and collect supernatant liquor after the centrifugal 20min of 4000r/min, and residue is for subsequent use.In supernatant liquor, add step by step a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach successively 40%, 60%, 80%, each 1h that fully stirs, after the centrifugal 10min of 8000r/min, collect precipitations at different levels, with the phosphate buffered saline buffer dissolving of pH7.0, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains water-soluble crude protein solution.
Step 2 is at-55 DEG C of temperature, and vacuum lyophilization, obtains peony pollen aqueous soluble protein.
Embodiment 6 peony pollen aqueous soluble protein preparations
Step 1, by peony pollen arbitrary embodiment 1-4, adds distilled water by material-water ratio at 1: 20, and 60 DEG C are stirred extraction 1h, and collect supernatant liquor after the centrifugal 10min of 8000r/min, and residue is for subsequent use.In supernatant liquor, add step by step a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach successively 40%, 60%, 80%, each 1h that fully stirs, after the centrifugal 20min of 4000r/min, collect precipitations at different levels, with the phosphate buffered saline buffer dissolving of pH5.0, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains water-soluble crude protein solution.
Step 2 is at-40 DEG C of temperature, and vacuum lyophilization, obtains peony pollen aqueous soluble protein.
Embodiment 7 peony pollen salting-in-protein preparations
Step 1, by the residue after extraction with aqueous solution arbitrary embodiment 5-6, adds 0.15mol/L sodium chloride solution by solid-liquid ratio at 1: 10, and 4 DEG C are stirred extraction 8h, and collect supernatant liquor after the centrifugal 20min of 4000r/min, and residue is for subsequent use.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 10min of 8000r/min, collecting precipitation, with the dissolving of 0.15mol/L sodium chloride solution, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of salt.
Step 2 is at-40 DEG C of temperature, and vacuum lyophilization, obtains peony pollen salting-in-protein.
Embodiment 8 peony pollen salting-in-protein preparations
Step 1, by the residue after extraction with aqueous solution arbitrary embodiment 5-6, adds 0.50mol/L sodium chloride solution by solid-liquid ratio at 1: 20, and 60 DEG C are stirred extraction 1h, and collect supernatant liquor after the centrifugal 10min of 8000r/min, and residue is for subsequent use.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 20min of 4000r/min, collecting precipitation, with the dissolving of 0.50mol/L sodium chloride solution, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of salt.
Step 2 is at-55 DEG C of temperature, and vacuum lyophilization, obtains peony pollen salting-in-protein.
Embodiment 9 peony pollen prolamine preparations
Step 1, by the residue after sodium chloride solution extraction arbitrary embodiment 7-8, adds 75% ethanolic soln by solid-liquid ratio at 1: 10, and 4 DEG C are stirred extraction 8h, and collect supernatant liquor after the centrifugal 20min of 4000r/min, and residue is for subsequent use.In supernatant liquor, add distilled water at 1: 30 by material-water ratio, fully stir 1h, after the centrifugal 10min of 8000r/min, collecting precipitation, uses 75% dissolve with ethanol,, with after distill water dialysis 24h, obtain the molten crude protein precipitation of alcohol at 4 DEG C.
Step 2 is at-55 DEG C of temperature, and vacuum lyophilization, obtains peony pollen prolamine.
Embodiment 10 peony pollen prolamine preparations
Step 1, by the residue after sodium chloride solution extraction arbitrary embodiment 7-8, adds 75% ethanolic soln by solid-liquid ratio at 1: 20, and 60 DEG C are stirred extraction 1h, and collect supernatant liquor after the centrifugal 10min of 8000r/min, and residue is for subsequent use.In supernatant liquor, add distilled water at 1: 20 by material-water ratio, fully stir 1h, after the centrifugal 20min of 4000r/min, collecting precipitation, uses 75% dissolve with ethanol,, with after distill water dialysis 24h, obtain the molten crude protein precipitation of alcohol at 4 DEG C.
Step 2 is at-40 DEG C of temperature, and vacuum lyophilization, obtains peony pollen prolamine.
Embodiment 11 peony pollen alkali-soluble protein preparations
Residue after step 1 is extracted ethanolic soln arbitrary embodiment 9-10, adds 0.10mol/L NaOH solution by solid-liquid ratio at 1: 10, and 4 DEG C are stirred extraction 8h, and collect supernatant liquor after the centrifugal 20min of 4000r/min.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 10min of 8000r/min, collecting precipitation, with 0.10mol/L NaOH dissolving, then the ultra-filtration membrane that is 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of alkali.
Step 2 is at-40 DEG C of temperature, and vacuum lyophilization, obtains peony pollen alkali-soluble protein.
Embodiment 12 peony pollen alkali-soluble protein preparations
Residue after step 1 is extracted ethanolic soln arbitrary embodiment 9-10, adds 0.75mol/L NaOH solution by solid-liquid ratio at 1: 20, and 60 DEG C are stirred extraction 1h, and collect supernatant liquor after the centrifugal 10min of 8000r/min.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 20min of 4000r/min, collecting precipitation, with 0.75mol/L NaOH dissolving, then the ultra-filtration membrane that is 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of alkali.
Step 2 is at-55 DEG C of temperature, and vacuum lyophilization, obtains peony pollen alkali-soluble protein.
Embodiment 13 peony pollen albumen preparations
By peony pollen aqueous soluble protein arbitrary embodiment 5-6, the arbitrary arbitrary arbitrary peony pollen alkali-soluble protein of peony pollen prolamine, embodiment 11-12 of peony pollen salting-in-protein, embodiment 9-10 of embodiment 7-8, choose 2 kinds or two or more mixing, make peony pollen albumen.
Embodiment 14 peony pollen polypeptide preparations
Step 1 enzymolysis, by peony pollen albumen arbitrary embodiment 5-13, adds distilled water by material-water ratio at 1: 10, adds the Sumizyme MP of peony pollen albumen butt 3%, and adjusting pH is 10.0,50 DEG C of insulation enzymolysis 2h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 80 DEG C, to the enzymolysis solution enzyme 15min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 40 DEG C, adds 0.5% gac stir process 30min.
The separation of step 4 filtrate is removed the solid impurity in enzymolysis solution by flame filter press, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 10KDa.
Step 6 is dried under 0.15 normal atmosphere, and 90 DEG C of sprayings are dry.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of peony pollen albumen is 42.3%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
Embodiment 15 peony pollen polypeptide preparations
Step 1 enzymolysis, by peony pollen albumen arbitrary embodiment 5-13, adds distilled water by material-water ratio at 1: 15, adds the papoid of peony pollen albumen butt 8%, and adjusting pH is 6.5,45 DEG C of insulation enzymolysis 3h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 95 DEG C, to the enzymolysis solution enzyme 8min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 50 DEG C, adds 0.2% gac stir process 40min.
Step 4 filtrate separates the centrifugal 25min of 4000r/min, gets supernatant liquor, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 5KDa.
Step 6 is dried under 0.2 normal atmosphere, and 80 DEG C of sprayings are dry.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of peony pollen albumen is 28.1%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
Embodiment 16 peony pollen polypeptide preparations
Step 1 enzymolysis, by peony pollen albumen arbitrary embodiment 5-13, adds distilled water by material-water ratio at 1: 20, adds the neutral protease of peony pollen albumen butt 10%, and adjusting pH is 7,40 DEG C of insulation enzymolysis 3h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 80 DEG C, to the enzymolysis solution enzyme 15min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 65 DEG C, adds 0.1% gac stir process 30min.
Step 4 filtrate separates the centrifugal 20min of 10000r/min, gets supernatant liquor, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 3KDa.
Step 6 is dried at-55 DEG C of temperature, vacuum lyophilization.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of peony pollen albumen is 30.4%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
Embodiment 17 peony pollen polypeptide preparations
Step 1 enzymolysis, by peony pollen albumen arbitrary embodiment 5-13, adds distilled water by material-water ratio at 1: 20, adds the flavor protease of peony pollen albumen butt 8%, and adjusting pH is 6,50 DEG C of insulation enzymolysis 12h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 95 DEG C, to the enzymolysis solution enzyme 8min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 40 DEG C, adds 0.5% gac stir process 30min.
The separation of step 4 filtrate is removed the solid impurity in enzymolysis solution by flame filter press, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 5KDa.
Step 6 is dried at-40 DEG C of temperature, vacuum lyophilization.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of Tree Peony flower pollen protein is 33.3%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
Embodiment 18 peony pollen polypeptide preparations
Step 1 enzymolysis is by peony pollen albumen arbitrary embodiment 5-13, add distilled water at 1: 10 by material-water ratio, add the mixing protease of peony pollen albumen butt 5%, described mixing protease is Sumizyme MP: neutral protease mass ratio is 1: 9, adjusting pH is 9,45 DEG C of insulation enzymolysis 2h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 80 DEG C, to the enzymolysis solution enzyme 15min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 50 DEG C, adds 0.2% gac stir process 40min.
Step 4 filtrate separates the centrifugal 25min of 4000r/min, gets supernatant liquor, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 10KDa.
Step 6 is dried under 0.15 normal atmosphere, and 90 DEG C of sprayings are dry.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of Tree Peony flower pollen protein is 45.7%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
Embodiment 19 peony pollen polypeptide preparations
Step 1 enzymolysis is by peony pollen albumen arbitrary embodiment 5-13, add distilled water at 1: 15 by material-water ratio, add the mixing protease of peony pollen albumen butt 8%, described mixing protease is Sumizyme MP: papoid mass ratio is 1: 1, adjusting pH is 9,45 DEG C of insulation enzymolysis 2h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 95 DEG C, to the enzymolysis solution enzyme 8min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 65 DEG C, adds 0.1% gac stir process 30min.
Step 4 filtrate separates the centrifugal 20min of 10000r/min, gets supernatant liquor, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 3KDa.
Step 6 is dried at-40 DEG C of temperature, vacuum lyophilization.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of Tree Peony flower pollen protein is 38.1%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
Embodiment 20 peony pollen polypeptide preparations
Step 1 enzymolysis is by peony pollen albumen arbitrary embodiment 5-13, add distilled water at 1: 20 by material-water ratio, add the mixing protease of peony pollen albumen butt 10%, described mixing protease is flavor protease: neutral protease mass ratio is 2: 1, adjusting pH is 7,40 DEG C of insulation enzymolysis 12h.
Step 2 is gone out after enzyme treats enzymolysis, adds rapidly thermal degradation to 80 DEG C, to the enzymolysis solution enzyme 15min that goes out.
Step 3 debitterizing and decoloring, in the time that enzymolysis solution is cooled to 40 DEG C, adds 0.5% gac stir process 30min.
The separation of step 4 filtrate is removed the solid impurity in enzymolysis solution by flame filter press, thereby obtains peony pollen polypeptide coarse filtration liquid.
Step 5 refining is carried out fractional separation with ultra-filtration membrane by molecular weight by peony pollen polypeptide coarse filtration liquid, and molecular weight cut-off is 10KDa.
Step 6 is dried at-55 DEG C of temperature, vacuum lyophilization.
140 DEG C of high-temperature short-time sterilization 8s of step 7 sterilizing, obtain peony pollen polypeptide.
Mix the peony pollen albumen making as example taking peony pollen aqueous soluble protein in embodiment 13 (embodiment 5), peony pollen salting-in-protein (embodiment 7), peony pollen prolamine (embodiment 9), 4 kinds of albumen of peony pollen alkali-soluble protein (embodiment 11), after measured, the degree of hydrolysis of Tree Peony flower pollen protein is 40%, and the polypeptide product obtaining is without bitter taste.
Said products is directly as nutritive food, protective foods, arsenic or protein drinks.
The preparation of embodiment 21 tree peony polypeptide granules
Take each component according to following weight percent: tree peony protein polypeptide powder 35.0%, sucrose 45.0% that embodiment 5-20 is arbitrary, modified starch 16.0%, citric acid 2.0%, oxysuccinic acid 2.0%.
First above-mentioned each material is pulverized or sieved; By formula ratio take successively pulverize or sieve after material; Load weighted material is dropped into mixing machine, mix; Add appropriate alcohol, granulate by nodulizer; The particle making is dried with drying plant, carries out whole grain by pelletizing machine subsequently; Last packing, makes peony pollen protein polypeptide granule of the present invention.
The preparation of embodiment 22 tree peony polypeptide tablets
Take each component according to following weight percent: tree peony protein polypeptide powder 35.0%, sucrose 28.5%, starch 10.0%, coated vitamin C 1.5%, Microcrystalline Cellulose 15.0%, dextrin 8.0%, film coating agent 1.0%, Magnesium Stearate 0.6%, silicon-dioxide 0.4% that embodiment 5-20 is arbitrary.
First above-mentioned each material is pulverized or sieved; By formula ratio take successively pulverize or sieve after material; Load weighted material is dropped into mixing machine, mix; Add appropriate alcohol, granulate by nodulizer; The particle making is dried with drying plant, carries out whole grain by pelletizing machine subsequently; Add Magnesium Stearate, silicon-dioxide always to mix, the material being always mixed after even is carried out to compressing tablet by tabletting machine, plain sheet is temporary, for subsequent use; Film coating agent is added in suitable quantity of water, stir, be mixed with the coating liquid that concentration is 5.0-15.0%, by seed-coating machine, the plain sheet pressing is carried out to Cotton seeds; Last packing, makes the tablet of peony pollen protein polypeptide of the present invention.
The preparation of embodiment 23 tree peony polypeptide capsules
Take each component according to following weight percent: tree peony protein polypeptide powder 54.0%, coated vitamin C 9.0%, Microcrystalline Cellulose 36.0%, Magnesium Stearate 0.6%, silicon-dioxide 0.4% that embodiment 5-20 is arbitrary.
First above-mentioned each material is pulverized, sieved; By formula ratio take successively pulverizing, material after sieving; Load weighted material is dropped into mixing machine, mix; Then by capsule filler, capsule is filled; Last packing, makes peony pollen protein polypeptide capsule of the present invention.
The preparation of embodiment 24 tree peony polypeptide oral liquors
Take each component according to following weight percent: tree peony protein polypeptide powder 50.0%, honey 10.0%, vitamin C sodium salt 5%, oxysuccinic acid 2.0%, citric acid 1.6%, carrageenin 1.0%, Steviosides 0.2%, acesulfame potassium 0.2%, 30% pure water that embodiment 5-20 is arbitrary.
Take above-mentioned each material by formula ratio, drop in water, fully dissolve; Then gained solution is transferred in oral liquid filling machine, injects in container; Sterilizing, makes peony pollen protein polypeptide oral liquid of the present invention.
One, for the relatively variation of peony pollen broken wall front and back Major Nutrient material, we are to broken pollen and broken pollen crude protein, amino acid, total polysaccharides, total flavones equal size are not evaluated.
Table 1: tree peony broken wall and the not comparison of broken pollen content
| Sample | Sporoderm-broken rate % | Crude protein g/100g | Amino acid g/100g | Total polysaccharides g/100g | Total flavones g/100g |
| Embodiment 1 | 100 | 39.8 | 31.3 | 2.92 | 2.73 |
| Embodiment 2 | 96 | 37.5 | 30.9 | 2.65 | 2.54 |
| Embodiment 3 | 100 | 39.1 | 32.8 | 2.85 | 2.68 |
| Embodiment 4 | 94 | 35.9 | 30.4 | 2.51 | 2.42 |
| Common peony pollen (contrast) | 0 | 32.3 | 25.1 | 1.94 | 2.02 |
Index explanation:
1, the calculating of rate of breaking pollen wall
Configuration suspension: accurately take the contrast pollen of seasoning and the each about 10mg of the pollen of broken wall treatment, be placed in respectively the mortar of dried and clean, add 1ml Chloral Hydrate test solution (to get Chloral Hydrate 50g, add water 15ml and glycerine 10ml is miscible), be ground to and be uniformly dispersed gently, immediately load.Film-making: select a capillary in the line of suitable position, as scale mark, quantitatively draw suspension load by this root capillary tube, cover with 18 × 18mm cover glass.Microscopic examination: use same microscope, magnification eyepiece 10 × object lens 10, with 9 visuals field of mechanically-propelled device position observation, record each visual field pollen granule number.Pollen number is the mean value of 9 visual field pollen granule observed values of pollen, the actual mass of alleged pollen when pollen quality is configuration suspension.
2, protein content adopts micro-Kjeldahl.
3, aminoacids content automatic analyzer for amino acids.
4, total polysaccharides is measured and is adopted anthrone colorimetry.
5, Determination of Total Flavonoids adopts liquid phase chromatography.
Result demonstration, the sporoderm-broken rate of the application's method is very high, and at least 94%.Compare with common peony pollen, after pollen broken wall, in pollen, crude protein, amino acid, total polysaccharides, total flavones have obtained significance raising (p < 0.05), so peony pollen is more conducive to the release of nutritive ingredient and activeconstituents after broken wall, improves its nutrient utilization.
Two, we have also measured the difference of the various albumen that extract with peony pollen.
Table 2: each protein content difference that peony pollen extracts
| Sample | Aqueous soluble protein g | Salting-in-protein g | Prolamine g | Alkali-soluble protein g |
| Embodiment 5 | 0.3813 | ? | ? | ? |
| Embodiment 6 | 0.3789 | ? | ? | ? |
| Embodiment 7 | ? | 0.0716 | ? | ? |
| Embodiment 8 | ? | 0.0684 | ? | ? |
| Embodiment 9 | ? | ? | 0.3510 | ? |
| Embodiment 10 | ? | ? | 0.3541 | ? |
| Embodiment 11 | ? | ? | ? | 1.1819 |
| Embodiment 12 | ? | ? | ? | 1.1749 |
| Common peony pollen (contrast) | 0.2980 | 0.0554 | 0.2761 | 0.9240 |
Index explanation:
1, in table the measuring method of protein content with reference to first part.
2, the peony pollen of embodiment 5-12 is taking the peony pollen of embodiment 1 as raw material illustrates, and the peony pollen that contrasts peony pollen and embodiment 1 is respectively chosen 5g mensuration.
Result shows, compares with the peony pollen of broken wall not, and in the peony pollen after broken wall, tree peony aqueous soluble protein, salting-in-protein, prolamine, alkali-soluble protein have all obtained significance raising (p < 0.05).So Tree Peony flower micronizing and pollen broken wall are more conducive to the release of nutritive ingredient and activeconstituents, improve its nutrient utilization.
Claims (10)
1. a preparation method for peony pollen protein polypeptide, is characterized in that, is prepared from taking the peony pollen that is dried as raw material.Preferably, described peony pollen derives from the red tree peony of phoenix or Paeonia papaveracea.
2. preparation method as claimed in claim 1, is characterized in that, described method comprises peony pollen process pollen broken wall, crude protein extraction, vacuum-drying, obtains peony pollen albumen; Or further, by peony pollen albumen process proteolysis, debitterizing and decoloring, centrifugal or filtration, obtain peony pollen polypeptide.
3. preparation method as claimed in claim 2, is characterized in that, described method also comprises the step of ultra-filtration membrane refining after centrifugal or filtration.Preferably, described method also comprises step dry, sterilizing after refining.
4. preparation method as claimed in claim 2 or claim 3, is characterized in that, described crude protein extracts and comprises the following steps:
(1) aqueous soluble protein extracts, and is the pollen after broken wall, and by material-water ratio 1: 10-20 adds distilled water, 4-60 DEG C is stirred extraction 1-8h, and collects supernatant liquor after the centrifugal 10-20min of 4000-8000r/min, and residue is for subsequent use.In supernatant liquor, add step by step a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach successively 40%, 60%, 80%, each 1h that fully stirs, after the centrifugal 10-20min of 4000-8000r/min, collect precipitations at different levels, with the phosphate buffered saline buffer dissolving of pH5.0-7.0, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains water-soluble crude protein solution;
(2) preferred, further comprise that salting-in-protein extracts, be the residue after above-mentioned extraction with aqueous solution, add 0.15-0.50mol/L sodium chloride solution by solid-liquid ratio 1: 10-20,4-60 DEG C is stirred extraction 1-8h, and collect supernatant liquor after the centrifugal 10-20min of 4000-8000r/min, residue is for subsequent use.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, with the sodium chloride solution dissolving of above-mentioned equal concentration, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of salt;
(3) preferred, further comprise that prolamine extracts, be the residue after above-mentioned sodium chloride solution extraction, add 75% ethanolic soln by solid-liquid ratio 1: 10-20,4-60 DEG C is stirred extraction 1-8h, and collect supernatant liquor after the centrifugal 10-20min of 4000-8000r/min, residue is for subsequent use.In supernatant liquor by material-water ratio 1: 20-30 adds distilled water, fully stirs 1h, and after the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, uses 75% dissolve with ethanol, with after distill water dialysis 24h, obtains the molten crude protein precipitation of alcohol at 4 DEG C; Or,
(4) preferred, further comprise that alkali-soluble protein extracts, be the residue after above-mentioned ethanolic soln is extracted, add 0.10-0.75mol/L NaOH solution by solid-liquid ratio 1: 10-20,4-60 DEG C is stirred extraction 1-8h, and collect supernatant liquor after the centrifugal 10-20min of 4000-8000r/min.In supernatant liquor, add a certain amount of solid ammonium sulfate, make ammonium sulfate saturation ratio reach 40%, fully stir 1h, after the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, with the NaOH dissolving of above-mentioned equal concentration, the ultra-filtration membrane that is then 100KDa with trapped molecular weight carries out ultrafiltration, obtains the molten crude protein solution of alkali.
5. the preparation method as described in as arbitrary in claim 2-4, is characterized in that, described proteolytic enzyme is selected from one or several in papoid, flavor protease, Sumizyme MP, neutral protease etc.Preferred, described proteolytic enzyme is Sumizyme MP, or the combination of flavor protease and neutral protease, or the combination of Sumizyme MP and neutral protease, or the combination of Sumizyme MP and papoid.
6. the preparation method as described in as arbitrary in claim 2-5, it is characterized in that, described ultrafiltration refinement step is that peony pollen polypeptide liquid ultra-filtration membrane centrifugal or that filtration obtains is carried out to fractional separation by molecular weight, obtains different molecular weight peony pollen polypeptide solution; Molecular weight cut-off is below 10KDa, preferably, below 5KDa, more preferably, below 3KDa, most preferably is below 1KDa.
7. the peony pollen protein polypeptide that prepared by claim 1-6 either method.
8. a peony pollen protein polypeptide preparation, is characterized in that, be directly prepared from by protein polypeptide claimed in claim 7, or be further processed into particle, the form such as tablet, capsule, oral liquid.
9. the application of peony pollen protein polypeptide preparation described in peony pollen protein polypeptide or claim 8 described in claim 7, is characterized in that, described application comprises as nutritive food, protective foods, arsenic or protein drinks.
10. application as claimed in claim 9, is characterized in that, described nutritive food is the nutritive food providing for patient or baby; Described protective foods is the protective foods providing for the elderly or sub-health population, and described protein drinks refers to the independent Instant Drinks of tree peony protein polypeptide or the companion's Instant Drinks as existing beverage.
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| CN112841639A (en) * | 2020-11-27 | 2021-05-28 | 张贵宾 | Preparation method of peony pollen collagen peptide |
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