CN115386611A - Peony pollen protein polypeptide and preparation method and application thereof - Google Patents
Peony pollen protein polypeptide and preparation method and application thereof Download PDFInfo
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- 235000006484 Paeonia officinalis Nutrition 0.000 title claims abstract description 129
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 90
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 90
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 60
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 60
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 244000170916 Paeonia officinalis Species 0.000 title 1
- 241000736199 Paeonia Species 0.000 claims abstract description 128
- 238000000855 fermentation Methods 0.000 claims abstract description 44
- 230000004151 fermentation Effects 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 26
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 20
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 20
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 20
- 235000019750 Crude protein Nutrition 0.000 claims abstract description 17
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 68
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 239000006228 supernatant Substances 0.000 claims description 23
- 238000000108 ultra-filtration Methods 0.000 claims description 23
- 239000012528 membrane Substances 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 20
- 238000001914 filtration Methods 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000002244 precipitate Substances 0.000 claims description 14
- 239000012460 protein solution Substances 0.000 claims description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 238000007710 freezing Methods 0.000 claims description 11
- 230000008014 freezing Effects 0.000 claims description 11
- 238000001694 spray drying Methods 0.000 claims description 11
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- MIOPJNTWMNEORI-GMSGAONNSA-N (S)-camphorsulfonic acid Chemical compound C1C[C@@]2(CS(O)(=O)=O)C(=O)C[C@@H]1C2(C)C MIOPJNTWMNEORI-GMSGAONNSA-N 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 238000009835 boiling Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 230000036772 blood pressure Effects 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 2
- 239000002131 composite material Substances 0.000 abstract description 2
- 239000002220 antihypertensive agent Substances 0.000 abstract 1
- 229940127088 antihypertensive drug Drugs 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 17
- 241000700159 Rattus Species 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 7
- 230000035488 systolic blood pressure Effects 0.000 description 7
- 238000000227 grinding Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 238000001291 vacuum drying Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000003304 gavage Methods 0.000 description 4
- 230000001603 reducing effect Effects 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 3
- 108091005658 Basic proteases Proteins 0.000 description 3
- 108091005507 Neutral proteases Proteins 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
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- 238000010297 mechanical methods and process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
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- 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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention discloses a peony pollen protein polypeptide and a preparation method and application thereof, wherein the preparation method of the peony pollen protein polypeptide comprises the following steps: breaking the wall of peony pollen, extracting crude protein, adding bacillus licheniformis and bacillus subtilis, fermenting, and drying to obtain peony pollen protein polypeptide. According to the method, a temperature difference and ultrasonic composite wall breaking mode is adopted, the wall breaking rate of the peony pollen is improved, peony pollen protein is conveniently and effectively extracted, and the peony pollen protein polypeptide is high in purity by adopting a mode of combined fermentation of bacillus licheniformis and bacillus subtilis. The peony pollen protein polypeptide prepared by the method can be used for preparing the antihypertensive drug.
Description
Technical Field
The invention relates to the technical field of pollen fermentation, and particularly relates to a peony pollen protein polypeptide and a preparation method and application thereof.
Background
The pollen treatment method comprises mechanical method, enzymolysis method and fermentation method. Among them, the mechanical method is very violent in process and has high requirements on equipment. Although the enzymatic method is simple, it requires strict control of reaction conditions. The fermentation method is used for treating the pollen, so that the effects of bacteriostasis and wall breaking can be achieved, desensitization can be achieved, and nutritional ingredients in the pollen are not damaged.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a peony pollen protein polypeptide and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a preparation method of a peony pollen protein polypeptide is provided, which comprises the following steps:
(1) Inoculating bacillus subtilis and bacillus licheniformis into a peony pollen protein fermentation culture solution, fermenting for 9 to 18h at the temperature of 30 to 40 ℃, and after fermentation is finished, sterilizing in a boiling water bath;
(2) Centrifuging the sterilized peony pollen protein fermentation culture solution, taking supernatant, filtering, taking filtrate, and drying to obtain peony pollen protein polypeptide;
preferably, in the step (1), the bacillus subtilis seed solution and the bacillus licheniformis seed solution are mixed in equal volume to obtain a mixed seed solution, and the mixed seed solution is inoculated into the peony pollen protein fermentation culture solution.
Further preferably, the inoculation amount of the mixed seed solution is 2-3% of the volume of the peony pollen protein fermentation culture solution.
More preferably, the viable cell count of Bacillus subtilis in the mixed seed solution is 1.6X 10 8 ~1.8×10 8 cfu/mL, viable count of Bacillus licheniformis is 2.0 × 10 8 ~2.2×10 8 cfu/mL。
Preferably, in the step (2), the centrifugal rotating speed is 6000 to 8000r/min, and the centrifugal time is 10 to 20min.
Preferably, in step (2), the drying method is spray drying or vacuum freeze drying.
Further preferably, the spray drying conditions are 0.08-0.2 atm, 70-130 ℃; the vacuum freeze drying condition is-55 to-40 ℃.
Preferably, in the step (2), the filtering method is as follows: filtration was performed using a 0.45 μm filter membrane and then ultrafiltration was performed using a 10kDa ultrafiltration membrane.
Preferably, in the step (1), the peony pollen protein fermentation culture solution is prepared from the following raw materials in parts by weight: 8 to 10 parts of peony pollen protein, 0.2 to 0.3 part of glucose, 0.5 to 0.7 part of sodium chloride, 0.01 to 0.02 part of camphorsulfonic acid and 160 to 180 parts of distilled water.
Further preferably, the peony pollen protein is prepared by the following method:
(A) Crushing peony pollen, freezing, adding hot water, stirring, and performing ultrasonic treatment to obtain a peony pollen solution;
(B) Stirring and centrifuging the peony pollen solution, collecting supernatant, adding ammonium sulfate solid into the supernatant, stirring and centrifuging, collecting precipitate, adding phosphate buffer solution into the precipitate for dissolving, and performing ultrafiltration to obtain a peony pollen crude protein solution;
(C) And (3) carrying out vacuum freeze drying on the peony pollen crude protein solution to obtain peony pollen protein.
Preferably, in the step (A), the freezing temperature is-30 to-60 ℃, and the freezing time is 12 to 24 hours.
Preferably, in the step (A), the ultrasonic intensity is 200 to 600w, and the ultrasonic time is 10 to 40min.
Preferably, in the step (A), the feed-liquid ratio of the peony pollen to the hot water is 1g (10-30) mL.
Preferably, in the step (B), the stirring temperature is 30-50 ℃, and the stirring time is 4-6 h.
Preferably, in the step (B), the centrifugal rotating speed is 6000 to 8000r/min, and the centrifugal time is 10 to 20min.
Preferably, in the step (B), the pH of the phosphate buffer is 5.0 to 7.0.
Preferably, in step (B), ultrafiltration is performed using a 100kDa ultrafiltration membrane.
Preferably, in the step (C), the temperature of vacuum freeze drying is-60 ℃ to-40 ℃, and the vacuum freeze drying time is 6 to 8h.
In a second aspect of the invention, a peony pollen protein polypeptide is provided.
In a third aspect of the invention, an application of a peony pollen protein polypeptide in preparing a product for reducing blood pressure is provided.
The invention has the beneficial effects that:
the invention adopts the steps of extracting crude proteins of peony pollen after wall breaking treatment of the peony pollen, fermenting and hydrolyzing the crude proteins of the peony pollen, and drying to obtain peony pollen protein polypeptide. In the fermentation process, a mixed fermentation mode of bacillus subtilis and bacillus licheniformis is adopted, so that the process of polypeptide formation by crude proteolysis is effectively accelerated, and meanwhile, the extraction rate of peony pollen protein polypeptide can be ensured.
Wherein, the bacillus subtilis can be fermented to produce neutral protease, the bacillus licheniformis can be fermented to produce alkaline protease, and crude peony pollen protein can be effectively converted to form polypeptide under the synergistic effect of the neutral protease and the alkaline protease. Meanwhile, the method adopts a temperature difference-ultrasonic composite wall breaking mode, can effectively separate proteins, lipid, cellulose and the like in the peony pollen, improves the wall breaking rate of the peony pollen, and enables the peony pollen proteins to be better extracted in subsequent operations.
The peony pollen protein polypeptide prepared by the invention well maintains the nutritional ingredients in the peony pollen protein polypeptide, converts substances which are difficult to be absorbed by a human body into small molecules which are easy to be digested and absorbed by the human body through a fermentation method, ensures the substances to be digested and absorbed by the human body quickly, and provides energy required by the body.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Specifically, the method comprises the following steps: the bacillus licheniformis is purchased from China center for culture collection and management of industrial microorganisms, and the strain number is CICC 25064; the bacillus subtilis is purchased from China center for culture collection and management of industrial microorganisms, and the strain number is CICC 10087.
Example 1
(1) Putting peony pollen into a grinder for grinding, freezing for 24h at-30 ℃, and then adding hot water, wherein the material-liquid ratio of the ground and frozen peony pollen to the hot water is 1g:10mL, and carrying out ultrasonic treatment for 40min under the ultrasonic intensity of 200w to obtain a peony pollen solution;
(2) Stirring the peony pollen solution at 30 ℃ for 6h, centrifuging at 6000r/min for 20min, filtering, taking supernatant, adding ammonium sulfate solid into the supernatant step by step to ensure that the saturation of the ammonium sulfate reaches 20%, 40%, 60% and 80% in sequence, centrifuging at 6000r/min for 20min after stirring each time, collecting precipitates at each stage, adding phosphate buffer solution with the pH value of 5.0 into the precipitates, and performing ultrafiltration by using a 100KDa ultrafiltration membrane to obtain a crude protein solution;
(3) Vacuum drying the crude protein solution at-60 deg.C for 8 hr to obtain peony pollen protein;
(4) Mixing the bacillus subtilis seed solution and the bacillus licheniformis seed solution in equal volume to obtain a mixed seed solution, inoculating the mixed seed solution into a peony pollen protein fermentation culture solution according to 2% of the volume of the peony pollen protein fermentation culture solution, fermenting in a constant-temperature water bath for 20 hours at the temperature of 30 ℃ and the rotating speed of a shaking table of 200r/min, and sterilizing in a boiling water bath after the fermentation is finished;
wherein the viable count of Bacillus subtilis in the mixed seed solution is 1.6 × 10 8 cfu/mL, viable count of Bacillus licheniformis of 2.0X 10 8 cfu/mL。
The peony pollen protein fermentation culture solution is prepared by the following method: dissolving 8 parts of peony pollen protein, 0.2 part of glucose, 0.5 part of sodium chloride and 0.01 part of camphorsulfonic acid in 160 parts of distilled water, adjusting the pH value to 6.6, and sterilizing at 110 ℃ for 30min.
(5) Centrifuging the sterilized fermentation culture solution at 6000r/min for 20min, collecting supernatant, filtering with 0.45 μm filter membrane, ultrafiltering with 10kDa ultrafiltration membrane, and spray drying the filtrate to obtain peony pollen protein polypeptide.
Wherein the spray drying conditions were 0.08 atm, 70 ℃.
Example 2
(1) Putting peony pollen into a grinder for grinding, freezing at-60 ℃ for 12h, and adding hot water, wherein the material-liquid ratio of the ground and frozen peony pollen to the hot water is 1g:30mL, and carrying out ultrasonic treatment for 10min under the ultrasonic intensity of 600w to obtain a peony pollen solution;
(2) Stirring the peony pollen solution at 50 ℃ for 4h, centrifuging at 8000r/min for 10min, filtering, taking supernatant, adding ammonium sulfate solid into the supernatant step by step to ensure that the saturation of the ammonium sulfate reaches 20%, 40%, 60% and 80% in sequence, centrifuging at 6000r/min for 20min after stirring every time, collecting precipitates at all stages, adding phosphate buffer solution with the pH value of 7.0 into the precipitates, and performing ultrafiltration by using an ultrafiltration membrane of 100KDa to obtain a crude protein solution;
(3) Vacuum drying the crude protein solution at-40 deg.C for 6h to obtain peony pollen protein;
(4) Mixing the bacillus subtilis seed solution and the bacillus licheniformis seed solution in equal volume to obtain a mixed seed solution, inoculating the mixed seed solution into a peony pollen protein fermentation culture solution according to 3% of the volume of the peony pollen protein fermentation culture solution, fermenting in a constant-temperature water bath for 9 hours at 38 ℃ under the condition that the rotating speed of a shaking table is 240r/min, and sterilizing in a boiling water bath after the fermentation is finished;
wherein the viable count of Bacillus subtilis in the mixed seed solution is 1.8 × 10 8 cfu/mL, viable count of Bacillus licheniformis is 2.2 × 10 8 cfu/ mL。
The peony pollen protein fermentation culture solution is prepared by the following method: dissolving 10 parts of peony pollen protein, 0.3 part of glucose, 0.7 part of sodium chloride and 0.02 part of camphorsulfonic acid in 180 parts of distilled water, adjusting the pH value to 6.8, and sterilizing at 120 ℃ for 20min.
(5) Centrifuging the sterilized fermentation culture solution at 8000r/min for 10min, collecting supernatant, filtering with 0.45 μm filter membrane, ultrafiltering with 10kDa ultrafiltration membrane, and vacuum freeze drying the filtrate at-55 deg.C to-40 deg.C to obtain peony pollen protein polypeptide.
Example 3
(1) Putting peony pollen into a grinder for grinding, freezing at-45 ℃ for 18h, and adding hot water, wherein the material-liquid ratio of the ground and frozen peony pollen to the hot water is 1g:20mL, and carrying out ultrasonic treatment for 25min under the ultrasonic intensity of 400w to obtain a peony pollen solution;
(2) Stirring the peony pollen solution for 5 hours at 40 ℃, centrifuging for 15min at 7000r/min, filtering, taking supernatant, adding ammonium sulfate solid into the supernatant step by step to ensure that the saturation of the ammonium sulfate reaches 20%, 40%, 60% and 80% in sequence, centrifuging for 20min at 6000r/min after stirring each time, collecting precipitates at each stage, adding phosphate buffer solution with the pH value of 6.0 into the precipitates, and performing ultrafiltration by using a 100KDa ultrafiltration membrane to obtain a crude protein solution;
(3) Vacuum drying the crude protein solution at-50 deg.C for 7h to obtain peony pollen protein;
(4) Mixing the bacillus subtilis seed solution and the bacillus licheniformis seed solution in equal volume to obtain a mixed seed solution, inoculating the mixed seed solution into a peony pollen protein fermentation culture solution according to 3% of the volume of the peony pollen protein fermentation culture solution, fermenting in a constant-temperature water bath for 14 hours at 34 ℃ under the condition that the rotating speed of a shaking table is 220r/min, and sterilizing in a boiling water bath after the fermentation is finished;
wherein the viable count of Bacillus subtilis in the mixed seed solution is 1.7 × 10 8 cfu/mL, viable count of Bacillus licheniformis is 2.1 × 10 8 cfu/ mL。
The peony pollen protein fermentation culture solution is prepared by the following method: dissolving 9 parts of peony pollen protein, 0.25 part of glucose, 0.6 part of sodium chloride and 0.015 part of camphorsulfonic acid in 170 parts of distilled water, adjusting the pH to 6.7, and sterilizing at 115 ℃ for 25min.
(5) Centrifuging the sterilized fermentation culture solution at 7000r/min for 15min, collecting supernatant, filtering with 0.45 μm filter membrane, ultrafiltering with 10kDa ultrafiltration membrane, and spray drying the filtrate to obtain peony pollen protein polypeptide.
Wherein the spray drying conditions are 0.2 atm, 130 ℃.
Comparative example 1
(1) Putting peony pollen into a grinder for grinding, freezing at-45 ℃ for 18h, and adding hot water, wherein the material-liquid ratio of the ground and frozen peony pollen to the hot water is 1g:20mL, and carrying out ultrasonic treatment for 25min under the ultrasonic intensity of 400w to obtain a peony pollen solution;
(2) Stirring the peony pollen solution for 5 hours at 40 ℃, centrifuging for 15min at 7000r/min, filtering, taking supernatant, adding ammonium sulfate solid into the supernatant step by step to ensure that the saturation of the ammonium sulfate reaches 20%, 40%, 60% and 80% in sequence, centrifuging for 20min at 6000r/min after stirring each time, collecting precipitates at each stage, adding phosphate buffer solution with the pH value of 6.0 into the precipitates, and performing ultrafiltration by using a 100KDa ultrafiltration membrane to obtain a crude protein solution;
(3) Vacuum drying the crude protein solution at-50 deg.C for 7h to obtain peony pollen protein;
(4) Inoculating the bacillus subtilis seed solution into a peony pollen protein fermentation culture solution according to 3% of the volume of the peony pollen protein fermentation culture solution, fermenting in a constant-temperature water bath for 14h at 34 ℃ and at a table shaking speed of 220r/min, and sterilizing in a boiling water bath after fermentation is finished;
wherein the viable count of the seed liquid of the bacillus subtilis is 1.7 multiplied by 10 8 cfu/ mL。
The peony pollen protein fermentation culture solution is prepared by the following method: dissolving 9 parts of peony pollen protein, 0.25 part of glucose, 0.6 part of sodium chloride and 0.015 part of camphorsulfonic acid in 170 parts of distilled water, adjusting the pH to 6.7, and sterilizing at 115 ℃ for 25min.
(5) Centrifuging the sterilized fermentation culture solution at 7000r/min for 15min, collecting supernatant, filtering with 0.45 μm filter membrane, ultrafiltering with 10kDa ultrafiltration membrane, and spray drying the filtrate to obtain peony pollen protein polypeptide.
Wherein the spray drying conditions are 0.2 atm, 130 ℃.
Comparative example 2
(1) Putting peony pollen into a grinder for grinding, freezing at-45 ℃ for 18 hours, and then adding hot water, wherein the material-liquid ratio of the ground and frozen peony pollen to the hot water is 1g: performing ultrasonic treatment for 25min at the ultrasonic intensity of 400w for 20mL to obtain a peony pollen solution;
(2) Stirring the peony pollen solution for 5 hours at 40 ℃, centrifuging for 15min at 7000r/min, filtering, taking supernatant, adding ammonium sulfate solid into the supernatant step by step to ensure that the saturation of the ammonium sulfate reaches 20%, 40%, 60% and 80% in sequence, centrifuging for 20min at 6000r/min after stirring each time, collecting precipitates at each stage, adding phosphate buffer solution with the pH value of 6.0 into the precipitates, and performing ultrafiltration by using a 100KDa ultrafiltration membrane to obtain a crude protein solution;
(3) Vacuum drying the crude protein solution at-50 deg.C for 7h to obtain peony pollen protein;
(4) Inoculating the bacillus licheniformis seed solution into the peony pollen protein fermentation culture solution according to 3% of the peony pollen protein fermentation culture solution in volume, fermenting in a constant-temperature water bath for 14h at 34 ℃ and with the rotating speed of a shaking table of 220r/min, and sterilizing in a boiling water bath after the fermentation is finished;
wherein the viable count of Bacillus licheniformis seed liquid is 2.1 × 10 8 cfu/ mL。
The preparation method of the peony pollen protein fermentation culture solution comprises the following steps: dissolving 9 parts of peony pollen protein, 0.25 part of glucose, 0.6 part of sodium chloride and 0.015 part of camphorsulfonic acid in 170 parts of distilled water, adjusting the pH to 6.7, and sterilizing at 115 ℃ for 25min.
(5) Centrifuging the sterilized fermentation culture solution at 7000r/min for 15min, collecting supernatant, filtering with 0.45 μm filter membrane, ultrafiltering with 10kDa ultrafiltration membrane, and spray drying the filtrate to obtain peony pollen protein polypeptide.
Wherein the spray drying conditions are 0.2 atm, 130 ℃.
Test example 1
(1) Adding water into the peony pollen protein polypeptides prepared in the example 3, the comparative example 1 and the comparative example 2 to prepare sample solutions with the same concentration;
(2) Respectively taking 2.5mL of sample solutions of example 3, comparative example 1 and comparative example 2, adding 2.5mL of 10% trichloroacetic acid (TCA) aqueous solution, uniformly mixing on a vortex mixer, standing for 10min, centrifuging for 15min at 4000r/min, and obtaining supernatant fluid to obtain active polypeptide;
(3) All the active polypeptides were transferred to a 50mL volumetric flask, and the volume was made up to the mark with 5% TCA and shaken up. And then respectively placing 6.0mL of the solution into another test tube, adding 4.0mL of biuret reagent, wherein the volume ratio of the sample solution to the biuret reagent is 3:2, uniformly mixing on a vortex mixer, standing for 10min, centrifuging at 2000r/min for 10min, taking supernatant, measuring OD value at 540min, and contrasting a standard curve to obtain the polypeptide concentration C (mg/mL) in the sample solution, thereby obtaining the polypeptide content in the sample solution.
Wherein, the standard curve is: y = 0.3681x +0.0013 (R) 2 =1)。
Polypeptide purity (%) = [ (polypeptide content in sample solution x volume of sample solution)/sample mass ] × 100%,
the sample mass is the mass of the peony pollen protein polypeptides prepared in example 3, comparative example 1 and comparative example 2.
TABLE 1 polypeptide content and polypeptide purity in example 3, comparative example and comparative example 2
As can be seen from Table 1, the content of the peony pollen polypeptide prepared by the peony pollen protein polypeptide in example 3 is much higher than that of the peony pollen polypeptide prepared by fermenting only Bacillus subtilis in comparative example 1 and that prepared by fermenting only Bacillus licheniformis in comparative example 2, and meanwhile, the purity of the peony pollen protein polypeptide prepared in example 3 is higher than that of the peony pollen protein polypeptides in comparative examples 1 and 2. Therefore, the purity of the peony pollen protein polypeptide is obviously improved under the synergistic enhancement effect of neutral protease produced by bacillus subtilis and alkaline protease produced by bacillus licheniformis in the fermentation process.
Test example 2
The following experiments were carried out with reference to the procedure in example 4 animal experiments in patent CN 107699599B.
14-17 week old male Spontaneous Hypertension (SHR) rats were selected for the test and weighed 280 + -20 g. The SHR rats were purchased from experimental animals Breeding of Jinnanpunyue, inc. The temperature of the breeding environment is 22 + -2 deg.C, and the humidity is 40-70%.12h, alternately illuminating in light and shade, and freely taking food and drinking water. After the SHR rats are adaptively fed for one week, the experiment is carried out, and the SHR rats with the Systolic Blood Pressure (SBP) of more than 170mmHg are selected for the pressure reduction experiment.
Selecting 30 SHR rats into 3 groups for carrying out the gavage test, and respectively gavage distilled water, the peony pollen protein polypeptide in the embodiment 3, the peony pollen protein polypeptide in the comparative example 1 and the peony pollen protein polypeptide in the comparative example 2 for 1 time every day, wherein the gavage dosage is 3mg/kg & BW. The gavage was continued for 6 weeks, and SBP (systolic blood pressure) was measured once a week, and the results are shown in table 2.
TABLE 2 SBP values of SHR rats after gastric lavage
As can be seen from the table 2, along with the increase of the intragastric administration time, the systolic pressure of SHR rats of the peony pollen protein polypeptides in the intragastric administration example 3 and the comparative examples 1-2 is obviously reduced compared with that of SHR rats of the intragastric administration distilled water group, and the systolic pressure value of the peony pollen protein polypeptide in the intragastric administration example 3 is lower than that of the peony pollen protein polypeptides in the comparative examples 1-2, so that the peony pollen protein polypeptide has a remarkable blood pressure reducing effect and can be used for preparing blood pressure reducing products, and meanwhile, the blood pressure reducing effect of the peony pollen protein polypeptide prepared by mixed fermentation of bacillus subtilis and bacillus licheniformis is better than that of the peony pollen protein polypeptide fermented by a single strain.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A preparation method of peony pollen protein polypeptide is characterized by comprising the following steps:
(1) Inoculating bacillus subtilis and bacillus licheniformis into a peony pollen protein fermentation culture solution, fermenting for 9 to 18h at the temperature of 30 to 40 ℃, and after the fermentation is finished, sterilizing in a boiling water bath;
(2) Centrifuging the sterilized peony pollen protein fermentation culture solution, taking supernatant, filtering, taking filtrate, and drying to obtain peony pollen protein polypeptide;
the peony pollen protein fermentation culture solution is composed of the following raw materials in parts by weight: 8 to 10 parts of peony pollen protein, 0.2 to 0.3 part of glucose, 0.5 to 0.7 part of sodium chloride, 0.01 to 0.02 part of camphorsulfonic acid and 160 to 180 parts of distilled water.
2. The method for preparing peony pollen protein polypeptide according to claim 1, wherein in step (2), the centrifugation speed is 6000 to 8000r/min, and the centrifugation time is 10 to 20min.
3. The method for preparing a peony pollen protein polypeptide according to claim 1, wherein in step (2), the drying method is spray drying or vacuum freeze drying.
4. The method for preparing a peony pollen protein polypeptide according to claim 1, wherein in the step (2), the filtration method comprises: filtration was performed using a 0.45 μm filter membrane and then ultrafiltration was performed using a 10kDa ultrafiltration membrane.
5. The method of claim 1, wherein the peony pollen protein polypeptide is prepared by the following steps:
(A) Crushing peony pollen, freezing, adding hot water, stirring, and performing ultrasonic treatment to obtain a peony pollen solution;
(B) Stirring and centrifuging the peony pollen solution, collecting supernatant, adding ammonium sulfate solid into the supernatant, stirring and centrifuging, collecting precipitate, adding phosphate buffer solution into the precipitate for dissolving, and performing ultrafiltration to obtain a peony pollen crude protein solution;
(C) And (3) carrying out vacuum freeze drying on the peony pollen crude protein solution to obtain peony pollen protein.
6. The method for preparing the peony pollen protein polypeptide according to claim 5, wherein in the step (A), the freezing temperature is-30 to-60 ℃, and the freezing time is 12 to 24h;
the ultrasonic treatment time is 10 to 40min, and the ultrasonic intensity is 200 to 600w.
7. The method for preparing a peony pollen protein polypeptide according to claim 5, wherein in the step (B), the stirring temperature is 30 to 50 ℃, and the stirring time is 4 to 6 hours;
the centrifugal speed is 6000 to 8000r/min, and the centrifugal time is 10 to 20min.
8. The method for preparing the peony pollen protein polypeptide as claimed in claim 5, wherein in step (C), the temperature of vacuum freeze drying is-60 to-40 ℃, and the time of vacuum freeze drying is 6 to 8h.
9. A peony pollen protein polypeptide produced by the method of claim 1~8.
10. Use of the peony pollen protein polypeptide of claim 9 in the preparation of a blood pressure lowering product.
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