CN117778511A - Preparation method and application of royal jelly protein hypoglycemic peptide powder - Google Patents
Preparation method and application of royal jelly protein hypoglycemic peptide powder Download PDFInfo
- Publication number
- CN117778511A CN117778511A CN202410201927.5A CN202410201927A CN117778511A CN 117778511 A CN117778511 A CN 117778511A CN 202410201927 A CN202410201927 A CN 202410201927A CN 117778511 A CN117778511 A CN 117778511A
- Authority
- CN
- China
- Prior art keywords
- royal jelly
- enzymolysis
- hypoglycemic
- peptide
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 85
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 85
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 84
- 229940109850 royal jelly Drugs 0.000 title claims abstract description 79
- 230000002218 hypoglycaemic effect Effects 0.000 title claims abstract description 70
- 239000000843 powder Substances 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 26
- 108090000790 Enzymes Proteins 0.000 claims abstract description 26
- 108010033276 Peptide Fragments Proteins 0.000 claims abstract description 22
- 102000007079 Peptide Fragments Human genes 0.000 claims abstract description 22
- 230000000694 effects Effects 0.000 claims abstract description 19
- 238000004108 freeze drying Methods 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 12
- 108010067902 Peptide Library Proteins 0.000 claims abstract description 11
- 230000001105 regulatory effect Effects 0.000 claims abstract description 10
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims abstract description 9
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 3
- 229940088598 enzyme Drugs 0.000 claims description 34
- 239000000463 material Substances 0.000 claims description 25
- 102100024295 Maltase-glucoamylase Human genes 0.000 claims description 22
- 102000057297 Pepsin A Human genes 0.000 claims description 22
- 108090000284 Pepsin A Proteins 0.000 claims description 22
- 108010028144 alpha-Glucosidases Proteins 0.000 claims description 22
- 229940111202 pepsin Drugs 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 108091005658 Basic proteases Proteins 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 102000004142 Trypsin Human genes 0.000 claims description 16
- 108090000631 Trypsin Proteins 0.000 claims description 16
- 230000002401 inhibitory effect Effects 0.000 claims description 16
- 239000012588 trypsin Substances 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 230000029087 digestion Effects 0.000 claims description 12
- 238000009835 boiling Methods 0.000 claims description 7
- 238000003760 magnetic stirring Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 235000013376 functional food Nutrition 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 239000012460 protein solution Substances 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 230000000415 inactivating effect Effects 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 16
- 238000005516 engineering process Methods 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 9
- 230000005764 inhibitory process Effects 0.000 abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000003513 alkali Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 37
- 239000000243 solution Substances 0.000 description 25
- 238000011282 treatment Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- 238000012549 training Methods 0.000 description 8
- 238000012545 processing Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 230000002496 gastric effect Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 238000005199 ultracentrifugation Methods 0.000 description 5
- 102000004157 Hydrolases Human genes 0.000 description 4
- 108090000604 Hydrolases Proteins 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 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 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000012827 research and development Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- TXBNDGDMWKVRQW-UHFFFAOYSA-M sodium;2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]azaniumyl]acetate;dodecyl sulfate Chemical compound [Na+].OCC(CO)(CO)NCC(O)=O.CCCCCCCCCCCCOS([O-])(=O)=O TXBNDGDMWKVRQW-UHFFFAOYSA-M 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 description 1
- 240000007054 Avena nuda Species 0.000 description 1
- 235000007317 Avena nuda Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003888 alpha glucosidase inhibitor Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004252 chorionic villi Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000002790 cross-validation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000000275 quality assurance Methods 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Landscapes
- Jellies, Jams, And Syrups (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method and application of royal jelly protein hypoglycemic peptide powder, and relates to the field of functional peptides and special medical foods. The preparation method of the royal jelly protein hypoglycemic peptide powder mainly comprises the steps of taking protein in fresh royal jelly as a raw material, sequentially stirring, regulating acid and alkali, adding enzyme, centrifuging, and freeze-drying an enzymolysis liquid supernatant to obtain the royal jelly peptide powder; further adopting various food enzyme-linked technologies to improve the inhibition activity of the royal jelly peptide powder on the alpha-glycosidase; and (3) calculating enzymolysis peptide fragments, and combining a glycosidase peptide library and an LBSPepPreactor tool to identify the key efficacy peptide fragments. Compared with the prior art, the prepared royal jelly protein hypoglycemic peptide powder has the advantages that the inhibition rate of alpha-glycosidase is improved by 8.59 times, the inhibition rate is far superior to that of single enzyme or double enzyme, 7 high-activity hypoglycemic peptides are identified, theoretical guidance is provided for the deep development and utilization of royal jelly protein resources and the research of hypoglycemic effect products, and the effect peptide is clear to provide basis for the application of the royal jelly protein hypoglycemic peptide powder in the fields of functionality and special medical foods.
Description
Technical Field
The invention relates to the technical field of functional peptide and special medical food processing, in particular to a preparation method and application of royal jelly protein hypoglycemic peptide powder.
Background
Type 2 diabetes (T2 DM) is a chronic metabolic disease affecting more than 90% of diabetics, where irregular life rhythm and increased working pressure are important factors in the pathogenesis of T2 DM. Alpha-glucosidase is considered as an effective target for preventing and treating T2DM, and is mainly distributed on the brush border of the small intestine epithelium chorionic villus and is responsible for decomposing polysaccharides in food, such as starch and glycoside, to produce glucose and other monosaccharides that can be absorbed by the small intestine. Thus, inhibition of α -glucosidase helps control and lower blood glucose.
Compared with the traditional alpha-glucosidase inhibitor, the polypeptide with the alpha-glucosidase inhibiting activity is more green, healthy and safer through enzymolysis treatment of the protein, and the raw materials can be directly eaten as common food, and can be used as food auxiliary materials for the research and development of the hypoglycemic peptide powder or functional food.
Royal jelly is a highly favored source of high-quality functional proteins in the insect food field, and the key active ingredients of royal jelly account for a significant proportion (over 80% of its total protein content), called royal jelly proteins. In recent years, many researches and reports mainly focus on the biological activity of royal jelly, and particularly, the royal jelly is popular in the market and consumer's favor as a functional food in enhancing immunity and resisting aging. However, royal jelly proteins are extremely sensitive to temperature and acid, spontaneously aggregate to form fibrous aggregates during food processing and storage, and further are digested by the gastrointestinal tract, which restricts nutrient absorption, high-value utilization and functional food development of the royal jelly proteins.
At present, the preparation method of the royal jelly protein hypoglycemic peptide powder is not yet researched and reported, and the deep processing utilization aspect of the royal jelly protein hypoglycemic peptide powder is basically in a stagnation state. The prior invention patent CN108949887B, CN107868810B, CN105713943B respectively provides preparation methods of soybean hypoglycemic peptide powder, sweet potato active peptide powder and hulless oat hypoglycemic peptide powder, the enzymolysis process in the methods is limited to simple single or double enzyme treatment, and the gradual multi-enzyme combination is not considered to improve the hypoglycemic efficiency of the peptide powder, and the prior patent does not identify hypoglycemic components in the peptide powder.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides royal jelly protein hypoglycemic peptide powder, a preparation method and application thereof, wherein the method aims at inhibiting the activity of alpha-glucosidase, improves the hypoglycemic efficiency of the peptide powder by a step-by-step multi-enzyme-linked technology, and promotes the deep processing and functional research and development of the royal jelly protein. Meanwhile, the invention also identifies the hypoglycemic component in the royal jelly protein hypoglycemic peptide powder, which provides reference value and powerful guarantee for the efficacy research of the royal jelly protein hypoglycemic peptide powder.
In order to achieve the above object, the technical scheme of the present invention is realized by the following technical scheme:
a preparation method of royal jelly protein hypoglycemic peptide powder comprises the following steps:
s1, pretreatment of queen bee protein: selecting queen bee protein, dissolving in water, stirring and mixing uniformly, and regulating the pH value to 3-9 to obtain a pretreated protein solution;
s2, preliminary enzymolysis: adding alkaline protease into the pretreated protein solution for preliminary digestion and enzymolysis to obtain a preliminary enzymolysis material for later use;
s3, secondary enzymolysis: adding sodium chloride and hydrochloric acid into the primary enzymolysis material, regulating the pH value to be 1-3, and then adding pepsin for continuous enzymolysis to obtain a secondary enzymolysis material for later use;
s4, three times of enzymolysis: adding sodium hydroxide and potassium dihydrogen phosphate into the secondary enzymolysis material, regulating the pH to 6.5-9, and adding trypsin to continue enzymolysis and digestion to obtain a tertiary enzymolysis material;
s5, inactivating enzymes by boiling water for the three enzymatic hydrolysis materials, and centrifuging and freeze-drying to obtain the royal jelly protein hypoglycemic peptide powder;
s6, calculating enzymolysis peptide fragments of the royal jelly protein hypoglycemic peptide powder, and identifying key efficacy peptide fragments by combining a glycosidase peptide library and an LBSPep predictor tool.
Preferably, in the step S1, the ratio of the queen bee protein dissolved in water to the water is 10-50:100-500, and the uniformly stirring and mixing mode is that magnetic stirring is used for stirring for 1-30 minutes at 400-1200 r/min so as to fully hydrate the queen bee protein.
Preferably, the amount of the alkaline protease added in the step S2 is 0.5-5 mg/ml, and the enzymolysis condition is that the pH is 8.5-9.5, the temperature is 35-50 ℃, and the enzymolysis is carried out for 2-6h.
Preferably, the amount of the added enzyme of the pepsin in the step S3 is 0.5-10 mg/mL, and the enzymolysis condition is that the enzymolysis is carried out for 2-6 hours at the temperature of 35-50 ℃.
Preferably, the trypsin in the step S4 is added in an amount of 10mg/ml, the temperature of trypsin enzymolysis is 35-50 ℃, and the enzymolysis time is 2-6h.
Preferably, the centrifugal freeze-drying mode in the step S5 is that the centrifugal freeze-drying is carried out for 10-45 min at the centrifugal speed of 6000-12000 rpm, and then the sediment is removed to freeze-dry the clear supernatant.
Preferably, in the step S6, proteomics information of fresh royal jelly is sequentially introduced into a PeptideMass tool to obtain a small molecule peptide library with a sequence length of less than 5-10.
Preferably, the glycosidase peptide library in the step S6 comprises a hypoglycemic peptide confirmed by experiments, and the glycosidase peptide library has the structure as follows:
Dataset={“Sequence”:X,
“Activate”:Y,
“label”:Q},
wherein the method comprises the steps of
X=[x1,x2,x3,x4,...,xi],Y=[y1,y2,y3,y4,...,yi],Q=[q1,q2,q3,q4,...,qi],
Wherein i represents the number of peptide fragments in the database; x1 represents the first peptide fragment, the sequence of which expresses 20 kinds of ammoniaAny two or more combinations of base acids; y1 represents the value of the alpha-glucosidase inhibitory activity of the first peptide fragment, which means in particular the inhibitory activity of the alpha-glucosidase inhibitory peptide on the alpha-glucosidase, in particular the half inhibitory concentration of the enzyme activity measured, i.e.IC 50 A value; q1 represents the activity class of the first peptide fragment.
Preferably, the LBSPeppredictor is a highly active alpha-glucosidase inhibitory peptide prediction tool developed using CNN and a transducer algorithm.
And the royal jelly protein hypoglycemic peptide powder is applied to preparing functional food.
The invention provides royal jelly protein hypoglycemic peptide powder and a preparation method and application thereof, and has the advantages compared with the prior art that:
the invention aims at inhibiting the activity of alpha-glucosidase, improves the blood glucose reducing efficiency of peptide powder by a step-by-step multiple enzyme-linked technology, and promotes the deep processing and functional research and development of royal jelly protein. Meanwhile, the invention also identifies the hypoglycemic component in the royal jelly protein hypoglycemic peptide powder, which provides a reference value for the efficacy research of the royal jelly protein hypoglycemic peptide powder and a technical controllable scheme for the quality assurance of the royal jelly protein hypoglycemic peptide powder.
Drawings
FIG. 1 shows the results of Tricine-SDS electrophoresis of multi-enzyme hydrolyzed and non-hydrolyzed royal jelly proteins according to the examples, wherein: the first lane on the left side is a protein molecular weight standard, and the 3 lanes on the right side of the first lane are samples after multi-enzyme enzymolysis; the 3 lanes on the far right are samples that were not digested;
FIG. 2 is a lyophilized sample after stepwise multi-enzyme enzymolysis in the example;
FIG. 3 is a reconstitution test of lyophilized samples after stepwise multi-enzyme enzymolysis in the examples;
FIG. 4 shows the inhibitory activity of the example multienzyme digested and unenzymatic samples on alpha-glucosidase.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
the royal jelly protein hypoglycemic peptide powder (alkaline protease + pepsin + trypsin) is prepared by a multienzyme enzymolysis technology:
s1, accurately weighing 30g of a sample, dissolving the sample in 300mL deionized water, and stirring the sample for 10 minutes under the condition of 400 rpm by using magnetic stirring to fully hydrate the sample;
s2, adding alkaline protease according to 5mg/ml by using 1M hydrochloric acid and sodium hydroxide to adjust the pH of a protein sample to 9.0, and respectively digesting for 2 hours at the temperature of 45 ℃ after uniformly mixing to obtain a first enzymolysis material;
s3, performing pepsin digestion on the first enzymolysis material for 2 hours: regulating the pH to 2 by adopting 2.0 g/L sodium chloride solution and 2.917 g/L hydrochloric acid solution, adding pepsin according to 10mg/mL, and continuing enzymolysis for 2 hours to obtain a second enzymolysis material;
s4, regulating the pH value of the second enzymolysis material to 7.5 by adopting 0.616 g/L sodium hydroxide solution and 6.8 g/L monopotassium phosphate solution, and then adding trypsin according to 10mg/mL, and continuing enzymolysis for 4 hours; after enzymolysis is combined, placing the enzymolysis liquid in boiling water to quickly inactivate hydrolase;
s5, performing ultracentrifugation treatment on the enzymolysis liquid at 10000rpm for 30 minutes, pouring the clarified enzymolysis liquid into a glass plate, and freeze-drying the plate in a freeze dryer at-80 ℃ for 48 hours to obtain the royal jelly protein hypoglycemic peptide powder.
Comparative example 1:
single enzyme enzymolysis technology for preparing royal jelly protein hypoglycemic peptide powder (alkaline protease):
s1, accurately weighing 30g of a sample, dissolving the sample in 300mL deionized water, and stirring the sample for 10 minutes under the condition of 400 rpm by using magnetic stirring to fully hydrate the sample;
s2, adding alkaline protease into a 1M hydrochloric acid and sodium hydroxide protein-regulating sample ph of 9.0 according to 5mg/ml, uniformly mixing and respectively digesting for 2 hours at 45 ℃ to obtain an enzymolysis material, and after enzymolysis is combined, placing the enzymolysis liquid into boiling water to quickly inactivate the hydrolase;
s3, performing ultracentrifugation treatment on the enzymolysis liquid at 10000rpm for 30 minutes, pouring the clarified enzymolysis liquid into a glass plate, and freeze-drying the plate in a freeze dryer at-80 ℃ for 48 hours to obtain the royal jelly protein hypoglycemic peptide powder.
Comparative example 2:
single enzyme enzymolysis technology for preparing royal jelly protein hypoglycemic peptide powder (pepsin):
s1, accurately weighing 30g of a sample, dissolving the sample in 300mL deionized water, and stirring the sample for 10 minutes under the condition of 400 rpm by using magnetic stirring to fully hydrate the sample;
s2, adopting 2.0 g/L sodium chloride solution and 2.917 g/L hydrochloric acid solution, regulating the pH value to be 2, adding pepsin according to 10mg/mL, carrying out enzymolysis digestion for 2 hours to obtain an enzymolysis material, and after enzymolysis is combined, placing the enzymolysis liquid in boiling water to quickly inactivate hydrolase;
s3, performing ultracentrifugation treatment on the enzymolysis liquid at 10000rpm for 30 minutes, pouring the clarified enzymolysis liquid into a glass plate, and freeze-drying the plate in a freeze dryer at-80 ℃ for 48 hours to obtain the royal jelly protein hypoglycemic peptide powder.
Comparative example 3:
the royal jelly protein hypoglycemic peptide powder (alkaline protease and pepsin) is prepared by a double-enzyme enzymolysis technology:
s1, accurately weighing 30g of a sample, dissolving the sample in 300mL of deionized water, and stirring the sample for 10 minutes under the condition of 400 rpm by using magnetic stirring to fully hydrate the sample;
s2, adding alkaline protease according to 5mg/ml by using 1M sodium hydroxide and a hydrochloric acid to adjust the pH value of a protein sample to 9.0, and respectively digesting for 2 hours at the temperature of 45 ℃ after uniformly mixing to obtain a first enzymolysis material;
s3, adding 2.0 g/L sodium chloride solution and 2.917 g/L hydrochloric acid solution into the first enzymolysis material, adjusting the pH to 2, adding pepsin according to 10mg/mL, performing enzymolysis and digestion for 2 hours to obtain an enzymolysis material, and after enzymolysis is combined, placing the enzymolysis liquid into boiling water to quickly inactivate hydrolytic enzymes;
s4, performing ultracentrifugation treatment on the enzymolysis liquid at 10000rpm for 30 minutes, pouring the clarified enzymolysis liquid into a glass plate, and freeze-drying the plate in a freeze dryer at-80 ℃ for 48 hours to obtain the royal jelly protein hypoglycemic peptide powder.
Comparative example 4:
the royal jelly protein hypoglycemic peptide powder (pepsin and trypsin) is prepared by a double-enzyme enzymolysis technology:
s1, accurately weighing 30g of a sample, dissolving the sample in 300mL of deionized water, and stirring the sample for 10 minutes under the condition of 400 rpm by using magnetic stirring to fully hydrate the sample;
s2, adding 2.0 g/L sodium chloride solution and 2.917 g/L hydrochloric acid solution, adjusting the pH to 2, adding pepsin according to 10mg/mL, and performing enzymolysis digestion for 2 hours to obtain an enzymolysis material;
s3, adding the enzymolysis material into 0.616 g/L sodium hydroxide solution and 6.8 g/L potassium dihydrogen phosphate solution, adjusting the pH to 7.5, adding trypsin according to 10mg/mL, and carrying out trypsin digestion for 4 hours; after enzymolysis is combined, placing the enzymolysis liquid in boiling water to quickly inactivate hydrolase;
s4, performing ultracentrifugation treatment on the enzymolysis liquid at 10000rpm for 30 minutes, pouring the clarified enzymolysis liquid into a glass plate, and freeze-drying the plate in a freeze dryer at-80 ℃ for 48 hours to obtain the royal jelly protein hypoglycemic peptide powder.
And (3) detecting the correlation performance:
1. the electrophoresis of the royal jelly protein and the untreated royal jelly protein under the enzymolysis treatment using the above-mentioned example 1 was verified:
Tricine-SDS electrophoresis detection is carried out on the enzymolysis material obtained in the step S3 in the experiment I, and the specific result is shown in figure 1 by taking the non-enzymolysis/digestion royal jelly protein as a control: compared with a sample which is not subjected to enzymolysis/digestion, the molecular weight of the royal jelly protein hypoglycemic peptide powder prepared by adopting the multienzyme enzymolysis technology is obviously reduced, and researches show that the peptide powder with low molecular weight is beneficial to gastrointestinal absorption of human bodies, namely the royal jelly protein hypoglycemic peptide powder prepared by adopting the multienzyme enzymolysis technology can be effectively improved into normal absorption efficiency.
2. The properties of the royal jelly protein hypoglycemic peptide powder prepared by the multi-enzyme enzymolysis treatment of the example 1 and the single-enzyme and double-enzyme treatments of the comparative examples 1-4 as non-enzymolysis/digestion royal jelly proteins are verified:
example 1 and comparative examples 1-4 and the non-enzymatically hydrolyzed/digested royal jelly protein properties are shown in fig. 2, in which the non-enzymatically hydrolyzed/digested sample is in the form of a viscous flocculent, and the single, double and triple enzyme-prepared royal jelly protein hypoglycemic peptide powder is in the form of a dispersed network structure (the left to right samples in the figure are respectively an alkaline protease single enzyme hydrolyzed sample, an alkaline protease + pepsin double enzyme hydrolyzed sample, an alkaline protease + pepsin + trypsin multi-enzyme hydrolyzed sample, an untreated sample, a pepsin single enzyme hydrolyzed sample, a pepsin + trypsin double enzyme hydrolyzed sample).
3. Solubility detection of the royal jelly protein hypoglycemic peptide powder:
taking the royal jelly protein hypoglycemic peptide powder of the example 1 and the comparative examples 1-4 and untreated royal jelly protein (control group) as samples; further accurately weighing each group of samples 48 and mg, and using ultrapure water of 1 mL for dissolution to evaluate the solubility of the peptide powder, wherein the result is shown in figure 3 (the dissolution effect graphs of the non-hydrolyzed protein samples, pepsin single enzyme hydrolysis samples, pepsin+trypsin double enzyme hydrolysis samples, alkaline protease single enzyme hydrolysis samples, alkaline protease+pepsin double enzyme hydrolysis samples, alkaline protease+pepsin+trypsin multi enzyme hydrolysis samples are respectively from left to right); as can be seen from FIG. 3, the royal jelly protein glycopeptide powders prepared in example 1 and comparative examples 1 to 4 have good water solubility and clarity, and the sample dissolution effect in example 1 is better than that in the case of non-enzymatic hydrolysis.
4. An inhibition activity experiment of alpha-glucosidase was performed on the royal jelly protein hypoglycemic peptide powder prepared in example 1 and comparative examples 1 to 4:
sample solutions (10.0 μl) of different concentrations were mixed in PBS solution (100.0 μl, ph=6.8) and α -glucosidase solution (10.0 μl, 7U/mL dissolved in PBS) and incubated for 15 min at 37 ℃. Then, 2.5 mmol/L of pNPG solution (10.0. Mu.L in PBS) was added thereto and incubated at 37℃for 30 minutes. Then Na was added to the reaction solution 2 CO 3 The solution (60.0. Mu.L, 20 mmol/L) was mixed well.
Absorbance values for each reaction solution were measured with a SpectraMax M5 microplate reader at 405 nm. PBS solution (10.0. Mu.L) was used as a sample blank, PBS solution (10.0. Mu.L) was used as a control, and PBS solution (20.0. Mu.L) was used as a control blank. The alpha-glucosidase inhibition rate was calculated as follows:
alpha-glucosidase inhibition ratio (%) = [1- (a sample group-a sample blank)/(a control group-a control blank) ]
Wherein A represents the absorbance.
FIG. 4 shows the results of inhibition activity of alpha-glucosidase by multienzyme enzymatically and unenzymatic samples, wherein preferential treatment with alkaline protease helps to increase the activity of royal jelly protein hypoglycemic peptide powder, wherein the activity of alkaline gastric double enzyme treated royal jelly protein hypoglycemic peptide powder is increased by 4.79 times, and the activity of alkaline gastric triple enzyme treated royal jelly protein hypoglycemic peptide powder is increased by 8.59 times, compared to the unenzymatic sample. Because pepsin and trypsin are human gastrointestinal digestive enzymes, the results show that the royal jelly protein hypoglycemic peptide powder prepared by the method provided by the invention has better hypoglycemic effect and gastrointestinal stability.
5. Based on the detection, peptide fragments obtained after enzymolysis in the example 1 are obtained by adopting PeptideMass, and alpha-glucosidase inhibitory peptides in the royal jelly protein hypoglycemic peptide powder obtained in the example 1 are identified and identified by using a glycosidase peptide library and an LBSPepPreactor tool;
wherein the architecture of the glycosidase peptide library is:
Dataset={“Sequence”:X,
“Activate”:Y,
“label”:Q},
wherein the method comprises the steps of
X=[x1,x2,x3,x4,...,xi],Y=[y1,y2,y3,y4,...,yi],Q=[q1,q2,q3,q4,...,qi],
i represents the number of peptide fragments in the database, x1 represents the first peptide fragment, its sequence expresses any two or more combinations of 20 amino acids (indicated by the capital letters of amino acids), y1 represents the value of alpha-glucosidase Inhibition (IC) corresponding to the first peptide fragment 50 Value), q1 represents the activity class of the first peptide fragment (tags 0 and 1, wherein a tag of 0 represents high activityPeptide fragment, 1 represents a low or no active peptide fragment).
The build of the LBSPepPreactor tool is:
(1) dividing the high activity peptide data set into a training set and a test set by using sklearn. Model_selection. StratifiedShuffleSplice hierarchical sampling, wherein the ratio of the training set to the test set is 8:2;
(2) the method comprises the steps of adopting ESM-2 to read the characteristics of polypeptides in a training set, generating 320 characteristic vectors for each polypeptide, generating 320 characteristic vector diagrams, creating a standardized example by using sklearn.preprocessing.Standard scaler, carrying out standardized processing on the characteristic vector diagrams of the training set, and mapping a standardized example model to a test set;
(3) further adopting CNN and a transducer algorithm model to carry out 5-time cross validation training on the training set;
(4) predicting and evaluating the test set by using a trained and optimized model, predicting the verification set by using a training set model after model training is finished to obtain a verification set prediction label, calculating the real label and the prediction label of the verification set by using a sklearn. Metrics. Fusion_matrix function to obtain a confusion matrix result, and calculating ACC and AUC values;
(5) and selecting an optimal model from the cross verification according to the ACC value and the AUC value, and predicting and evaluating the test set so as to screen out a final model which is LBSPeppredictor.
The results are shown in Table 1:
table 1 shows the identification of active peptide fragments in the hypoglycemic peptide powder
As is clear from the above table, 7 high activity peptide fragments, GSR, LW, IW, TW, IF, PR, CL respectively, were identified in total in royal jelly protein hypoglycemic peptide powder, wherein PR had the strongest inhibitory activity on alpha-glucosidase of 19.79. Mu.M.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The preparation method of the royal jelly protein hypoglycemic peptide powder is characterized by comprising the following steps of:
s1, pretreatment of queen bee protein: selecting queen bee protein, dissolving in water, stirring and mixing uniformly, and regulating the pH value to 3-9 to obtain a pretreated protein solution;
s2, preliminary enzymolysis: adding alkaline protease into the pretreated protein solution for preliminary digestion and enzymolysis to obtain a preliminary enzymolysis material for later use;
s3, secondary enzymolysis: adding sodium chloride and hydrochloric acid into the primary enzymolysis material, regulating the pH value to be 1-3, and then adding pepsin for continuous enzymolysis to obtain a secondary enzymolysis material for later use;
s4, three times of enzymolysis: adding sodium hydroxide and potassium dihydrogen phosphate into the secondary enzymolysis material, regulating the pH to 6.5-9, and adding trypsin to continue enzymolysis and digestion to obtain a tertiary enzymolysis material;
s5, inactivating enzymes by boiling water for the three enzymatic hydrolysis materials, and centrifuging and freeze-drying to obtain the royal jelly protein hypoglycemic peptide powder;
s6, calculating enzymolysis peptide fragments of the royal jelly protein hypoglycemic peptide powder, and identifying key efficacy peptide fragments by combining a glycosidase peptide library and an LBSPep predictor tool.
2. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 1, which is characterized in that: in the step S1, the ratio of the queen bee protein dissolved in water is 10-50:100-500, and the uniformly stirring and mixing mode is that magnetic stirring is adopted for stirring for 1-30 minutes at 400-1200 r/min so as to fully hydrate the queen bee protein.
3. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 1, which is characterized in that: the addition amount of alkaline protease in the step S2 is 0.5-5 mg/ml, and the enzymolysis condition is that the pH is 8.5-9.5, the temperature is 35-50 ℃, and the enzymolysis is carried out for 2-6h.
4. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 1, which is characterized in that: the added enzyme amount of pepsin in the step S3 is 0.5-10 mg/mL, and the enzymolysis condition is that the enzymolysis is carried out for 2-6h at the temperature of 35-50 ℃.
5. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 1, which is characterized in that: the adding amount of trypsin in the step S4 is 10mg/ml, the enzymolysis temperature of trypsin is 35-50 ℃, and the enzymolysis time is 2-6h.
6. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 1, which is characterized in that: and in the step S5, the centrifugal freeze-drying mode is that the centrifugal freeze-drying is carried out for 10-45 min at the centrifugal speed of 6000-12000 rpm, and then the sediment is removed to freeze-dry the clear supernatant.
7. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 1, which is characterized in that: and in the step S6, proteomics information of fresh royal jelly is sequentially imported into a PeptideMass tool to obtain a small molecule peptide library with the sequence length of less than 5-10.
8. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 7, wherein the steps of: the glycosidase peptide library in the step S6 comprises the hypoglycemic peptide confirmed by experiments, and the glycosidase peptide library has the structure as follows:
Dataset={“Sequence”:X,
“Activate”:Y,
“label”:Q},
wherein the method comprises the steps of
X=[x1,x2,x3,x4,...,xi],Y=[y1,y2,y3,y4,...,yi],Q=[q1,q2,q3,q4,...,qi],
Wherein i represents the number of peptide fragments in the database; x1 represents a first peptide fragment whose sequence expresses any two or more combinations of 20 amino acids; y1 represents the value of the alpha-glucosidase inhibitory activity of the first peptide fragment, which means in particular the inhibitory activity of the alpha-glucosidase inhibitory peptide on the alpha-glucosidase, in particular the half inhibitory concentration of the enzyme activity measured, i.e.IC 50 A value; q1 represents the activity class of the first peptide fragment.
9. The method for preparing the royal jelly protein hypoglycemic peptide powder according to claim 8, wherein the method is characterized by comprising the following steps: the LBSPeppredictor is a highly active alpha-glucosidase inhibitory peptide prediction tool developed using CNN and the transducer algorithm.
10. A royal jelly protein hypoglycemic peptide powder prepared by the preparation method of the royal jelly protein hypoglycemic peptide powder according to any one of claims 1-6 is applied to preparing functional food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410201927.5A CN117778511A (en) | 2024-02-23 | 2024-02-23 | Preparation method and application of royal jelly protein hypoglycemic peptide powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410201927.5A CN117778511A (en) | 2024-02-23 | 2024-02-23 | Preparation method and application of royal jelly protein hypoglycemic peptide powder |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117778511A true CN117778511A (en) | 2024-03-29 |
Family
ID=90392968
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410201927.5A Pending CN117778511A (en) | 2024-02-23 | 2024-02-23 | Preparation method and application of royal jelly protein hypoglycemic peptide powder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117778511A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1070804A (en) * | 1991-09-09 | 1993-04-14 | 拜奥斯有限公司 | Process for preparing transparent royal jelly solution |
| JP2001002576A (en) * | 1999-06-22 | 2001-01-09 | Biox:Kk | Suppressant for weight increase |
| JP2001002583A (en) * | 1999-06-22 | 2001-01-09 | Biox:Kk | Blood sugar level increase inhibitor |
| JP2014068553A (en) * | 2012-09-27 | 2014-04-21 | Morikawa Kenkodo Kk | METHOD OF PRODUCING ROYAL JELLY HAVING α-GLUCOSIDASE INHIBITORY ACTION |
| CN114190561A (en) * | 2021-12-09 | 2022-03-18 | 北京中蜜科技发展有限公司 | Microencapsulated royal jelly enzymolysis polypeptide, preparation method thereof and solid powder containing microencapsulated royal jelly enzymolysis polypeptide |
| CN116479081A (en) * | 2023-06-07 | 2023-07-25 | 北京金王健康科技有限公司 | Preparation method of microencapsulated royal jelly functional peptide |
| CN117089590A (en) * | 2023-10-07 | 2023-11-21 | 暨南大学 | Royal jelly protein peptide and preparation method and application thereof |
-
2024
- 2024-02-23 CN CN202410201927.5A patent/CN117778511A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1070804A (en) * | 1991-09-09 | 1993-04-14 | 拜奥斯有限公司 | Process for preparing transparent royal jelly solution |
| JP2001002576A (en) * | 1999-06-22 | 2001-01-09 | Biox:Kk | Suppressant for weight increase |
| JP2001002583A (en) * | 1999-06-22 | 2001-01-09 | Biox:Kk | Blood sugar level increase inhibitor |
| JP2014068553A (en) * | 2012-09-27 | 2014-04-21 | Morikawa Kenkodo Kk | METHOD OF PRODUCING ROYAL JELLY HAVING α-GLUCOSIDASE INHIBITORY ACTION |
| CN114190561A (en) * | 2021-12-09 | 2022-03-18 | 北京中蜜科技发展有限公司 | Microencapsulated royal jelly enzymolysis polypeptide, preparation method thereof and solid powder containing microencapsulated royal jelly enzymolysis polypeptide |
| CN116479081A (en) * | 2023-06-07 | 2023-07-25 | 北京金王健康科技有限公司 | Preparation method of microencapsulated royal jelly functional peptide |
| CN117089590A (en) * | 2023-10-07 | 2023-11-21 | 暨南大学 | Royal jelly protein peptide and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 励建荣等: ""蜂王浆水溶性蛋白质及其水解多肽对自由基的清除能力和 对胰脂肪酶和 α-葡萄糖苷酶的抑制活性"", 《中国食品学报》, vol. 12, no. 9, 30 September 2012 (2012-09-30), pages 8 - 15 * |
| 戚向阳等: ""酶改性蛋白制备水溶性蜂王浆的研究"", 《食品工业科技》, vol. 30, no. 4, 31 December 2009 (2009-12-31), pages 144 - 147 * |
| 朱作艺;张玉;王君虹;李雪;王伟;: "蜂王浆蛋白肽的制备及其降血糖和抗氧化活性研究", 食品工业科技, vol. 41, no. 17, 31 December 2020 (2020-12-31), pages 45 - 57 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kleywegt et al. | The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 Å resolution, and a comparison with related enzymes | |
| US9689010B2 (en) | Microbial-derived chondroitin sulfate | |
| Theodore et al. | Angiotensin converting enzyme inhibition of fish protein hydrolysates prepared from alkaline‐aided channel catfish protein isolate | |
| CN108823270A (en) | A kind of extracting method of oyster peptide | |
| Katayama et al. | Solubility improvement of shellfish muscle proteins by reaction with glucose and its soluble state in low-ionic-strength medium | |
| CN102809541B (en) | Determination method of enzyme activity of collagenase | |
| Zhao et al. | Effects of soy protein hydrolysates on the growth and fermentation performances of brewer's yeast | |
| CN105473607A (en) | Bacterial hyaluronidase and process for its production | |
| CN107557422A (en) | A kind of cottonseed protein polypeptide with high ACE inhibitory activity and preparation method thereof | |
| CN116589533A (en) | Euphausia superba small molecule active peptide, and preparation method and application thereof | |
| Guo et al. | Characterization of an intracellular aspartic protease (PsAPA) from Penicillium sp. XT7 and its application in collagen extraction | |
| Yao et al. | Purification of Angiotensin‐I‐Converting Enzyme Inhibitory Peptides Derived from Camellia oleifera Abel Seed Meal Hydrolysate | |
| CN103589705A (en) | Keratinase and its coding gene | |
| Qu et al. | A novel GH family 20 β-N-acetylhexosaminidase with both chitosanase and chitinase activity from Aspergillus oryzae | |
| CN117778511A (en) | Preparation method and application of royal jelly protein hypoglycemic peptide powder | |
| US20220007682A1 (en) | Method for preparing an ipp- and vpp-rich hydrolysate from wheat gluten protein by enzymatic hydrolysis | |
| Lu et al. | Alpha-Glucosidase inhibitory peptides: Sources, preparations, identifications, and action mechanisms | |
| Tovar-Herrera et al. | Analysis of the Binding of Expansin Exl1, from Pectobacterium carotovorum, to Plant Xylem and Comparison to EXLX1 from Bacillus subtilis | |
| Liu et al. | Differential proteomic analysis using a tandem-mass-tag-based strategy to identify proteins associated with the quality indicators of Penaeus vannamei after high-pressure treatment | |
| Cui et al. | Effects of high solid concentrations on the efficacy of enzymatic hydrolysis of yeast cells and the taste characteristics of the resulting hydrolysates | |
| CN104774893A (en) | Method of gluten enzymolysis through countercurrent ultrasound treatment | |
| CN110195048B (en) | Method for improving performance of pepsin enzymatic hydrolysis whey protein product through dynamic/static high-pressure combined treatment | |
| CN116426433B (en) | Lactobacillus plantarum and application thereof in preparation of whey protein cholesterol-reducing peptide | |
| CN113735942B (en) | Recombinant hypoglycemic polypeptide and preparation method and application thereof | |
| TW202035688A (en) | Engineered chitosanase and method of producing chitobiose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |