CN110183512B - Patinopecten yessoensis dipeptide, virtual screening method thereof and preparation method of composite gel of patinopecten yessoensis dipeptide - Google Patents
Patinopecten yessoensis dipeptide, virtual screening method thereof and preparation method of composite gel of patinopecten yessoensis dipeptide Download PDFInfo
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- CN110183512B CN110183512B CN201910391757.0A CN201910391757A CN110183512B CN 110183512 B CN110183512 B CN 110183512B CN 201910391757 A CN201910391757 A CN 201910391757A CN 110183512 B CN110183512 B CN 110183512B
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- carrageenan
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a comb scallop dipeptide, which consists of M, K amino acid residues, and has the molecular weight of 277.38Da; mixing the patinopecten yessoensis dipeptide and the iota-carrageenan according to a certain ratio, and preparing a composite gel by heating, swelling and cooling the gel; meanwhile, the invention discloses a virtual screening method of the patinopecten yessoensis dipeptide, which comprises the following steps: preprocessing raw materials, analyzing SDS-PAGE, identifying protein mass spectrum, screening protein sequences, analyzing in silico, screening target peptides, and preparing synthetic peptides to obtain the patinopecten yessoensis dipeptide. The preparation method has the advantages of simple steps, convenient operation, economy, time and labor saving, and the prepared composite gel has the characteristics of high elastic performance and strong gel strength, has high nutritional value, can be used as a raw material of edible colloid industry, enriches the types of edible colloid products, is easy to be absorbed by human bodies, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of ocean resource development and utilization and processing, in particular to patinopecten yessoensis dipeptide, a virtual screening method thereof and a preparation method of composite gel thereof.
Background
The food system is a multi-component complex system, and protein and polysaccharide are two important natural high molecular polymers. The protein has amphiphilic unique structure and surface activity, but has certain limitation in industrial production application, because the protein can be denatured when being heated, and the denaturation leads to random polymerization and coagulation, so that the stability of food is reduced, and the quality and shelf life of the food are further influenced. As a food additive, the food gum has excellent characteristics of gelling, water retention, thickening, emulsion stability promotion and the like, is mainly derived from algae, plants, animals and microorganisms, has chemical compositions of natural polysaccharides, proteins and derivatives, and is widely distributed in the nature. In recent years, the research on polysaccharides and proteins has been intensive, and new analytical means such as rheology and microstructure have been introduced to develop a highly effective study on the reaction mechanism, structure, functional properties, etc. of protein-polysaccharide complexes.
The scallop is a precious sea food with high economic and nutritional values, tastes delicious and is rich in various nutrients. The artificial culture of scallops in China starts in 1968, the scale of scallop culture is continuously enlarged along with the continuous progress of culture increasing technology, the yield is increased year by year, the scallop culture becomes a key economic shellfish variety for coastal aquaculture in China, and the culture has obtained obvious economic benefit. In 2017, the scallop culture yield in China is up to 201 ten thousand tons. With the expansion of scallop cultivation scale and the improvement of yield, the demand of scallop processed products is also increased. The protein content in the male gonad of the Japanese scallop can reach about 87 percent of the dry basis of the Japanese scallop, and the Japanese scallop can be used as one of protein intake sources, and the discovery has important social and economic values for the development of the gonad function of the scallop; in addition, the content of essential amino acid is also very rich, which accounts for about 42 percent of the whole amino acid content, and the peptide is a good source for developing active functional peptide.
Carrageenans (carrageenans), also known as carrageenans, belong to the group of hydrophilic gel polysaccharides. It is a linear glycan with 1,3-glycosidic bond connecting beta-D-galactopyranyl and 1,4-glycosidic bond connecting alpha-D-galactopyranyl as basic unit. Carrageenan can be classified into seven types, kappa-, mu-, iota-, theta-, lambda-, epsilon-, v-depending on whether galactose contains the number and the linking position of the lactone group and the sulfate group on galactose. The most common and highest yielding carrageenans are the kappa-, iota-and lambda-types. Wherein the content of iota-sulfate groups is high, the gel is a random coil in the solution, and the formed gel is transparent and elastic.
Meanwhile, the patinopecten yessoensis protein has the phenomenon of accelerating gel formation after enzymolysis, and the enzymolysis degree and the generation amount of product peptide have positive regulation effect on the gel strength, which indicates that the patinopecten yessoensis hydrolysate is a gel system taking peptide molecules as a main body. The carrageenan serving as a typical effective procoagulant factor has a synergistic effect with the patinopecten yessoensis zymolyte to form gel. However, the process of preparing the zymolyte is complicated, the purity of the obtained peptide is low, and the key peptide molecules determining the gelation behavior of the peptide are in urgent need of further research.
In recent years, with the development of bioinformatics technology, in silico (in) analysis has been widely used in the identification of active peptide molecules. The method omits the steps of purification and the like in the traditional natural product extraction process, greatly improves the efficiency of finding new peptides, and makes the whole research of a peptide molecule research system changed from fine decomposition research possible. The use of the analog (in silico) assay is premised on two aspects, namely, the presence of a known protein sequence on the one hand and a well-defined cleavage site for the selected tool protease on the other hand. At present, the genomic sequence of Japanese scallop is disclosed, and protein sequence information can be obtained by a Nano-LC-MS/MS means; trypsin is a commonly used tool enzyme in bioinformatics software and has a well-defined cleavage site.
Disclosure of Invention
The invention aims to develop a method for obtaining male comb shell composite gel peptide by using an in silico technology. According to the driving force of the gelation formed by the patinopecten yessoensis genome sequence and the peptide, a computer online simulation method capable of obtaining the gel peptide is developed, the gel peptide and the iota-carrageenan are reacted to form the composite gel, the steps of purification and the like in the traditional natural product extraction process are omitted, the efficiency of finding new peptides is greatly improved, the nutritional value of a mixed colloid is improved, and the defect of single nutrition of the existing colloid is overcome.
In order to achieve the aim, the invention provides patinopecten yessoensis dipeptide which consists of M, K2 amino acid residues, has the molecular weight of 277.38Da, and has the amino acid sequence shown as SEQ ID: 1; the patinopecten yessoensis dipeptide is derived from the gonad of male patinopecten yessoensis.
The simulated screening method of the patinopecten yessoensis dipeptide comprises the following steps:
s1, raw material pretreatment: taking the gonad of male Japanese scallop, homogenizing until no granules exist to obtain homogenate;
s2, SDS-PAGE analysis: taking the homogenate obtained in the step S1 to perform SDS-PAGE electrophoresis;
s3, protein mass spectrum identification: cutting off a clear single protein band on the SDS-PAGE gel obtained in the step S2 to obtain patinopecten yessoensis protein, and performing protein mass spectrum identification;
s4, protein sequence screening: according to the result of the protein mass spectrum identification in the step S3, screening out protein sequences which belong to the species of the patinopecten yessoensis and have the molecular weights of 47.97KDa-67.97KDa, 6.99KDa-26.99KDa, 5.46KDa-25.46KDa and 4.07KDa-24.07KDa respectively;
s5, in silico analysis: selecting Trypsin (Trypsin) as a parameter to perform simulated enzymolysis by using a BIOPEP on-line tool (www.uwm.edu.pl/biochemia/index. Php/pl/BIOPEP) to obtain a product peptide;
s6, screening of target peptides: screening cationic peptide with isoelectric point pI of more than 7.0 and positive charge and good solubility at pH of 7.0 to obtain target peptide sequence, wherein the amino acid sequence of the target peptide is shown as SEQ ID: 1;
s7, synthetic peptide: and (4) performing solid phase method in chemical method on the target peptide sequence obtained in the step (S6) to synthesize the peptide, wherein the purity of the peptide is more than 90%, so as to obtain the patinopecten yessoensis dipeptide.
Preferably, the homogenate of step S1 is specifically: 2000-4000 rpm for 10-15 min.
Preferably, the SDS-PAGE analysis in step S2 specifically is: mixing the homogenate obtained in the step S1 with a sample loading buffer solution in an equal volume, heating at 100 ℃ for 5-10min, oscillating at 200-500 rpm for 10-14h at 25 ℃, centrifuging at 10000g for 5-10 min, and taking supernate; taking the supernatant, diluting the supernatant by 2 times, 4 times and 8 times respectively by using a sample loading buffer solution, and carrying out SDS-PAGE detection on 10 mu L of the obtained diluted sample; performing SDS-PAGE detection by using 10% separation gel and 5% concentrated gel, wherein the current of the concentrated gel is 8mA, and the current of the separation gel is 15mA, after electrophoresis is finished, performing overnight dyeing by using 0.05% Coomassie brilliant blue R-250, and decoloring by using 9% glacial acetic acid and 50% ethanol solution;
wherein the loading buffer comprises the following components: 950 mu L A liquid, 50 mu L beta-mercaptoethanol; the liquid A comprises the following components: 1.25mL Tris-HCl (pH 6.8,0.5M), 2.5mL glycerol, 2mL mass fraction w/v 10% SDS;
the patinopecten yessoensis dipeptide can be used for preparing composite gel; a preparation method of patinopecten yessoensis dipeptide/iota-carrageenan composite gel comprises the following steps:
s1, preparing an iota-carrageenan aqueous solution: dissolving iota-carrageenan in water to prepare a carrageenan aqueous solution with the concentration of 12-15 mg/mL;
s2, preparing composite gel: adding patinopecten yessoensis dipeptide into the iota-carrageenan aqueous solution prepared in the step (S1), and uniformly mixing to obtain patinopecten yessoensis dipeptide/iota-carrageenan composite gel; the concentration of the patinopecten yessoensis dipeptide/iota-carrageenan composite gel is 0.1-0.3 mol/L; wherein the amino acid sequence of the patinopecten yessoensis dipeptide is shown as SEQ ID: 1;
s3, exhausting bubbles: and (3) removing air bubbles from the patinopecten yessoensis dipeptide/iota-carrageenan composite gel prepared in the step (S2) to obtain a final product.
Preferably, the temperature for the dissolution in step S1 is 60 to 70 ℃.
Preferably, the method for discharging bubbles in step S3 includes: centrifuging the composite gel obtained in the step S2 at 2500-5000 rpm for 5-10 min to remove bubbles in the mixed solution; standing at 4-7 ℃ for 8-16 h to obtain the final product.
The beneficial effects of the invention are:
1. the gonad is a by-product in the processing and production process of the patinopecten yessoensis, although the patinopecten yessoensis is edible, the utilization rate is lower, the invention improves the utilization rate of the gonad of the patinopecten yessoensis, and makes full use of the components of the protein of the patinopecten yessoensis, and the patinopecten yessoensis dipeptide/iota-carrageenan mixed gel prepared by the method can be used as a nutritional gel preparation to be applied to various foods.
2. The analog (in silico) analysis technology selected by the method of the invention is widely applied to the identification of active peptide molecules. The method omits the steps of purification and the like in the traditional natural product extraction process, and greatly improves the efficiency of discovering new peptides.
3. In the method, the prepared patinopecten yessoensis dipeptide is mixed with iota-carrageenan, so that the gel property is obviously enhanced.
4. The invention improves the gel property of male Japanese scallop dipeptide, because the protein contained in the gonad of male Japanese scallop is subjected to in silico simulated enzymolysis to generate a large amount of small molecular peptide, the small molecular peptide is easier to be absorbed by human body, and the product has trophism. In addition, the mixed colloid has unique food functionality-gel property, and can be used as a novel gel agent to be added into food in food processing production to improve the gel property and nutrition of the product.
5. The invention has simple operation process, does not need complex equipment and has better effect of improving the gel property of male patinopecten yessoensis dipeptide.
The method utilizes in silico trypsin to simulate enzymolysis of the gonad of the patinopecten yessoensis, effectively improves the gel characteristic of the dipeptide of the patinopecten yessoensis by the methods of screening peptide sequences and mixing with carrageenan solution, and has the characteristics of simple method and good effect.
Drawings
FIG. 1 is an SDS-PAGE analysis of the gonad of Japanese scallop prepared according to the embodiment of the present invention; HM: high marker, LM: low marker;
FIG. 2 is the number distribution of peptides with positive and negative charges obtained from in silico of the gonad of Japanese scallop in example 1 of the present invention;
FIG. 3 is the number distribution of different soluble peptides in the positive charged peptide obtained from the gonad of Patinopecten yessoensis through in silico in example 1 of the present invention;
FIG. 4 shows the molecular weight distribution of the cationic peptide with good solubility obtained from in silico of gonad of Japanese scallop in example 1 of the present invention;
fig. 5 is a frequency scanning mode rheological property of patinopecten yessoensis dipeptide/iota-carrageenan composite gel prepared in example 3 of the present invention;
FIG. 6 is a schematic representation of the arginine/iota-carrageenan complex gel prepared in example 2 of the present invention;
FIG. 7 is a schematic representation of the lysine/iota-carrageenan complex gel prepared in example 2 of the present invention;
FIG. 8 is a pictorial representation of the TK/iota-carrageenan complex gel prepared in example 3 of the present invention;
FIG. 9 is a pictorial representation of a WK/iota carrageenan complex gel made in example 3 of the present invention;
fig. 10 is a physical representation of LK/iota-carrageenan complex gel prepared in example 3 of the present invention;
fig. 11 is a pictorial representation of the GK/iota-carrageenan complex gel prepared in example 3 of the present invention;
FIG. 12 is a schematic representation of a VK/iota-carrageenan complex gel prepared in example 3 of the present invention;
FIG. 13 is a pictorial representation of a CK/iota-carrageenan complex gel prepared in example 3 of the present invention;
FIG. 14 is a pictorial representation of the NK/iota-carrageenan complex gel prepared in example 3 of this invention;
fig. 15 is a pictorial view of the FK/iota-carrageenan complex gel prepared in example 3 of the present invention;
FIG. 16 is a pictorial representation of an MK/iota carrageenan complex gel prepared in example 3 of the invention;
fig. 17 is a physical representation of the iota-carrageenan aqueous solution prepared in comparative example 1 of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples.
The experimental procedures used in the following examples are conventional unless otherwise specified.
Materials and the like used in the following examples are commercially available unless otherwise specified.
In order to achieve the above purpose, the invention provides a method for obtaining comb shell composite gel peptide based on in silico, comprising the following steps:
1. a method for obtaining comb shell composite gel peptide based on in silico comprises the following steps:
s1, raw material pretreatment: taking the gonad of male Japanese scallop out of the whole scallop, and homogenizing.
S2, SDS-PAGE analysis: pretreating the homogenate of the gonad of the male patinopecten yessoensis, injecting the pretreated sample into a sample hole, carrying out SDS-PAGE analysis by a certain current, taking down the gel, and dyeing and decoloring;
s3, cutting the glue: cutting off clear bands on the gel prepared in the step S2, and performing protein mass spectrometry identification;
s4, protein sequence screening: screening the protein sequence identified in the step S3 according to species, protein name and molecular weight;
s5, in silico analysis: carrying out simulated enzymolysis on the protein sequence obtained after the screening in the step S4 by using a BIOPEP online tool;
s6, screening of target peptides: screening the product peptide obtained in the step S5 according to isoelectric points, electrostatic charges and solubility to obtain a target peptide sequence;
s7, synthetic peptide: and (4) performing peptide synthesis on the target peptide sequence obtained in the step S6.
S8, preparing an iota-carrageenan aqueous solution: adding water into iota-carrageenan to prepare a solution;
s9, preparing composite gel: adding the synthetic peptide powder obtained in the step S7 into the iota-carrageenan aqueous solution obtained in the step S8 respectively, and uniformly mixing to obtain patinopecten yessoensis dipeptide/iota-carrageenan composite gel;
s10, exhausting bubbles: and (4) removing air bubbles from the patinopecten yessoensis dipeptide/iota-carrageenan composite gel prepared in the step (S9) to obtain a final product.
Preferably, the SDS-PAGE analysis in step S2 specifically is: mixing 10% of separation glue and 5% of concentrated glue; mixing the homogenate of the male Japanese scallop prepared in the step S1 with the sample buffer solution in equal volume, boiling the sample for 5-10 min, shaking up overnight at room temperature, and centrifuging for 5-10 min (10000 Xg); taking supernatant, diluting by 2, 4 and 8 times respectively, injecting into a sample hole, concentrating gel current by 8mA, and separating gel current by 15mA to perform SDS-PAGE detection; after completion of the electrophoresis, the gel was stained with 0.05% Coomassie Brilliant blue R-250 overnight, and then decolorized with 9% glacial acetic acid and 50% ethanol solution.
Wherein the loading buffer is: 950. Mu. L A, 50. Mu.L of beta-mercaptoethanol.
Wherein the solution A is: 1.25mL Tris-HCl (pH 6.8,0.5M), 2.5mL glycerol, 2mL SDS (10%).
In a preferable mode, the step S3 of cutting the rubber specifically includes: selecting the more clearly defined protein from the gel prepared in step S2The strips (Patinopecten yessoensis-1, patinopecten yessoensis-2, patinopecten yessoensis-3, patinopecten yessoensis-4) are marked with standard markers to estimate molecular weights (57.97 KDa, 16.99KDa, 15.46KDa, 14.07 KDa), and cut into small pieces (1 mm) with a blade 3 ) And performing mass spectrum identification.
Preferably, the protein sequence screening in step S4 specifically comprises: identifying protein sequence information from the step S3, and screening out protein sequences of the same species (Japanese scallop), wherein the molecular weights of the protein sequences are respectively in the standard molecular weight ranges of the Japanese scallop-1 (47.97 KDa-67.97 KDa), the Japanese scallop-2 (6.99 KDa-26.99 KDa), the Japanese scallop-3 (5.46 KDa-25.46 KDa) and the Japanese scallop-4 (4.07 KDa-24.07 KDa) in the step S2;
in a preferred embodiment, the step S6 of screening the target peptide specifically includes: the product Peptide obtained in step S5 was analyzed for isoelectric point, net charge and solubility using computer pI/Mw (http:// web. Expay. Org/computer _ pI /) in ExPASY and Peptide property calculator on-line tool of INNOVAGEN (http:// www.innovagen.com/proteomics-tools), and the cationic Peptide with good solubility was screened. The screened peptide sequences all contain arginine (R) or lysine (K), and peptides containing lysine (K) are further screened according to the action of arginine or lysine/iota-carrageenan.
Preferably, the synthetic peptide of step S7 is specifically: and (4) submitting the sequence information of the target peptide screened in the step (S6) to Shanghai Qiangyao biotechnology limited, and entrusting peptide synthesis to ensure that the purity is over 90-95%.
In a preferred mode, the step S8 of preparing the iota-carrageenan aqueous solution specifically comprises the following steps: adding water to dissolve the carrageenan, controlling the dissolving temperature at 60-70 ℃ and preparing the carrageenan aqueous solution with the concentration of 12-15 mg/mL.
Preferably, in the preparation of the patinopecten yessoensis dipeptide/iota-carrageenan composite gel in the step S9, the concentration of the patinopecten yessoensis dipeptide is 0.1 to 0.3mol/L.
Preferably, the method for discharging bubbles in step S10 includes: centrifuging the composite gel prepared in the step S9 at 2500-5000 rpm for 5-10 min to remove bubbles in the mixed solution; standing at 4-7 ℃ for 8-16 h to obtain the composite gel.
Example 1
The simulated screening method of the patinopecten yessoensis dipeptide comprises the following steps:
s1, raw material pretreatment: taking the gonad of male Japanese scallop, homogenizing at 2000rpm for 10min until no granules exist to obtain homogenate;
s2, SDS-PAGE analysis: mixing the homogenate obtained in the step S1 with a sample loading buffer solution in the same volume, heating at 100 ℃ for 5-10min, vibrating at 200rpm for 12h at 25 ℃, centrifuging at 10000g for 10min, and taking supernate; taking the supernatant, diluting the supernatant by 2 times, 4 times and 8 times respectively by using a sample loading buffer solution, and carrying out SDS-PAGE detection on 10 mu l of the obtained diluted sample; performing SDS-PAGE detection by using 10% separation gel and 5% concentrated gel, wherein the current of the concentrated gel is 8mA, and the current of the separation gel is 15mA, after electrophoresis is finished, performing overnight dyeing by using 0.05% Coomassie brilliant blue R-250, and decoloring by using 9% glacial acetic acid and 50% ethanol solution; wherein the loading buffer is: 950 mu L A liquid, 50 mu L beta-mercaptoethanol; the solution A is as follows: 1.25mL Tris-HCl (pH 6.8,0.5M), 2.5mL glycerol, 2mL SDS (10%); the electrophoresis result is shown in FIG. 1, where HM and LM are high marker and low marker, respectively;
s3, protein mass spectrum identification: cutting a clear single protein strip on the SDS-PAGE gel obtained in the step (2) to obtain Patinopecten yessoensis protein, wherein the Patinopecten yessoensis protein has four strips which are respectively named as Patinopecten yessoensis-1, patinopecten yessoensis-2, patinopecten yessoensis-3 and Patinopecten yessoensis-4, and the molecular weights of the Patinopecten yessoensis-1, the Patinopecten yessoensis-2, the Patinopecten yessoensis-3 and the Patinopecten yessoensis-4 are respectively as follows: 57.97KDa, 16.99KDa, 15.46KDa and 14.07KDa, and performing protein mass spectrum identification on the Japanese scallops-1, 2, 3 and 4;
s4, protein sequence screening: according to the protein mass spectrum identification result obtained in the step S3, protein sequences belonging to the patinopecten yessoensis protein and having molecular weights respectively in the standard molecular weight ranges of patinopecten yessoensis-1 (47.97 KDa-67.97 KDa), patinopecten yessoensis-2 (6.99 KDa-26.99 KDa), patinopecten yessoensis-3 (5.46 KDa-25.46 KDa) and patinopecten yessoensis-4 (4.07 KDa-24.07 KDa) are screened out; the screening results are shown in table 1:
TABLE 1 Mass Spectrometry results
S5, in silico analysis: selecting Trypsin (Trypsin) as a parameter to carry out simulated enzymolysis on the protein sequence obtained after screening in the step S4 by using a BIOPEP on-line tool (www.uwm.edu.pl/biochemia/index. Php/pl/BIOPEP), so as to obtain a product peptide;
s6, screening of target peptides: respectively analyzing isoelectric points, net charges and solubility of the product Peptide by utilizing computer pI/Mw (http:// web. Expay. Org/computer _ pI /) in ExPASY and Peptide property calculator online tools (http:// www.innovagen.com/properties-tools) of INNOVAGEN, screening cationic peptides with isoelectric points pI of 7.0, positive charges at pH of 7.0 and good solubility; the peptide sequences selected all contained arginine (R) or lysine (K). The screening results are shown in table 2:
TABLE 2 Patinopecten yessoensis dipeptide screening results
Example 2
1. Preparing lysine/iota-carrageenan composite gel by using lysine, comprising the following steps:
s1, preparing an iota-carrageenan aqueous solution: 0.0213g iota-carrageenan, adding 1.7mL deionized water, heating at 65 deg.C for 15min to obtain 12.5mg/mL iota-carrageenan aqueous solution;
s2, preparing composite gel: adding 0.0497g of lysine (K) screened in the step S6 in the example 1 into the iota-carrageenan aqueous solution prepared in the step S1, and uniformly mixing to prepare lysine/iota-carrageenan composite gel with the patinopecten yessoensis dipeptide concentration of 0.2 mol/L;
s3, exhausting bubbles: centrifuging the composite gel prepared in the step S2 at 5000rpm for 10min to remove air bubbles in the mixed solution, and standing at 4 ℃ for 16h to obtain a lysine/iota-carrageenan composite gel product (K/iota-C).
2. Preparing arginine/iota-carrageenan composite gel by using arginine, comprising the following steps:
s1, preparing an iota-carrageenan aqueous solution: 0.0213g iota-carrageenan, adding 1.7mL deionized water, heating at 65 deg.C for 15min to obtain 12.5mg/mL iota-carrageenan aqueous solution;
s2, preparing composite gel: adding 0.0497g of arginine (R) screened in the step S6 in the embodiment 1 into the iota-carrageenan aqueous solution prepared in the step S1, and uniformly mixing to prepare the arginine/iota-carrageenan composite gel with the patinopecten yessoensis dipeptide concentration of 0.2 mol/L;
s3, exhausting bubbles: centrifuging the composite gel prepared in the step S2 at 5000rpm for 10min to remove air bubbles in the mixed solution, and standing at 4 ℃ for 16h to obtain an arginine/iota-carrageenan composite gel product.
Through the observation of fig. 6 and 7, the gel effect of the lysine/iota-carrageenan composite gel is much higher than that of the arginine/iota-carrageenan composite gel, so that the gel characteristic of the patinopecten yessoensis dipeptide/iota-carrageenan composite gel is further judged by selecting dipeptide with lysine (K).
According to analysis of a rheometer, the lysine/iota-carrageenan composite gel prepared in the embodiment has the storage modulus G' (frequency scanning mode) of 141Pa and shows elastic characteristics under the frequency scanning and the fixed stress of 0.5% and the frequency range of 0.1-10 Hz. Therefore, according to the dipeptide sequence of Patinopecten yessoensis in Table 3, 9 Patinopecten yessoensis dipeptides were synthesized using a chemical solid phase synthesis method, and the purity of the peptides was more than 90%.
TABLE 3 Patinopecten yessoensis dipeptide screening results
Example 3
The preparation method for preparing patinopecten yessoensis dipeptide/iota-carrageenan composite gel by respectively using the 9 patinopecten yessoensis dipeptides prepared in the embodiment 2 comprises the following steps:
s1, preparing an iota-carrageenan aqueous solution: 0.0213g iota-carrageenan, adding 1.7mL deionized water, heating at 65 deg.C for 15min to obtain 12.5mg/mL iota-carrageenan aqueous solution;
s2, preparing composite gel: adding patinopecten yessoensis dipeptide into the iota-carrageenan aqueous solution obtained in the step S1 to prepare patinopecten yessoensis dipeptide/iota-carrageenan composite gel with the patinopecten yessoensis dipeptide concentration of 0.2 mol/L; wherein the patinopecten yessoensis dipeptide is one of TK, WK, LK, GK, VK, CK, NK, FK or MK;
s3, exhausting bubbles: centrifuging the composite gel prepared in the step S2 at 5000rpm for 10min to remove air bubbles in the mixed solution, and standing at 4 ℃ for 16h to obtain the patinopecten yessoensis dipeptide/iota-carrageenan composite gel product.
The gel characteristics of the patinopecten yessoensis dipeptide/iota-carrageenan composite gel product are shown in table 4, and the MK/iota-carrageenan composite gel has the highest gel strength.
TABLE 4 gel characteristics of patinopecten yessoensis dipeptide/iota-carrageenan complex gel
According to analysis of a rheometer, the patinopecten yessoensis dipeptide/iota-carrageenan composite gel prepared in the embodiment has the storage modulus G' (frequency scanning mode) of 4332Pa and shows elastic characteristics under the frequency scanning and at the fixed stress of 0.5% and the frequency range of 0.1-10 Hz; .
Comparative example 1
S1, preparing an iota-carrageenan aqueous solution: 0.0213g iota-carrageenan, adding 1.7mL deionized water, heating at 65 deg.C for 15min to obtain 12.5mg/mL iota-carrageenan aqueous solution;
s2, exhausting bubbles: and (3) centrifuging the iota-carrageenan aqueous solution prepared in the step (S1) at 6000rpm for 5min to remove air bubbles, and standing at 4 ℃ for 12h to obtain the iota-carrageenan gel product (iota-C).
The iota-carrageenan gel prepared in this example exhibited an elastic behavior with a storage modulus G' (frequency sweep mode) of 13Pa under a frequency sweep at a fixed stress of 0.5% and a frequency in the range of 0.1 to 10Hz, as analyzed by a rheometer.
The SDS-PAGE method is adopted to obtain the protein sequence of Japanese scallop in the embodiment of the invention, and the result is shown in figure 1: four strips are obtained, and 14 protein sequences are obtained through mass spectrum identification and screening according to NanoLC-ESI-MS/MS; the gel formation is carried out on the premise that the gel is soluble in water, and because the iota-carrageenan tape is negatively charged, the gel property is generated by electrostatic interaction with the comb shell dipeptide which is positively charged, as can be proved by the graphs in fig. 2 and 3, the comb shell dipeptide is formed into a composite gel due to the action of the cationic peptide with good solubility and the iota-carrageenan; fig. 4 found that the content of peptides in the range of 0.2 to 0.5KDa was large, and the peptides in this range consisted of 2 to 4 amino acids, and thus the complex coacervation of dipeptide and iota-carrageenan was intensively studied.
The invention adopts in silico to screen out that all peptide sequences contain arginine (R) or lysine (K), and iota-carrageenan is respectively compared with the two for observing the gel strength, and the results are shown in figures 6 and 7: lysine/iota carrageenan produces significant gelling upon interaction, while arginine/iota carrageenan does not exhibit gelling; the strength of the lysine-bearing comb shell dipeptide/iota-carrageenan composite gel prepared in the embodiment of the invention is measured, and the result is shown in fig. 6: the gel strength of the composite gel MK/iota-carrageenan prepared in the embodiment 3 of the invention is obviously greater than that of the iota-carrageenan. The patinopecten yessoensis dipeptide MK and the iota-carrageenan have a certain synergistic effect, so that the gel property of the patinopecten yessoensis dipeptide MK and the iota-carrageenan is enhanced.
According to analysis of a rheometer, under a frequency scanning mode, when the fixed stress is 0.5% and the fixed frequency is 0.1-10Hz, the rheological property response values of iota-carrageenan are very low, the storage modulus G 'value is 13Pa, the storage modulus G' value of lysine/iota-carrageenan is 141Pa, the experiments show elastic characteristics, and the rheological property of the patinopecten yessoensis dipeptide can be enhanced by adding iota-carrageenan; the storage modulus G' (frequency scanning mode) of the patinopecten yessoensis dipeptide MK/iota-carrageenan composite gel prepared in the embodiment 3 of the invention is 4332Pa. The rheological property of the patinopecten yessoensis dipeptide MK/iota-carrageenan composite gel prepared by the invention is obviously higher than that of the single iota-carrageenan and lysine/iota-carrageenan composite gel.
Rheological properties of the complex gel prepared in the example of the present invention and iota-carrageenan alone were measured, respectively, and the storage modulus G' under frequency sweep, which is an indicator of the elasticity of the reaction substance, was measured, while the MK contains lysine (K), so the rheological properties of lysine/iota-carrageenan were used as a control. The results are shown in fig. 5, the rheological properties of the composite gel prepared by the invention are significantly higher than those of iota-carrageenan and lysine/iota-carrageenan, which indicates that the comb shell dipeptide MK and iota-carrageenan have a synergistic effect, and iota-carrageenan can greatly enhance the rheological properties of comb shell dipeptide, namely the gel properties.
At present, the research on the aspect of the gel characteristics of peptide/carrageenan is less, wei Peng uses natural edible gums with certain physiological functions, namely carrageenan, konjac glucomannan and locust bean gum, as raw materials, and selects a sol-gel system capable of stabilizing soybean polypeptide by researching the gel performance and rheological performance of a compound system and utilizing the interaction between polysaccharide and polypeptide; in the experiment, the kappa-carrageenan commonly used for preparing the jelly is selected and compounded with the konjac gum and the locust bean gum, and through orthogonal experiments, the water holding capacity, the gel strength and the viscosity are used as the same important evaluation indexes, so that the better mass ratio of the kappa-carrageenan, the konjac gum and the locust bean gum is determined; the stress control rheometer is used for researching the fluid types of the mixed colloidal solutions with different concentrations, and the influence of concentration, temperature, pH value, sucrose, sodium chloride, sodium citrate, soybean polypeptide and freeze-thaw treatment on the viscosity of the system is systematically examined; a chemical method capable of detecting the content of the soybean polypeptide in the soybean polypeptide raw material and the jelly is established, and the stability of the soybean polypeptide in the jelly in the preparation and storage processes of the jelly is evaluated on the basis. Selig et al, treated whey protein isolate with whey protransglutaminase in heated kappa-carrageenan slurry, observed that the strength of the gel, deformability and translucency may be related to the size of the peptides present at the time of gel formation; as the size of the peptide decreases, all three aspects increase. As a result of the above analysis, the complex gel prepared by the in silico method of the present invention is simpler, more easily available with peptides, and has more remarkable improvement in gel properties than the complex gel prepared by the method of Wei Peng or Selig.
In conclusion, the rheological property of the patinopecten yessoensis dipeptide MK/iota-carrageenan mixed gel prepared by the invention is obviously higher than that of a comparison sample.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Sequence listing
<110> university of Dalian Industrial university
<120> Patinopecten yessoensis dipeptide, virtual screening method thereof and preparation method of composite gel thereof
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<170> SIPOSequenceListing 1.0
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Claims (3)
1. A preparation method of patinopecten yessoensis dipeptide/iota-carrageenan composite gel is characterized by comprising the following steps:
s1, preparing an iota-carrageenan aqueous solution: dissolving iota-carrageenan in water to prepare a carrageenan aqueous solution with the concentration of 12-15 mg/mL;
s2, preparing composite gel: adding patinopecten yessoensis dipeptide into the iota-carrageenan aqueous solution prepared in the step (S1), and uniformly mixing to obtain patinopecten yessoensis dipeptide/iota-carrageenan composite gel; the concentration of the patinopecten yessoensis dipeptide/iota-carrageenan composite gel is 0.1-0.3 mol/L; the amino acid sequence of the patinopecten yessoensis dipeptide is shown as SEQ ID: 1;
s3, exhausting bubbles: and (3) removing air bubbles from the patinopecten yessoensis dipeptide/iota-carrageenan composite gel prepared in the step (S2) to obtain a final product.
2. The method for preparing patinopecten yessoensis dipeptide composite gel/iota-carrageenan composite gel according to claim 1, wherein the dissolving temperature in the step S1 is 60-70 ℃.
3. The preparation method of the patinopecten yessoensis dipeptide composite gel/iota-carrageenan composite gel according to claim 1, wherein the air bubbles discharged in the step S3 are specifically: centrifuging the patinopecten yessoensis dipeptide/iota-carrageenan composite gel obtained in the step S2 at 2500-5000 rpm for 5-10min, and standing at 4-7 ℃ for 8-16 h to obtain a final product.
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