CN107190040B - Antioxidant peptide of penaeus japonicus and preparation method thereof - Google Patents
Antioxidant peptide of penaeus japonicus and preparation method thereof Download PDFInfo
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- CN107190040B CN107190040B CN201710412246.3A CN201710412246A CN107190040B CN 107190040 B CN107190040 B CN 107190040B CN 201710412246 A CN201710412246 A CN 201710412246A CN 107190040 B CN107190040 B CN 107190040B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Engineering & Computer Science (AREA)
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Abstract
The invention relates to a shrimp cracker antioxidative peptide, the amino acid sequence of which is Gly-Asn-Gly-Leu-Pro; also relates to a preparation method of the antioxidant peptide of the Japanese shrimp, which comprises the following steps: pretreating raw materials; carrying out ultrasonic-assisted enzymolysis; performing ultrafiltration treatment; gel column treatment; and reversed phase high performance liquid chromatography treatment. Compared with the prior art, the antioxidant peptide of the bunchy shrimp has stronger antioxidant activity, takes the heads of the bunchy shrimp as a raw material, has wide sources and low cost, solves the problem of leftovers generated in the processing process of the bunchy shrimp, and has theoretical significance for further improving the economic value of the bunchy shrimp.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a shrimp nodosum antioxidant peptide and a preparation method thereof.
Background
The famous Japanese prawn (Penaeus japonicus) belongs to the family Penaeidae of the order of Tenpoda, is the prawn breeding variety with the largest breeding area and yield among three breeding shrimps in the world, and is famous and reputed as postprawns due to the gorgeous color and the speckled shape. The bamboo-joint shrimp is large in size, delicious in meat quality and is a fine product in marine products, so that the bamboo-joint shrimp is very suitable for being used as sushi shrimp and other products.
At present, the bamboo shrimp is mostly processed into the head-removed quick-frozen shrimp meat product for internal marketing or export, so that about 35 percent of the shrimp head leftovers are produced in the processing process, wherein the shrimp head meat accounts for about 30 percent of the weight of the shrimp head leftovers. Because the heads of the shrimps contain the hard sandstone components such as gastric sacs, mouthparts and the like and can not be directly eaten, how to deeply process the by-products of the heads of the shrimps in the processing of the bunchy shrimps to improve the added value of the products is a problem to be solved urgently at present. At present, most of researches on the bamboo-joint shrimps by domestic and foreign scholars are focused on the aspects of seedling culture, cultivation, biological morphology, habit and the like, for example, the invention patent of China with the application number of 201410346224.8 (the publication number of CN104115772A) discloses a freshwater cultivation pond management method for the bamboo-joint shrimps.
The antioxidant property is a new subject in the research of polypeptide bioactivity by scientists at home and abroad in recent years, and the antioxidant peptide derived from food protein not only has good antioxidant activity, but also has extremely high safety, so the antioxidant peptide has very wide application prospect in the fields of health food and biomedicine. For example: antioxidant peptide derived from food protein is disclosed in patents such as 'shark protein antioxidant peptide and preparation method and application thereof' in Chinese patent No. ZL201210230756.6 (No. CN103524596A) and 'pea protein enzymolysis antioxidant peptide preparation method and application' in Chinese patent No. ZL201310152590.5 (No. CN 103194519A). However, at present, no research report of extracting and separating antioxidant peptide by using the heads of the shrimps of the nodded shrimps is provided, and the research on the antioxidant components of the heads of the nodded shrimps is carried out, so that the leftovers of the heads of the nodded shrimps can be fully utilized, the added value of the products of the nodded shrimps is improved, and the important significance on the research of expanding the antioxidant peptide is realized.
Disclosure of Invention
The first technical problem to be solved by the invention is to provide the antioxidant peptide of the penaeus japonicus, which has strong antioxidant activity, aiming at the prior art.
The second technical problem to be solved by the invention is to provide a preparation method of the antioxidant peptide of the shrimp.
The third technical problem to be solved by the invention is to provide a method for improving the stability of the antioxidant peptide of the shrimps in the organism aiming at the prior art.
The technical scheme adopted by the invention for solving the first technical problem is as follows: the antioxidant peptide of the juvenile prawns is characterized in that the amino acid sequence of the antioxidant peptide of the juvenile prawns is Gly-Asn-Gly-Leu-Pro.
The technical scheme adopted by the invention for solving the second technical problem is as follows: the preparation method of the antioxidant peptide of the phaeodacea japonica is characterized by comprising the following steps:
(1) pretreatment of raw materials: cleaning heads of the bamboo-joint shrimp, removing lipid components, homogenizing and mincing into slurry for later use;
(2) ultrasonic-assisted enzymolysis: adding ultrapure water into the slurry to prepare a solution with a material-liquid ratio of 1: 6-14 w/v, adjusting the pH value to be neutral, adding neutral protease, performing ultrasonic-assisted enzymolysis at 45-65 ℃ for 20-60 min, inactivating with boiling water after enzymolysis is finished, and centrifuging to obtain a supernatant to obtain an enzymolysis solution;
(3) and (3) ultrafiltration treatment: ultrafiltering and separating the enzymolysis solution to obtain a group with the molecular weight of more than 10kDa, a group with the molecular weight of 5-10 kDa, a group with the molecular weight of 3-15 kDa and a group with the molecular weight of less than 3kDa, and respectively measuring DPPH clearance and reducing power of each group after freeze-drying;
(4) gel column treatment: selecting the component with the highest DPPH clearance from each group obtained after ultrafiltration separation by a filter membrane, adding ultrapure water to prepare a solution, desalting, separating and purifying by a Sephadex G-25 gel column, collecting each peak, and measuring the DPPH clearance of each group after freeze-drying;
(5) reversed-phase high performance liquid chromatography treatment: and (3) selecting the component with the highest DPPH removal rate from the groups obtained after the gel column separation, adding ultrapure water to prepare a solution, filtering the solution by using a filter membrane, separating and purifying the solution by using a reverse phase high performance liquid phase, collecting each elution peak, and measuring the DPPH removal rate of each group after concentration, wherein the group with the highest DPPH removal rate is the required antioxidant peptide of the lobster.
Preferably, in the step (2), the ultrasonic frequency is 10-30 kHz.
Preferably, in the step (4), the eluent is ultrapure water, and the flow rate is 1.0 mL/min-1。
Preferably, in the step (5), the chromatographic conditions of the reversed-phase high performance liquid chromatography are as follows: by C18Chromatographic column 4.6X 250mm, 5 μm, phase A of acetonitrile containing 0.1% trifluoroacetic acid, phase B of ultrapure water containing 0.1% trifluoroacetic acid, column temperature 25 deg.C, flow rate 1.0 mL/min-1The elution process is as follows: 0-100% B for 8min, 15-85% B for 17min, 30-70% B for 10min, and 0-100% B for 5 min.
The technical scheme adopted by the invention for solving the third technical problem is as follows: the method for improving the stability of the antioxidant peptides of the shrimps in the living body is characterized in that the antioxidant peptides of the shrimps and the egg white protein are respectively prepared into solutions with the same concentration, the egg white protein solution is added into the prepared antioxidant peptides of the shrimps, and the volume ratio of the egg white protein solution to the antioxidant peptides of the shrimps is 1: 4-6.
Compared with the prior art, the invention has the advantages that: the invention provides a brand new antioxidant peptide of a bunchy shrimp, which has stronger antioxidant activity and DPPH clearance rate of more than 85 percent and can be widely applied to various antioxidant products; the method takes the heads of the shrimps of the bunchy shrimps as the raw material, has wide sources and low cost, solves the problem of leftovers generated in the processing process of the bunchy shrimps, and has theoretical significance for further improving the economic value of the bunchy shrimps.
Drawings
FIG. 1 is a schematic diagram showing the change in the degree of hydrolysis of different hydrolyzing proteases in a certain period of time in example 1 of the present invention;
FIG. 2 is a graph showing the change in DPPH clearance of different hydrolytic proteases over a period of time in example 1 of the present invention;
FIG. 3 shows the effect of ultrasonic temperature on DPPH clearance and hydrolysis degree of shrimp heads enzymatic hydrolysate of shrimps of a Japanese shrimp in example 1 of the present invention;
FIG. 4 shows the effect of the ultrasonic time on the DPPH clearance rate and the hydrolysis degree of the shrimp head enzymatic hydrolysate of the shrimps of the Japanese shrimps in example 1 of the present invention;
FIG. 5 shows the effect of ultrasonic frequency on DPPH removal rate and hydrolysis degree of shrimp heads enzymatic hydrolysate of shrimps of a penaeus japonicus in example 1 of the present invention;
FIG. 6 shows the effect of feed-liquid ratio on DPPH clearance and hydrolysis degree of shrimp heads enzymatic hydrolysate of shrimps of a Japanese shrimp in example 1 of the present invention;
FIG. 7 is a graph showing the results of response analysis of the influence of the ultrasonic time and ultrasonic temperature on DPPH clearance of enzymatic products in example 1 of the present invention;
FIG. 8 is a graph showing the results of response analysis of the influence of ultrasonic frequency and ultrasonic temperature on DPPH clearance of enzymatic products in example 1 of the present invention;
FIG. 9 is a graph showing the results of response analysis of the influence of the feed-to-liquid ratio and the ultrasonic temperature on the DPPH clearance of the enzymatic hydrolysis products in example 1 of the present invention;
FIG. 10 is a graph showing the results of response analysis of the influence of ultrasonic frequency and ultrasonic time on DPPH clearance of enzymatic products in example 1 of the present invention;
FIG. 11 is a graph showing the results of response analysis of the influence of the feed-to-liquid ratio and the ultrasonic time on the DPPH clearance of the enzymatic hydrolysis products in example 1 of the present invention;
FIG. 12 is a graph showing the results of response analysis of the influence of the liquid-feed ratio and the ultrasonic frequency on the DPPH clearance of the enzymatic hydrolysate in example 1 of the present invention;
FIG. 13 is a Sephadex G-25 chromatogram of the SHP4 fraction of example 1 of the present invention;
FIG. 14 shows DPPH clearance of each peak after Sephadex G-25 chromatography of SHP4 fraction in example 1 of the present invention;
FIG. 15 is a reversed-phase high performance liquid separation profile of SHP4-II component in example 1 of the present invention;
FIG. 16 is a reversed-phase high performance liquid separation profile of the SHP-D fraction of example 1 of this invention;
FIG. 17 is a one-stage mass spectrometry total ion flow diagram of the SHP-D component in example 2 of the present invention;
FIG. 18 is the mass spectrum of the polypeptide of example 2 of the present invention after the separation and purification of SHP-D fraction.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1: preparation of antioxidant peptide of bunchy shrimp
1.1 starting materials and reagents
Raw materials: heads of Pandalus japonicus, purchased from Holothuria living organisms, Inc., Zhoushan.
Reagent: trypsin, bromelain, pepsin, alkaline protease and neutral protease, all of which are available from Pompe bioengineering, Inc. of southwestern Guangxi; gly, Gly-Gly-Gly, vitamin B12, aprotinin, cytochrome C, bovine serum albumin, acetonitrile (chromatographic grade), trifluoroacetic acid (chromatographic grade), formic acid (chromatographic grade), chromatographic column packing Sephadex G-25 (Sigma company, USA); reagents such as absolute ethyl alcohol, 2, 6-di-tert-butyl-4-methylphenol (BHT), hydrochloric acid, sodium hydroxide and the like are analytically pure (chemical reagents of national drug group, Ltd.).
1.2 instruments and devices
WATERS SYNAPT ultra high pressure liquid chromatography quadrupole tandem time-of-flight mass spectrometer (Waters corporation, USA); UPLC BEH C18 column (2.l ID)X 120mm) (Waters, USA); sunfireTMC18 column (4.6X 250mm) (Waters, USA); alliance e2695 high performance liquid chromatograph (Waters corporation, USA); an AKTA purifier 100 protein purification system (Amersham Biosciences, Sweden); HiTrapTMDesaling prepacked columns (GE corporation, usa); 2.6X 100cm column (Beijing Kangchun technology Co., Ltd.); pelliconTM-2Mini Holder Catalog XX42PMINI ultrafiltration unit (MILLIPORE, USA); a model BT4K-ZL freeze dryer (VIRTIS, USA); DL-720A ultrasonic cleaner (Shanghai Shuichang electronic Co., Ltd.); UV-8900 ultraviolet-visible spectrophotometer (Shanghai chromatography instruments, Inc.); FJ300SH digital display high speed dispersion homogenizer (Shanghai Longdu Instrument Co., Ltd.).
1.3 extraction and separation of antioxidant peptides from Japanese shrimp
(1) Pretreatment of raw materials: cleaning heads of the bamboo joint shrimps, removing lipid components such as shrimp yellow and the like, and homogenizing and mincing the heads into slurry for later use by a high-speed homogenizer.
(2) Ultrasonic-assisted enzymolysis: the continuous ultrasonic-assisted enzymolysis mode is adopted, and the ultrasonic-assisted enzymolysis temperature is controlled in real time through a super constant-temperature water bath, and the specific process comprises the following steps: adding ultrapure water into the slurry to prepare a solution, wherein the material-liquid ratio is 1: 6-14 w/v, adjusting the pH value to be neutral, adding neutral protease, performing ultrasonic-assisted enzymolysis at 45-65 ℃ for 20-60 min, inactivating in a boiling water bath for 10min after the enzymolysis is finished, centrifuging (4000r/min, 10min), and taking the supernatant to obtain an enzymolysis solution (named as SHP), wherein if a small amount of grease floats in the supernatant after centrifuging, the supernatant can be sucked by a suction tube.
(3) And (3) ultrafiltration treatment: and (3) carrying out microfiltration separation on the enzymolysis solution through a 0.45-micrometer filter membrane, sequentially intercepting ultrafiltration components with the molecular weights of 10kDa, 5kDa and 3kDa, and respectively determining the DPPH clearance of each group after freeze-drying, wherein the ultrafiltration components comprise a group with the molecular weight of more than 10kDa, a group with the molecular weight of 5-10 kDa, a group with the molecular weight of 3-15 kDa and a group with the molecular weight of less than 3 kDa. The DPPH clearance rate is determined by the following method: adding 1mL of newly prepared DPPH ethanol solution with concentration of 0.1mmol/L into 1mL of hydrolysate, uniformly mixing, reacting for 20min at room temperature in a dark place, and measuring the light absorption value at 517nm to be As; the blank group is 1mL of absolute ethyl alcohol instead of DPPH solution, and the light absorption value is measured as Ax; the contrast group is 1mL of distilled water instead of the sample, and the light absorption value is determined to be Ao; blank zero adjustment is carried out by using a mixed solution of distilled water and absolute ethyl alcohol with the same volume. All measurements were averaged three times and the DPPH radical clearance (DSA) was calculated as:
DSA＝[1-(As-Ax)/Ao]×100％
(4) gel column treatment: selecting the group with highest DPPH clearance from the groups obtained after ultrafiltration separation, adding ultrapure water to prepare a solution, and performing HiTrapTMDesalting with desaling pre-packed column, separating and purifying with Sephadex G-25 gel column, collecting each peak, lyophilizing, and determining DPPH clearance of each group, wherein the eluent in the gel column separation and purification is ultrapure water with flow rate of 1.0 mL/min-1。
(5) Reversed-phase high performance liquid chromatography treatment: and (3) selecting the component with the highest DPPH removal rate from the groups obtained after the gel column separation, adding ultrapure water to prepare a solution, filtering the solution by using a filter membrane, separating and purifying the solution by using a reverse phase high performance liquid phase, collecting each elution peak, and measuring the DPPH removal rate of each group after concentration, wherein the group with the highest DPPH removal rate is the required antioxidant peptide of the lobster. The chromatographic conditions of the reversed-phase high-performance liquid chromatography are as follows: by C18Chromatographic column 4.6X 250mm, 5 μm, phase A of acetonitrile containing 0.1% trifluoroacetic acid, phase B of ultrapure water containing 0.1% trifluoroacetic acid, column temperature 25 deg.C, flow rate 1.0 mL/min-1The elution conditions are shown in Table 1.
TABLE 1 RP-HPLC elution conditions
| Time/min | Flow rate/(mL. min)-1) | A/％ | B/％ |
| 0 | 1.0 | 0 | 100 |
| 8 | 1.0 | 0 | 100 |
| 25 | 1.0 | 15 | 85 |
| 35 | 1.0 | 30 | 70 |
| 40 | 1.0 | 0 | 100 |
1.3.1 screening of hydrolases
Selecting five proteolytic enzymes including trypsin, bromelain, pepsin, alkaline protease and neutral protease, wherein the five proteolytic enzymes are used for respectively carrying out enzymolysis on the head of the lobster under respective optimal conditions, and the change of the hydrolysis degree and the DPPH free radical scavenging capacity of the enzymolysis of the head of the lobster at each time point within the range of 1-8 h is respectively shown in a figure 1 and a figure 2.
As can be seen from FIG. 1, the degree of hydrolysis of the enzymatic hydrolysate obtained by the neutral protease and the enzymatic hydrolysate obtained by the alkaline protease are equivalent. As can be seen from FIG. 2, the neutral protease has the best DPPH free radical scavenging ability in the enzymolysis solution under the optimal condition. In conclusion, the neutral protease is the ideal protease for the antioxidant active peptide of the heads of the shrimps.
1.3.2 determination of ultrasound conditions
(1) Influence of ultrasonic temperature on antioxidant activity of shrimp head enzymatic hydrolysate of the shrimps of the Japanese shrimp: under the conditions of ultrasonic time of 40min, ultrasonic frequency of 20kHz and material-liquid ratio of 1:8(w/v), ultrasonic temperatures are respectively set to 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃.
(2) Influence of ultrasonic time on antioxidant activity of shrimp head enzymatic hydrolysate of the shrimps of the Japanese shrimp: the ultrasonic time is respectively set to be 20min, 30min, 40min, 50min and 60min under the conditions that the ultrasonic temperature is 55 ℃, the ultrasonic frequency is 20kHz and the material-liquid ratio is 1:8 (w/v).
(3) Influence of ultrasonic frequency on antioxidant activity of shrimp head enzymatic hydrolysate of the shrimps of the bunchy shrimps: the ultrasonic frequency was set to 10kHz, 15kHz, 20kHz, 25kHz, 30kHz, respectively, at an ultrasonic temperature of 55 deg.C, a material-to-liquid ratio of 1:8(w/v), and an ultrasonic time of 40 min.
(4) The influence of the feed liquid on the antioxidant activity of the shrimp head enzymatic hydrolysate of the shrimp head of the shrimp is as follows: under the conditions that the ultrasonic temperature is 55 ℃, the ultrasonic time is 40min and the ultrasonic frequency is 20kHz, the material-liquid ratio is respectively set to be 1:6, 1:8, 1:10, 1:12 and 1:14 (w/v). Each set of optimized experiments was performed in triplicate and the average was finally taken.
On the basis of a single-factor test, Design-expert.8.0.5b software is adopted for further optimization, 4 factors of ultrasonic temperature, ultrasonic time, ultrasonic frequency and feed-liquid ratio are used as independent variables, the DPPH clearance of enzymolysis products is used as a response value, a four-factor three-level test is designed, and the factor levels are shown in Table 2.
TABLE 2 response surface test design
The influence of the ultrasonic temperature on the DPPH clearance and the hydrolysis degree of the shrimp heads enzymatic hydrolysate of the shrimps is shown in figure 3, and as can be seen from figure 3, the ultrasonic temperature has a large influence on the DPPH clearance and the hydrolysis degree. When the ultrasonic temperature is less than 55 ℃, the hydrolysis degree of the enzymolysis product increases along with the increase of the temperature, when the temperature reaches 55 ℃, the hydrolysis degree reaches the highest value (54.05%), and when the temperature exceeds 55 ℃, the hydrolysis degree is in a descending trend along with the increase of the temperature. When the ultrasonic temperature is 55 ℃ and 60 ℃, the DPPH clearance rate is 61.26 percent and 62.00 percent respectively, and the DPPH clearance rate have no significant difference. This is because an appropriate temperature rise can promote the activity of the enzyme and make the hydrolysis more sufficient, but an excessively high temperature can inhibit the activity of the enzyme. In general terms, the invention selects the proper ultrasonic temperature of 55 ℃.
The influence of the ultrasonic time on the DPPH removal rate and the hydrolysis degree of the shrimp head enzymatic hydrolysate of the shrimps is shown in FIG. 4, and it can be known from FIG. 4 that the DPPH removal rate and the hydrolysis degree of the shrimp head enzymatic hydrolysate are in a trend of increasing and then decreasing within the time range of 20-60 min of the ultrasonic time. This is because too short a time will result in insufficient enzymatic reaction, while too long a time will result in easy conversion of the enzymatic products into other by-products, which will affect the yield. When the ultrasonic treatment time is 40min, the ultrasonic treatment time and the ultrasonic treatment time reach the highest values, the hydrolysis degree is 51.28%, and the DPPH clearance rate is 67.72%. Therefore, the invention selects 40min as the optimal ultrasonic time.
The influence of the ultrasonic frequency on the DPPH clearance and the degree of hydrolysis of the shrimp heads of the shrimps is shown in FIG. 5, and it can be seen from FIG. 5 that the DPPH clearance and the degree of hydrolysis of the shrimp heads of the shrimps are firstly increased and then decreased with the increase of the ultrasonic frequency, and when the ultrasonic frequency is 20kHz, the degree of hydrolysis and the DPPH clearance are highest and are 55.15% and 62.21% respectively. This is because the collision frequency of enzyme and substrate is increased with the increase of ultrasonic frequency, and the enzymolysis reaction rate is finally accelerated, but if the ultrasonic frequency is too high, the enzyme conformation is changed, and the enzyme activity is reduced, so the enzymolysis effect is influenced. Therefore, the present invention selects 20kHz as the optimum ultrasonic frequency.
The influence of the feed liquid ratio on the DPPH clearance and the hydrolysis degree of the shrimp heads of the shrimps is shown in FIG. 6, and it can be seen from FIG. 6 that when the feed liquid ratio is less than 1:10(w/v), the DPPH clearance of the shrimp heads of the shrimps is continuously increased along with the increase of the feed liquid ratio, and when the feed liquid ratio is greater than 1:10(w/v), the DPPH clearance is continuously decreased. The hydrolysis degree of the shrimp head enzymolysis product is in an ascending trend within the range of the feed-liquid ratio of 1: 6-1: 8(w/v), and when the feed-liquid ratio is more than 1:8(w/v), the hydrolysis degree is hardly increased. Generally, too large or too small a feed-to-liquid ratio affects the speed of enzyme catalysis and the diffusion of product molecules. In general, the ratio of feed to liquid is preferably 1:10(w/v), and when the ratio of feed to liquid is 1:10(w/v), the DPPH clearance rate is 64.66%, and the hydrolysis degree is 55.67%.
1.3.3 response surface optimization of shrimp head enzymolysis conditions of bunchy shrimps
(1) Response surface analysis and regression model
According to the results of single-factor tests in the early stage, ultrasonic temperature (A), ultrasonic time (B), ultrasonic frequency (C) and a material-liquid ratio (D) are selected as variables, a Design-expert.8.0.5B software Design center is used for combined tests, the optimal enzymolysis process is explored, the test times are 29, 5 of the test times are central points, and the test designs and results are shown in table 3. Carrying out polynomial regression analysis on the enzymolysis data, and fitting to obtain a quadratic regression equation:
Y＝68.40+1.27A+0.80B+3.81C-3.94D+3.52AC-11.68CD-14.43A2-11.57B2-11.23C2-14.63D2。
TABLE 3 center combination design and results
The quadratic regression equation is subjected to variance analysis and inspection, and the results of the influencing factors of the variance analysis in the table 4 show that 4 factors of the ultrasonic temperature (A), the ultrasonic time (B), the ultrasonic frequency (C) and the feed-liquid ratio (D) play important roles in the enzymolysis process. Model F value 42.27(p <0.0001) indicates that the quadratic equation is significant modeled, and mismatching term p 0.1353>0.05 indicates that mismatching is not significant, and in conclusion, the regression equation has high fitting degree to the actual test, and the equation can be applied to the actual test.
The influence degree of each factor on the response value can be more clearly compared through the 3D response surface graph. The maximum value of the model is shown by the response surface analysis results obtained by treating Design-expert.8.0.5b software of fig. 7 to 12. Optimizing all factors according to the obtained model, and obtaining the optimal process conditions as follows: the ultrasonic temperature is 55.35 ℃, the ultrasonic time is 40.60min, the ultrasonic frequency is 21.55kHz, the material-liquid ratio is 1:9.48(w/v), and the predicted value of the DPPH clearance rate under the condition is 69.58%.
In general, if the mathematical model created by the response surface analysis method does not fit the actual experimental value well, the solution obtained by the optimization analysis by the model estimation may deviate from the actual value to some extent, and it is necessary to test the estimation solution in order to verify the reliability of the solution. The test conditions are corrected to be ultrasonic temperature of 55 ℃, ultrasonic time of 40min, ultrasonic frequency of 20kHz and material-liquid ratio of 1:10(w/v), enzymolysis verification is carried out 3 times in parallel on the conditions, the result shows that the average value of DPPH clearance is 69.50% +/-1.58%, calculation is carried out through SPSS staticistics 19 software, and on the premise that the significance level p is set to be less than 0.01, no significant difference exists between a predicted value and an actual value, which shows that the model equation has good fitting test data and the prediction model is feasible.
TABLE 4 regression coefficient model and analysis of variance
Note that the difference in expression is extremely significant (P < 0.01); indicates significant differences (P < 0.05).
However, in practical experiment operation, a thermal effect is generated, and particularly, the thermal effect generated in the ultrasonic process has a great influence on the activity of the enzymatic hydrolysate, which may be caused by that the molecular activity is enhanced along with the increase of temperature, the chemical bond is unstable, and the polypeptide is easy to dehydrogenate and cannot be combined with the free radical, so that the antioxidant activity of the enzymatic hydrolysate is reduced. Specifically, for the present invention, as shown in the following example 2, the amino acid sequence of the antioxidant peptide of the shrimp cracker is Gly-Asn-Gly-Leu-Pro (as shown in SEQ 1), with increasing temperature, the molecular activity is enhanced, the chemical bond is unstable, the enzymolysis product is easy to dehydrogenate to generate hydrogen ions, and cannot be combined with free radicals, thereby reducing the antioxidant activity of the polypeptide, and meanwhile, the amino groups in the polypeptide structures Gly and Asn and the carboxyl groups at the end are easy to dehydrogenate to generate hydrogen ions, further reducing the antioxidant activity of the enzymolysis product.
Therefore, on the basis of the optimal process conditions, the ultrasonic-assisted enzymolysis conditions are adjusted to be as follows: the ultrasonic temperature is 52 ℃, the ultrasonic time is 35min, the material-liquid ratio is 1:9, and the influence of the thermal effect on the polypeptide can be reduced to a certain extent by adjusting the ultrasonic temperature and the ultrasonic time, so that the antioxidant activity of the enzymolysis product is further ensured, the reduction of the biological activity of the enzymolysis product is effectively avoided, and the biological value of the enzymolysis product is ensured.
1.3.4 comparison of ultrasound-assisted enzymatic hydrolysis and non-ultrasound-assisted enzymatic hydrolysis
As can be seen from Table 5, under the condition of non-ultrasonic-assisted enzymolysis, when the enzymolysis time is 2 hours, the hydrolysis degree is the highest and is 34.19%; the hydrolysis degree reaches 52.14% only after 40min of ultrasonic-assisted enzymolysis, which shows that the ultrasonic-assisted enzymolysis can not only improve the enzymolysis degree, but also shorten the enzymolysis time and save energy. In the design range of the test, the DPPH clearance of the enzymolysis product obtained by carrying out ultrasonic-assisted enzymolysis on the shrimp heads is up to 68.34%, which is 18.21% higher than that of non-ultrasonic-assisted enzymolysis, and the result shows that the antioxidant activity of the enzymolysis product can be improved by carrying out ultrasonic-assisted enzymolysis.
TABLE 5 comparison of ultrasonic-assisted and non-ultrasonic-assisted enzymatic hydrolysis
1.3.5 Ultrafiltration treatment
Preparing an enzymolysis product SHP under the optimized conditions of ultrasonic temperature of 55 ℃, ultrasonic time of 40min, ultrasonic frequency of 20kHz and material-liquid ratio of 1:10(w/v), performing primary separation on the SHP by using ultrafiltration separation equipment to obtain four components of SHP1, SHP2, SHP3 and SHP4 respectively, and determining the antioxidant activity of different relative molecular masses respectively, wherein the results show that the relative molecular mass of the enzymolysis product has significant influence on the antioxidant activity of the enzymolysis product. Among them, SHP4(<3kDa) fraction showed the highest DPPH clearance and reduction, 73.35% and 0.66%, respectively (as shown in table 6). Thus, SHP4 was identified as the subject of further study.
TABLE 6 antioxidant Activity of different relative molecular mass fractions
Note: DPPH clearance and reducing force determination peptide and BHT concentrations were 2 mg/mL-1。
1.3.6 gel column treatment
Separating SHP4 component by Sephadex G-25 gel chromatography column, eluting with ultrapure water, and collecting sample with concentration of 20 mg/mL-1The sample loading amount was 5.0mL, and the flow rate was 1.0 mL. multidot.min-1Collected with an automatic collector, 2.0mL per tube. The collected liquid of the same peak is combined, and the DPPH clearance rate of each peak is respectively measured after freeze drying.
As can be seen from FIG. 13, after Sephadex G-25 gel chromatography separation, 4 components, namely SHP4-I, SHP4-II, SHP4-III and SHP4-IV, are obtained by sample separation, wherein the component SHP4-I is eluted first, which means that the molecular weight of the polypeptide of the component is the largest, the component SHP4-IV is eluted last, which means that the molecular weight is the smallest, and the peak area of the component SHP4-II is the largest, which means that the content of the polypeptide of the component is the highest among the 4 components. As shown in FIG. 14, the content of the polypeptide was 2.0 mg/mL-1In this case, the DPPH clearance rates of the 4 components are 43.51% + -3.45%, 83.04% + -2.61%, 58.29% + -1.67%, and 24.61% + -3.71%, respectively, wherein the DPPH clearance rate of the component SHP4-II is the highest, indicating that the component contains more polypeptides with antioxidant activity. Compared with ultrafiltrate, DPPH clearance of the component is improved by 7.83 percent, which is equivalent to standard sample BHT, and the Sephadex G-25 gel chromatography achieves the further purification effect.
1.3.7 reverse phase high performance liquid chromatography treatment
The active component SHP4-II obtained by Sephadex G-25 gel chromatography is further purified and separated by RP-HPLC to obtain SHP-A, SHP-B, SHP-C, SHP-D4 main peptide peaks (as shown in FIG. 15). Collecting 4 peaks respectively, freeze-drying, measuring antioxidant activity with DPPH clearance rate as index, and measuring polypeptide content at 1.5 mg/mL-1When the composition is used, the DPPH clearance rates of 4 components are 45.21% + -1.25%, 52.66% + -2.19%, 63.73% + -2.01% and 85.69% + -3.12%, respectively, and the component SHP4-D has stronger antioxidant activity. The fraction was collected for purity detection and analysis, and acetonitrile containing 0.1% trifluoroacetic acid/ultrapure water containing 0.1% trifluoroacetic acid was used as a mobile phase at a flow rate of 1.0 mL-min-1The elution mode is a linear elution mode in which the concentration of acetonitrile containing 0.1% trifluoroacetic acid is changed from 0 to 20% within 20min, and according to the detection result, the component has better separation degree (as shown in fig. 16).
Example 2: structure identification of antioxidant peptide
The antioxidant component SHP-D after reversed-phase high performance liquid separation and purification is subjected to structure identification by UPLC-TOF-MS/MS technology, and the total ion current spectrum of the primary mass spectrum is shown in figure 17. As can be seen from FIG. 17, the fraction shows a single peak, and therefore the peptide fragment can be considered to be relatively pure, the peak is subjected to secondary mass spectrometry, and the peptide sequencing in Masslynx software is used for analyzing the structure of the peak, and the result shows that 1 polypeptide sequence with reliability of more than 90% is identified from the SHP-D fraction, and the structure of the polypeptide sequence is Gly-Asn-Gly-Leu-Pro (as shown in FIG. 18).
Example 3: determination of biological stability of antioxidant peptide of Japanese shrimp
Preparing artificial gastric juice and artificial intestinal juice for standby, wherein the preparation process of the artificial gastric juice comprises the following steps: respectively measuring 16.4mL of dilute hydrochloric acid and 800mL of distilled water, mixing the dilute hydrochloric acid and the distilled water, adding 10g of pepsin, shaking uniformly, placing in a volumetric flask, and fixing the volume to 1000 mL. Preparing artificial intestinal juice: 6.8g of monopotassium phosphate is weighed and dissolved in 500mL of distilled water, the pH value is adjusted to 6.8 by using 0.1mol/L NaOH solution, 10g of trypsin is taken and dissolved in a certain amount of distilled water, the mixture is shaken uniformly, and the volume is fixed to 1000 mL.
Preparing a chicken egg albumin solution and a shrimp nodus antioxidant peptide solution with the same concentration (10mg/ml), mixing the solutions according to the proportion of (1:4, 1:5 and 1:6), adding an equivalent amount of prepared artificial gastric juice or intestinal juice, adding the chicken egg albumin solution and the shrimp nodus antioxidant peptide solution of the artificial gastric juice (intestinal juice), standing the mixture in a water bath kettle at 37 ℃ for 3 hours, then performing centrifugation in a boiling water bath for 15 minutes at the rotation speed of 5000r/min and the temperature of-4 ℃ for 10 minutes, measuring the DPPH free radical clearance and ABTS free radical clearance, and repeating the experiment for 3 times, wherein the influence of the chicken egg albumin on the DPPH clearance of the shrimp antioxidant peptide is shown in a table 7, and the influence of the chicken egg albumin on the ABTS clearance of the shrimp antioxidant peptide is shown in a table 8.
TABLE 7 Activity retention/% of antioxidant peptides DPPH clearance of Pandalus japonicus
| Ratio of egg white protein to antioxidant peptide solution of Japanese shrimp | Pepsin | Trypsin |
| Without addition of egg white protein solution | 84.12±1.04 | 65.51±0.81 |
| 1:4 | 90.59±0.47 | 68.83±0.55 |
| 1:5 | 95.19±0.66 | 73.12±1.34 |
| 1:6 | 88.60±0.64 | 66.33±0.79 |
TABLE 8 Activity retention of the antioxidant peptides ABTS in Pandalus japonicus Activity%
As can be seen from the above tables 7 and 8, the addition of the chicken ovalbumin has a better protective effect on the antioxidant peptides of the shrimps in the environment simulating gastric juice and intestinal juice, and compared with the environment simulating intestinal juice, the chicken ovalbumin has a better protective effect on the biological activity of the antioxidant peptides of the shrimps in the environment simulating gastric juice.
SEQUENCE LISTING
<110> Zhejiang international maritime professional technology college
<120> antioxidative peptide of Japanese shrimp and preparation method thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
<213> bamboo shrimp (Penaeus japonica)
<400> 1
Gly Asn Gly Leu Pro
1 5
Claims (4)
1. A method for improving the stability of the antioxidant peptide of the shrimps in a living body is characterized in that the amino acid sequence of the antioxidant peptide of the shrimps is Gly-Asn-Gly-Leu-Pro, and the antioxidant peptide of the shrimps is prepared by the following method:
(1) pretreatment of raw materials: cleaning heads of the bamboo-joint shrimp, removing lipid components, homogenizing and mincing into slurry for later use;
(2) ultrasonic-assisted enzymolysis: adding ultrapure water into the slurry to prepare a solution, wherein the material-liquid ratio is 1: 6-14 w/v, adjusting the pH value to be neutral, adding neutral protease, performing ultrasonic-assisted enzymolysis at 45-65 ℃ for 20-60 min, inactivating with boiling water after enzymolysis is finished, and centrifuging to obtain a supernatant to obtain an enzymolysis solution;
(3) and (3) ultrafiltration treatment: after the enzymolysis solution is subjected to ultrafiltration separation, groups with molecular weights of more than 10kDa, groups with molecular weights of 5-10 kDa, groups with molecular weights of 3-15 kDa and groups with molecular weights of less than 3kDa are obtained, and DPPH clearance of each group is respectively determined after freeze-drying;
(4) gel column treatment: selecting the group with the highest DPPH clearance from all groups obtained after the ultrafiltration separation of the filter membrane, adding ultrapure water to prepare a solution, desalting, separating and purifying by a Sephadex G-25 gel column, collecting all peaks, and measuring the DPPH clearance of all groups after freeze-drying;
(5) reversed-phase high performance liquid chromatography treatment: selecting the component with the highest DPPH clearance rate from each group obtained after gel column separation, adding ultrapure water to prepare a solution, filtering the solution by using a filter membrane, separating and purifying the solution by using a reverse phase high performance liquid phase, collecting each elution peak, and measuring the DPPH clearance rate of each group after concentration, wherein the group with the highest DPPH clearance rate is the required antioxidant peptide of the lobster;
the method for stabilizing the antioxidant peptides of the penaeus japonicus in the organism comprises the following steps: respectively preparing the antioxidant peptides of the shrimps and the chicken egg white protein into solutions with the same concentration, and adding the chicken egg white protein solution into the prepared antioxidant peptides of the shrimps, wherein the volume ratio of the chicken egg white protein solution to the antioxidant peptides of the shrimps is 1: 4-6.
2. The method according to claim 1, wherein in the step (2), the ultrasonic frequency is 10 to 30 kHz.
3. The method of claim 1The method is characterized in that in the step (4), the eluent is ultrapure water, and the flow rate is 1.0 mL/min-1。
4. The method according to claim 1, wherein in the step (5), the chromatographic conditions of the reversed-phase high-performance liquid chromatography are as follows: by C18Chromatographic column 4.6X 250mm5 μm, phase A being acetonitrile containing 0.1% trifluoroacetic acid, phase B being ultrapure water containing 0.1% trifluoroacetic acid, column temperature 25 deg.C, flow rate 1.0 mL/min-1The elution process is as follows: 0-100% B for 8min, 15-85% B for 17min, 30-70% B for 10min, and 0-100% B for 5 min.
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| CN104026556A (en) * | 2013-11-15 | 2014-09-10 | 广西中医药大学 | Production technology for ultrafine low-salt low-fishy smell prawn paste |
| CN104886389A (en) * | 2015-05-27 | 2015-09-09 | 蚌埠市淮景绿湾生态农业有限公司 | Elopichthys bambusa output improving aquaculture feed |
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| CN104886389A (en) * | 2015-05-27 | 2015-09-09 | 蚌埠市淮景绿湾生态农业有限公司 | Elopichthys bambusa output improving aquaculture feed |
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