CN111248413B - Biological inhibitor for silver carp minced fillet gel deterioration - Google Patents
Biological inhibitor for silver carp minced fillet gel deterioration Download PDFInfo
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Abstract
The invention relates to the field of aquatic products, and discloses a biological inhibitor for silver carp minced fillet gel degradation, which is characterized in that myofibril-bound serine protease (MBSP) causing silver carp minced fillet gel degradation is used as an antigen immune hinged shark, a single domain antibody gene is separated from the serum of the hinged shark, a single domain antibody gene text library is established, a high-specificity single domain antibody target gene is obtained through a phage display technology, and then a silver carp MBSP specificity shark origin single domain antibody with high purity, high activity and high expression is obtained through separation by utilizing a clone expression technology and an affinity chromatography technology; the biological inhibitor can obviously inhibit the activity of the myofibril-bound serine protease of the silver carp and effectively inhibit the gel deterioration of the silver carp surimi. The biological inhibitor provided by the invention is safe, specific and efficient, is simple to operate in the using process, and can be applied to inhibition of gel deterioration of silver carp surimi.
Description
Technical Field
The invention relates to the field of aquatic products, in particular to a biological inhibitor for silver carp minced fillet gel degradation.
Background
The minced fillet product is an important direction for processing freshwater fish in China, and the gel deterioration is one of the main factors influencing the quality of the minced fillet product at present. The Japanese scholars have found that the myogen-binding serine protease (MBSP) in fish muscle tissue is the main cause of the gel strength reduction of minced fillet products after being heated for a long time at 50-65 ℃. MBSP can degrade the heavy chains of myosin in myofibrils under a certain temperature condition, and can also degrade alpha-actinin, actin and tropomyosin. Because the MBSP is widely existed in fish muscle tissues, the MBSP cannot be removed by rinsing in the production process of minced fillet, and the development of the minced fillet product industry is severely restricted.
Current studies on the inhibition of MBSP fall into two main categories: aim at the inhibition of the serine protease family in which MBSP is located: such as ovalbumin, animal plasma protein, potato starch, bean trypsin inhibitor and the like can obviously inhibit the activity of serine protease, but the application of the serine protease in the actual production is limited due to the defects of high cost, influence on the color and flavor of the minced fillet product, no contribution to the digestion and absorption of human bodies and the like. ② for specific MBSP inhibitors: the inhibitor has less research, mainly has the inhibitory action of endogenous glucose-6-phosphate isomerase (GPI) in fish bodies on MBSP, and the GPI only has the inhibitory action on the MBSP of the same type of fish and does not have broad-spectrum specificity. Due to the shortage of MBSP inhibitor, the gel strength of minced fillet products is improved by adding glutamine Transaminase (TG) enzyme in most of the minced fillet production at the present stage, but the TG enzyme catalysis has higher requirements on processing conditions, and non-muscle protein is required to be added for combined action to improve the effect, thereby influencing the property of the minced fillet. Therefore, the novel broad-spectrum specific inhibitor which is safe and efficient to research and develop and can directly act on MBSP in the processing process realizes the control of gel degradation of the minced fillet product and has an important promoting effect on the development of the whole minced fillet product industry.
Single domain antibodies (sdAbs) are novel antibodies that have been cloned in recent years from camelids and cartilaginous fish sera by genetic engineering techniques, retaining only the heavy chain variable region and having antigen binding activity. The antibody has the molecular weight of only 12-15kDa, and has the characteristics of high specificity, strong affinity, high stability, strong penetrating power and the like. Compared with the traditional IgG, the CDR3 area is longer, and the formed finger-shaped convex ring can be embedded into the groove or cleft of an antigen molecule and can directly inhibit the activity of the antigen after being specifically combined with the antigen. At present, a great deal of research reports exist on the application of single domain antibodies in medical detection and drug development, the single domain antibodies are still in a blank stage in the field of food processing, if specific single domain antibodies can be prepared aiming at endogenous enzymes in food, the single domain antibodies can be used for realizing the inhibition of enzyme activity by utilizing the specific binding of the single domain antibodies to the endogenous enzymes in the food, and the single domain antibodies have strong targeting property, low preparation cost and good repeatability, and have great application prospects as an enzyme inhibitor in food processing.
Disclosure of Invention
Aiming at the problems of the chub fish meat gel degradation inhibitor in the market in the using process, the invention provides a safe, specific and efficient biological inhibitor which can effectively inhibit the chub surimi gel degradation.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a shark-derived single domain antibody gene with high specificity for silver carp myofibril-binding serine protease, and the nucleotide sequence is shown as SEQ ID NO:1, and the following components:
in a second aspect, the present invention provides a shark-derived single domain antibody with high specificity for a silver carp myofibril-binding serine protease, comprising the amino acid sequence as set forth in SEQ ID NO: 2:
in a third aspect, the invention provides a biological inhibitor for chub surimi gel deterioration, comprising the shark-derived single domain antibody.
The preparation method of the shark-derived single-domain antibody comprises the following steps:
1) establishing a silver carp myofibril combined serine protease shark source single-domain antibody gene text library.
2) And (3) panning and identifying the single-domain antibody genes in the text library by taking the silver carp myofibril-bound serine protease causing the deterioration of the silver carp minced fillet gel as an antigen to obtain the shark-derived single-domain antibody gene with the highest specificity to the silver carp myofibril-bound serine protease.
3) The shark source single-domain antibody with high specificity to the silver carp myofibril-binding serine protease is obtained by cloning, expressing, purifying and separating.
The preparation method comprises the following specific steps:
1) taking silver carp flesh myofibril combined serine protease as antigen immune shark reaming, and determining the immune response condition of the shark; collecting high-immunity shark peripheral blood lymphocytes, extracting RNA (ribonucleic acid) and synthesizing a cDNA (complementary deoxyribonucleic acid) library, constructing a phagemid vector by using a gene recombination technology, introducing escherichia coli to construct a shark source single-domain antibody gene textbook and identifying the textbook capacity;
2) infecting a single domain antibody gene text library by using auxiliary phagemids, taking silver carp fish myofibril combined serine protease as an antigen, performing multiple rounds of panning by using a phage display technology, and identifying by ELISA to obtain a single domain antibody gene IgNAR B7 with the highest specificity;
3) in order to obtain a single-domain antibody with higher activity, through multiple experimental researches, a recombinant plasmid pET28a-SUMO carrying an SUMO fusion tag is determined to be used as an expression vector, so that the single-domain antibody can be expressed in a soluble manner, and the yield is higher. Therefore, the invention takes pET28a-SUMO as an expression vector, takes BamHI and SalI as enzyme cutting sites, constructs pET28a-SUMO-IgNAR B7 recombinant expression plasmid through double enzyme cutting verification, carries out mass expression after the optimal induction condition is determined through experiments, carries out nickel agarose affinity chromatography purification on the fusion protein obtained by expression, carries out enzymolysis on the expression fusion protein by SUMO protease Ulp1, and further obtains the shark-derived single domain antibody through further purification.
Compared with the traditional technology of inhibiting the degradation of the minced fillet gel by using the enzyme inhibitor, the invention has the following beneficial effects:
(1) the single domain antibody acts by specifically binding to Myofibrillar Binding Serine Protease (MBSP), and has high specificity, low usage amount, and high efficiency.
(2) The single-domain antibody has high thermal stability and high acid and alkali resistance, and the processing conditions of the minced fillet processing process have little influence on the activity of the antibody.
(3) The gene of the specific single domain antibody is derived from the shark of the hand-shake, and the single domain antibody is produced in large quantities by means of bioengineering, and the single domain antibody is clinically proved to be safe and has no potential risk.
Drawings
FIG. 1 is a diagram of shark serum binding reaction analysis;
FIG. 2 is a SDS-PAGE validation of fusion proteolysis and purification of the protein of interest;
FIG. 3 is the fusion protein P2-B7 map;
FIG. 4 is an affinity verification diagram of fusion protein P2-B7 (CK: PBS; NC: empty plasmid expression product; AC: sharp blood; 1: P2-B7(0.557mg/m 1); 2: P2-B7(0.1mg/m 1); 3: fusion protein B7-SUMO (0.1mg/m 1); and 4: purified SUMO tag protein);
FIG. 5 is a graph showing the inhibitory effect of different concentrations of single-domain antibody P2-B7 on the enzyme activity of silver carp MBSP (1: PBS + enzyme substrate; 2: MBSP + enzyme substrate; 3: 0.5mg/ml single-domain antibody P2-B7; 4: 0.1mg/ml single-domain antibody P2-B7);
FIG. 6 is a graph of temperature scan results for different surimi gel systems (G': storage modulus; G ": loss modulus).
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A shark-derived single-domain antibody gene with high specificity to silver carp myofibril-binding serine protease has a nucleotide sequence shown as SEQ ID NO:1, and the following steps:
a shark-derived single domain antibody with high specificity for silver carp myofibril-binding serine protease, comprising the amino acid sequence shown in SEQ ID NO: 2:
a biological inhibitor for the deterioration of chub surimi gel comprises the shark-derived single-domain antibody. The preparation method of the shark-derived single domain antibody comprises the following steps:
1) taking silver carp flesh myofibril combined serine protease as antigen immune shark reaming, and determining the immune response condition of the shark; collecting high-immunity shark peripheral blood lymphocytes, extracting RNA and synthesizing a cDNA library, constructing a phagemid vector by using a gene recombination technology, introducing escherichia coli to construct a shark-derived single-domain antibody gene textbank, and identifying the textbank capacity.
2) And infecting a single domain antibody gene text library by using auxiliary phagemids, taking silver carp fish myofibril-binding serine protease as an antigen, performing multiple rounds of panning by using a phage display technology, and identifying by ELISA to obtain the single domain antibody gene IgNAR B7 with the highest specificity.
3) pET28a-SUMO is used as an expression vector, BamHI and SalI are used as enzyme cutting sites, double enzyme cutting verification is carried out, pET28a-SUMO-IgNAR B7 recombinant expression plasmids are constructed and induced by IPTG inducer, the fusion protein obtained by expression is purified by nickel agarose affinity chromatography, SUMO protease Ulp1 is used for carrying out enzymolysis on the expressed fusion protein, and the shark-derived single domain antibody is obtained by further purification.
The specific embodiment is as follows: preparation of silver carp flesh myofibril-binding type serine protease (MBSP) specific shark-derived single-domain antibody and inhibition of silver carp minced fillet gel degradation effect
1. Establishment of silver carp fish MBSP shark source single-domain antibody text library
Fully mixing the MBSP of chub fish prepared in a laboratory with Freund's adjuvant at a ratio of 1: 1, injecting immune shark subcutaneously into dorsal fin, enhancing the immunity once every two weeks, collecting shark serum after immunizing for four times, and evaluating the response effect of the MBSP-resistant serum by using indirect ELISA; collecting shark peripheral blood lymphocytes which efficiently respond after immunization, extracting total RNA by a TRIzol method, identifying RNA quality by Experion, processing by TURBO TMDnase, synthesizing a cDNA library by a SuperScript system, designing a single-domain antibody bidirectional primer, amplifying a DNA product by PCR, carrying out agarose electrophoresis, cutting gel, purifying a target DNA, carrying out restriction enzyme digestion by restriction enzymes NcoI and NotI, constructing a vector with pHEN2 phagemid, carrying out electrotransformation, introducing into competent Escherichia coli TG1 cells, constructing a single-domain antibody textbank, and carrying out plate culture counting to evaluate the volume of the textbank.
As shown in FIG. 1, the binding reaction of shark plasma to MBSP occurred with three immunizations of the reamed shark. The immune function of the grated shark through MBSP is proved to generate specific antibody aiming at the MBSP.
2. Elutriation and identification of silver carp MBSP (MBSP) specific shark-derived single-domain antibody target gene
Culturing and amplifying a single-domain antibody text library in a 2-YT culture medium containing ampicillin at 37 ℃, adding M13K07 to assist phage infection host bacteria (TG1) to activate recombinant phagemid expression, culturing overnight at 30 ℃, centrifuging and collecting culture supernatant, precipitating phage by polyethylene glycol/sodium chloride solution (PEG8000/NaCl), coating an immunoassay tube with MBSP to carry out panning of the single-domain antibody, infecting the host bacteria again by the panned positive phagemid (TG1), measuring the phagemid titer by using polyclonal phage ELISA after circulating for four times of panning, sequencing and analyzing the nucleotide sequence (shown as SEQ ID NO: 1) and the amino acid sequence (shown as SEQ ID NO: 2) of the positive phagemid, and determining the best silver carp MBSP specific phagemid gene.
3. Preparation of silver carp MBSP (MBSP) specific shark-derived single-domain antibody
3.1 construction and identification of pET28a-SUMO-IgNAR B7 recombinant expression plasmid
The IgNAR B7 gene sequence is taken as a template, the target gene B7 and pET28a-SUMO plasmid are analyzed by SnapGeen gene sequence analysis software to determine the appropriate restriction enzyme sites BamHI and SalI, and a stop codon is added at the 3' end of the target gene for facilitating the purification of the later-stage fusion protein. Extraction of pET28a-SUMO Plasmid was performed according to the TIANprep Rapid Mini Plasmid Kit instructions. The IgNAR B7 target gene and plasmid pET28a-SUMO DNA are used as templates, and BamHI and SalI restriction enzymes are selected for double enzyme digestion. The specific digestion system is shown in Table 1, and the digestion is carried out overnight at 37 ℃, and the gel is identified by 1.0% agarose gel electrophoresis and recovered.
TABLE 1 double enzyme digestion System
| Reaction components | Volume (mu L) |
| pET28a+sumo/VNAR-B7 | 2μg |
| |
5 |
| BamHI(10μ/μ1) | 1 |
| SalI(10μ/μl) | 1 |
| ddH 2 O | To 50 |
The pET28a-SUMO plasmid after double enzyme digestion and purification is connected with VNAR-B7 target gene according to the molar ratio of 1: 10, and the specific reaction system is shown in Table 2 and reacts for 20min at 50 ℃. Immediately after the reaction was completed, the centrifuge tube was cooled on ice for 2min for transformation into competent cells DH5 α.
TABLE 2 ligation reaction System
| Reaction components | Volume (μ L) | |
| pET28a+sumo | 100ng | |
| | 40ng | |
| 2×Seamless |
1 | |
| ddH 2 O | To 20 |
10 positive cloning bacteria are selected to extract recombinant plasmids according to the instruction of a TIAnprep Rapid Mini Plasmid Kit. The extracted 10 recombinant plasmids are used as templates, gene amplification is carried out according to a PCR reaction system and PCR reaction conditions in the table 3, amplification products are identified in 1% agarose gel electrophoresis, and the plasmid capable of amplifying a target band is the positive transformation strain. The constructed pET28a + -VNAR-B7 vector was extracted using a TIANGEN rapid plasmid miniprep kit (DP-150), and verified by double digestion with BamHI and SalI restriction enzymes to verify that the target gene was ligated in the correct position, the digestion system is shown in Table 4. And (3) sending the positive recombinant plasmid to Shanghai workers for sequencing, and analyzing the insertion position, direction and size of the gene of the sequencing result.
TABLE 3PCR reaction System
| Reaction components | Volume/. mu.L |
| 10X pfu |
5 |
| Primer-1 | 2 |
| Primer-2 | 2 |
| |
1 |
| DNTP(25mM each) | 1 |
| Pfu(5μ/μL) | 0.4 |
| ddH 2 O | To 50 |
The PCR reaction conditions were:
95℃-3min
95℃-30s
55℃-30s
72℃-40s
72℃-5min
4 deg.C-preservation
Wherein, the circulation is carried out for 25 times in the degrees of 95-30 s, 55-30 s and 72-40 s.
TABLE 4 restriction enzyme system
| Reaction reagent | Reaction volume (μ L) |
| pET 28a + VNAR-B7 vector | 200ng |
| |
5 |
| NdeI(10u/μl) | 1 |
| SalI(10u/μl) | 1 |
| ddH 2 O | To 50 |
3.2 inducible expression of a specific shark Single Domain antibody of silver carp MBSP
To improve the expression efficiency of the fusion protein, the recombinant plasmid was transformed into the expression strain e.coli BL21 competition Cells, and then induced to express.
(1) 20ml of overnight-cultured broth was inoculated at a rate of 1: 100 portions of the cells were inoculated into 4L of LB medium, added with 50. mu.g/mL kanamycin to a final concentration, and cultured at 37 ℃ and 220 rpm.
(2) When the OD reached 0.6, IPTG was added to a final concentration of 0.3mM, 220rpm, and induction was carried out at 16 ℃ for 20 hours, and a negative control in which IPTG inducer was not added was used.
(3) Centrifuging at 4000rpm for 15min to collect thallus, discarding supernatant, suspending thallus with 60mL Tris-HCl (pH8.0) buffer solution, crushing at high pressure for 3min, collecting supernatant and precipitate, dissolving the precipitate with 10mL Tris-HCl (pH8.0), and purifying the supernatant.
(4) Purifying the fusion protein by nickel ion affinity chromatography, respectively collecting eluates of each component, performing SDS-PAGE detection, desalting and dialyzing the completely purified eluates into ultrapure water for 16h, changing dialysate every 8h, and centrifuging at 12000rpm for 20 min.
3.3 enzymatic hydrolysis and purification of fusion protein B7-SUMO
The SUMO protein label of the purified fusion protein B7-SUMO is cut off by SUMO protease (Ulp) according to the following ratio: SUMO protease and target protein to be cut are 1: 100, and the reaction system is shown in Table 4 and is carried out overnight at 4 ℃. Under the enzymatic hydrolysis system in Table 4, the SUMO tag on fusion protein B7-SUMO was separated from the protein of interest P2-B7. Separating and purifying the enzymolysis product by using a nickel ion affinity chromatography technology.
TABLE 4 fusion protein B7-SUMO enzymolysis system
| Reaction components | Volume (μ L) |
| Fusion proteins | 1000μg |
| 10× |
20 |
| |
2 |
| ddH 2 O | To 1000 |
As shown in FIG. 2, the SUMO protease was successful in completely digesting the expression product. As shown in FIG. 3, after the enzymolysis system is purified by a nickel ion affinity chromatography column, the target protein P2-B7 with the size of 14kDa can be obtained.
4. Analysis of Effect of specific shark-derived Single Domain antibody on inhibition of Myxoplasma serine protease (MBSP) Activity
4.1 affinity verification of specific shark-derived single-domain antibody P2-B7 with silver carp MBSP
In the ELISA test sample design scheme of Table 5, indirect ELISA validation was performed using silver carp MBSP as antigen, and the affinity of the specific shark-derived single-domain antibody P2-B7 to silver carp MBSP was analyzed.
As shown in the figure 4, the indirect ELISA proves that the ELISA light absorption values of the target protein P2-B7 with the concentrations of 0.557mg/ml (stock solution) and 0.1mg/ml are significantly higher than those of a positive control group, and the indirect ELISA has good affinity with chub MBSP.
TABLE 5 ELISA sample design
4.2 analysis of inhibitory Effect of specific shark-derived Single-Domain antibody P2-B7 on the enzymatic Activity of silver carp MBSP
Since the myofibrillar bound serine protease MBSP decomposes the fluorogenic substrate Boc-Phe-Ser-Arg-MCA at 55 ℃ the amount of the product AMC released by the reaction can be determined with a fluorospectrophotometer at the excitation wavelength of 380nm and the emission wavelength of 450 nm. Therefore, the inhibitory effect of different concentrations of the shark-derived single domain antibody on MBSP can be analyzed by the fluorescence intensity measured by the reaction of the shark-derived single domain antibody and fish MBSP.
The specific method comprises the following steps: 50. mu.L (about 0.03units) of suitably diluted highly purified silver carp MBSP in 20mmol/L Tris-HCl buffer (pH8.0) was added to each of shark-derived single domain antibodies (0mg/mL, 0.5mg/mL, 0.1 mg/mL) at different concentrations, reacted at room temperature for 30min, immediately added to 50. mu.L 10. mu. mol/L Boc-Phe-Ser-Arg-MCA, reacted at 55 ℃ for 30min, and then the reaction was terminated with 1.5mL of a terminator. The fluorescence values were measured with a fluorescence spectrophotometer at an excitation wavelength of 380nm and an emission wavelength of 450 nm.
As shown in FIG. 5, after the shark-derived single-domain antibody with different concentrations and the enzyme substrate are added to fully act under appropriate conditions, the fluorescence intensity is significantly different from that of a blank control group, which indicates that the adding of the shark-derived single-domain antibody with different concentrations has a good inhibition effect on the enzymatic activity of the silver carp MBSP, and about 50% of inhibition rate can be generated on the enzymatic activity of the MBSP under the antibody concentration of 0.1 mg/ml.
4.3 analysis of inhibitory Effect of specific shark-derived Single Domain antibody on deterioration of minced silver carp gel
4.3.1 preparation of silver carp minced fillet gel
200g of frozen silver carp minced fillet is taken, semi-thawed, cut into strips and placed into a vacuum cutting and mixing machine (with a condensation cycle) for cutting and mixing for 5min, then 2.5% of salt and 0.01 per thousand of specific single domain antibody are added (meanwhile, a reference group is set according to the existing research literature, the positive control is 0.03% of soybean trypsin inhibitor STI, the negative control is 0.01 per thousand BSA, a blank group is PBS, the adding volume is 20m1, the vacuum cutting and mixing is carried out for 5min, the temperature is controlled to be about 4 ℃ in the experimental operation, then the cut and mixed composite sol is poured into a 30mm nylon casing, the nylon casing is sealed, and the nylon casing is placed into a refrigerator with 4 ℃ for 3 days (the pure minced fillet forms a stable gel system).
4.3.1 dynamic rheological measurements of surimi gels
After correcting the MCR101 rheometer, the prepared minced fillet gel slice is placed on a platform of the minced fillet gel slice, and the minced fillet gel slice is sealed by using dimethyl silicone oil so as to prevent the volatilization of moisture. Parallel plates with a diameter of 50mm and a PP50 rotor were selected, and the distance between the rotor and the stage was set to 1 mm. Firstly, strain scanning test is carried out in the range of 0.01-100% at 25 ℃ and 1Hz, and the relation curve between the storage modulus (G ') and the loss modulus (G') and the strain is recorded and analyzed to determine the linear viscoelasticity range of the sample. The temperature dependence of storage modulus, loss modulus and loss factor was determined by heating from 10 ℃ to 100 ℃ at a heating rate of 5/min, then holding at 100 ℃ for 5min, setting the oscillation frequency to 1Hz, setting the strain to 2%.
As shown in FIG. 6, MBSP in fish meat is activated at 50 ℃ to maximize gel deterioration. After 0.01 per mill of silver carp P2-B7 specific shark source single-domain antibody is added, when the temperature reaches 50-60 ℃, the change range of the G 'value is small, the G' value is obviously improved at 90 ℃, the activity of MBSP is effectively inhibited, and the gel degradation degree of minced fillet is reduced to about 56.2%.
The specific analysis is as follows:
the storage modulus G' of all experimental groups showed a tendency to decrease and then increase throughout the temperature scan and to bottom out around 50 ℃. Studies have shown that endogenous cathepsin is activated in the 50-60 ℃ range, resulting in degradation of myosin heavy chain, exposure of hydrophobic groups, and thus breakdown deterioration of the overall gel structure.
1. Compared with other sample groups, the control group has the most drastic change of G 'and the smallest G' at the end of temperature scanning, which indicates that the endogenous cathepsin activity is the highest, the deterioration degree of the internal gel structure is the most serious, and the gel strength of the formed gel network is lower. According to the existing research, 0.03% STI and 0.01 ‰ BSA are selected as negative and positive controls respectively, and the experimental result shows that the change of G' in a BSA sample group is gentler than that of a control group, but the value is still smaller, which shows that the BSA can inhibit the degradation of myosin to a certain extent but has a smaller effect of improving the strength of the whole gel system. Compared with the sample group added with STI, the G' value of the sample group is obviously increased, and the change amplitude is small, which shows that STI can effectively inhibit the degradation of the heavy chain of the surimi-myosin and increase the gel strength of the system.
2. The change trend of G ' of a sample group added with 0.01 per thousand single domain antibody P2-B7 is similar to that of an STI group, the change degree of G ' is small in the range of 50-60 ℃, and the G ' value is remarkably improved in the whole temperature scanning range, which shows that the addition of P2-B7 can effectively inhibit the activity of endogenous cathepsin of silver carp surimi, so that the deterioration degree of a formed gel system is reduced, and the gel strength of the system is increased.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.
Sequence listing
<110> China oceanic university
<120> biological inhibitor for chub surimi gel deterioration
<130> 2019
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 363
<212> DNA
<213> hinged shark (Ginglymostoma cirratum)
<400> 1
ataataagga attccatggc tcgagtggac caaacaccgc aaacaataac aaaggagacg 60
ggcgaatcac tgaccatcaa ctgtgtccta cgagatagta actgtgcatt gtccagcacg 120
tactggtatc gcaaaaaatc gggctcaaca aacgaggaga gcatatcgaa aggtggacga 180
tatgttgaaa cagttaacag cggatcaaag tccttttctt tgagaattaa tgatctaaca 240
gttgaagaca gtggcacgta tcgatgcaag gtatacggcc ggactccgat gatacgggat 300
agctgggctt ggtgtcgaac tggcgaagat gtatacggag atggcactgc cgtgactgtg 360
aat 363
<210> 2
<211> 129
<212> PRT
<213> hinged shark (Ginglymostoma cirratum)
<400> 2
Gly Ser Ile Ile Arg Asn Ser Met Ala Arg Val Asp Gln Thr Pro Gln
1 5 10 15
Thr Ile Thr Lys Glu Thr Gly Glu Ser Leu Thr Ile Asn Cys Val Leu
20 25 30
Arg Asp Ser Asn Cys Ala Leu Ser Ser Thr Tyr Trp Tyr Arg Lys Lys
35 40 45
Ser Gly Ser Thr Asn Glu Glu Ser Ile Ser Lys Gly Gly Arg Tyr Val
50 55 60
Glu Thr Val Asn Ser Gly Ser Lys Ser Phe Ser Leu Arg Ile Asn Asp
65 70 75 80
Leu Thr Val Glu Asp Ser Gly Thr Tyr Arg Cys Lys Val Tyr Gly Arg
85 90 95
Thr Pro Met Ile Arg Asp Ser Trp Ala Trp Cys Arg Thr Gly Glu Asp
100 105 110
Val Tyr Gly Asp Gly Thr Ala Val Thr Val Asn Ala Ala Ala Ser Leu
115 120 125
Ile
Claims (4)
1. A shark origin single domain antibody gene with high specificity to silver carp myofibril-binding serine protease is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1: ATAATAAGGAATTCCATGGCTCGAGTGGACCAAACACCGCAAACAATAACAAAGGAGACGGGCGAATCACTGACCATCAACTGTGTCCTACGAGATAGTAACTGTGCATTGTCCAGCACGTACTGGTATCGCAAAAAATCGGGCTCAACAAACGAGGAGAGCATATCGAAAGGTGGACGATATGTTGAAACAGTTAACAGCGGATCAAAGTCCTTTTCTTTGAGAATTAATGATCTAACAGTTGAAGACAGTGGCACGTATCGATGCAAGGTATACGGCCGGACTCCGATGATACGGGATAGCTGGGCTTGGTGTCGAACTGGCGAAGATGTATACGGAGATGGCACTGCCGTGACTGTGAAT are provided.
2. A shark-derived single-domain antibody having high specificity for a silver carp myofibril-binding serine protease, which comprises an amino acid sequence shown in SEQ ID NO. 2: GSIIRNSMARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEESISKGGRYVETVNSGSKSFSLRINDLTVEDSGTYRCKVYGRTPMIRDSWAWCRTGEDVYGDGTAVTVNAAASLI are provided.
3. A biostatic agent for the deterioration of minced silver carp gel, comprising the shark-derived single domain antibody according to claim 2.
4. The use of the shark-derived single domain antibody of claim 2 in the preparation of a biological inhibitor for inhibiting the deterioration of chub surimi gel.
Priority Applications (1)
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| CN103102413A (en) * | 2013-01-21 | 2013-05-15 | 集美大学 | Soybean trypsin inhibitor antibody as well as preparation method and application thereof |
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| Shark IgNAR-derived binding domains as potential diagnostic and therapeutic agents;Hanover Matz et al;《Developmental and Comparative Immunology》;20180917;100-107 * |
| 超高压对鲢鱼中关键酶与结构蛋白质构影响的研究;邱春江;《中国优秀博硕士学位论文全文数据库(博士) 工程科技Ⅰ辑(月刊)》;20141215;24-44 * |
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