CN109486901B

CN109486901B - Method for detecting glutamine transaminase in minced fillet product

Info

Publication number: CN109486901B
Application number: CN201811338788.1A
Authority: CN
Inventors: 张凌晶; 曹敏杰; 刘光明; 孙乐常; 章骞; 靳小莹
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2018-11-12
Filing date: 2018-11-12
Publication date: 2022-08-05
Anticipated expiration: 2038-11-12
Also published as: CN109486901A

Abstract

The invention relates to a detection method of a food additive, in particular to a method for detecting glutamine transaminase in a minced fillet product, which comprises four steps of separating and purifying the glutamine transaminase food additive, measuring the concentration of the glutamine transaminase, detecting and analyzing Western-blotting sensitivity, detecting the commercial minced fillet product by adopting an established Western-blotting method and the like.

Description

Method for detecting glutamine transaminase in minced fillet product

Technical Field

The invention relates to a detection method of a food additive, in particular to a method for detecting glutamine transaminase in a minced fillet product.

Background

Transglutaminase has the functions of catalyzing the cross-linking of protein molecules and between molecules, the connection between protein and amino acid and the hydrolysis of glutamine in protein molecules, and is widely applied to the fields of food, medicine, textile and the like. Protein is the main factor for maintaining the texture characteristics of meat products, and the cross-linking and coagulation of protein molecules endow the food with mechanical properties. The cross-linking of protein molecules imparts a number of properties to the food product, including viscosity, solubility, emulsifiability, and gelling properties. Transglutaminase is of interest because it is capable of cross-linking proteins, catalyzing the acyl transfer reaction of primary amines with the carboxyamino group on the peptide bond of the glutamine residue. The detection method provided by the invention can effectively detect the content of glutamine transaminase in commercially available minced fillet products.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for detecting glutamine transaminase in a minced fillet product, which is simple to operate and accurate in result.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that:

a method for detecting glutamine transaminase in minced fillet products comprises the following steps:

(1) separation and purification of glutamine transaminase (MTGase) food additive: weighing 6g of glutamine transaminase (MTGase) food additive by using a two-aqueous phase extraction method, dissolving the food additive in 400mL of 20mmol/L Tris-HCl, and extracting the food additive with polyethylene glycol 6000/(NH4)2SO4 according to the proportion of 1: 5, standing for 2 hours, and taking a lower-layer liquid phase;

(2) determination of the concentration of transglutaminase (MTGase): determining protein concentration by using a method of a Bio-Rad kit, establishing a standard curve according to the light absorption values of BSA with different concentrations at 595nm, and calculating the concentration of TGase;

(3) detection and analysis of Western-blotting sensitivity: using 1/50mg/mL MTGase as the initial concentration, adopting a 2-fold dilution method, gradually diluting to 1/800mg/mL, and adding an anti-MTGase antibody in a ratio of 1: 10000 of the reagent is diluted, 10 mu L of the reagent is loaded, and the lowest detection limit of Western-blotting is determined;

(4) detecting the commercial minced fillet products by adopting an established Western-blotting method: performing MTGase extraction and protein concentration determination on the minced fillet product by using 20mmol/L Tris-HCl buffer solution; the MTGase added to the fish slurry was detected by SDS-PAGE analysis using the purified MTGase as a semi-quantitative control.

Preferably, the Tris-HCl buffer solution contains 8moL/L urea and 0.1mmol/L DTT, pH8.0 in Tris-HCl buffer.

Preferably, in the step (4) of measuring the protein concentration, the concentration of the Tris-HCl buffer solution is too high, and appropriate dilution is required.

Drawings

FIG. 1 is an electrophoretogram of SDS-PAGE of an MTGase extract according to the present invention;

wherein, M, standard molecular weight protein; 1. commercial transglutaminase food additive, 2, purified MTGase.

FIG. 2 is an immunoblot analysis of MTGase extract.

FIG. 3 is a standard curve of bovine serum albumin concentration.

FIG. 4 is an analysis graph of the sensitivity of the method;

wherein, the MTGase concentration of each lane is: 1. 1/50mg/mL, 2, 1/100mg/mL, 3, 1/200mg/mL, 4, 1/400mg/mL, 5, 1/800 mg/mL.

FIG. 5 shows the results of detection of MTGase in commercially available fish paste;

wherein, C, purified MTGase, 1, trash fish paste, 2, anoectochilus formosanus paste, and 3, freshwater fish paste.

FIG. 6 shows the detection result of MTGase in commercially available fish balls;

wherein, c, purified MTGase, 1, fish balls, 2, fish tofu, 3 and crisp balls.

FIG. 7 shows the results of SDS-PAGE and Western-blotting analyses of antibody specificity;

wherein, c, 1/200mg/mL TGase, 1, chub crude extract protein, 2, balang fish crude extract protein, 3, grass carp crude extract protein.

FIG. 8 is a comparison of the endogenous glutamine transaminase content of fish;

wherein, c, 1/200mg/mLTGase, 1, crude protein of grass carp, 2, crude protein of silver carp, 3, crude protein of Palangyu, 4, crude protein of weever, and 5, crude protein of crucian.

FIG. 9 is a comparison of fish ball and endogenous TGase antibody specificity;

wherein, c, a control (1/200mg/mL MTGase), 1, crude extracted protein of weever, No. 2 and No. 1 fish balls, No. 3 and No. 2 fish bean curd, No. 4 and No. 3 crisp balls, No. 5 and No. 4 shrimp dumplings, and No. 6 and No. 5 fish bean curd.

Detailed Description

In order to make the content of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

(1) separation and purification of glutamine transaminase (MTGase) food additive: using the aqueous two-phase extraction method, 6g of glutamine transaminase (MTGase) food additive was weighed, dissolved in 400mL of 20mmol/L Tris-HCl (pH8.0), and extracted with polyethylene glycol 6000/(NH4)2SO4 in a ratio of 1: 5, standing for 2 hours, and taking a lower-layer liquid phase;

the resulting solution was subjected to SDS-PAGE analysis, and the final results are shown in FIG. 1. There is only a single band between 35kDa and 45kDa, consistent with the molecular weight of MTGase reported in the reference standard literature. The protein is further verified by Western-blotting, and the result is shown in figure 2, the protein and rabbit anti-MTGase have immunoreaction, which indicates that the lower liquid phase is purified MTGase.

as shown in fig. 3, the calculation equation is: y is 0. 8899x + 0. 5187 the correlation coefficient is 0. 9924, it shows that the curve is linear, and the calculated TGase concentration is 0. 12 mg/mL.

(3) Analysis of Western-blotting sensitivity: using 1/50mg/mL MTGase as the initial concentration, adopting a 2-fold dilution method, gradually diluting to 1/800mg/mL, and adding an anti-MTGase antibody in a ratio of 1: 10000 of the sample is loaded by 10 mu L under the dilution of the proportion, and the lowest detection limit of Western-blotting is determined;

as a result, as shown in FIG. 4, SDS-PAGE showed that the minimum detection limit was 1/200mg/mLMTGase, which corresponds to 25mg of TGase diet. Western-blotting results show that the bands show a decreasing trend with the decrease of TGase concentration; when the TGase concentration is 1/400mg/mL, clear bands still remain, and finally the lowest detection limit of Western-blotting is determined to be 1/400mg/mL MTGase, which is equivalent to 5mg of TGase food.

(4) Detecting the commercial minced fillet products by adopting an established Western-blotting method: the solution contains 8mol/L urea and 0. Extracting minced fillet product with 20mmol/L Tris-HCl (pH8.0) buffer solution of 1mmol/L DTT by use of transglutaminase (MTGase), and determining protein concentration by diluting if the concentration is too high; the MTGase added to the fish slurry was detected by SDS-PAGE analysis using the purified MTGase as a semi-quantitative control.

In the process, three commercially available fish pastes and three commercially available fish balls are respectively subjected to grouping detection, and the results are as follows:

MTGase was detected in 3 commercially available fish pulps by the method described in the procedure (4), and as shown in FIG. 5-I, proteins were shown in the positions of the target protein bands for all three fish pulps. In the anti-MTGase antibody at 1: 10000 dilution, and the results are shown in FIGS. 5-II, and strong immunoreactions exist in the

lanes

2 and 3, which proves that the method can detect MTGase added in fish paste.

MTGase was detected in 3 commercially available fish balls by the method described in the process (4), and as shown in FIG. 6-I, the extracted protein in lane 1 was significantly higher than that in the other 2 lanes at the position of the molecular weight of the target protein. In the anti-MTGase antibody at 1: 10000 dilution, as shown in FIG. 6-II, the Western blotting analysis showed that the immunoreactions were present in all of

lanes

1, 2 and 3, and the immunoreactions in lane 1 were weak due to the small amount of the reagent added, indicating that the method can detect glutamine transaminase (MTGase) added to fish pellets.

Note that:

firstly, before detection, 0 containing 8moL/L urea needs to be prepared. 1mmoL/L DTT 20mmoL/L Tris-HCl buffer solution;

to extract more glutamine transaminase (MTGase), two buffers were chosen for the extraction alignment of MTGase:

buffer a: contains 0. 5mol/L NaCl in 20mmol/L Tris-HCl (pH8.0) buffer;

and (b) buffer solution: contains 8moL/L urea and 0.1mmoL/L DTT of 20mmoL/L Tris-HCl;

taking purified MTGase as a positive control, selecting 6 groups of commercially available minced fillet products as extracted raw materials, and obtaining the following products through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) experiments: at the target protein of 38kDa, the amount of protein extracted by using the two buffers is equivalent; the Western-blotting experiment shows that: the extraction rate of the target protein by using the buffer solution b is obviously higher than that of the buffer solution a.

Therefore, the MTGase extraction efficiency of the minced fillet product by using the buffer b is higher.

Secondly, interference of endogenous TGase needs to be eliminated in the detection process, the difficulty of detecting the glutamine transaminase (MTGase) in the minced fillet product lies in the interference of the endogenous TGase in fish meat, although the endogenous TGase in fish meat and the MTGase produced by microorganisms have different amino acid compositions around the center of an active site, the secondary structures are similar, and the optimal pH and the optimal temperature of the endogenous TGase and the MTGase are basically the same, so that the endogenous TGase and the MTGase are difficult to distinguish only by depending on the detection of the activity of the endogenous TGase.

Since the prepared anti-MTGase antibody is a polyclonal antibody, the antibody has non-specificity, and the immune reaction of the antibody and endogenous TGase in fish is not excluded.

In order to verify the specificity of the antibody to the endogenous TGase in fish and judge whether the antibody has immunoreaction with the TGase in fish, chub, grass carp and Palang fish are selected in experiments, and the ratio of the anti-MTGase antibody is 1: the dilution was carried out at a ratio of 1000 by Western-blotting. As shown in FIG. 7, significant immunoreaction occurred in both chub and balang, and thus the antibody reacted with TGase present in fish meat.

1. Selection of endogenous TGase: the raw materials are freshwater fish and low-value seawater fish which are often used as raw materials of minced fillet products, and comprise: grass carp, silver carp, Paeonia japonica, weever and crucian carp. The extraction of TGase was performed with Tris-HCl containing buffer, and the ratio of anti-MTGase antibody to 1: 1000, judging the content of the glutamine transaminase in the fish meat by an immunoblotting method. The Western-blotting result is shown in FIG. 8, the chub, the baggy fish, the weever and the crucian all show strips, which indicate that the immunoreaction occurs, and the strip shown at the weever is the clearest, which indicates that the weever has higher content of endogenous glutamine transaminase, so the weever is selected as a representative of the endogenous TGase of the fish.

2. Comparison of endogenous and exogenous TGase antibody specificities: crude protein extraction was performed on 6 groups of commercially available fish pellets using Tris-HCl buffer, immunoblot analysis was performed at different antibody concentrations using TGase in weever as endogenous reference and 1/200mg/mL MTGase as semi-quantitative comparison, and the Western-blotting results are shown in FIG. 9, in which FIG. 9-I the antibody was diluted at a ratio of 1:3000, and in which FIG. 9-II the antibody was diluted at a ratio of 1: dilution ratio of 5000, fig. 9-iii is antibody at 1: 10000 of diluted. From the results, it was found that the immune band of the commercially available fish balls became gradually lighter as the antibody concentration decreased, and the concentration thereof was preliminarily confirmed by comparing with the band of MTGase purified in the control group. When the concentration of the antibody is gradually reduced, the endogenous TGase immune bands of the fish gradually disappear. When the antibody is expressed as a 1: 10000 of the fish meat, endogenous TGase of the fish meat has no immunoreaction, and the fish ball still has immunoreaction.

In conclusion, the maximum protein amount can be obtained by using 20mmoL/L Tris-HCl (pH8.0) buffer solution containing 8moL/L urea and 0.1mmoL/L DTT for sample preparation; selecting the weever TGase with the highest content as an endogenous reference, and when the antibody is expressed by 1: 10000, the detection method eliminates the interference of endogenous TGase and can specifically detect the MTGase added in the surimi product.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting glutamine transaminase in minced fillet products is characterized by comprising the following steps: (1) separating and purifying the glutamine transaminase MTGase food additive: weighing 6g of glutamine transaminase MTGase food additive by using a two-aqueous-phase extraction method, dissolving the food additive in 400mL of 20mmol/L Tris-HCl buffer solution, and mixing with 6000/(NH) polyethylene glycol ₄ ) ₂ SO ₄ The extraction liquid is prepared according to the following steps of 1: 5, standing for 2 hours, and taking a lower-layer liquid phase;

(2) determination of the concentration of transglutaminase MTGase: determining protein concentration by using a Bio-Rad kit method, establishing a standard curve according to the light absorption values of BSA with different concentrations at 595nm, and calculating the concentration of glutamine transaminase TGase;

(3) detection and analysis of Western-blotting sensitivity: using 1/50mg/mL MTGase as initial concentration, adopting 2-fold dilution method to dilute to 1/800mg/mL step by step, and adding 1: 10000 of the sample is loaded by 10 mu L under the dilution of the proportion, and the lowest detection limit of Western-blotting is determined;

(4) detecting the commercial minced fillet products by adopting an established Western-blotting method: extracting the minced fillet product by using 20mmol/L Tris-HCl buffer solution to obtain transglutaminase (MTGase), measuring the protein concentration, and performing SDS-PAGE analysis by using the purified MTGase as a semi-quantitative control to detect the added MTGase in the fish paste;

wherein the 20mmol/L Tris-HCl buffer solution in the step (1) and the step (4) is a Tris-HCl buffer solution containing 8moL/L urea, 0.1mmol/L DTT and pH 8.0.