Nitric oxide-based fusion enzyme-based hair colorant and use

Abstract

The application discloses a one-oxygen-based fusion enzyme color developing agent and application. The color developing agent comprises a one-oxygen-based fusion enzyme, and the fusion enzyme is combined by a nitric oxide synthase, a flavoprotein and a flavoprotein reductase in sequence and two by two through a connecting peptide. The fusion enzyme provided by the application has high enzyme activity, can catalyze a large amount of one-oxygen, and can effectively combine myoglobin in meat products to generate nitroso-myoglobin, effectively improve the red color of the meat products, and obtain a color developing effect equivalent to that of sodium nitrite, thereby providing a very practically applicable solution for replacing the sodium nitrite color developing of the meat products and improving the safety of the meat products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of meat science and technology, and particularly relates to a color-developing agent based on a nitric oxide-producing fusion enzyme and application thereof. BACKGROUND

[0002] The color formation of fermented sausages usually relies on the addition of nitrite, the reduction product of which, nitric oxide (NO), interacts with myoglobin in meat to form a red pigment, nitrosylmyoglobin. However, due to its potential to cause teratogenicity and carcinogenicity, its use in meat products has attracted much attention. In order to reduce the use of nitrite in meat products, it is crucial to find a functional alternative. In recent years, the color-developing effect of coagulase-negative staphylococci (CNS) in fermented meat products has been studied, in which bacterial nitric oxide synthase can convert arginine into NO and citrulline, thereby promoting the formation of nitrosylmyoglobin in meat products.

[0003] Among the many alternatives to nitrite color development, microbial fermentation has great potential. Patent application No. CN110800913A discloses a color-developing agent that replaces nitrite in processed meat products. One of the color-developing agents is a bacterial powder or bacterial suspension of coagulase-negative staphylococci, which is used at an inoculation amount of 10 6 ~ 10 7 CFU / g meat in the processing of meat products. Another color-developing agent consists of a bacterial powder or bacterial suspension of coagulase-negative staphylococci and L-arginine. When used, the coagulase-negative staphylococci are inoculated in the processing of meat products at an amount of 10 6 ~ 10 7 CFU / g meat, and the L-arginine is added in the processing of meat products at an amount of 0.6-1.2% of its mass. The bacterial powder or bacterial suspension is prepared by culturing coagulase-negative staphylococci in a liquid medium. By using coagulase-negative staphylococci with nitric oxide synthase, patent application No. CN110800913A can produce NO in processed meat products, thereby forming red nitrosylmyoglobin and making the processed meat products appear red. Meanwhile, the addition of L-arginine can further enhance the red color. However, due to the low expression of nitric oxide synthase in coagulase-negative staphylococci, its color-developing effect is generally weaker than that of nitrite. Patent application No. CN114568644A discloses a processing method for promoting the red color enhancement of fermented sausages. The method involves subjecting fermented sausages inoculated with coagulase-negative staphylococci to low-intensity high hydrostatic pressure treatment and then to fermentation, so that the redness value of the finished sausages reaches 6.5-7.0. When used, the coagulase-negative staphylococci are inoculated in the sausages at an amount of 10 6 ~ 10 7CFU / g meat; the high hydrostatic pressure treatment pressure is 200-300 MPa, and the treatment time is 3-7 min. The low-intensity high hydrostatic pressure treatment promotes the production of nitrosylmyoglobin in fermented sausages by increasing the expression amount of nitric oxide synthase in coagulase-negative staphylococcus, thereby improving the color of fermented sausages. The processing method can improve the color of fermented sausages, provides a feasible solution for the color development of nitrite in fermented sausages, and meets the demand of people for healthy and safe fermented sausage products. However, since high hydrostatic pressure can cause the volume of food to shrink, this technology is only suitable for soft-packaged food, and high hydrostatic pressure technology cannot treat a large amount of raw materials at one time, and the cost is very high, so high hydrostatic pressure is mainly used in the processing of high-end products such as seafood and precious fruits in commercial products. The patent application with the publication number CN115232830A discloses a meat colorant based on recombinant bacterial nitric oxide synthase. A large amount of bacterial nitric oxide synthase is obtained by recombinant expression. When used, the inoculation amount of coagulase-negative staphylococcus suspension is 6.0-7.5 log CFU / g meat; the addition amount of recombinant bacterial crude extract is 3.8-4.2 mg / 100 g meat. The recombinant bacterial nitric oxide synthase has high enzyme activity and can catalyze a large amount of NO, but when it is applied to fermented sausages, it cannot produce enough NO due to the lack of sufficient reductase and various cofactors. In order to solve the problem of the lack of cofactors and reductase, the staphylococcus succinii is inoculated in the sausage making process, and the bacterial inoculation provides the cofactors and other substances required for the catalytic reaction of nitric oxide synthase. Compared with the simple recombinant bacterial nitric oxide synthase, the inoculation of staphylococcus succinii can produce a large amount of NO, effectively combine with myoglobin in meat products to produce nitrosylmyoglobin, and achieve good color development effect. The recombinant enzyme described in the application with the publication number CN115232830A has insufficient NO production capacity when used alone due to the lack of necessary cofactors and reductase, resulting in a nitrosylmyoglobin content in meat products far lower than that of meat products added with nitrite.

[0004] Although the above-mentioned recombinant bacterial nitric oxide synthase has a promoting effect on the formation of meat product color, it has insufficient NO production capacity in meat products, resulting in a nitrosylmyoglobin content in meat products far lower than that of meat products added with nitrite, and the meat product color development effect is far weaker than that of meat products added with sodium nitrite. In addition, the inoculation of a large amount of staphylococcus succinii will force the meat product to change the production process, especially when making non-fermented meat products, which is quite inconvenient. Therefore, it is necessary to find a new method to obtain nitric oxide synthase with higher activity and apply it to meat products. SUMMARY

[0005] The main purpose of the present application is to provide a color developer based on nitric oxide-producing fusion enzyme and application to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned technical purposes, the technical scheme adopted by the present application comprises the following.

[0007] The embodiment of the present application provides a color-developing agent based on a nitric oxide-producing fusion enzyme, which comprises the nitric oxide-producing fusion enzyme; the fusion enzyme is combined by nitric oxide synthase, flavoprotein and flavoprotein reductase in sequence two by two through a connecting peptide.

[0008] The embodiment of the present application also provides a use of a nitric oxide-producing fusion enzyme in the preparation of a color-developing agent, which is combined by nitric oxide synthase, flavoprotein and flavoprotein reductase in sequence two by two through a connecting peptide.

[0009] The embodiment of the present application also provides an application of the aforementioned color-developing agent based on a nitric oxide-producing fusion enzyme in the color development of meat products.

[0010] The embodiment of the present application also provides a color development method of a color-developing agent, which comprises: mixing and incubating the color-developing agent with an action substrate, so as to realize the color development of the action substrate.

[0011] The color-developing agent comprises the aforementioned color-developing agent based on a nitric oxide-producing fusion enzyme; and the action substrate comprises high-iron myoglobin or a meat product.

[0012] Compared with the prior art, the present application has the beneficial effects that: the present application directly connects the reductase domain YkuN-YumC to the bacterial nitric oxide synthase through gene fusion, reduces the complexity of the oxidation-reduction chain, improves the efficiency of electron transfer, provides the reductase domain required for the catalysis of the bacterial nitric oxide synthase, and thus significantly improves the efficiency of NO production of the fusion enzyme, effectively combines the myoglobin in the meat product to produce nitrosylmyoglobin, and achieves the color development effect equivalent to that of adding sodium nitrite. The fusion enzyme prepared by the present application can effectively replace sodium nitrite in the color development of meat products, and provides a very practical application solution for the replacement of sodium nitrite color development of meat products and the improvement of safety. The present application proposes the concept of enzymatic color development, which can directly develop the color of meat products by using only enzymes, retains the original process and traditional flavor of meat product production, reduces the use of sodium nitrite in meat products, is conducive to creating green food, and promotes food safety. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creating laborious work.

[0014] Figure 1 SDS-PAGE analysis diagram of the crude extract of the recombinant bacteria prepared in the present application comparative example 1, the recombinase enzyme solution and the crude extract of the fusion bacteria prepared in example 1, the fusion enzyme solution are shown; wherein lane 1, lane 2 correspond to the crude extract of the recombinant bacteria prepared in the present application comparative example 1, the recombinase enzyme solution respectively, lane 3, lane 4 correspond to the crude extract of the fusion bacteria prepared in example 1, the fusion enzyme solution respectively;

[0015] Figures 2a-2b The hematin absorption spectrum curve diagram of different processing groups of adding the present application test example 4 or comparative example 1 or example 1 is shown.

[0016] Figures 3a-3b The hematin absorption spectrum curve diagram of different processing groups of the present application example 2 and comparative examples 2-4 meat paste is shown. DETAILED DESCRIPTION

[0017] In view of the defects of the prior art, the present inventors have long-term research and a large number of practices, and have proposed the technical scheme of the present application. The technical scheme of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0018] Specifically, as one aspect of the technical scheme of the present application, a kind of chromogenic agent based on one oxygen production of fusion enzyme of the present application includes one oxygen production of fusion enzyme;The fusion enzyme is that nitric oxide synthase, flavoprotein, flavoprotein reductase are combined in order in turn two by two through connecting peptide.

[0019] Further, the amino acid sequence of the fusion enzyme is combined by Bacillus subtilis nitric oxide synthase, flavoprotein, flavoprotein reductase and two connecting peptides, specifically, nitric oxide synthase, flavoprotein and flavoprotein reductase are combined in order in turn two by two through connecting peptide into a fusion enzyme.

[0020] The recombinase described in the application with publication number CN115232830A is insufficient in NO production when used alone, due to the lack of necessary cofactors and reductase, resulting in a much lower content of nitrosylmyoglobin in meat products than meat products with added nitrite. YkuN and YumC are flavoprotein and flavoprotein reductase derived from Bacillus subtilis. Flavoprotein is an electron carrier that can provide electrons for various enzymes, including p450. The present inventors found that by gene fusion, YkuN-YumC can be directly connected to nitric oxide synthase, which can enhance the enzyme activity of nitric oxide synthase and significantly improve the color development effect.

[0021] In some preferred embodiments, the method for preparing the fusion enzyme comprises:

[0022] providing a fusion plasmid containing at least a nitric oxide synthase gene, a flavoprotein gene, and a flavoprotein reductase gene;

[0023] transforming the fusion plasmid into Bacillus subtilis to obtain a fusion strain;

[0024] and culturing the fusion strain, then incubating and separating after breaking the cell wall and mixing with Ni-NTA agarose purification resin to obtain the fusion enzyme.

[0025] Further, the method for preparing the fusion enzyme specifically comprises:

[0026] a1. amplifying the nitric oxide synthase gene, the flavoprotein gene, and the flavoprotein reductase gene fragments in a PCR system using Bacillus subtilis 168 genomic DNA as the template; wherein the nitric oxide synthase upstream primer used in the PCR system has the sequence shown in SEQ ID NO. 1, the nitric oxide synthase downstream primer has the sequence shown in SEQ ID. NO. 2 or SEQ ID NO. 3; the flavoprotein upstream primer used has the sequence shown in SEQ ID NO. 4, and the flavoprotein downstream primer has the sequence shown in SEQ ID NO. 5; the flavoprotein reductase upstream primer used has the sequence shown in SEQ ID NO. 6, and the flavoprotein reductase downstream primer has the sequence shown in SEQ ID NO. 7;

[0027] a2. amplifying the linearized plasmid in a PCR system using pP43NMK as the template to obtain the linearized pP43NMK plasmid; wherein the pP43NMK upstream primer used in the PCR system has the sequence shown in SEQ ID NO. 8, and the pP43NMK downstream primer has the sequence shown in SEQ ID NO. 9;

[0028] a3. adding FastDigest DpnI enzyme to the linearized pP43NMK plasmid obtained in step a2, and processing on a PCR instrument to eliminate circular plasmids, and then inactivating the FastDigest DpnI enzyme to obtain the pP43NMK linear plasmid;

[0029] a4. fusing the nitric oxide synthase gene, the flavoprotein gene, and the flavoprotein reductase gene fragments obtained in step a1 by triple fusion PCR in a PCR1 system to obtain a fusion fragment, and then further amplifying the fusion fragment in a PCR2 system to obtain a large amount of fusion fragment amplification product;

[0030] a5, the fusion fragment amplification product obtained in step a4 and the linear pP43NMK plasmid obtained in step a3 are subjected to seamless ligation to obtain a fusion plasmid A; then the fusion plasmid A is mixed with E. coli JM109 competent cells to obtain E. coli JM109 containing the fusion plasmid A;

[0031] a6, the E. coli JM109 containing the fusion plasmid A is cultured in LB medium containing ampicillin to obtain bacterial cells, and then the bacterial cells are subjected to plasmid extraction to obtain a fusion plasmid B;

[0032] a7, a histidine tag is added to the end of the flavoprotein reductase gene in the fusion plasmid B obtained in step a6 to obtain E. coli TOP10 containing the fusion plasmid;

[0033] a8, the E. coli TOP10 containing the fusion plasmid obtained in step a7 is cultured in LB medium containing ampicillin to obtain bacterial cells, and then the bacterial cells are subjected to plasmid extraction to obtain a fusion plasmid.

[0034] Further, the preparation method of the fusion enzyme specifically comprises: thawing the B. subtilis 168 competent cells at room temperature, and then adding the obtained fusion plasmid to incubate, culture and screen to obtain a fusion strain.

[0035] Further, the preparation method of the fusion enzyme specifically comprises:

[0036] c1, a single colony of the obtained fusion strain is inoculated into a culture medium containing kanamycin, and then the strain in the culture system is isolated;

[0037] c2, the strain isolated in step c1 is washed and broken, and then the supernatant is separated to obtain a fusion bacteria crude extract;

[0038] c3, the fusion bacteria crude extract obtained in step c2 is mixed with Ni-NTA agarose purification resin to incubate, and then the protein is eluted to obtain a fusion enzyme enzyme solution; preferably, the concentration of the fusion enzyme in the fusion enzyme enzyme solution is 0.1-0.5 mg / mL.

[0039] In some more specific embodiments, the preparation method of the fusion enzyme mainly comprises:

[0040] Y1, providing a fusion plasmid containing a nitric oxide synthase gene, a flavoprotein gene and a flavoprotein reductase gene fragment;

[0041] Y2, transforming B. subtilis with the fusion plasmid in step Y1 to obtain a fusion strain;

[0042] Y3, culturing the fusion strain obtained in step Y2, breaking the wall, and then mixing with Ni-NTA agarose purification resin to incubate, and then isolating to obtain the fusion enzyme.

[0043] In one embodiment, the preparation method of the fusion plasmid in step Y1 specifically comprises the following steps:

[0044] a1, amplifying nitric oxide synthase gene, flavoprotein gene and flavoprotein reductase gene fragments in a PCR system with B. subtilis 168 genomic DNA as a template;

[0045] a2, amplifying linearized plasmid in a PCR system with pP43NMK as a template, and the obtained PCR product is the linearized pP43NMK plasmid;

[0046] a3, adding Fast Digest DpnI enzyme to the linearized pP43NMK plasmid PCR product obtained in step a2, treating at 37°C for 10 min on a PCR instrument to eliminate circular plasmid, and then inactivating the Fast Digest DpnI enzyme at 85°C for 10 min to obtain the pP43NMK linear plasmid;

[0047] a4, fusing the nitric oxide synthase gene, flavoprotein gene and flavoprotein reductase gene fragments obtained in step a1 by triple fusion PCR in PCR1 system to obtain a fusion fragment, and then further amplifying the fusion fragment in PCR2 system to obtain a large amount of fusion fragment amplification product;

[0048] a5, performing seamless ligation on the fusion fragment amplification product obtained in step a4 and the pP43NMK linear plasmid obtained in step a3 to obtain a fusion plasmid A; taking 10 μL of the fusion plasmid A and adding it into 100 μL of E. coli JM109 competent cells, ice-bathing for 30 min, then heat-shocking at 42°C for 45 s, and then immediately placing on ice for 3 min; adding 1 mL of LB medium, and incubating at 37°C for 1 h, then centrifuging to discard 900 μL of supernatant, resuspending the remaining culture with the remaining medium and spreading on an LB plate containing ampicillin, and culturing at 37°C for 16 h to obtain E. coli JM109 containing the fusion plasmid A;

[0049] a6, culturing the E. coli JM109 containing the fusion plasmid A obtained in step a5 in LB medium containing ampicillin for 12 h, centrifuging the culture at 8000g for 2 min at 0-4°C to obtain bacterial cells, and using a DiaSpin column plasmid DNA small-scale extraction kit to extract the plasmid to obtain the fusion plasmid B;

[0050] a7, adding a histidine tag to the end of the flavoprotein reductase gene in the fusion plasmid B obtained in step a6 to obtain E. coli TOP10 containing the fusion plasmid;

[0051] a8. E. coli TOP10 containing the fusion plasmid was cultured in LB medium containing ampicillin for 12 h. The culture was centrifuged at 8000g for 2 min at 0-4℃ to obtain bacterial cells. The plasmid was extracted using the DiaSpin column-based plasmid DNA mini-extraction kit to obtain the fusion plasmid.

[0052] Furthermore, the PCR system described in step a1 includes: 2 μL each of 10 mM upstream and downstream primers, 25 μL PrimeSTARMax DNA Polymerase, 21 μL sterile water, and 0.5 μL 25 mmol / L MgCl2. A small amount of Bacillus subtilis 168 single colony is picked up with an inoculation loop and inoculated into the system and stirred well. The PCR reaction program is as follows: 95℃ for 15 min pre-denaturation, 95℃ for 30 s, 56℃ for 30 s and 72℃ for 2 min as one cycle, for a total of 35 cycles, and finally extension for 5 min.

[0053] Furthermore, the PCR system described in step a2 includes: 2 μL each of 10 mM upstream and downstream primers, 2 μL of 20–100 mg / μL pP43NMK plasmid, 25 μL of PrimeSTAR Max DNA Polymerase, and 19 μL of sterile water; the PCR reaction program is as follows: 95℃ for 5 min pre-denaturation, 95℃ for 30 s, 56℃ for 30 s and 72℃ for 2 min as one cycle, for a total of 35 cycles, and finally extension for 5 min.

[0054] Further, the PCR1 system described in step a4 includes: 10 μL PrimeSTAR Max DNA Polymerase, 10 μL nitric oxide synthase sequence, flavoprotein sequence, and flavoprotein reductase sequence (concentration ratio of 1:2:1); the PCR1 reaction program is: 95℃ for 3 min pre-denaturation; 95℃ for 30 s, 56.8℃ for 30 s, and 72℃ for 2 min as one cycle, for a total of 15 cycles; and finally, extension for 5 min; the PCR2 system includes: 2 μL fusion fragment, 2 μL each of 10 mM forward and reverse primers, 25 μL PrimeSTAR Max DNA Polymerase, and 19 μL sterile water; the PCR2 reaction program is: 95℃ for 3 min pre-denaturation; 95℃ for 30 s, 56℃ for 30 s, and 72℃ for 2 min as one cycle, for a total of 35 cycles; and finally, extension for 5 min.

[0055] In one embodiment, the preparation method of the fusion strain described in step Y2 specifically includes the following steps: thawing the prepared Bacillus subtilis 168 competent cells at room temperature, adding the fusion plasmid from step Y1, incubating at 37°C with shaking at 200 rpm for 2 hours, then plating on LB plates containing kanamycin and culturing for 18 hours to obtain the fusion strain.

[0056] In one embodiment, the fusion enzyme in step Y3 is a purified fusion enzyme solution, and the preparation method comprises the following steps:

[0057] c1. picking the single colony of the fusion strain obtained in step Y2 and inoculating it into a culture medium containing kanamycin for culture, and then separating the strain in the culture system;

[0058] c2. washing the strain separated in step c1 thoroughly, then performing wall breaking treatment, and then separating the supernatant to obtain a crude fusion enzyme solution;

[0059] c3. mixing the crude fusion enzyme solution obtained in step c2 with Ni-NTA agarose purification resin for incubation, and then obtaining a purified fusion enzyme solution after eluting the protein.

[0060] Further, the preparation method of the fusion enzyme solution comprises the following steps:

[0061] c1. picking the single colony of the fusion strain, inoculating it into a TB culture medium containing kanamycin, and culturing it at 36-38°C for 18-20h, and then taking the precipitate to obtain the bacterial body;

[0062] c2. washing the bacterial body obtained in step c1 with 15-20mM phosphate buffer with a pH value of 7.4-7.5 for 3-5 times, performing ultrasonic wall breaking at 0-4°C for 12-15min, then centrifuging at 0-4°C at a speed of 10000-12000rpm for 5-8min, and then taking the supernatant to obtain a crude fusion enzyme solution;

[0063] c3. incubating the crude fusion enzyme solution obtained in step c2 with Ni-NTA agarose purification resin at 0-4°C for 1.8-2.2h, then eluting the protein with Elution Buffer with a pH value of 7.8-8.2, and eluting the protein 8-12 times with 750ul Elution Buffer, so as to obtain the fusion enzyme solution, and the concentration of the target enzyme solution is 0.1-0.5mg / mL.

[0064] In one embodiment, the wall breaking treatment in step c2 is ultrasonic wall breaking, and the specific conditions are as follows: working for 2-3s at an interval of 2-3s at a frequency of 20-25kHz and a power of 100-500w.

[0065] The present application uses fusion and recombinant expression technology to express a large amount of fusion enzyme in an edible grade bacillus subtilis expression system, and adds the expression product to meat products, which can effectively promote the generation of nitroso-myoglobin and better improve the color development effect of meat products.

[0066] Another aspect of the embodiments of the present application also provides a use of a nitric oxide-producing fusion enzyme in preparing a color-developing agent, wherein the fusion enzyme is formed by combining nitric oxide synthase, flavoprotein and flavoprotein reductase in sequence via a connecting peptide.

[0067] The fusion enzyme provided in the present application has high enzyme activity, can catalyze the production of a large amount of nitric oxide, and can effectively combine myoglobin in meat products to produce nitrosylmyoglobin, effectively improve the red color of meat products, and obtain a color-developing effect comparable to sodium nitrite, thereby providing a very practically significant solution for the replacement of sodium nitrite color development and the improvement of the safety of meat products.

[0068] Another aspect of the embodiments of the present application also provides an application of the aforementioned color-developing agent based on the nitric oxide-producing fusion enzyme in the color development of meat products.

[0069] Further, the meat products include fermented and / or non-fermented meat products.

[0070] Further, the meat products include sausages, hams, luncheon meats, bacon, meat biscuits, etc.

[0071] Another aspect of the embodiments of the present application also provides a color development method of a color-developing agent, which comprises: mixing and incubating the color-developing agent with an action substrate, so as to realize the color development of the action substrate.

[0072] In the color development method, the color-developing agent includes the aforementioned color-developing agent based on the nitric oxide-producing fusion enzyme, and the action substrate includes high-iron myoglobin or meat products.

[0073] In some preferred embodiments, the NO produced by the color-developing agent can convert brown high-iron myoglobin into bright red nitrosylmyoglobin.

[0074] In some preferred embodiments, the color development method specifically comprises: mixing the color-developing agent with the action substrate and incubating at 4-42℃ for 0.5-30h.

[0075] In some preferred embodiments, the concentration of the fusion enzyme in the color-developing agent is 0.1-0.5mg / mL.

[0076] In some preferred embodiments, the color development method specifically comprises: adding the color-developing agent into LB medium containing 4-6me / mL high-iron myoglobin and 8-12mmol / L L-arginine, immediately covering the top of the medium with sterile paraffin oil, and anaerobically incubating at 4-42℃ for 0.5-16h; wherein the volume ratio of the color-developing agent, the LB medium and the sterile paraffin oil is 100-200μL:1-5mL:100-300μL.

[0077] In some preferred embodiments, the color-developing method specifically comprises: uniformly mixing the color-developing agent with the meat product and the auxiliary material to form a prepared meat paste, and tray packaging the prepared meat paste at 15-42℃, covering it with a preservative film and placing it for 6-30h, and then storing and processing the obtained prepared meat paste; wherein the auxiliary material comprises L-arginine, sodium chloride and glucose.

[0078] Further, the mass ratio of the color-developing agent to the meat product is 8-12mg:100g.

[0079] Further, the mass ratio of the meat product, L-arginine, sodium chloride and glucose is 100:0.5-1.0:0.5-5.0:0.5-10.

[0080] In one embodiment, the color-developing method specifically comprises the following steps:

[0081] (1) taking pork hind leg meat, removing fat and connective tissue under the condition of 0-4℃, cutting it into small pieces and stirring it in a meat grinder with a pore diameter of 1-5mm to obtain a meat paste;

[0082] (2) adding auxiliary material to the meat paste obtained in step (1) and stirring to mix, and then adding enzyme solution of fusion enzyme and stirring to mix, to obtain a prepared meat paste;

[0083] (3) storing or processing the prepared meat paste obtained in step (2) under certain conditions to obtain a meat product.

[0084] Further, the auxiliary material in step (1) comprises, but is not limited to, the following components by weight based on 100 parts of meat: 0.5-1.0 parts of L-arginine, 0.5-5.0 parts of sodium chloride and 0.5-10 parts of glucose.

[0085] Further, the storage method in step (3) specifically comprises: tray packaging the prepared meat paste at a temperature of 15-42℃, covering it with a preservative film and placing it for 6-30h.

[0086] The technical solutions of the present application will be further described in detail below in combination with several preferred embodiments and the accompanying drawings, and the embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0087] In the following examples, the experimental materials used in the examples are commercially available from conventional biochemical reagent companies unless otherwise specified.

[0088] The sources and preparation methods of some raw materials used in the following examples and comparative examples are as follows:

[0089] (1) Experimental medium

[0090] LB medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., which was used for the activation and subculture of E. coli and B. subtilis.

[0091] LB agar medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., which was used for resistance screening and counting of E. coli and B. subtilis.

[0092] TB medium was used for the expansion culture of B. subtilis. The formula was 10.0 g glycerol, 24.0 g yeast powder, 12.0 g tryptone, 16.4 g K2HPO4·3H2O, 2.3 g KH2PO4 per liter of water, and the final pH was 7.4±0.2.

[0093] GM medium mother liquor: 10× minimal salt solution, 50% glucose, 5% hydrolyzed casein, 10% yeast juice, calcium magnesium ion solution. Except that the hydrolyzed casein needs to be sterilized in a sterile environment by sterile filtration membrane, other solution components are sterilized by 121 ℃ high pressure steam for 15 min for standby use. 100 mL 10× minimal salt solution: dissolve 18.34 g K2HPO4·H2O, 6.0 g KH2PO4, 2.0 g (NH4)2SO4, 1.0 g trisodium citrate dihydrate, 0.2 g MgSO4·7H2O with distilled water, and make up to 100 mL. 20 mL calcium magnesium ion solution: dissolve 2.03 g MgCl2·6H2O, 0.22 g CaCl2 with distilled water, and make up to 20 mL.

[0094] 5 mL GM I medium: 500 μL 10× minimal salt solution, 50 μL 50% glucose, 20 μL 5% hydrolyzed casein, 50 μL 10% yeast juice, and 4.38 mL sterile water.

[0095] 90 mL GM II medium: 8.8 mL 10× minimal salt solution, 900 μL 50% glucose, 72 μL 5% hydrolyzed casein, 36 μL 10% yeast juice, 450 μL calcium magnesium ion solution, and 79.742 mL sterile water.

[0096] Ampicillin and kanamycin were selectively added to the medium. Ampicillin was added when E. coli was cultured after transformation, and kanamycin was added when B. subtilis was cultured after transformation. The working concentration was 100 μg / mL.

[0097] Competent Cell Preparation Kit was purchased from Bioron Biotech (Beijing) Co., Ltd., which was used for plasmid construction.

[0098] ClonExpress II One Step Cloning Kit was purchased from Nanjing Nvigen Biotech Co., Ltd., which was used for plasmid construction.

[0099] DiaSpin Plasmid DNA Miniprep Kit was purchased from Shanghai Biomed Co., Ltd., which was used for extracting plasmid.

[0100] SDS-PAGE Gel Preparation Kit was purchased from Beijing Solyle Bio-technology Co., Ltd., which was used for preparing gel.

[0101] Bradford Protein Concentration Assay Kit was purchased from Shanghai Blue Sky Bio-technology Co., Ltd., which was used for determining protein concentration.

[0102] His-tag was added to the end of xanthine reductase gene in fusion plasmid B by commissioning GenScript Biotech Co., Ltd., and the fusion plasmid was obtained. His-tag was added to the end of nitric oxide synthase gene in recombinant plasmid B by commissioning GenScript Biotech Co., Ltd., and the recombinant plasmid was obtained.

[0103] (2) Preparation of E. coli JM109 competence

[0104] E. coli JM109 preserved in glycerol tube was inoculated into 10 mL LB liquid medium, and the strain was activated by oscillating culture at 37℃ for 12 h at a rotation speed of 200 rpm. The E. coli competence was prepared according to the instructions of Competent Cell Preparation Kit. The prepared competence was stored at -80℃ for standby.

[0105] (3) Preparation of B. subtilis 168 competence

[0106] B. subtilis 168 preserved in slant was inoculated into 5 mL GMI medium, and the strain was activated by oscillating culture at 37℃ for 13 h at a rotation speed of 200 rpm. 1 mL of the culture was added to 9 mL of GMI medium, and the culture was continued under the same conditions for 3.5 h. 10 mL of the above culture was inoculated into 90 mL of GMII medium, and the culture was continued under the same conditions for 1.5 h. 10 mL of the above GMII culture was divided into 50 mL sterile centrifuge tubes, and centrifuged at 4℃ and 3000 rpm for 5 min. 9 mL of supernatant was removed, and 1 mL of supernatant was resuspended with bacterial cells, which was divided into 1.5 mL centrifuge tubes, each containing 500 μL, and 250 μL of 30% glycerol was added, and stored at -80℃ for standby.

[0107] (4) The primers used in the following examples are shown in Table 1.

[0108] Table 1 Primers used in the examples of the present application

[0109]

[0110]

[0111] (5) Experimental strain: Escherichia coli JM109, item number: C1300, Beijing Zhuangmeng International Biotechnology Co., Ltd. Bacillus subtilis 168, ATCC 23857, BioVector NTCC typical culture preservation center.

[0112] (6) Preparation method of high-iron myoglobin: 50 mg / mL myoglobin stock solution in 50 mM phosphate buffer (pH 6.0) was heated at 50°C for 30 min to inactivate high-iron myoglobin reductase, denatured protein was removed by centrifugation (10,000g, 5 min, 4°C), the obtained supernatant contained about 80% or more high-iron myoglobin, and the high-iron myoglobin solution was filtered through a 0.22 μm filter membrane to remove bacteria.

[0113] Example 1

[0114] The nitric oxide-producing fusion enzyme-based color developer mainly comprises a nitric oxide-producing fusion enzyme, and the preparation method of the fusion enzyme comprises the following steps:

[0115] Y1, providing a fusion plasmid containing a nitric oxide synthase gene, a flavoprotein gene and a flavoprotein reductase gene, and the preparation method thereof specifically comprises:

[0116] a1, amplifying the nitric oxide synthase gene, the flavoprotein gene and the flavoprotein reductase gene fragments in a 50 μL PCR system with Bacillus subtilis 168 genomic DNA as the template; the PCR system is: 2 μL of 10 mM upper and lower primers, 25 μL of PrimeSTAR Max DNA Polymerase, 21 μL of sterile water, 0.5 μL of 25 mmol / L MgCl2, and a small amount of Bacillus subtilis 168 single colony is inoculated into the system with a inoculation loop and stirred; the PCR reaction program is: 95°C for 15 min pre-denaturation, 95°C for 30 s, 56°C for 30 s and 72°C for 2 min for one cycle, a total of 35 cycles, and finally continue to extend for 5 min;

[0117] a2, amplifying the linearized plasmid in a 50 μL PCR system with pP43NMK as the template, and the obtained PCR product is the linearized pP43NMK plasmid; the PCR system is: 2 μL of 10 mM upper and lower primers, 2 μL of 50 mg / μL pP43NMK plasmid, 25 μL of PrimeSTAR Max DNA Polymerase, and 19 μL of sterile water; the PCR reaction program is: 95°C for 5 min pre-denaturation, 95°C for 30 s, 56°C for 30 s and 72°C for 2 min for one cycle, a total of 35 cycles, and finally continue to extend for 5 min;

[0118] a3、To 50 μL of the PCR product of the linearized pP43NMK plasmid from step a2, 1 μL of FastDigest DpnI enzyme was added, and the mixture was treated at 37 °C for 10 min on a PCR machine to eliminate the circular plasmid, and then inactivated at 85 °C for 10 min, to obtain the pP43NMK plasmid;

[0119] a4、In a 20 μL PCR1 system, the nitric oxide synthase gene, the flavoprotein gene and the flavoprotein reductase gene fragments obtained in step a1 were fused by triple fusion PCR to obtain a fusion fragment; the PCR1 system was as follows: 10 μL of Prime STAR Max DNA Polymerase, 10 μL of nitric oxide synthase sequence, flavoprotein sequence and flavoprotein reductase sequence (at a concentration ratio of 1:2:1); the PCR1 reaction program was as follows: pre-denaturation at 95 °C for 3 min; 95 °C for 30 s, 56.8 °C for 30 s and 72 °C for 2 min for one cycle, a total of 15 cycles; and then continued extension for 5 min; the fusion fragment was further amplified, and the PCR2 system was 50 μL, including: 2 μL of the fusion fragment, 2 μL of 10 mM upper and lower primers, 25 μL of PrimeSTAR Max DNA Polymerase, and 19 μL of sterile water; the PCR2 reaction program was as follows: pre-denaturation at 95 °C for 3 min; 95 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min for one cycle, a total of 35 cycles; and then continued extension for 5 min; a large amount of fusion fragment amplification product was obtained;

[0120] a5、The fusion fragment amplification product obtained in step a4 and the pP43NMK plasmid obtained in step a3 were subjected to seamless ligation using ClonExpress II One Step Cloning Kit to obtain a fusion plasmid A; 10 μL of the fusion plasmid A was added to 100 μL of E. coli JM109 competent cells, and the mixture was subjected to ice bath for 30 min, then heat shocked at 42 °C for 45 s, and then immediately placed on ice for 3 min; 900 μL of LB medium was added, and the mixture was incubated at 37 °C for 1 h, then centrifuged to discard 900 μL of supernatant, and the remaining culture was resuspended with the remaining medium and plated on an LB plate containing ampicillin, and incubated at 37 °C for 16 h, to obtain E. coli JM109 containing the fusion plasmid A;

[0121] a6、The E. coli JM109 containing the fusion plasmid A was cultured in LB medium containing ampicillin for 12 h, and the culture was centrifuged at 8000 g for 2 min at 0-4 °C to obtain bacterial cells, and a DiaSpin column plasmid DNA small-scale extraction kit was used to extract the plasmid, to obtain the fusion plasmid B.

[0122] a7, adding a histidine tag at the end of the flavin reductase gene in the fusion plasmid B obtained in step a6 to obtain E. coli TOP10 containing the fusion plasmid; the histidine tag sequence is: CATCACCATCACCATCAC;

[0123] a8, culturing the E. coli TOP10 containing the fusion plasmid in 5 mL LB medium containing ampicillin for 12 h, centrifuging the culture at 8000 g at 4°C for 2 min to obtain the bacterial cells, and using a DiaSpin column plasmid DNA small-scale extraction kit to extract the plasmid to obtain the fusion plasmid;

[0124] Y2, transforming the fusion plasmid described in step Y1 into B. subtilis to obtain a fusion strain, specifically including: thawing the B. subtilis 168 competent cells at room temperature, adding the fusion plasmid extracted in step b1, and incubating at 37°C with shaking at 200 rpm for 2 h, then spreading on a LB plate containing kanamycin and culturing for 18 h, to obtain the fusion strain by transforming the fusion plasmid into B. subtilis 168;

[0125] Y3, culturing the fusion strain obtained in step Y2, and then isolating the fusion enzyme, specifically including:

[0126] c1, picking a single colony of the fusion bacteria prepared in step Y2, inoculating into 600 mL TB medium containing kanamycin, and culturing at 37°C with shaking at 200 rpm for 20 h, then centrifuging the culture at 10000 rpm at 4°C for 3 min, and taking the precipitate to obtain the bacterial cells;

[0127] c2, washing the bacterial cells obtained in step c1 with 20 mM phosphate buffer at pH 7.4 for three times, and then performing ultrasonic disruption at 0°C with a frequency of 25 kHz and a power of 300 W for 15 min, with an interval of 3 s working for 2 s, and then centrifuging at 10000 rpm at 0°C for 5 min, and taking the supernatant to obtain the crude extract of the fusion bacteria;

[0128] c3, incubating the crude extract of the fusion bacteria obtained in step c2 with Ni-NTA agarose purification resin at 4°C for 2 h, and then eluting the protein with Elution Buffer at pH 8, and eluting the protein with 750 μL Elution Buffer for ten times to obtain the fusion enzyme solution, and the concentration of the target enzyme solution is 0.5 mg / mL.

[0129] Example 2

[0130] The method for realizing meat coloration based on the nitric oxide-producing fusion enzyme is as follows:

[0131] (1) Take the pork ham, remove the fat and connective tissue at 4°C, cut into small pieces and stir in a meat grinder with a hole diameter of 3 mm to obtain meat paste;

[0132] (2) Add 0.8 g L-arginine, 2.5 g sodium chloride and 5 g glucose to 100 g of the meat paste obtained in step (1) and stir to mix, then add the fusion enzyme solution prepared in Example 1, with an addition amount of 10 mg / 100 g meat, and stir to obtain a prepared meat paste;

[0133] (3) Tray-pack the prepared meat paste obtained in step (2) at a temperature of 25°C, cover with preservative film and place for 20 h to improve the red color of the meat paste.

[0134] Example 3

[0135] The method for realizing meat coloration based on the nitric oxide-producing fusion enzyme colorant is as follows:

[0136] (1) Take the pork ham, remove the fat and connective tissue at 4°C, cut into small pieces and stir in a meat grinder with a hole diameter of 3 mm to obtain meat paste;

[0137] (2) Add 0.8 g L-arginine, 2.5 g sodium chloride and 5 g glucose to 100 g of the meat paste obtained in step (1) and stir to mix, then add the fusion enzyme solution prepared in Example 1, with an addition amount of 10 mg / 100 g meat, and stir to obtain a prepared meat paste;

[0138] (3) Tray-pack the prepared meat paste obtained in step (2) at a temperature of 37°C, cover with preservative film and place for 8 h to improve the red color of the meat paste.

[0139] Example 4

[0140] The method for promoting high ferric metmyoglobin coloration based on the nitric oxide-producing fusion enzyme colorant includes:

[0141] Add 100 μL of 0.5 mg / mL colorant (fusion enzyme solution) prepared in Example 1 to 2 mL of LB medium containing 5 mg / mL high ferric metmyoglobin and 10 mM L-arginine, immediately cover the top of the medium with 300 μL of sterile paraffin oil, and incubate anaerobically at 37°C for 8 h to promote high ferric metmyoglobin coloration.

[0142] Example 5

[0143] The method for promoting high ferric metmyoglobin coloration based on the nitric oxide-producing fusion enzyme colorant includes:

[0144] The color development of high ferric metmyoglobin was promoted by adding 200 μL of 0.5 mg / mL color developing agent prepared in Example 1 (fusion enzyme solution) to 2 mL of LB medium containing 5 mg / mL high ferric metmyoglobin and 10 mM L-arginine, immediately covering the top of the medium with 300 μL of sterile paraffin oil, and incubating anaerobically at 37°C for 8 h.

[0145] Example 6

[0146] A method for promoting the color development of high ferric metmyoglobin based on a nitric oxide synthase-producing fusion enzyme, comprising:

[0147] The color development of high ferric metmyoglobin was promoted by adding 100 μL of 0.5 mg / mL color developing agent prepared in Example 1 (fusion enzyme solution) to 2 mL of LB medium containing 5 mg / mL high ferric metmyoglobin and 10 mM L-arginine, immediately covering the top of the medium with 300 μL of sterile paraffin oil, and incubating anaerobically at 37°C for 0.5 h.

[0148] Example 7

[0149] A method for promoting the color development of high ferric metmyoglobin based on a nitric oxide synthase-producing fusion enzyme, comprising:

[0150] The color development of high ferric metmyoglobin was promoted by adding 100 μL of 0.5 mg / mL color developing agent prepared in Example 1 (fusion enzyme solution) to 2 mL of LB medium containing 5 mg / mL high ferric metmyoglobin and 10 mM L-arginine, immediately covering the top of the medium with 300 μL of sterile paraffin oil, and incubating anaerobically at 7°C for 16 h.

[0151] Comparative Example 1

[0152] A method for preparing a meat color developing agent based on a recombinant enzyme, comprising the following steps:

[0153] A nitric oxide synthase recombinant bacterium was prepared according to the method of Example 1 in CN115232830A and was plasmid-modified.

[0154] Y1, a recombinant plasmid containing a nitric oxide synthase gene was provided, and the preparation method thereof specifically comprises:

[0155] a1, a nitric oxide synthase gene fragment was amplified in a 50 μL PCR system using Bacillus subtilis 168 genomic DNA as a template; the PCR system was as follows: 2 μL of each of 10 mM upper and lower primers, 25 μL of PrimeSTAR Max DNA Polymerase, 21 μL of sterile water, and a small amount of Bacillus subtilis 168 single colony was inoculated into the system with a inoculation loop and stirred; the PCR reaction program was as follows: 95°C for 15 min for pre-denaturation, 95°C for 30 s, 56°C for 30 s, and 72°C for 2 min for one cycle, a total of 35 cycles, and finally continued to extend for 5 min.

[0156] a2, in a 50 μL PCR system, linearized plasmid was amplified by taking pP43NMK as a template, and the PCR product was the linearized pP43NMK plasmid; the PCR system was as follows: 10 mM of each of the upstream and downstream primers, 2 μL of 50 mg / μL pP43NMK plasmid, 25 μL PrimeSTAR Max DNA Polymerase, and 19 μL sterile water; the PCR reaction program was as follows: pre-denaturation at 95 °C for 5 min, 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 2 min for one cycle, a total of 35 cycles, and then continued to extend for 5 min;

[0157] a3, 1 μL of FastDigest DpnI enzyme was added to the 50 μL linearized pP43NMK plasmid PCR product obtained in step a2, and the mixture was treated at 37 °C for 10 min on a PCR instrument to eliminate the circular plasmid, and then the FastDigest DpnI enzyme was inactivated at 85 °C for 10 min, thereby obtaining the pP43NMK plasmid;

[0158] a4, the nitric oxide synthase gene fragment obtained in step a1 and the pP43NMK plasmid obtained in step a3 were subjected to seamless ligation using ClonExpress II One Step Cloning Kit to obtain a recombinant plasmid A; 10 μL of the recombinant plasmid A was added to 100 μL of E. coli JM109 competent cells, and the mixture was subjected to ice bath for 30 min, then heat shock at 42 °C for 45 s, and then immediately placed on ice for 3 min; 900 μL of LB medium was added, and the mixture was incubated at 37 °C for 1 h, then centrifuged to discard 900 μL of supernatant, the remaining culture was resuspended with the remaining medium and plated on an LB plate containing ampicillin, and incubated at 37 °C for 16 h, thereby obtaining E. coli JM109 containing the recombinant plasmid A;

[0159] a5, the E. coli JM109 containing the recombinant plasmid A obtained in step a4 was cultured in LB medium containing ampicillin for 12 h, and the culture was centrifuged at 8000 g for 2 min at 0-4 °C to obtain bacterial cells, and a DiaSpin column plasmid DNA small-scale extraction kit was used to extract the plasmid, thereby obtaining the recombinant plasmid B.

[0160] a6, a histidine tag was added to the end of the nitric oxide synthase gene in the recombinant plasmid B obtained in step a5, thereby obtaining E. coli TOP10 containing the recombinant plasmid; the sequence of the histidine tag was as follows: CATCACCATCACCATCAC;

[0161] a7. The E. coli TOP10 containing the recombinant plasmid obtained in step a5 was cultured in 5 mL of LB medium containing ampicillin for 12 h. The culture was centrifuged at 8000 g for 2 min at 4 °C to obtain bacterial cells. The plasmid was extracted using the DiaSpin column-based plasmid DNA mini-extraction kit to obtain the recombinant plasmid.

[0162] Y2. Transform Bacillus subtilis with the recombinant plasmid described in step Y1 to obtain a recombinant strain. Specifically, this includes: thawing Bacillus subtilis 168 competent cells at room temperature, adding the recombinant plasmid extracted in step b1, incubating at 37°C with shaking at 200 rpm for 2 h, then plating on LB plates containing kanamycin and culturing for 18 h, and transforming Bacillus subtilis 168 with the recombinant plasmid to obtain a recombinant strain.

[0163] Y3. Cultivate the recombinant strain obtained in step Y2, and then isolate the recombinase, specifically including:

[0164] c1. Pick a single colony of the recombinant bacteria prepared in step b2, inoculate it into 600 mL of TB medium containing kanamycin, and culture it at 37°C with shaking at 200 rpm for 20 h. Take the culture solution and centrifuge it at 10000 rpm for 3 min at 4°C. Take the precipitate to obtain the bacterial cells.

[0165] c2. Wash the bacterial cells three times with 20mM phosphate buffer at pH 7.4, and sonicate them at 25kHz frequency and 300W power at 0℃ for 15min, with a 3s interval between 2s. Then centrifuge at 10000rpm at 0℃ for 5min, and take the supernatant to obtain the crude extract of recombinant bacteria.

[0166] c3. The crude extract of recombinant bacteria obtained in step X3 is incubated with Ni-NTA agarose purification resin at 4°C for 2 hours. Then, the protein is eluted with Elution Buffer at pH 8. The protein is eluted ten times with 750 μL of Elution Buffer to obtain the recombinant enzyme solution. The concentration of the target enzyme solution is 0.5 mg / mL.

[0167] Comparative Example 2

[0168] The method for achieving meat color development using recombinase-based meat color-developing agents comprises the following steps:

[0169] (1) Take pork hind leg meat, remove fat and connective tissue at 4℃, cut into small pieces and grind in a meat grinder with a 3mm aperture to obtain minced meat;

[0170] (2) In 100 g of the meat paste obtained in step (1), 0.8 g of L-arginine, 2.5 g of sodium chloride and 5 g of glucose were added and stirred to mix, and 5 mg of the recombinant enzyme solution per 100 g of meat was added and stirred to obtain a prepared meat paste;

[0171] (3) The prepared meat paste obtained in step (2) was tray-packed at a temperature of 25°C, covered with a preservative film and left to stand for 20 h.

[0172] Comparative Example 3

[0173] A method for coloring meat, comprising the following steps:

[0174] (1) Pig hind leg meat was taken, and fat and connective tissue were removed at 4°C, and cut into small pieces and stirred in a meat grinder with a hole diameter of 3 mm to obtain a meat paste;

[0175] (2) In 100 g of the meat paste obtained in step (1), 0.8 g of L-arginine, 2.5 g of sodium chloride and 5 g of glucose were added and stirred to mix to obtain a prepared meat paste;

[0176] (3) The prepared meat paste obtained in step (2) was tray-packed at a temperature of 25°C, covered with a preservative film and left to stand for 20 h.

[0177] Comparative Example 4

[0178] A method for coloring meat using nitrite, comprising the following steps:

[0179] (1) Pig hind leg meat was taken, and fat and connective tissue were removed at 4°C, and cut into small pieces and stirred in a meat grinder with a hole diameter of 3 mm to obtain a meat paste;

[0180] (2) In 100 g of the meat paste obtained in step (1), 9 mg of sodium nitrite, 0.8 g of L-arginine, 2.5 g of sodium chloride and 5 g of glucose were added and stirred to mix to obtain a prepared meat paste;

[0181] (3) The prepared meat paste obtained in step (2) was tray-packed at a temperature of 25°C, covered with a preservative film and left to stand for 20 h.

[0182] Comparative Example 5

[0183] A method for promoting the coloring of high ferric metmyoglobin using a nitric oxide-producing fusion enzyme-based colorant, comprising:

[0184] 100 μL of phosphate buffer (20 mM, pH 7.4) was added to 2 mL of LB medium containing 5 mg / mL of high ferric metmyoglobin and 10 mM of L-arginine, 300 μL of sterile paraffin oil was immediately overlaid on top of the medium, and the medium was incubated anaerobically at 37°C for 8 h.

[0185] Comparative Example 6

[0186] A method for promoting the color development of high ferric metmyoglobin based on a nitric oxide synthase fusion enzyme, comprising:

[0187] 100 μL of the recombinant enzyme solution prepared in Comparative Example 1 was added to 2 mL of LB medium containing 5 mg / mL of high ferric metmyoglobin and 10 mM of L-arginine, 300 μL of sterile paraffin oil was immediately overlaid on top of the medium, and the medium was incubated anaerobically at 37°C for 8 h.

[0188] The concentration of the recombinant enzyme solution was 0.5 mg / mL.

[0189] Test Example 1

[0190] Identification of the fusion plasmid A and the recombinant plasmid A:

[0191] Single colonies of E. coli JM109 containing the fusion plasmid A and the recombinant plasmid A with ampicillin resistance in Example 1 and Comparative Example 1 were inoculated into 10 mL of LB liquid medium containing ampicillin and cultured for 12 h, and plasmids were extracted using a GenScript DiaSpin column plasmid DNA small-scale extraction kit. The plasmids were subjected to gene sequencing.

[0192] The base sequences and positions of the fusion gene fragment and the nitric oxide synthase gene fragment prepared in Example 1 and Comparative Example 1 were measured to be correct, proving that the fusion plasmid A and the recombinant plasmid A were successfully constructed.

[0193] Test Example 2

[0194] SDS-PAGE electrophoresis of the fusion enzyme solution prepared from the crude extract of the fusion bacteria in Example 1 and the recombinant enzyme solution prepared from the crude extract of the recombinant bacteria in Comparative Example 1:

[0195] A gel was prepared according to the instructions of an SDS-PAGE gel preparation kit, with a concentrated gel concentration of 5% and a separation gel concentration of 10%. The crude extracts prepared in Comparative Example 1 and Example 1 were diluted to 1 mg / mL with 20 mM phosphate buffer at pH 7.4, 20 μL of which was added to 5 μL of 5x protein loading buffer, followed by boiling for 5 min and centrifugation at 4000 g for 5 min. 20 μL of the supernatant was loaded together with a protein molecular weight marker for SDS-PAGE electrophoresis. The electrophoresis conditions were as follows: first set the voltage to 80 V, and when the separation gel was reached, adjust the voltage to 120 V until the end of the electrophoresis, and then remove the gel. The gel was stained in a coomassie brilliant blue staining solution for 30 min, and then rinsed several times with a destaining solution prepared by mixing 100 mL of acetic acid and 900 mL of water, until clear bands appeared. The test results are shown in Figure 1Lane 1 and lane 2 correspond to the crude extract of recombinant bacteria and recombinase enzyme solution prepared in Comparative Example 1, and lane 3 and lane 4 correspond to the crude extract of fusion bacteria and fusion enzyme solution prepared in Example 1.

[0196] Theoretically, the crude extract of recombinant bacteria and fusion bacteria will have a band of crude protein at 48 kDa and 101 kDa, respectively, i.e. recombinase and fusion enzyme. The results are shown in Figure 1. Figure 1 As can be seen, the bands at these two positions are significantly wider compared with other bands in the crude extract lane, so it is preliminarily judged that the two proteins have been successfully and efficiently expressed. After the protein binds to and is eluted from the Ni-NTA agarose purification resin, the target bands in lane 2 and lane 4 are clear without any scattered bands, proving that the recombinase and fusion enzyme have been successfully obtained.

[0197] Test Example 3

[0198] Bacterial nitric oxide synthase activity analysis:

[0199] The enzyme activity of the recombinase enzyme solution prepared in Comparative Example 1 and the fusion enzyme solution prepared in Example 1 was analyzed. Nitric oxide synthase has the function of catalyzing the production of NO, and the NOS enzyme activity is indirectly determined by measuring the oxidation product of NO, nitrite. As a free radical with a high degree of reduction, NO is easily oxidized to nitrite ion, and the NOS enzyme activity can be quantitatively analyzed by detecting the nitrite ion through Griess reagent. The activity of the enzyme was determined using a nitric oxide synthase assay kit.

[0200] Table 2 Nitric oxide synthase enzyme activity

[0201]

[0202] Note: Different letters indicate significant differences (P < 0.05) between different groups.

[0203] From Table 2, the nitric oxide synthase and fusion enzyme activities measured by the kit were 1.7133 ± 0.2641 U / mg prot and 20.193 ± 0.6809 U / mg prot, respectively. The enzyme activity after fusion expression was about 12 times that of the unmodified enzyme, which indicated that the covalently linked flavoprotein and flavoprotein reductase retained the ability to transfer the required electrons to nitric oxide synthase. YkuN and YumC are flavoprotein and flavoprotein reductase derived from Bacillus subtilis. Flavoprotein is an electron carrier that can provide electrons for various enzymes including p450. Flavoprotein YkuN was fused to the N-terminus of flavoprotein reductase YumC because the C-terminal aromatic residue, which is important for NADPH binding and electron exchange, can be interfered with if the flavin reductase is connected to the C-terminus of YumC. The interior of the fusion protein is connected by a rigid linker (EPPPP-EPPPP-LPPPP-LPPPP-LPPPP), and the proline-rich linker can more effectively separate the domains of the fusion protein. By directly linking YkuN-YumC to nitric oxide synthase through gene fusion, the enzyme activity of nitric oxide synthase was increased by 12 times, indicating that the reductase domain we supplemented helped electron transfer. The present application uses the Bacillus subtilis fusion expression system to obtain a fusion enzyme with extremely high enzyme activity, providing a feasible solution for the coloration of fermented sausages.

[0204] Test Example 4

[0205] Promoting the metmyoglobin color development effect:

[0206] The color of the culture was observed and the redness value of each group was measured using a ZE7700 electric color difference meter (Nippon Denshoku, Kogyo Co., Tokyo, Japan) in transmission mode using a quartz cuvette (2 mm light path) to measure the color of the LB culture. The test results are shown in Table 3. The culture was centrifuged and precipitated, and the supernatant of the culture medium was used for ultraviolet-visible spectrum analysis. A 75% (v / v) pre-cooled acetone solution was used to extract nitrosylmyoglobin, and the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was the NO-Mb extract, which was scanned for absorption at an interval of 1 nm from 350 nm to 650 nm. The test results are shown in Figures 2a-2b .

[0207] Table 3 Redness values of the culture medium of each group

[0208]

[0209] Note: Different letters indicate significant differences (P < 0.05) between different groups.

[0210] NO produced by the catalysis of recombinant nitric oxide synthase can convert brownish ferric metmyoglobin to bright red nitrosyl myoglobin. However, as shown in Table 3 and Figure 2, the use of recombinant nitric oxide synthase alone is not very effective, probably because the system lacks the reductase and cofactors required for the catalysis of nitric oxide synthase. After incubation at 37°C for 8 h, the redness values of Example 4 and Example 5 were significantly higher (P < 0.05), indicating that a large amount of ferric metmyoglobin was converted to other red myoglobin derivatives in the system. According to the results of UV-visible spectrum scanning, the samples with added fusion enzyme all had maximum peaks at 394 and 540 nm, which are typical absorption peaks of nitrosyl myoglobin. Among them, Example 5 had the highest absorption peak value, indicating that the content of nitrosyl myoglobin in the system was the highest. At this time, the system with added recombinant nitric oxide synthase had almost no nitrosyl myoglobin formed. This shows that the fusion enzyme solution prepared in Example 1 has a better conversion ability of ferric metmyoglobin than the recombinant enzyme solution prepared in Comparative Example 1.

[0211] Test Example 5

[0212] The redness values of the meat batters prepared in Example 2 and Comparative Examples 2-4 were measured. 5 g of each meat batter sample was measured for redness value using a ZE7700 electric color difference meter (Nippon Denshoku, Kogyo Co., Tokyo, Japan), and the test results are shown in Table 4.

[0213] Table 4 Redness values of meat batters prepared in Example 2 and Comparative Examples 2-4

[0214]

[0215]

[0216] Note: Different letters indicate significant differences in survival rate between different groups (P < 0.05).

[0217] The results of the bacterial nitric oxide synthase enzyme activity assay showed that the recombinant enzyme and the fusion enzyme expressed in the Bacillus subtilis expression system had complete nitric oxide synthase activity. Therefore, the recombinant enzyme solution and the fusion enzyme solution of Comparative Example 2 and Example 2 were extracted and applied to the meat paste, but as shown in Table 4, compared with Comparative Example 3, Comparative Example 4 and Example 2 had obvious promoting effects on the formation of red color of the meat paste by adding sodium nitrite and the fusion enzyme, and there was no significant difference between Comparative Example 4 and Example 2 (P>0.05), indicating that the fusion enzyme prepared in Example 2 could achieve a similar color development effect as sodium nitrite. However, compared with Comparative Example 3 and Example 2, Comparative Example 2 showed that the addition of only the recombinant enzyme solution could not achieve the ideal color development effect. This is because the structure of bacterial nitric oxide synthase is different from that of mammalian nitric oxide synthase, and bacterial nitric oxide synthase lacks a reductase domain. Therefore, in bacteria, the catalytic reaction of nitric oxide synthase often needs the assistance of other reductases, and in addition, the assistance of various cofactors such as NADPH, FMN, FAD, H4B / H4F, etc. is also needed. Therefore, in the meat paste system supplemented with only nitric oxide synthase, due to the lack of sufficient reductase and various cofactors, it is not possible to produce sufficient amounts of NO. Examples 3 and 6-7 used different incubation temperatures and times, and the color development effect was similar to that under other incubation conditions, which will not be described here.

[0218] Example 2 of the present application added the fusion enzyme solution, which can provide more cofactors and reductases. The meat paste prepared in Example 2 had a higher redness value than the fermented sausages prepared in Comparative Example 2 and Comparative Example 3, which indicates that the fusion enzyme has a considerable promoting effect on the improvement of the redness value of the sausage. The redness value data show that the addition of the fusion enzyme can effectively improve the formation of red color of the fermented sausage, and even reach a level comparable to the addition of sodium nitrite, which provides a new and extremely practical method for the replacement of sodium nitrite color development in fermented sausages and the improvement of the safety of fermented sausages.

[0219] Test Example 6

[0220] Color development effect of meat paste:

[0221] UV-visible spectroscopy analysis was performed on the systems of Example 2 and Comparative Examples 2-4. 5.0 g of each meat paste was accurately weighed and added to 45 mL of 75% (v / v) pre-cooled acetone solution, homogenized in a high-speed disperser for 1 min (under ice bath conditions), centrifuged at 4000g for 5 min at 4°C, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was the nitrosylmyoglobin extract solution. The nitrosylmyoglobin extract solution was analyzed using a UV-visible spectrophotometer, the baseline was adjusted with 75% acetone solution, and scanning was performed at an interval of 1 nm from 350 nm to 650 nm. The test results are shown in Figure 3.

[0222] The formation of red color of meat paste is mainly due to the presence of nitroso myoglobin, in order to further analyze the possible reasons for the fusion enzyme to promote the redness value of meat paste, the nitroso pigment of each group of meat paste is extracted and analyzed. According to the enzyme activity test of example 3, the fusion enzyme has high enzyme activity and can catalyze the production of a large amount of NO. As shown in table 4, the high iron myoglobin cannot be effectively converted into nitroso myoglobin by the action of recombinant enzyme alone, and the possible reason is that the meat system lacks the reductase and other cofactors required for the catalytic action of nitric oxide synthase. Therefore, the fusion enzyme is added to the meat paste prepared in example 2, and the reduction domain structure of nitric oxide synthase is supplemented by fusion expression, so as to supplement the substances required for the catalytic reaction of nitric oxide synthase.

[0223] According to Figures 3a-3b As can be seen from the results, except for comparative example 3, the rest of the groups all have peak values at 394nm and 540nm, which are typical nitroso myoglobin absorption peaks, indicating that nitroso myoglobin is produced in the meat paste prepared in comparative example 2, comparative example 4 and example 2. According to the absorption peak, the content of NO-Mb formed in the meat paste prepared in comparative example 4 is the highest, followed by example 2, which is higher than that of comparative examples 2-3, which corresponds to the trend of the redness value of each group of meat paste. Combined with the test results of color change and UV-Vis analysis in table 4, compared with the recombinant enzyme solution added in comparative example 2, the fusion enzyme solution added in example 2 can greatly improve the formation efficiency of nitroso myoglobin. The fusion enzyme solution added in the meat paste prepared in example 2 contains sufficient nitric oxide synthase enzyme activity, which can fully catalyze the reaction of L-arginine to produce sufficient NO, and NO combines with myoglobin in meat paste to form a large amount of nitroso myoglobin, which is equivalent to the effect of sodium nitrite treatment, which provides a method with great practical application significance for the replacement of nitrite color development of meat products and the improvement of meat safety.

[0224] In addition, the present inventors have also carried out tests with other raw materials, process operations and process conditions described in the present specification with reference to the foregoing examples, and have obtained relatively ideal results.

[0225] It should be understood that the technical solutions of the present application are not limited to the above specific implementation cases, and any technical modification made according to the technical solutions of the present application without departing from the purpose of the present application and the scope protected by the claims falls within the protection scope of the present application.

Claims

1. A chromogenic agent based on a nitric oxide-producing fusion enzyme, characterized in that, The color-developing agent includes a nitric oxide-producing fusion enzyme, which is formed by sequentially combining nitric oxide synthase, flavoprotein, and flavoprotein reductase in pairs via linking peptides. The nitric oxide synthase is a bacterial nitric oxide synthase, and the flavoprotein is fused to the N-terminus of the flavoprotein reductase.

2. The color-developing agent according to claim 1, characterized in that, The preparation method of the fusion enzyme includes: Provide a fusion plasmid containing at least the nitric oxide synthase gene, the flavoprotein gene, and the flavoprotein reductase gene; The fusion plasmid was used to transform Bacillus subtilis to obtain a fusion strain; Furthermore, the fusion strain is cultured, then its cell wall is broken, and it is mixed with Ni-NTA agarose purification resin for incubation and separation to obtain the fusion enzyme.

3. The color-developing agent according to claim 2, characterized in that, The preparation method of the fusion enzyme specifically includes: a1. In a PCR system, using Bacillus subtilis 168 genomic DNA as a template, fragments of the nitric oxide synthase gene, flavoprotein gene, and flavoprotein reductase gene are amplified; wherein, the upstream primer for nitric oxide synthase used in the PCR system has the sequence shown in SEQ ID NO.1, and the downstream primer for nitric oxide synthase has the sequence shown in SEQ ID NO.2 or SEQ ID NO.3; the upstream primer for flavoprotein has the sequence shown in SEQ ID NO.4, and the downstream primer for flavoprotein has the sequence shown in SEQ ID NO.5; the upstream primer for flavoprotein reductase has the sequence shown in SEQ ID NO.6, and the downstream primer for flavoprotein reductase has the sequence shown in SEQ ID NO.7; a2. In the PCR system, using pP43NMK as a template, the linearized plasmid was amplified to obtain the linearized pP43NMK plasmid. a3. Add FastDigest DpnI enzyme to the linearized pP43NMK plasmid obtained in step a2, and process it on a PCR instrument to eliminate the circular plasmid. Then inactivate FastDigest DpnI enzyme to obtain pP43NMK linear plasmid. a4. The nitric oxide synthase gene, flavoprotein gene and flavoprotein reductase gene fragments obtained in step a1 are fused by triple fusion PCR to obtain fusion fragments. Then, the fusion fragments are further amplified by PCR to obtain a large number of fusion fragment amplification products. a5. Seamlessly ligate the amplified product of the fusion fragment obtained in step a4 and the pP43NMK linear plasmid obtained in step a3 to obtain fusion plasmid A; then mix and incubate the fusion plasmid A with Escherichia coli JM109 competent cells to obtain Escherichia coli JM109 containing fusion plasmid A. a6. Escherichia coli JM109 containing fusion plasmid A was cultured in LB medium containing ampicillin to obtain bacterial cells, which were then processed by plasmid extraction to obtain fusion plasmid B. a7. Add a histidine tag to the end of the flavoprotein reductase gene in the fusion plasmid B obtained in step a6 to obtain E. coli TOP10 containing the fusion plasmid. a8. The E. coli TOP10 containing the fusion plasmid obtained in step a7 is cultured in LB medium containing ampicillin to obtain bacterial cells, and then the plasmid is extracted to obtain the fusion plasmid.

4. The color-developing agent according to claim 3, characterized in that, The preparation method of the fusion enzyme specifically includes: thawing Bacillus subtilis 168 competent cells at room temperature, adding the obtained fusion plasmid for incubation, culture, and screening to obtain the fusion strain.

5. The color-developing agent according to claim 4, characterized in that, The preparation method of the fusion enzyme specifically includes: c1. Inoculate single colonies of the obtained fusion strain into a culture medium containing kanamycin and then isolate the strains from the culture system. c2. After thoroughly washing the strains isolated in step c1, the cell walls were broken, and then the supernatant was obtained to obtain the crude extract of fusion bacteria. c3. The crude extract of fusion bacteria obtained in step c2 is mixed with Ni-NTA agarose purification resin and incubated to elute proteins and obtain fusion enzyme solution; wherein the concentration of fusion enzyme in the fusion enzyme solution is 0.1~0.5 mg / mL.

6. The use of a nitric oxide-producing fusion enzyme in the preparation of a chromogenic agent, characterized in that: The fusion enzyme is formed by sequentially combining nitric oxide synthase, flavoprotein, and flavoprotein reductase in pairs via linking peptides. The nitric oxide synthase is bacterial nitric oxide synthase, and the flavoprotein is fused to the N-terminus of the flavoprotein reductase.

7. The application of the color-developing agent based on the nitric oxide-producing fusion enzyme according to any one of claims 1-5 in the color development of meat products, characterized in that: The meat products include fermented and / or non-fermented meat products.

8. A method for developing color with a color-developing agent, characterized in that, include: The color-developing agent is mixed with the substrate and incubated to achieve the color development of the substrate; The color-developing agent includes the color-developing agent based on the nitric oxide-producing fusion enzyme according to any one of claims 1-5; the substrate includes metmyoglobin or meat products.

9. The coloring method according to claim 8, characterized in that, Specifically, it includes: Mix the color-developing agent with the substrate and incubate at 4-42°C for 0.5-30 hours; And / or, the concentration of the fusion enzyme in the chromogenic agent is 0.1~0.5 mg / mL.

10. The color-developing method according to claim 8, characterized in that, Specifically, it includes: Add the color-developing agent containing 4-6 mg / mL metmyoglobin and 8-12 mmol / L L In LB medium containing arginine, sterile paraffin oil was immediately placed on top of the medium and anaerobic incubated at 4-42 °C for 0.5-16 h; wherein the volume ratio of the color-developing agent, LB medium and sterile paraffin oil was 100-200 µL: 1-5 mL: 100-300 µL. Alternatively, the color-developing method specifically includes: uniformly mixing the color-developing agent with meat products and auxiliary materials to form a modified meat paste, packaging it on a tray at 15~42 ℃, covering it with plastic wrap and leaving it for 6~30 h, and then storing and processing the obtained modified meat paste; wherein, the auxiliary materials include L -Arginine, sodium chloride, and glucose; the color-developing agent is in a mass ratio of 8-12 mg:100g to the meat product; the meat product, L - The mass ratio of arginine, sodium chloride and glucose is 100:0.5~1.0:0.5~5.0:0.5~10.

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