CN115232830B

CN115232830B - Meat color former based on recombinant bacterial nitric oxide synthase, method and application thereof

Info

Publication number: CN115232830B
Application number: CN202210972602.8A
Authority: CN
Inventors: 李沛军; 潘琼; 朱苗苗; 徐宝才; 厉冰玉; 肖晴
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2022-08-12
Filing date: 2022-08-12
Publication date: 2024-01-09
Anticipated expiration: 2042-08-12
Also published as: CN115232830A

Abstract

The invention discloses a meat color former based on recombinant bacterial nitric oxide synthase, a method and application thereof. The meat color former comprises recombinant bacterial nitric oxide synthase and coagulase negative staphylococci. The recombinant bacterial nitric oxide synthase is obtained by culturing a nitric oxide synthase recombinant strain formed by transforming bacillus subtilis with a recombinant expression vector, wherein the recombinant expression vector contains a nitric oxide synthase gene. The recombinant bacterial nitric oxide synthase has higher enzyme activity, can catalyze and generate a large amount of nitric oxide, can effectively combine myoglobin in meat products to generate nitrosomyoglobin, realizes better color development effect, can more effectively improve the color of meat fuchsin particularly when being used together with coagulase negative staphylococci, obtains the color development effect equivalent to sodium nitrite, and provides a solution with practical application significance for nitrite color development substitution of meat products and improvement of the safety of the nitrite color development substitution.

Description

Meat color former based on recombinant bacterial nitric oxide synthase, method and application thereof

Technical Field

The invention relates to a meat product color development method, in particular to a method for realizing meat color development by utilizing recombinant bacterial nitric oxide synthase, belonging to the technical field of meat science.

Background

Color is an important index for measuring the quality of meat products. The long-term storage of meat products results in the formation of high-iron myoglobin in large amounts due to oxidation of myoglobin, which gives the meat an undesirable tan, and so one usually solves this problem by adding nitrite. After nitrite is added into meat, the nitrite is reduced into nitric oxide by microbial nitrite reductase in the meat, and the nitric oxide is combined with myoglobin to generate nitrosomyoglobin with typical salted meat red color, so that the meat product presents ideal red color. In industrial production, nitrite is widely used for the color development of meat products, but its potential teratogenic carcinogenicity causes great concern to consumers. Although the addition of nitrite in meat products is currently severely limited in the market, the long-term intake of such meat products still raises the associated risks. This raises a great concern and thus reduces its use in meat products and improves the safety of the meat products.

Among the many alternatives to nitrite chromogenic action, microbial fermentation has great potential. CN110800913a discloses a color former for replacing nitrite in processed meat products. One of the chromophores is bacteria powder or bacteria suspension of coagulase negative staphylococcus, and the inoculation amount of the chromophores in meat product processing is 10 when the chromophores are used ⁶ ～10 ⁷ CFU/g meat. The other chromogenic agent consists of bacterial powder or bacterial suspension of coagulase negative staphylococcus and L-arginine. When in use, the inoculation amount of coagulase negative staphylococcus in meat product processing is 10 ⁶ ～10 ⁷ The addition amount of CFU/g meat and L-arginine in meat product processing is 0.6-1.2% of the mass of the meat product. The bacterial powder or bacterial suspension is prepared by culturing coagulase negative staphylococcus through a liquid culture medium. CN110800913a can generate nitric oxide in processed meat products by using coagulase-negative staphylococci with nitric oxide synthase, thereby forming red nitrosomyoglobin, which gives the processed meat products a red color; meanwhile, the L-arginine is added, so that the red color can be further improved.

CN114568644a discloses a processing method for promoting red color enhancement of fermented sausage. The method comprises the steps of inoculating the fermented sausage after coagulase negative staphylococcus through low-strength high hydrostatic pressure treatment, and fermenting to ensure that the redness value of a sausage finished product reaches 6.5-7.0. When in use, the inoculation amount of coagulase negative staphylococcus in sausage meat is 10 ⁶ ～10 ⁷ CFU/g meat; the high hydrostatic pressure treatment pressure is 200-300 MPa, and the treatment time is 3-7 min. The low-strength high hydrostatic pressure treatment promotes the yield of nitrosomyoglobin in the fermented sausage by improving the expression level of nitric oxide synthase in coagulase negative staphylococci, and improves the color of the fermented sausage. The processing method can improve the color of the fermented sausage, provides a feasible scheme for replacing the color development of nitrite in the fermented sausage, and meets the requirements of people on healthy and safe fermented sausage products.

Although the coagulase-negative staphylococcus strain has a promoting effect on the formation of the color of the meat product, the nitric oxide synthase activity of the coagulase-negative staphylococcus strain is generally weak, and the capability of producing nitric oxide in the meat product is insufficient, so that the nitrosomyoglobin content in the meat product is far lower than that of the meat product added with nitrite, and the color development effect of the meat product is far weaker than that of the meat product added with sodium nitrite.

Disclosure of Invention

The invention mainly aims to provide a meat color former based on recombinant bacterial nitric oxide synthase, a method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

In one aspect, the present invention provides a recombinant bacterial nitric oxide synthase-based meat color former comprising a recombinant bacterial nitric oxide synthase, the recombinant bacterial nitric oxide synthase being prepared by a process comprising:

providing a recombinant expression vector containing a nitric oxide synthase gene, wherein the nitric oxide synthase gene is obtained by taking bacillus subtilis 168 genome DNA as a template for amplification;

transforming bacillus subtilis by using the recombinant expression vector to obtain a nitric oxide synthase recombinant strain;

culturing the recombinant strain of nitric oxide synthase, and then isolating to obtain the recombinant bacterial nitric oxide synthase.

In one embodiment, the method of preparing the recombinant bacterial nitric oxide synthase comprises the steps of:

y1, constructing a nitric oxide synthase recombinant plasmid;

and transforming the bacillus subtilis by the Y2 and nitric oxide synthase recombinant plasmid to obtain the nitric oxide synthase recombinant strain.

In one embodiment, step Y1 specifically includes the steps of:

a1, in a PCR system, using bacillus subtilis 168 genome DNA as a template to amplify a nitric oxide synthase gene sequence;

a2, in a PCR system, using pP43NMK as a template, amplifying the linearization plasmid, and obtaining a PCR product, namely the linearization pP43NMK plasmid;

a3, adding FastDiget DpnI enzyme into the linearized pP43NMK plasmid PCR product obtained in the step a2, treating at 37 ℃ for 10min on a PCR instrument to eliminate the circular plasmid, and inactivating the FastDiget DpnI enzyme at 85 ℃ for 10min to obtain pP43NMK linear plasmid;

a4, performing seamless connection on the target gene nitric oxide synthase sequence obtained in the step a1 and the pP43NMK linear plasmid obtained in the step a3 to obtain a recombinant plasmid; the recombinant plasmid was taken and added to E.coli JM109 competent, ice-bathed for 30min, followed by heat shock in a water bath at 42℃for 45s, immediately thereafter placed on ice for 3min. Adding LB culture medium, shaking at 37 ℃ for incubation for 1h, centrifuging, discarding supernatant, re-suspending and coating the rest culture medium on an LB plate containing ampicillin, and culturing at 37 ℃ for 16h to obtain the nitric oxide synthase recombinant plasmid.

Further, the PCR system described in the step a1 comprises: 2 mu L, 25 mu L PrimeSTAR Max DNAPolymerase and 21 mu L of sterile water of 10mM upstream and downstream primers respectively, and a small amount of bacillus subtilis 168 single colony is picked up by an inoculating loop to be connected into the system and stirred uniformly; the PCR reaction procedure was: the pre-denaturation is carried out at 95 ℃ for 15min, one cycle is carried out at 95 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 2min, the total cycle is 35 times, and finally the extension is continued for 5min.

Further, the PCR system described in step a2 includes: 10mM upstream and downstream primer each 2. Mu.L, 2. Mu.L 20-100 mg/. Mu.L pP43NMK plasmid, 25. Mu. L PrimeSTAR Max DNA Polymerase, 19. Mu.L sterile water; the PCR reaction procedure was: the pre-denaturation is carried out at 95 ℃ for 5min, one cycle is carried out at 95 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 2min, the total cycle is 35 times, and finally the extension is continued for 5min.

In one embodiment, step Y2 specifically includes the steps of:

b1, culturing escherichia coli JM109 containing recombinant plasmids in LB culture medium containing ampicillin for 12 hours, centrifuging 8000g of culture solution at 4 ℃ for 2min to obtain thalli, and extracting plasmids to obtain nitric oxide synthase recombinant plasmids;

b2, thawing the competent cells of the bacillus subtilis 168 prepared in advance at room temperature, adding the nitric oxide synthase recombinant plasmid extracted in the step b1, incubating for 2 hours at 37 ℃ under shaking at a speed of 200rpm, then coating the competent cells on a kanamycin-containing LB plate, and culturing for 18 hours to obtain the nitric oxide synthase recombinant strain.

According to the invention, a recombinant expression technology is used, a large amount of nitric oxide synthase is expressed in an edible bacillus subtilis expression system, and the expression product is added into the meat product, so that the generation of nitrosomyoglobin can be effectively promoted, and the color development effect of the meat product is better improved.

In one embodiment, the meat color former further comprises coagulase-negative staphylococci.

In one embodiment, the meat color former comprises a recombinant bacterial nitric oxide synthase crude extract and a coagulase negative staphylococcal suspensionThe solution, the recombinant bacterial nitric oxide synthase crude extract and coagulase negative staphylococcal suspension respectively contain 0.1-1.2 mg/mL of recombinant bacterial nitric oxide synthase and 10 ⁹ ～10 ¹⁰ CFU/mL of the coagulase-negative staphylococci.

In one embodiment, the method for preparing the recombinant bacterial nitric oxide synthase crude extract comprises the following steps:

x1, picking a single colony of the nitric oxide synthase recombinant bacteria, inoculating the single colony into an LB culture medium containing kanamycin, and culturing for 12-14 h at 36-38 ℃ to obtain a culture solution;

x2, inoculating the culture solution obtained in the step X1 into a TB culture medium containing kanamycin, enabling the OD600 to reach 0.08-0.12 during inoculation, culturing for 24-26 h at 36-38 ℃, and then separating thalli in a culture system;

and X3, fully washing the thalli separated in the step X2 by using phosphate buffer solution with the pH value of 7.4-7.5 and the concentration of 15-20 mM, re-suspending by using lysozyme solution with the concentration of 18-20 mg/mL, performing wall breaking treatment, and separating to obtain supernatant, namely the recombinant bacterial nitric oxide synthase crude extract.

x1, picking a single colony of the nitric oxide synthase recombinant bacteria, and inoculating the single colony into an LB culture medium containing kanamycin for shake culture to obtain a culture solution;

x2, inoculating the culture solution obtained in the step X1 into a TB culture medium containing kanamycin for shake culture, and centrifuging the culture solution to obtain a precipitate to obtain thalli;

and X3, washing thalli with phosphate buffer solution, re-suspending thalli with lysozyme solution, performing water bath, ultrasonic wall breaking and centrifugation, and taking supernatant to obtain recombinant bacterial nitric oxide synthase crude extract.

Further, the preparation method of the recombinant bacterial nitric oxide synthase crude extract comprises the following steps:

x1, picking a single colony of the nitric oxide synthase recombinant bacteria, inoculating the single colony into an LB culture medium containing kanamycin, and carrying out shake culture at a speed of 180-220 rpm and a temperature of 36-38 ℃ for 12-14 h to obtain a culture solution;

x2, inoculating the culture solution obtained in the step X1 into a TB culture medium containing kanamycin, enabling OD600 to reach 0.08-0.12 during inoculation, carrying out shake culture at a speed of 150-250 rpm and a temperature of 36-38 ℃ for 24-26 h, taking the culture solution, centrifuging at a speed of 10000-12000 rpm for 3-5 min at a temperature of 0-4 ℃, and taking a precipitate to obtain thalli;

X3, washing the thalli for 3-5 times by using 15-20 mM phosphate buffer solution with the pH value of 7.4-7.5, re-suspending the thalli by using 18-20 mg/mL lysozyme solution, carrying out ultrasonic wall breaking for 12-15 min at the temperature of 0-4 ℃ in a water bath of 25-35 min at the temperature of 36-38 ℃, centrifuging for 5-8 min at the speed of 10000-12000 rpm at the temperature of 0-4 ℃, and taking the supernatant to obtain the recombinant bacterial nitric oxide synthase crude extract.

In one embodiment, the lysozyme solution of step X3 is a phosphate buffer having a pH of 7.4 and a concentration of 20 mM.

In one embodiment, the wall breaking treatment in step X3 is ultrasonic wall breaking, and the specific conditions are: the working is carried out for 2 to 3 seconds at intervals of 2 to 3 seconds under the conditions of 20 to 25kHz and 100 to 500W of power.

In one embodiment, the method of preparing the coagulase-negative staphylococcal suspension comprises the steps of:

s1, inoculating coagulase negative staphylococcus into an SCB culture medium for culturing to obtain first-generation bacteria;

s2, inoculating the first-generation bacteria obtained in the step S1 into a new SCB culture medium for culture to obtain second-generation bacteria;

s3, inoculating the second-generation bacteria obtained in the step S2 into a new SCB culture medium for culture to obtain third-generation bacteria;

s4, separating thalli from the third-generation fungus culture solution finally obtained in the step S3;

S5, washing and resuspension the thalli separated in the step S4 with a sterile phosphate buffer solution with the pH value of 7.0-7.2 and the concentration of 15-20 mM to obtain coagulase negative staphylococcal suspension.

In one embodiment, the coagulase-negative staphylococcal suspension preparation method comprises the steps of:

s1, inoculating coagulase negative staphylococci into an SCB culture medium, uniformly mixing, and carrying out shake culture to obtain first-generation bacteria;

s2, sucking the first-generation bacteria obtained in the step S1, and inoculating the first-generation bacteria into a new SCB culture medium for shake culture to obtain second-generation bacteria;

s3, inoculating the second-generation bacterial liquid obtained in the step S2 into an SCB culture medium for shake culture to obtain third-generation bacteria;

s4, centrifuging the third-generation fungus culture solution obtained in the step S3 to remove the supernatant to obtain a fungus precipitate;

s5, washing and re-suspending the bacterial sediment by using a buffer solution to obtain coagulase negative staphylococcus suspension.

Further, the preparation method of the coagulase-negative staphylococcus suspension comprises the following steps:

s1, taking an experimental strain coagulase negative staphylococcal inclined plane, picking bacterial sludge in a sterile environment, inoculating the bacterial sludge into a sterilized SCB culture medium, uniformly mixing, and carrying out shake culture at a speed of 150-250 rpm for 18-20 h at 37 ℃ to obtain first-generation bacteria;

s2, sucking the first-generation bacteria obtained in the step S1, and inoculating the first-generation bacteria into a novel sterilized SCB culture medium, wherein the inoculation concentration is 10 ⁶ ～10 ⁸ CFU/mL, shake culturing at the speed of 150-250 rpm and the temperature of 36-38 ℃ for 18-20 h to obtain second-generation bacteria;

s3, inoculating the second-generation bacterial liquid obtained in the step S2 into an SCB culture medium, wherein the inoculation concentration is 10 ⁶ ～10 ⁸ CFU/mL, shake culturing at the speed of 150-250 rpm and the temperature of 36-38 ℃ for 18-20 h to obtain third-generation bacteria;

s4, centrifuging the third-generation bacterial culture solution obtained in the step S3 for 5-8 min under the conditions that the centrifugal force is 10000-12000 g and the temperature is 0-4 ℃, and removing the supernatant to obtain bacterial sediment;

s5, washing the bacterial sediment for 3-5 times by using a sterile phosphate buffer solution with the pH value of 7.0-7.2 and the concentration of 15-20 mM, and re-suspending to obtain coagulase negative staphylococcus suspension.

In one embodiment, the coagulase-negative staphylococci include any one or a combination of a plurality of staphylococcus equi, staphylococcus botulinum and staphylococcus calf, and are not limited thereto.

In another aspect, the present invention provides a method for coloring meat based on recombinant bacterial nitric oxide synthase comprising: uniformly mixing the meat color former based on recombinant bacterial nitric oxide synthase, meat emulsion and auxiliary materials to form prepared meat emulsion, and then fermenting and maturing the prepared meat emulsion.

In one embodiment, the method for coloring meat specifically comprises the following steps: fermenting the prepared meat emulsion for 1-2 d under the conditions of the temperature of 23-27 ℃ and the humidity of 84-86%, and immediately after finishing, placing the prepared meat emulsion for 7-12 d under the conditions of the temperature of 23-27 ℃ and the humidity of 69-71%.

In one embodiment, the coagulase-negative staphylococcal suspension is inoculated at 6.0-7.5 log CFU/g meat.

In one embodiment, the amount of the recombinant nitric oxide synthase crude extract added is 3.8-4.2 mg/100g meat.

In one embodiment, the method for coloring meat comprises the following steps:

(1) Mincing pork leg meat to obtain minced meat;

(2) Adding auxiliary materials and a recombinant bacterial nitric oxide synthase crude extract into the meat emulsion obtained in the step (1), and uniformly stirring to obtain a prepared meat emulsion;

(3) And (3) processing the prepared meat emulsion obtained in the step (2) under a certain condition to obtain the meat product.

In one embodiment, the method for coloring meat comprises the following steps:

(1) Mincing pork leg meat to obtain minced meat;

(2) Adding auxiliary materials and a recombinant bacterial nitric oxide synthase crude extract into the minced meat obtained in the step (1), uniformly stirring, and then, pouring with a pig casing to obtain sausage;

(3) Fermenting and maturing the sausage obtained in the step (2) under a certain condition to obtain the fermented sausage.

In one embodiment, the method for coloring meat comprises the following steps:

(1) Mincing pork leg meat to obtain minced meat;

(2) Adding auxiliary materials, coagulase negative staphylococcus suspension and recombinant bacterial nitric oxide synthase crude extract into the minced meat obtained in the step (1), uniformly stirring, and then pouring with a pig casing to obtain sausage;

In one embodiment, the method for coloring meat comprises the following steps:

(1) Removing fat and connective tissue from pork hind leg meat at 0-4 ℃, cutting into small pieces, and mincing in a meat mincer with the aperture of 1-5 mm to obtain minced meat;

(2) Adding auxiliary materials into the minced meat obtained in the step (1), stirring and mixing uniformly, adding coagulase negative staphylococcal suspension with the inoculation amount of 6.0-7.5 log CFU/g meat and/or recombinant bacterial nitric oxide synthase crude extract with the addition amount of 3.8-4.2 mg/100g meat, stirring uniformly, then pouring with pig casing, and bundling into one section with cotton threads every 8-12 cm to obtain sausage;

(3) Fermenting the sausage obtained in the step (2) for 1-2 d at the temperature of 23-27 ℃ and the humidity of 84-86%, and immediately after finishing, placing the sausage into the temperature of 23-27 ℃ and the humidity of 69-71% for maturation for 7-12 d to obtain the fermented sausage.

In one embodiment, the auxiliary materials comprise the following components in parts by weight based on 100 parts by weight of meat: 0.5 to 1.0 weight part of L-arginine, 0.5 to 5.0 weight parts of sodium chloride and 0.5 to 10 weight parts of sucrose.

In the present invention, the recombinant bacterial nitric oxide synthase, when used alone, is less effective in color development of meat products than nitrite addition. Surprisingly, the combination of the recombinant bacterial nitric oxide synthase with coagulase-negative staphylococci significantly increases the color development, probably because the addition of coagulase-negative staphylococci provides the bacterial nitric oxide synthase with cofactors required for the catalytic reaction, in particular with host reductase involved in the transfer of electrons, and thus significantly increases the efficiency of the recombinant bacterial nitric oxide synthase in the conversion of high-iron myoglobin.

In a further aspect the present invention provides a meat product, said meat product being produced by said recombinant bacterial nitric oxide synthase based meat coloring method.

In some embodiments, the meat product includes, but is not limited to, sausage, ham sausage, luncheon meat, bacon, dried meat, and the like.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The provided recombinant bacterial nitric oxide synthase has higher enzyme activity, can catalyze the generation of a large amount of nitric oxide, and can effectively combine myoglobin in meat products to generate nitrosomyoglobin when being applied to the color development of the meat products.

(2) The recombinant bacterial nitric oxide synthase and coagulase negative staphylococcus are used cooperatively, so that the color of the meat fuchsin can be improved more effectively, the color development effect equivalent to that of sodium nitrite is achieved, and a novel method with practical application significance is provided for nitrite color development substitution of meat products and improvement of safety of the nitrite color development substitution.

Drawings

FIG. 1 shows SDS-PAGE analysis of crude liquid of the empty fungus prepared in comparative example 3 and crude extract of recombinant bacterial nitric oxide synthase prepared in example 3.

FIG. 2 shows the absorption spectra of heme in the fermented sausages of the different treatment groups of examples 2-3 and comparative examples 2-6, mature 7 d.

Detailed Description

The technical scheme of the invention is explained in more detail below with reference to a plurality of embodiments.

The sources and preparation methods of the partial raw materials used in the following examples and comparative examples are as follows:

(1) Experimental culture medium

SCB medium: 7.5% sodium chloride broth was purchased from Guangdong Cryptographic microorganisms Inc. for activation and subculture of Staphylococcus aureus.

MSA medium: mannitol sodium chloride agar medium was purchased from Guangdong Cryptographic microorganisms Inc. for counting and slope preservation of staphylococci.

LB medium was purchased from Qingdao sea Bo biotechnology Co., ltd for activation and subculture of E.coli and Bacillus subtilis.

LB agar medium was purchased from Qingdao sea Bo biotechnology Co., ltd for resistance screening and counting of E.coli and B.subtilis.

TB medium was used for the expansion of Bacillus subtilis. The formula comprises 10.0g glycerin, 24.0g yeast powder, 12.0g tryptone and 16.4. 16.4g K per liter of water ₂ HPO4·3H ₂ O、2.3g KH ₂ PO ₄ Final ph7.4±0.2.

GM medium stock: 10 x lowest salt solution, 50% glucose, 5% hydrolyzed casein, 10% yeast juice, calcium magnesium ion solution. Wherein, except the hydrolyzed casein needs to be sterilized by a sterile filter membrane in a sterile environment, other solution components are sterilized by high-pressure steam at 121 ℃ for 15min for standby. 100mL of 10 Xthe lowest salt solution dissolved 18.34g K in distilled water ₂ HPO ₄ ·H ₂ O、6.0g KH ₂ PO ₄ 、2.0g(NH ₄ )2SO ₄ 1.0g trisodium citrate dihydrate, 0.2g MgSO ₄ ·7H ₂ O, and constant volume to 100mL.20mL of calcium magnesium ion solution: 2.03g MgCl was dissolved in distilled water ₂ ·6H ₂ O、0.22g CaCl ₂ And the volume was fixed to 20mL.

5mL GM I medium: 500. Mu.L of 10 Xminimum salt solution, 50. Mu.L of 50% glucose, 20. Mu.L of 5% hydrolyzed casein, 50. Mu.L of 10% yeast extract, and 4.38mL of sterile water.

90mL GM II medium: 8.8mL of 10 Xminimum salt solution, 900. Mu.L of 50% glucose, 72. Mu.L of 5% hydrolyzed casein, 36. Mu.L of 10% yeast extract, 450. Mu.L of calcium magnesium ion solution, and 79.742mL of sterile water.

The selective addition of antibiotics to the medium consisted essentially of ampicillin, kanamycin and spectinomycin at working concentrations of 100. Mu.g/mL, 100. Mu.g/mL and 50. Mu.g/mL, respectively.

(2) Preparation of E.coli JM109 competent

E.coli JM109 deposited with the glycerol tube was picked up and inoculated into 10mL of LB liquid medium, and the activated strain was cultured with shaking at 37℃for 12 hours at a rotation speed of 200 rpm. Coli competence was prepared using the Competent Cell Preparation kit according to the instructions. The prepared competent cells were stored at-80℃for further use.

(3) Preparation of Bacillus subtilis 168 competent

The slant deposited Bacillus subtilis 168 was taken, and the strain was picked up and activated in 5mL of GM I medium, and cultured with shaking at 37℃for 13 hours at a rotation speed of 200 rpm. 1mL of the culture broth was added to 9mL of GM I medium, and the culture was continued under the same conditions for 3.5 hours. The 10mL of the culture solution was inoculated into 90mL of GM II medium, and cultured under the same conditions for 1.5 hours. The 10mL GM II culture broth was dispensed into 50mL sterile centrifuge tubes and centrifuged at 860g for 5min at 4 ℃. Removing 9mL of supernatant, collecting 1mL of supernatant, re-suspending thallus, subpackaging into 1.5mL centrifuge tubes, packaging 500 μl each tube, adding 250 μl 30% glycerol, and freezing at-80deg.C.

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

TABLE 1 primers used in the examples of the present invention

(5) Experimental strains: staphylococcus aureus CICC 10850, china center for type culture Collection of Industrial microorganisms.

Coli JM109, accession number: c1300, beijing village allied biogenic gene technologies Co.

Bacillus subtilis 168,ATCC 23857,BioVectorNTCC collection of classical cultures.

Example 1 preparation of a single colony of nitric oxide synthase recombinant bacteria the following steps are followed:

y1, constructing a nitric oxide synthase recombinant plasmid, which specifically comprises:

a1, amplifying a nitric oxide synthase gene sequence in a 50 mu L PCR system by taking bacillus subtilis 168 genome DNA as a template; the PCR system is as follows: 2 mu L, 25 mu L PrimeSTAR Max DNA Polymerase and 21 mu L of sterile water of 10mM upstream and downstream primers respectively, and a small amount of bacillus subtilis 168 single colony is picked up by an inoculating loop to be connected into the system and stirred uniformly; the PCR reaction procedure was: pre-denaturation at 95 ℃ for 15min, one cycle of 30s at 95 ℃, 30s at 56 ℃ and 2min at 72 ℃ for 35 times, and finally continuous extension for 5min;

a2, amplifying the linearization plasmid by taking the pP43NMK as a template in a 50 mu L PCR system, wherein the obtained PCR product is the linearization pP43NMK plasmid; the PCR system is as follows: 10mM upstream and downstream primer each 2. Mu.L, 2. Mu.L 20-100 mg/. Mu.L pP43NMK plasmid, 25. Mu. L PrimeSTAR Max DNA Polymerase, 19. Mu.L sterile water; the PCR reaction procedure was: pre-denaturation at 95 ℃ for 5min, one cycle of 95 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 2min, total 35 times of cycles, and finally continuous extension for 5min:

a3, adding 1 mu L FastDigest DpnI enzyme into 50 mu L of the linearized pP43NMK plasmid PCR product obtained in the step a2, treating at 37 ℃ for 10min on a PCR instrument to eliminate the circular plasmid, and inactivating FastDiget DpnI enzyme at 85 ℃ for 10min to obtain pP43NMK plasmid;

a4, seamlessly connecting the target gene nitric oxide synthase sequence obtained in the step a1 and the pP43NMK plasmid obtained in the step a3 according to a specification by using ClonExpress II One Step Cloning Kit to obtain a recombinant plasmid; 10. Mu.L of the recombinant plasmid was added to 100. Mu.L of E.coli JM109 competent, and the mixture was subjected to ice bath for 30min, followed by heat shock in a water bath at 42℃for 45s, and immediately thereafter placed on ice for 3min. 900. Mu.L of the supernatant was removed by centrifugation after adding 900. Mu.LLB medium and incubating at 37℃for 1 hour, and the remaining medium was resuspended and spread on LB plates containing ampicillin and incubated at 37℃for 16 hours to obtain a recombinant plasmid of nitric oxide synthase.

Converting the bacillus subtilis by the Y2 and nitric oxide synthase recombinant plasmid to obtain a nitric oxide synthase recombinant strain, which specifically comprises the following steps:

b1, culturing escherichia coli JM109 containing the nitric oxide synthase recombinant plasmid in 5mL LB culture medium containing ampicillin for 12h, centrifuging 8000g of culture solution at 4 ℃ for 2min to obtain thalli, and extracting the plasmid according to the specification by using a small amount of extraction kit of an engineering Diaspin column type plasmid DNA to obtain the nitric oxide synthase recombinant plasmid;

b2, thawing competent cells of the bacillus subtilis 168 prepared in advance at room temperature, adding the nitric oxide synthase recombinant plasmid extracted in the step b1, incubating for 2 hours at 37 ℃ under shaking at a speed of 200rpm, then coating the mixture on a kanamycin-containing LB plate, culturing for 18 hours, and transforming the bacillus subtilis 168 with the nitric oxide synthase recombinant plasmid to obtain the nitric oxide synthase recombinant strain.

Example 2 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(1) Removing fat and connective tissue from pork leg meat at 4deg.C, cutting into small pieces, and mincing in a meat mincer with aperture of 3mm to obtain meat emulsion;

(2) Adding 0.8g L-arginine, 2.5g sodium chloride and 5g sucrose into 100g meat emulsion obtained in the step (1), stirring and mixing uniformly, adding recombinant bacterial nitric oxide synthase crude extract with the addition amount of 4mg/100g meat, stirring uniformly, then pouring with a pig casing, and bundling into one section every 10cm by using cotton threads to obtain sausage;

(3) Fermenting the sausage obtained in the step (2) for 1d at the temperature of 25 ℃ and the humidity of 85%, and immediately after the fermentation, putting the sausage into the temperature of 25 ℃ and the humidity of 70% for 7d to obtain the fermented sausage.

The preparation method of the recombinant bacterial nitric oxide synthase crude extract in the step (2) comprises the following steps:

X1, picking a single colony of the nitric oxide synthase recombinant bacteria prepared in the example 1, inoculating the single colony into 20mL of LB culture medium containing kanamycin, and carrying out shake culture at a speed of 200rpm at about 37 ℃ for 12 hours to obtain a culture solution;

x2, inoculating the culture solution obtained in the step X1 into 50mL of TB culture medium containing kanamycin, enabling OD600 to reach 0.1 during inoculation, carrying out shake culture at 37 ℃ for 24 hours at a speed of 200rpm, centrifuging the culture solution at 4 ℃ for 3min at a speed of 10000rpm, and taking a precipitate to obtain thalli;

x3, washing thalli three times by using 20mM phosphate buffer solution with pH value of 7.4, re-suspending thalli by using 20mg/mL lysozyme solution, carrying out ultrasonic wall breaking for 15min at the temperature of 37 ℃ in water bath for 30min, centrifuging for 5min at the temperature of 0 ℃ at the rotating speed of 10000rpm, and taking supernatant to obtain recombinant bacterial nitric oxide synthase crude extract.

The lysozyme solution in the step X3 is a 20mM phosphate buffer solution with pH value of 7.4.

The ultrasonic wall breaking condition in the step X3 is that the ultrasonic wall breaking condition works for 2s at intervals of 3s under the power of 300W at the frequency of 25 kHz.

Example 3 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(2) Adding 0.8g L-arginine, 2.5g sodium chloride and 5g sucrose into 100g meat emulsion obtained in the step (1), stirring and mixing uniformly, adding staphylococcus calf suspension with the inoculation amount of 7.0log CFU/g meat and recombinant bacterial nitric oxide synthase crude extract with the addition amount of 4mg/100g meat, stirring uniformly, then pouring with pig casing, and bundling into one section every 10cm by cotton threads to obtain sausage;

The preparation method of the staphylococcus calf suspension in the step (2) comprises the following steps:

s1, taking an experiment strain staphylococcus aureus inclined plane, picking bacterial mud in a sterile environment, inoculating the bacterial mud into 10mL of sterilized SCB culture medium, uniformly mixing, and carrying out shake culture at a speed of 200rpm at 37 ℃ for 18 hours to obtain first-generation bacteria;

s2, sucking the first-generation bacteria obtained in the step S1, inoculating the first-generation bacteria into 10mL of a new sterilized SCB culture medium, wherein the inoculation concentration is 10 ⁷ CFU/mL, shake culturing at 37 ℃ for 18h at 200rpm to obtain second-generation bacteria;

s3, inoculating the second-generation bacterial liquid obtained in the step S2 into 100mL of SCB culture medium, wherein the inoculation concentration is 10 ⁷ CFU/mL, shake culturing at 37 deg.C for 18h at 200rpm to obtain third-generation bacteria;

s4, centrifuging the third-generation bacterial culture solution obtained in the step S3 for 5min at the centrifugal force of 10000g and the temperature of 4 ℃, and removing the supernatant to obtain bacterial precipitate;

s5, washing the bacterial sediment 3 times by using a sterile 20mM phosphate buffer solution with the pH value of 7.0, and re-suspending to obtain the staphylococcus aureus suspension.

x1, picking a single colony of the nitric oxide synthase recombinant bacteria prepared in the example 1, inoculating the single colony into 20mL of LB culture medium containing kanamycin, and carrying out shake culture at a speed of 200rpm at 37 ℃ for 12 hours to obtain a culture solution;

x3, washing thalli three times by using 20mM phosphate buffer solution with pH value of 7.4, re-suspending thalli by using 20mg/mL lysozyme solution, carrying out ultrasonic wall breaking for 15min at the temperature of 37 ℃ in water bath for 30min, centrifuging for 5min at the temperature of 4 ℃ at the speed of 10000rpm, and taking supernatant to obtain recombinant bacterial nitric oxide synthase crude extract.

Comparative example 1 preparation method of empty vector single colony comprises the following steps:

b1, culturing escherichia coli JM109 containing empty-load pP43NMK plasmid in 5mL LB culture medium containing ampicillin for 12h, centrifuging 8000g of culture solution at 4 ℃ for 2min to obtain thalli, and extracting the plasmid according to the specification by using a small amount of extraction kit of a crude Diaspin column type plasmid DNA to obtain the empty-load plasmid;

b2, thawing competent cells of the bacillus subtilis 168 prepared in advance at room temperature, adding the empty plasmid extracted in the step b1, incubating for 2 hours at 37 ℃ under shaking at a speed of 200rpm, then coating the cells on a kanamycin-containing LB plate, and culturing for 18 hours, wherein the empty plasmid converts the bacillus subtilis 168 to obtain an empty strain.

Comparative example 2 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(2) Adding 0.8g L-arginine, 2.5g sodium chloride and 5g sucrose into 100g meat paste obtained in the step (1), stirring and mixing uniformly, adding 4mg/100g empty-carrier crude extract of meat, stirring uniformly, then pouring with pig casing, and bundling into one section every 10cm by cotton threads to obtain sausage;

The preparation method of the no-load bacteria nitric oxide synthase crude extract in the step (2) comprises the following steps:

x1, picking single colonies of the empty-load bacteria prepared in the comparative example 1, respectively inoculating the single colonies into 20mL of LB culture medium containing kanamycin, and carrying out shake culture at a speed of 180-220 rpm at 36-38 ℃ for 12h to obtain a culture solution;

x3, washing thalli three times by using 20mM phosphate buffer solution with pH value of 7.4, re-suspending thalli by using 20mg/mL lysozyme solution, carrying out ultrasonic wall breaking for 15min at the temperature of 0-4 ℃ in a water bath of 37 ℃, centrifuging for 5min at the speed of 10000rpm at the temperature of 0-4 ℃, and taking the supernatant to obtain the crude extract of the bacteria-free nitric oxide synthase.

Comparative example 3 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(2) Adding 0.8g L-arginine, 2.5g sodium chloride and 5g sucrose into 100g meat emulsion obtained in the step (1), stirring and mixing uniformly, adding staphylococcus calf suspension with the inoculation amount of 7.0log CFU/g meat and the empty fungus nitric oxide synthase crude extract with the addition amount of 4mg/100g meat, stirring uniformly, then pouring with pig casing, and bundling into one section every 10cm by cotton threads to obtain sausage;

The preparation method of the empty fungus crude extract in the step (2) comprises the following steps:

x1, picking a single colony of the empty fungus prepared in the comparative example 1, inoculating the single colony into 20mL of LB medium containing kanamycin, and carrying out shake culture at a speed of 200rpm for 12 hours at 37 ℃ to obtain a culture solution;

X3, washing the thalli three times by using 20mM phosphate buffer solution with the pH value of 7.4, re-suspending the thalli by using 20mg/mL lysozyme solution, carrying out ultrasonic wall breaking for 15min at the temperature of 37 ℃ in water bath for 30min, centrifuging for 5min at the temperature of 4 ℃ at the speed of 10000rpm, and taking the supernatant to obtain the crude extract of the bacteria-free nitric oxide synthase.

Comparative example 4 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(2) Adding 0.8g L-arginine, 2.5g sodium chloride and 5g sucrose into 100g of the meat emulsion obtained in the step (1), stirring and mixing uniformly, then pouring with a pig casing, and bundling into one section every 10cm by using cotton threads to obtain sausage;

Comparative example 5 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(2) Adding 0.8g L-arginine, 2.5g sodium chloride and 5g sucrose into 100g of the minced meat obtained in the step (1), stirring and mixing uniformly, adding the calf staphylococcal suspension with the inoculation amount of 7.0log CFU/g meat, stirring uniformly, then pouring with a pig casing, and bundling into one section every 10cm by using cotton threads to obtain sausage;

Comparative example 6 a method for achieving color development of meat using recombinant bacterial nitric oxide synthase, the steps are as follows:

(2) Adding 9mg of sodium nitrite, 0.8g g L-arginine, 2.5g of sodium chloride and 5g of sucrose into 100g of the meat emulsion obtained in the step (1), stirring and mixing uniformly, then pouring with a pig casing, and bundling into a section every 10cm by using cotton threads to obtain sausage;

Test example 1 identification of nitric oxide synthase recombinant plasmids:

single colonies with ampicillin resistance in example 1 were picked, inoculated into 10mL of LB liquid medium containing ampicillin, cultured for 12 hours, and plasmids were extracted using a small extraction kit for the plasmid DNA of the Diaspin column, and sent to Shanghai Bioengineering Co., ltd for gene sequencing.

The base sequence and the position of the nitric oxide synthase prepared in the example 1 are determined to be correct, and the construction of the recombinant plasmid of the nitric oxide synthase is proved to be successful.

Test example 2 SDS-PAGE electrophoresis of crude extracts of recombinant bacterial nitric oxide synthase prepared in comparative example 3 and example 3:

gel was prepared according to the instructions using SDS-PAGE gel preparation kit, the concentration of the concentrated gel was 5% and the concentration of the separation gel was 10%. The crude extracts prepared in comparative example 3 and example 3 were diluted to 1mg/mL with 20mM PBS having pH=7.4, 20. Mu.L was added to 5. Mu.L of 5 Xprotein loading buffer, and then boiled for 5min, centrifuged at 4000g for 5min, 20. Mu.L of the supernatant was taken and SDS-PAGE was performed separately from the protein molecular weight standard. The electrophoresis conditions were: setting the voltage at 80V, adjusting the voltage to 120V until the electrophoresis is finished when the gel is separated, and taking out the gel. The gel was stained in coomassie blue staining solution for 30min, followed by rinsing several times with a destaining solution mixed with 400mL of ethanol, 100mL of acetic acid and 500mL of water until clear bands appeared. The test results are shown in FIG. 1, wherein lanes 1 and 2 correspond to the crude liquid of the empty fungus prepared in comparative example 3 and the crude extract of the recombinant bacterial nitric oxide synthase prepared in example 3, respectively.

In theory, compared with the empty fungus crude extract, the recombinant fungus crude extract has one more protein, namely recombinant bacterial nitric oxide synthase protein, and when the two crude extract proteins are electrophoresed, the band with difference is the recombinant bacterial nitric oxide synthase protein band. As shown in the results of FIG. 1, the recombinant bacterial nitric oxide synthase crude extract had a band of about 48kDa more than the empty bacterial crude extract, thus preliminarily judging that the recombinant bacterial nitric oxide synthase protein had been successfully expressed and that the protein had a molecular weight of about 48kDa. Moreover, as can be seen from the figure, the color of the band is obviously darker compared with other bands, which indicates that the expression efficiency of the recombinant protein is high.

Test example 3 analysis of recombinant bacterial nitric oxide synthase enzyme activity:

enzyme activity assays were performed on recombinant bacterial nitric oxide synthases of the empty bacteria and the recombinant bacteria of nitric oxide synthase prepared in comparative example 1 and example 1. In order to eliminate the influence of nitrite reductase in host bacteria on the enzyme activity measurement of recombinant bacterial nitric oxide synthase, the nitrite content is accurately measured, and the nitrite reductase gene on the genome of the nitrite reductase needs to be knocked out, which comprises the following specific steps:

(1) Constructing a knockout frame to obtain a large number of knockout frame sequence amplification products:

a1, amplifying 1000bp gene sequences at the upstream and downstream of the nasD gene by using bacillus subtilis 168 genome DNA as a template in a 20 mu L PCR system for amplifying the upstream and downstream sequences of the nasD gene, and simultaneously amplifying a lox71-spc-lox66 sequence from a plasmid P7s/P43 in a 20 mu L PCR system for amplifying the lox71-spc-lox66 box; the PCR system for amplifying the up-and-down stream sequence of the nas gene is as follows: 10mM upstream and downstream primers of 0.8 mu L, 10 mu L PrimeSTAR Max DNA Polymerase and 8.4 mu L of sterile water respectively, and a small amount of bacillus subtilis 168 single colony is picked up by an inoculating loop to be accessed into the system and stirred uniformly; the PCR system for amplifying the lox71-spc-lox66 box is as follows: 10mM upstream and downstream primers are respectively 0.8 mu L, 10 mu L PrimeSTAR Max DNA Polymerase and 8.4 mu L of sterile water, and P7 s/P43-escherichia coli single colonies are respectively picked up by a small amount by an inoculating loop, connected into the system and stirred uniformly; the PCR reaction procedures were as follows: pre-denaturation at 95 ℃ for 15min, one cycle of 30s at 95 ℃, 30s at 55 ℃ and 1min at 72 ℃ for 35 times, and finally continuous extension for 5min;

a2, fusing the nasD upstream and downstream sequences with the lox71-spc-lox66 sequence obtained in the step al in a 20 mu L PCR system through triple fusion PCR to obtain a knockout frame sequence; the PCR system is as follows: 10 mu L PrimeSTAR Max DNA Polymerase, 10 mu L of the left and right wall sequences of the knockout frame and the concentration ratio of lox71-spc-lox66 sequences is 1:1:2; the PCR reaction procedures were as follows: pre-denaturation at 95 ℃ for 3min, one cycle of 30s at 95 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 15 times, and finally continuous extension for 5min;

a3, in a 50 mu L PCR system, taking the knockout frame sequence obtained in the step a2 for further amplification to obtain a large number of knockout frame sequence amplification products; the PCR system is as follows: 2. Mu.L of the knockout frame sequence, 2. Mu.L of 10mM upstream and downstream primers, 25. Mu. L PrimeSTAR Max DNA Polymerase and 19. Mu.L of sterile water, respectively; the PCR reaction procedures were as follows: pre-denaturation at 95 ℃ for 3min, one cycle of 30s at 95 ℃, 30s at 55 ℃ and 2min at 72 ℃ for 35 times, and finally continuous extension for 5min;

(2) Transforming the knockout frame to obtain a gene-defective bacillus subtilis 168 strain:

transforming the knockout frame fragment into bacillus subtilis 168 competent cells by a chemical transformation method; adding amplified nasD knockout frame fragments into bacillus subtilis 168 competent cells, uniformly mixing, shaking and incubating for 2 hours in a 37 ℃ incubator, centrifuging 8000g of bacterial liquid at 4 ℃ for 5 minutes after incubation, removing redundant supernatant, reserving 100 mu L of supernatant to resuspend thalli, respectively coating the supernatant on LB plates containing spectinomycin, and culturing overnight at 37 ℃ to obtain the delta nasD gene defective bacillus subtilis 168 strain;

(3) The empty plasmid and the nitric oxide synthase recombinant plasmid are transformed into the delta nasD gene-defective bacillus subtilis 168 strain to respectively obtain gene-defective empty bacteria and gene-defective nitric oxide synthase recombinant strain.

c1, taking a delta nasD gene-defective bacillus subtilis 168 strain, picking the strain, activating in 5mL of GM I culture medium, and carrying out shake culture at 37 ℃ for 13h at a rotating speed of 200 rpm; 1mL of the culture solution is added into 9mL of GM I culture medium, and the culture is continued for 3.5h under the same conditions; inoculating the 10mL culture solution into 90mL GM II culture medium, and culturing for 1.5h under the same conditions; taking 10mL of the GM II culture solution, subpackaging the GM II culture solution into a 50mL sterile centrifuge tube, and centrifuging for 5min at 4 ℃ and 860 g; removing 9mL of supernatant, reserving 1mL of supernatant to resuspend thalli, subpackaging into 1.5mL centrifuge tubes, filling 500 mu L of each tube, adding 250 mu L of 30% glycerol, and freezing at-80 ℃ for standby to obtain delta nasD gene defective bacillus subtilis 168 competent cells;

and c2, thawing the delta nasD gene-defective bacillus subtilis 168 competent cells obtained in the step c1 at room temperature, and adding the extracted empty plasmid and recombinant plasmid. Incubation was performed at 200rpm with shaking at 37℃for 2h, followed by plating on LB plates containing kanamycin and incubation for 18h. Transforming the delta nasD gene-defective bacillus subtilis 168 competent cells by using the empty vector to obtain gene-defective empty vector; and transforming the genetically defective bacillus subtilis 168 competent cells by the recombinant plasmid of the nitric oxide synthase to obtain the recombinant bacterium of the genetically defective nitric oxide synthase.

Single colonies of the gene-deficient no-load bacteria and the gene-deficient nitric oxide synthase recombinant bacteria are picked and respectively inoculated into 20mL of kanamycin-containing LB culture medium, and the culture is carried out for 12 hours at 37 ℃ by shaking at the speed of 200 rpm. The strain culture broth was inoculated into 50mL kanamycin-containing LB medium containing 20mM L-arginine, the initial OD600 was allowed to reach 0.1, the culture was performed at 37℃with shaking at 200rpm for 24 hours, the kanamycin-containing LB medium without the strain culture broth was used as a control group, the nitric oxide yield in the medium before and after the culture was detected using the Biyun nitrogen oxide detection kit, and the nitric oxide synthase activity was analyzed, and the analysis results were shown in Table 2.

TABLE 2 recombinant bacterial nitric oxide synthase Activity

	Control group	Comparative example 1	Example 1
				Nitrite content (mu mol/L)	18 ^b	37 ^b	1592 ^a

Note that: the different letters indicate that the data difference between the different groups is significant (P < 0.05).

Nitric oxide synthase has the function of catalyzing the generation of nitric oxide, and nitric oxide, as a hydrophobic molecule, can easily penetrate cell walls and cell membranes. In the culture medium system, nitric oxide produced by nitric oxide synthase catalysis in bacillus subtilis 168 thalli penetrates cells into the culture medium and can be rapidly oxidized into nitrite and nitrate, so that the content of nitrite and nitrate in the culture medium can indirectly reflect the activity of the nitric oxide synthase. However, the expression host bacillus subtilis 168 has nitrite reductase, which does not affect the nitric oxide synthase, but can degrade nitrite, and has interference with the measurement of the enzyme activity of the nitric oxide synthase. Therefore, the nitrite reductase gene delta nasD of the host bacillus subtilis 168 is knocked out to obtain nitrite reductase defective host bacteria, and the nitrite reductase defective host bacteria and the recombinant plasmid are respectively introduced to obtain gene defective empty bacteria and gene defective nitric oxide synthase recombinant bacterial strains, and the activity of the recombinant bacterial nitric oxide synthase is judged by measuring the capability of the two bacterial strains for catalyzing and producing nitric oxide. The recombinant bacteria of the gene-defective no-load bacteria and the recombinant bacteria of the gene-defective nitric oxide synthase are respectively inoculated into LB culture medium containing 20mM L-arginine in equal quantity, and the nitrite content in the supernatant of the culture medium after 24 hours is measured to indirectly react with the activity of the recombinant bacteria of the nitric oxide synthase.

As can be seen from Table 2, the gene-deficient no-load bacteria produced about 37. Mu. Mol/L nitrite after 24 hours of cultivation, which may be the result of the catalytic production of nitric oxide synthase contained in the host bacteria themselves; the gene-defective nitric oxide synthase recombinant strain is cultured for 24 hours to catalyze and produce about 1580 mu mol/L nitrite which is 58 times of that of the gene-defective empty bacteria, which shows that the recombinant expression product nitric oxide synthase has extremely high enzyme activity. The invention utilizes a bacillus subtilis recombinant expression system to obtain recombinant bacterial nitric oxide synthase with extremely high enzymatic activity, and provides a feasible scheme for the color development of the fermented sausage.

Test example 4 the measurement of the redness value was carried out on 7 sets of sausages prepared in examples 2 to 3 and comparative examples 2 to 6. Each group of sausage samples was chopped at 5g and the measurement of the redness value was carried out with a 3nh spectrocolorimeter, and the test results are shown in Table 3.

TABLE 3 redness values of sausages prepared in examples 2 to 3 and comparative examples 2 to 6

Group of	Sausage redness value after 7 days of fermentation
		Comparative example 2	3.86±0.20 ^f
Comparative example 3	5.76±0.15 ^d
		Comparative example 4	3.10±0.43 ^g
Comparative example 5	6.32±0.12 ^c
		Comparative example 6	6.94±0.08 ^b
Example 2	4.29±0.14 ^e
		Example 3	7.27±0.16 ^a

Note that: different letters indicate significant differences in survival between the different groups (P < 0.05).

The result of the enzyme activity test of the recombinant bacterial nitric oxide synthase shows that the recombinant bacterial nitric oxide synthase produced by expression in the bacillus subtilis expression system has complete nitric oxide synthase activity, and a great amount of nitric oxide is produced by catalysis, which is theoretically enough to replace the chromogenic application of nitrite in the fermented sausage. Thus, the comparative example 2 and example 2 extract a crude recombinant bacterial nitric oxide synthase extract and were applied to the production of fermented sausage, but as shown in table 3, the addition of the comparative example 2 and example 2 by the crude recombinant bacterial nitric oxide synthase extract has a remarkable promoting effect on the formation of red color of fermented sausage, but it is difficult to achieve an ideal color development effect by the addition of the crude recombinant bacterial nitric oxide synthase extract as compared with the comparative examples 5 and 6. Bacterial nitric oxide synthase has a different structure from mammalian nitric oxide synthase, and bacterial nitric oxide synthase lacks a reductase domain, so that in bacteria, catalytic reactions of nitric oxide synthase often require other reductase partners instead of the reductase partners, and various cofactors such as NADPH, FMN, FAD, H B/H4F and the like are needed for assistance. Therefore, in a fermented sausage system added with only the recombinant bacterial nitric oxide synthase crude extract, the nitric oxide synthase enzyme activity is enough, but enough reductase and various cofactors are lacking, and the yield of nitric oxide still has room for further improvement.

In example 3 of the present invention, not only was the recombinant bacterial nitric oxide synthase crude extract added, but also staphylococcus calfsii was inoculated, which provided more cofactors and reductase. At 7d, the red value of the inoculated fermented sausage added with the recombinant bacterial nitric oxide synthase crude extract of the example 3 is higher than that of the comparative examples 3-5, and slightly higher than that of the sausage added with sodium nitrite of the comparative example 6. Wherein the fermented sausage prepared in example 3 has a higher redness value than the fermented sausage prepared in example 2, which demonstrates that the recombinant bacterial nitric oxide synthase has a considerable promoting effect on the increase of the redness value of the sausage. The redness value data show that the addition of the recombinant bacterial nitric oxide synthase and the starter staphylococcus aureus can effectively promote the formation of red color of the fermented sausage, even reach the level equivalent to or higher than the addition of nitrite, and the method provides a brand-new and very practical method for improving the safety of the fermented sausage for substituting nitrite color of the fermented sausage.

Test example 5 sausage color effect:

ultraviolet-visible spectrum analysis was performed on the systems of examples 2 to 3 and comparative examples 2 to 6. Baseline was adjusted with LB medium liquid, and after centrifugation and precipitation removal of each set of medium systems, scanning was performed at 1nm intervals over the wavelength range from 350nm to 650nm, and the test results are shown in fig. 2.

The formation of the red color of the fermented sausage is mainly due to the existence of nitrosomyoglobin, and in order to further analyze the possible reason that the recombinant bacterial nitric oxide synthase promotes the improvement of the sausage redness value, the nitrosopigments of each group of sausage are extracted and analyzed. The enzyme activity of test example 1 proves that the recombinant bacterial nitric oxide synthase has higher enzyme activity, can catalyze the generation of a large amount of nitric oxide, and can be theoretically and effectively combined with myoglobin in the fermented sausage to generate enough nitrosomyoglobin. However, as can be seen from Table 3, the sausages prepared in comparative example 2 and example 2, by recombinant bacterial nitric oxide synthase, alone, did not effectively convert high iron myoglobin to nitrosomyoglobin, probably due to the lack of reductase and other cofactors required for the catalysis of nitric oxide synthase in the meat system. Thus, the sausage prepared in example 3 was incubated by adding recombinant bacterial nitric oxide synthase together with staphylococcus calf to LB medium containing ferritin in order to provide the substances required for the nitric oxide synthase catalytic reaction by the addition of live bacteria.

As can be seen from the results of fig. 2, comparative example 4 has absorption peaks at 505 and 635nm, which are typical characteristic absorption peaks of high-iron myoglobin; except for comparative example 4, the groups each exhibited the highest peaks at 394, 480, 540 and 565nm, which are typical absorption peaks of nitrosomyoglobin, indicating the production of nitrosomyoglobin in the fermented sausages prepared in comparative examples 2 to 3, comparative examples 5 to 6 and examples 2 to 3. According to the peak value of the absorption peak, the sausage prepared in example 3 has the maximum content of nitrosomyoglobin, and the second time of comparative example 6 is higher than the fermented sausage prepared in example 2, comparative examples 2-3 and comparative example 5, which corresponds to the red value trend of each group of sausage. Combining the color change of table 3 with the experimental results of UV-Vis analysis, example 3 was able to increase the efficiency of conversion of high-iron myoglobin to nitrosomyoglobin by staphylococcus calves to a greater extent by the addition of recombinant bacterial nitric oxide synthase than comparative examples 2-3 and example 2 inoculated with staphylococcus calves alone. The crude extract of recombinant bacterial nitric oxide synthase added to sausage prepared in example 3 contains sufficient nitric oxide synthase activity to fully catalyze the reaction of L-arginine to generate sufficient nitric oxide, and the nitric oxide is combined with myoglobin in sausage to form a large amount of nitrosomyoglobin. By combining the nitrosomyoglobin absorption spectrum curve and the rule of the sausage redness value, the nitric oxide synthase obtained by recombinant expression and the starter staphylococcus calf are added into the sausage together, so that the red color of the sausage can be effectively improved, the processing effect is equivalent to that of sodium nitrite, and the method has great practical application significance for replacing nitrite color of meat and improving the safety of the meat.

In addition, the applicant carried out experiments with other types of coagulase-negative staphylococci such as Staphylococcus equi and Staphylococcus sarcochrous instead of Staphylococcus equi in example 3, and finally obtained similar experimental results as in example 3.

It should be noted that, the foregoing description is only a preferred embodiment of the present application, and although the present application has been described in detail with reference to the foregoing embodiment, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, and any modifications, equivalents, improvements and modifications made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A meat color former based on recombinant bacterial nitric oxide synthase comprising recombinant bacterial nitric oxide synthase and coagulase negative staphylococci; wherein, the preparation method of the recombinant bacterial nitric oxide synthase comprises the following steps:

2. The meat color former based on recombinant bacterial nitric oxide synthase according to claim 1, comprising a crude recombinant bacterial nitric oxide synthase extract and a coagulase-negative staphylococcal suspension, wherein the crude recombinant bacterial nitric oxide synthase extract and the coagulase-negative staphylococcal suspension respectively contain 0.1-1.2 mg/mL of the recombinant bacterial nitric oxide synthase and 10 g/mL of the recombinant bacterial nitric oxide synthase ⁹ ~10 ¹⁰ CFU/mL of the coagulase-negative staphylococci.

3. The recombinant bacterial nitric oxide synthase based meat colorant according to any of claims 1-2, wherein said coagulase-negative staphylococci are selected from any one or more of the group consisting of staphylococcus equine, staphylococcus botulinum, and staphylococcus calf.

4. The recombinant bacterial nitric oxide synthase based meat color former of claim 2, wherein the method for preparing the crude recombinant bacterial nitric oxide synthase extract comprises the steps of:

x1, picking a single colony of the nitric oxide synthase recombinant bacteria, inoculating the single colony to an LB culture medium containing kanamycin, and culturing for 12-14 h at 36-38 ℃ to obtain a culture solution;

X2, inoculating the culture solution obtained in the step X1 into a TB culture medium containing kanamycin, enabling the OD600 to reach 0.08-0.12 during inoculation, culturing for 24-26 hours at 36-38 ℃, and then separating thalli in a culture system;

and X3, fully washing the thalli separated in the step X2 with phosphate buffer solution with the pH value of 7.4-7.5 and the concentration of 15-20 mM, re-suspending with lysozyme solution with the concentration of 18-20 mg/mL, performing wall breaking treatment, and separating to obtain supernatant, namely the recombinant bacterial nitric oxide synthase crude extract.

5. The recombinant bacterial nitric oxide synthase-based meat color former according to claim 4, wherein the wall breaking treatment in step X3 is ultrasonic wall breaking, and the specific conditions are: working for 2-3 s at the frequency of 20-25 kHz and the power of 100-500W at intervals of 2-3 s.

6. The recombinant bacterial nitric oxide synthase based meat color former according to claim 2, wherein said coagulase negative staphylococcal suspension is prepared by a method comprising the steps of:

s5, washing and resuspension the thalli separated in the step S4 with a sterile phosphate buffer solution with the pH value of 7.0-7.2 and the concentration of 15-20 mM, and obtaining coagulase negative staphylococcal suspension.

7. A method of coloring meat based on recombinant bacterial nitric oxide synthase comprising: uniformly mixing the recombinant bacterial nitric oxide synthase-based meat color former, meat emulsion, and adjuvants of any one of claims 1-6 to form a blended meat emulsion, and then fermenting and ripening the blended meat emulsion.

8. The method for coloring meat based on recombinant bacterial nitric oxide synthase according to claim 7, characterized in that it comprises in particular: fermenting the prepared meat emulsion for 1-2 d at the temperature of 23-27 ℃ and the humidity of 84-86%, and immediately after the fermentation, placing the meat emulsion at the temperature of 23-27 ℃ and the humidity of 69-71% for maturation for 7-12 d.

9. The method for coloring meat based on recombinant bacterial nitric oxide synthase according to claim 7, comprising: wherein the addition amount of the recombinant nitric oxide synthase crude extract is 3.8-4.2 mg/100g meat.

10. The method for coloring meat based on recombinant bacterial nitric oxide synthase according to claim 7, comprising: wherein the inoculation amount of the coagulase-negative staphylococcus suspension is 6.0-7.5 log CFU/g meat.

11. The method for coloring meat based on recombinant bacterial nitric oxide synthase according to claim 7, wherein: calculated by taking 100 parts by weight of meat emulsion as a reference, the auxiliary materials comprise the following components in parts by weight: 0.5 to 1.0 part by weight of L-arginine, 0.5 to 5.0 parts by weight of sodium chloride and 0.5 to 10 parts by weight of sucrose.

12. A meat product, characterized in that it is produced by the method for colouring meat based on recombinant bacterial nitric oxide synthase according to any one of claims 7 to 11.