Application of reagent containing multi-copper oxidase in reducing biogenic amine content of fermented meat product

Abstract

The invention discloses an application of a reagent containing multi-copper oxidase in reducing the biogenic amine content of a fermented meat product, recombinant multi-copper oxidase is obtained through a genetic engineering technology, and the multi-copper oxidase is developed into an enzyme reagent for degrading biogenic amine in Sichuan sausages. Results show that the addition of the reagent containing the multi-copper oxidase and the diamine oxidase has no influence on the eating quality and the sensory quality of the Sichuan sausages, in terms of the biogenic amine content, after the reagent is added, the degradation rates of histamine, tyramine, cadaverine and putrescine are at least 49.63%, 38.51%, 38.26% and 35.40%, and the degradation rates of the reagent containing the multi-copper oxidase and the diamine oxidase are at least 49.63%, 38.51%, 38.26% and 35.40%. The reagent provided by the invention has a good degradation effect on four main biogenic amines in Sichuan sausages.

Description

Technical Field

[0001] This invention relates to the fields of microbial and genetic engineering technology, specifically to the application of reagents containing polycopper oxidases in reducing the biogenic amine content of fermented meat products. Background Technology

[0002] Sichuan fermented sausage, also known as Sichuan-style sausage, is a traditional fermented meat product in my country, popular for its spicy and refreshing flavor. However, during the processing and storage of traditionally naturally fermented sausages, some substances harmful to human health are easily produced, with biogenic amines being the most representative. Biogenic amines are biologically active nitrogen-containing organic compounds formed by the decarboxylation of amino acids by amino acid decarboxylases. Four common biogenic amines are histamine, tyramine, cadaverine, and putrescine. Studies have reported that the average content of histamine, tyramine, cadaverine, and putrescine in traditionally naturally fermented sausages exceeds 100 mg / kg, posing a significant safety hazard.

[0003] Currently, the main approach to controlling biogenic amines in food is to inhibit their formation through physical, chemical, and biological methods. However, these methods cannot eliminate already formed biogenic amines. Sichuan sausage has a more complex composition than a pure culture system, and no method has yet been found that can effectively degrade biogenic amines in fermented Sichuan sausage without affecting its quality. Therefore, it is necessary to further develop safer, more reliable, controllable, and economical methods for degrading biogenic amines in Sichuan sausage. Summary of the Invention

[0004] The purpose of this invention is to explore new, safe, reliable, effective, and economically feasible methods for reducing the biogenic amine content in fermented meat products, and to provide the application of multi-copper oxidases in reducing the biogenic amine content in fermented meat products.

[0005] To address the problems existing in the prior art, the technical solution adopted in this invention is: In a first aspect, the present invention provides the application of polycopper oxidase in reducing the biogenic amine content in fermented meat products.

[0006] Furthermore, the fermented meat product is Sichuan sausage.

[0007] Furthermore, the biogenic amine includes at least one of histamine, tyramine, cadaverine, and putrescine.

[0008] Secondly, the present invention provides a reagent containing a polycopper oxidase.

[0009] Furthermore, the reagent containing polycopper oxidase contains diamine oxidase.

[0010] Furthermore, the mass ratio of polycopper oxidase to diamine oxidase in the reagent containing polycopper oxidase is 1-4:1-3.

[0011] Furthermore, the amount of the reagent containing polycopper oxidase added to the fermented meat product is (20-50) mg / kg.

[0012] Furthermore, the multi-copper oxidase was obtained through heterologous expression and then isolated and purified.

[0013] Furthermore, the heterologous expression process includes the following steps: First, genomic DNA was extracted from *Lactobacillus plantarum*, and primers were designed to amplify the multi-copper oxidase gene; simultaneously, using... EcoR I and Hind III. The plasmid pET28a was double-digested, and the amplified MCO gene was recombined with the double-digested plasmid. The recombinant gene was then identified and transformed into the expression strain. E. coli BL21 induces the expression of polycopper oxidase.

[0014] Furthermore, the separation and purification of the polycopper oxidase includes the following steps: First, crude protein is prepared by sonication, then purified by nickel column affinity chromatography, and finally desalted by dialysis.

[0015] Secondly, the present invention provides the application of the reagent containing polycopper oxidase in reducing the biogenic amine content of fermented meat products.

[0016] Furthermore, the fermented meat product is Sichuan sausage.

[0017] Furthermore, the biogenic amine includes at least one of histamine, tyramine, cadaverine, and putrescine.

[0018] The advantages and beneficial effects of this invention are: This invention utilizes genetic engineering to obtain recombinant multicopper oxidase (MCO). Studies in pure culture systems have shown that adding MCO obtained through heterologous expression to Sichuan sausage results in high activity against four major biogenic amines: histamine, tyramine, cadaverine, and putrescine. Therefore, adding MCO to fermented meat products can effectively reduce their biogenic amine content.

[0019] Furthermore, this invention also developed a reagent containing polycopper oxidase, which is a compound enzyme formed by combining polycopper oxidase (MCO) and diamine oxidase (DAO). It did not affect the sensory or edible quality of Sichuan sausage. In terms of biogenic amine content, after adding the reagent of this invention, the degradation rates of histamine, tyramine, cadaverine, and putrescine were at least 49.63%, 38.51%, 38.26%, and 35.40%, respectively, further indicating that MCO and DAO can synergistically enhance each other and have a good degradation effect on Sichuan sausage. Attached Figure Description

[0020] Figure 1 Construction of MCO recombinant bacteria; Figure 2 This is a PCR amplification diagram of the MCO gene. In the diagram: M: DNA Marker, 1-7 are... MCO Gene PCR amplification bands; Figure 3 For the enzyme digestion verification of recombinant plasmid and PCR verification of positive transformants, in the figure: A, M: DNA Maker; 1, 2 are double enzyme digestion identification of recombinant plasmid; B, M: DNA Maker; 1, 4, 5, 6, 8 are amplification products of positive transformants; 2, 3, 7 are negative controls. Figure 4 The expression of recombinant MCO is shown in the figure. M: Protein Maker; 1-7 are crude proteins from recombinant bacteria induced at 2, 4, 6, 8, 10, 12, and 14 h. Figure 5 The first-order mass spectrometry of peptide mass fingerprint of MCO is shown in the figure. In the figure, A, B and C are queries 8500, 11252 and 8481, respectively. Figure 6 The image shows the secondary mass spectrum of MCO. In the image, A, B, and C represent queries 8500, 11252, and 8481, respectively. Figure 7 For reorganization MCO Purification, in the figure: M: Protein Maker; 1 and 2 are crude protein and purified MCO, respectively; Figure 8 This is an SDS-PAGE protein electrophoresis image. In the image, A and B are myofibrillar proteins, and C and D are sarcoplasmic proteins. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Example 1

[0024] This embodiment provides a method for preparing a multi-copper oxidase (MCO). The multi-copper oxidase of this application is prepared by heterologous expression and then isolated and purified, specifically including the following steps: The heterologous expression process was as follows: First, genomic DNA was extracted from *Lactobacillus plantarum*, and primers were designed to amplify the MCO gene. Simultaneously, using... EcoR I and Hind III. The plasmid pET28a was double-digested, and the amplified MCO gene was recombined with the double-digested plasmid. The recombinant gene was then identified and transformed into the expression strain. E. coli BL21 induces the expression of MCO.

[0025] Finally, the MCO was separated and purified. The specific process was as follows: first, crude protein was prepared by ultrasonication, then purified by nickel column affinity chromatography, and finally desalted by dialysis.

[0026] The specific process of heterologous expression includes the following steps: 1. Prokaryotic expression of polycopper oxidase (MCO) 1.1 Extraction of genomic DNA Lactobacillus plantarum LPZN19 was screened from Sichuan sausage.

[0027] Single colonies of *Lactobacillus plantarum* LPZN19 were picked from MRS solid medium and inoculated into MRS liquid medium, and incubated statically at 37°C for 18–24 h. Genomic DNA extraction was performed according to the instructions of a commercially available bacterial genomic DNA extraction kit.

[0028] 1.2 Amplification of the MCO gene The MCO gene sequence was determined by analyzing the whole genome sequence of LPZN19 obtained from whole genome sequencing. Specific primers with homologous arms flanking the pET28a restriction site were designed using the MCO sequence as a template (Table 1). The PCR reaction system and conditions for the MCO gene are shown in Tables 2 and 3.

[0029] Table 1 Primers for the polycopper oxidase gene

[0030] Table 2 25µl reaction system

[0031] Table 3 PCR reaction procedure

[0032] 1.3 Construction of Recombinant Vectors use EcoR I and Hind III. Plasmid pET28a was double-digested with enzymes at 37℃ for 2 hours. The reaction system is shown in Table 4. The recombinant vector was constructed according to the reaction system in Table 5, and ligated at 50℃ for 15 minutes to obtain the recombinant vector.

[0033] Table 4 pET28a plasmid digestion reaction system

[0034] Table 5. Ligation system of recombinant plasmids

[0035] 1.4 Transformation Thawed competent cells were mixed with the recombinant vector, incubated in an ice-water bath for 30 min, subjected to a water bath heat shock for 45 s, and then incubated in an ice-water bath for 2 min. The transformation product was cultured in 500 µL LB liquid medium at 37°C and 200 r / min for 60 min, then centrifuged. The cells were then evenly spread on solid medium plates containing 30 mg / L kanamycin and incubated overnight at 37°C.

[0036] 1.5 Screening of recombinant plasmids Successfully transformed single colonies were cultured in LB liquid medium at 37°C for 8–12 h, followed by bacterial PCR. The PCR products of successfully recombinant plasmids were sent to Shanghai Sangon Biotech for sequencing. Strains expressing the recombinant plasmids with the correct sequencing results were then screened. E. coli BL21 was plated on LB agar plates containing 30 mg / L kanamycin and incubated overnight at 37°C. Single colonies were then verified by colony PCR.

[0037] 1.6 Inducible expression of MCO The recombinant bacteria were cultured overnight on LB liquid medium. 0.1 mL of the culture was then transferred to 20 mL of LB medium containing 30 mg / L kanamycin and cultured until OD (dose-free survival) was achieved. 600 nm When the value is between 0.6 and 0.8, 0.1 mmol / L IPTG is added to induce expression, and the induction temperature and time are 25℃ and 12 h, respectively.

[0038] 1.7 Identification of MCO The purified MCO was detected by SDS-PAGE gel electrophoresis. The target protein band was cut off with a sterile scalpel and dissolved in sterile water. The MCO was then analyzed and identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0039] 2. MCO separation and purification includes the following steps: 2.1 Preparation of crude protein The induced bacterial cells were centrifuged, resuspended in PBS at pH 7.0, and sonicated on ice. The lysed system was collected, centrifuged at 12000 r / min for 15 min, and the supernatant was collected, filtered through a 0.22 µm filter membrane, and analyzed by SDS-PAGE.

[0040] 2.2 Nickel column affinity chromatography Since recombinant multicopper oxidase contains 6 histidine tags, the target protein is purified using nickel column affinity chromatography. For specific procedures, please refer to the instructions of commercially available nickel column affinity chromatography kits.

[0041] 2.3 Polyacrylamide gel electrophoresis (SDS-PAGE) analysis First, thoroughly mix 30 μL of sample and 10 μL of loading buffer, then heat in a 100℃ metal bath for 10 min. Load 10 μL of protein marker and 15 μL of denatured sample onto the gel. Run the electrophoresis apparatus at 100 V for approximately 1.5 h. Peel off the electrophoresis gel, stain with Coomassie Brilliant Blue for 0.5 h, and then destain until protein bands are clearly visible.

[0042] 2.4 Dialysis Desalination Place the recombinant MCO to be purified into a dialysis bag, add dialysis buffer, and stir at 4°C. Replace the dialysis buffer at 2–4 h, 6–8 h, and 10–14 h after dialysis.

[0043] 3 Results 3.1 Cloning and Expression of MCO The recombinant plasmid was constructed using homologous recombination. The flowchart for the construction of the recombinant bacteria is shown below. Figure 1 As shown in Table 1, using the primers in Table 1 and the genomic DNA of Lactobacillus plantarum LPZN19 as a template, PCR amplification was performed to obtain... MCO Gene( Figure 2 The recovered MCO was ligated with double-digested and recovered linear pET28a and transformed into the cloning host. E. coli DH5α. Recombinant plasmid was extracted and processed... EcoR I and HindIII double digestion, 1% agarose gel electrophoresis analysis showed a vector fragment of approximately 5300 bp and a target gene band of 1500 bp ( ). Figure 3 A). The recombinant strain successfully recombined after PCR verification ( Figure 3 B), the plasmid was extracted and sent to Shanghai Sangon Biotech for sequencing, verifying the successful construction of the recombinant plasmid pET28a-MCO. The recombinant plasmid pET28a-MCO was successfully constructed and then transformed into the expression host. E. coli BL21 was successfully constructed as a recombinant bacterium. E. coli BL21-pET28a-MCO.

[0044] The successfully constructed recombinant bacteria were induced to express protein using IPTG. After induced recombinant bacterial cell disruption, the protein was analyzed by SDS-PAGE protein electrophoresis. The results are as follows: Figure 4 As shown, the recombinant strain exhibits an MCO band above 63 kDa, and its molecular weight is estimated to be 67 kDa using software. Since the recombinant plasmid contains a promoter and six histidine tags, its molecular weight is 6.68 kDa. Therefore, the molecular weight of the recombinant MCO can be concluded to be approximately 60 kDa, consistent with the predicted theoretical value of 56.8 kDa.

[0045] 3.2 Identification of the target protein After Q-TOF identification of the target band, the relevant information of the detected band and the matched peptide sequences are shown in Table 6. The top three peptides by protein score (queries: 8500, 11252, 8481) were selected, and their primary and secondary mass spectra were obtained. Figure 5 , 6 As shown in Table 6, the identified protein-related molecular weight is 57.10 kDa, which is consistent with the predicted theoretical value.

[0046] Table 6. Identification Results of MCO

[0047] 3.3 Purification of Recombinant MCO The SDS-PAGE image of the recombinant MCO purified by nickel column affinity chromatography is shown in Figure 7. As can be seen from the figure, the purified MCO showed a single band on electrophoresis compared to the crude protein. Table 7 shows that the purification recovery rate of recombinant MCO was approximately 33.04%, with a purification fold of 7, indicating good purification effect.

[0048] Table 7 Purification effect of MCO

[0049] Example 2 This embodiment provides a reagent containing polycopper oxidase, which contains polycopper oxidase and diamine oxidase (DAO) prepared in Example 1, with a mass ratio of polycopper oxidase to diamine oxidase (DAO) of 1:1.

[0050] Example 3

[0051] This embodiment provides a reagent containing polycopper oxidase, which contains polycopper oxidase and diamine oxidase (DAO) prepared in Example 1, with a mass ratio of polycopper oxidase to diamine oxidase (DAO) of 1:2.

[0052] Example 4

[0053] This embodiment provides a reagent containing polycopper oxidase, which contains polycopper oxidase and diamine oxidase (DAO) prepared in Example 1, with a mass ratio of polycopper oxidase to diamine oxidase (DAO) of 1:3.

[0054] Example 5

[0055] This embodiment provides a reagent containing polycopper oxidase, which contains polycopper oxidase and diamine oxidase (DAO) prepared in Example 1, with a mass ratio of polycopper oxidase to diamine oxidase (DAO) of 2:1.

[0056] Example 6

[0057] This embodiment provides a reagent containing polycopper oxidase, which contains polycopper oxidase and diamine oxidase (DAO) prepared in Example 1, with a mass ratio of polycopper oxidase to diamine oxidase (DAO) of 3:1.

[0058] Example 7

[0059] This comparative example provides a reagent containing polycopper oxidase, which contains polycopper oxidase and diamine oxidase (DAO) prepared in Example 1, with a mass ratio of polycopper oxidase to diamine oxidase (DAO) of 4:1.

[0060] Example 8

[0061] This embodiment provides a reagent containing polycopper oxidase, which contains only the polycopper oxidase prepared in Example 1.

[0062] Comparative Example 1 This comparative example provides a reagent containing only diamine oxidase (DAO).

[0063] Comparative Example 2 This comparative example provides a reagent containing polycopper oxidase. The only difference between this comparative example and Example 2 is that the mass ratio of polycopper oxidase to diamine oxidase is 0.5:1.

[0064] Experimental Example 1 1. Experimental Methods: 1) Experimental Groups: Groups 1-7 used the reagents prepared in Examples 2-8, respectively; Groups 8-9 used the reagents prepared in Comparative Examples 1-2, respectively; The dosage of the reagents used in Groups 1-9 was 25 mg / kg.

[0065] Group 10 involved inoculating sausages with the target bacterial strain to reduce biogenic amines. The target strain contained the MCO gene and was derived from patent number CN202110849825.0. The target strain was activated three times, each time for 8-12 hours. The activated bacterial solution was centrifuged at 4℃ and 4000-5000 rpm for 10-15 minutes, washed with physiological saline, and then centrifuged again to collect the bacterial cells.

[0066] Group 11 was the control group, which did not contain any biodegradation reagents.

[0067] 2) Experimental methods (preparation of fermented sausage and sample collection): Experimental methods for groups 1-9: Cleaned and pre-cooled raw meat at 4℃ was mixed evenly with seasonings, and then the reagents prepared for groups 1-9 were added at a concentration of 25 mg / kg. The mixture was marinated at 4℃ for 4 hours, and then stuffed into sausages. The fermentation temperature of the sausages was 18℃, the relative humidity was 70%, and the fermentation time was 2 days. The maturation temperature was 13℃, the maturation relative humidity was 60%, and the maturation time was 28 days. Five sampling points were selected during the sausage production process: 0 days (raw meat), 2 days (end of fermentation), 5 days (early maturation), 16 days (mid-maturation), and 30 days (end of maturation). Sausages without any added biodegrading amines served as a control group.

[0068] Experimental method for group 10: The cleaned and pre-cooled raw meat was mixed evenly with the seasonings. Then, the mycelium obtained from group 10 was directly inoculated into the sausage, with an inoculation amount of 10. 7 CFU / g. The sausages were marinated at 4℃ for 4 hours, then stuffed into sausages. The fermentation temperature was 18℃, relative humidity 70%, and fermentation time was 2 days. The maturation temperature was 13℃, maturation relative humidity 60%, and maturation time was 28 days. Five sampling points were selected during the sausage production process: 0 days (raw meat), 2 days (end of fermentation), 5 days (early maturation), 16 days (mid-maturation), and 30 days (end of maturation).

[0069] 2. Results and Analysis 2.1 Color difference Color was measured for Example 3 and the control group. The method for color measurement was as follows: 14 replicate samples were prepared for each group, and the samples were cut into 1 cm pieces. 3 The colorimeter of the sample was measured using a Linshang LS173 colorimeter. L value, a* value, b* value.

[0070] Color is an important indicator for evaluating the quality of meat products and influencing their commercial value. Because of its visual appeal, it significantly impacts consumer choice. According to Table 8, during fermentation from 0 to 16 days, the color of the meat products in Example 3 group... L * The degree of change in the value was similar to that of the control group, indicating that the addition of the compound enzyme did not affect the brightness of the Sichuan sausage. After 16 days, the brightness of the sample in Example 3 group... L * The change in values ​​was less than that in the control group, possibly due to the reduced moisture content and decreased light scattering ability. The slower rate of decrease in Example 3 group compared to the control group may be due to the oxidation reaction of biogenic amines catalyzed by the complex enzyme. L * The value was higher than that of the control group. From 0 to 30 days, a * Value and b * The values ​​all showed a downward trend, and the control group and Example 3 group showed the same trend over the same period of time. a * Value and b * The values ​​showed no significant differences. In conclusion, the addition of compound enzymes has little impact on the color of Sichuan sausage during fermentation, but it can play a certain role in protecting the color.

[0071] Table 8 Color difference values ​​of Sichuan sausage

[0072] Note: The same uppercase letter indicates that the difference in the same column is not significant, and the same lowercase letter indicates that the difference in the same row within the same indicator is not significant.

[0073] 2.2 Texture Texture analysis was performed on Example 3 and the control group using texture profile analysis (TPA). The TPA parameters were set as follows: probe: P / 5R; deformation: 50%; pre-measurement speed: 2.00 mm / s; measurement speed: 1.00 mm / s; post-measurement speed: 5.00 mm / s; interval between measurements: 3.00 s; trigger: automatic; measurement distance: 10.0 mm; trigger force: 5.0 g. The results were analyzed using Texture Expert software.

[0074] Texture is an important physicochemical indicator for consumers to perceive the taste and quality of meat products, and it is affected by multiple factors such as moisture content and ionic strength. As shown in Table 9, there were no significant differences between the Example 3 group and the control group in terms of hardness, adhesiveness, elasticity, cohesiveness, chewiness, and resilience. This indicates that adding the compound enzyme does not significantly affect the texture of Sichuan sausage. The slightly higher hardness of the 30-day sample in Example 3 compared to the control group may be due to the lower fat content in the measurement area of ​​the Sichuan sausage compared to the control group, resulting in a more compact structure and thus a slightly higher hardness.

[0075] Table 9 Texture data of Sichuan sausage

[0076] Note: s represents Example 3 group, k represents control group; the same uppercase letter indicates that the difference in the same row is not significant.

[0077] 2.3 Sensory rating Sensory evaluations were conducted on Example 3 and the control group using a blind scoring method. A panel of 10 food professionals with relevant training evaluated the samples from five aspects: appearance, color, texture, aroma, and taste. The specific sensory evaluation criteria are shown in Table 10.

[0078] Table 10 Sensory Evaluation Criteria

[0079] Sensory evaluation plays a crucial role in the research, development, production, and quality control of meat products. The sensory evaluation results are shown in Table 11. There were no significant differences between the two groups of Sichuan sausages in terms of appearance, texture, and total score. Regarding taste and aroma, Group 3 of Example 3 had a richer flavor, possibly due to the compound enzyme inducing lipid oxidation, producing aldehydes and ketones as aroma compounds. In conclusion, the addition of the compound enzyme has a positive effect on the taste and aroma of Sichuan sausages, but has no significant impact on their elasticity and firmness.

[0080] Table 11 Sensory ratings of Sichuan sausage

[0081] 2.4 Amino acid nitrogen Amino acid nitrogen was determined in Example 3 and the control group according to the acidity meter method specified in GB 5009.235-2016.

[0082] Amino acid nitrogen refers to the content of nitrogen element existing in the form of amino acids. Amino acid nitrogen is a characteristic indicator for determining the degree of fermentation of fermented products; the higher the amino acid nitrogen content, the more beneficial it is to the formation of the flavor of Sichuan sausage. According to Table 12, the amino acid nitrogen content in Sichuan sausage gradually increases with the extension of fermentation time. Within 0–30 days, there was no significant difference in amino acid nitrogen content between the Example 3 group and the control group, indicating that the addition of the compound enzyme had no significant effect on the amino acid nitrogen content. This further demonstrates that multi-copper oxidase (MCO) and diamine oxidase (DAO) are not proteases and will not affect the nutrients in Sichuan sausage.

[0083] Table 12 Amino acid nitrogen content (g / 100g)

[0084] 2.5 Free amino acids Free amino acids were determined in Example 3 and the control group according to the method specified in GB / T5009.124-2016.

[0085] Free amino acids in food originate from protein hydrolysis. A higher content indicates a higher degree of protein hydrolysis. Moderate protein hydrolysis can improve the flavor and quality of a product, but excessive degradation will negatively impact its nutritional quality. As shown in Table 13, no significant differences were found between the Example 3 group and the control group in either the content of a single amino acid or the total amino acid content, indicating that the addition of the compound enzyme did not promote protein hydrolysis in Sichuan sausage.

[0086] Table 13 Free amino acid content in Sichuan sausage (g / 100g)

[0087] 2.6 SDS-PAGE protein electrophoresis SDS-PAGE protein electrophoresis was performed on the proteins from Example 3 and the control group. Myofibrillar proteins are the main proteins that make up muscle fibers, while sarcoplasmic proteins are proteins dissolved in the cytoplasm of muscle cells. The SDS-PAGE protein electrophoresis results are shown in the images. Figure 8 As can be seen, during the entire fermentation and maturation period, the molecular weights of myofibrillar protein and sarcoplasmic protein in Sichuan sausages from the five sampling points were all around 25–135 kDa. As shown in the figure, there was no significant difference in the brightness of any band between the Example 3 group and the control group for either sarcoplasmic protein or myofibrillar protein, further confirming that polycopper oxidase (MCO) and diamine oxidase (DAO) do not significantly degrade proteins.

[0088] 2.7 Biogenic amine content The biogenic amine content of groups 1-10 and the control group was determined according to Method I in GB5009.208-2016, and the average biogenic amine degradation rate of the five sampling points was calculated. The biogenic amine degradation rates are as follows: .

[0089] Biogenic amine content is an important parameter for monitoring sausage safety; excessive accumulation can adversely affect the quality and safety of meat products. Table 14 shows the contents of the four main biogenic amines in Sichuan sausages. As can be seen from the table, the degradation rates of histamine, tyramine, cadaverine, and putrescine by MCO were 38.65%, 28.83%, 21.19%, and 22.83%, respectively. However, after adding the reagent of this invention, the degradation rates of histamine, tyramine, cadaverine, and putrescine were at least 49.63%, 38.51%, 38.26%, and 35.40%, respectively, indicating that the reagent of this invention has a high degradation rate for the four main biogenic amines in Sichuan sausages.

[0090] Table 14 Degradation rate of biogenic amines in fermented sausages after adding the reagent containing polycopper oxidase of the present invention

[0091] III. Conclusion As shown in Table 14, the addition of the reagent of this invention has no significant effect on the sensory quality, amino acid nitrogen, and free amino acid content of Sichuan fermented sausage, and will not have any adverse effect on the edible quality of Sichuan sausage. Meanwhile, the degradation rates of histamine, tyramine, cadaverine, and putrescine using MCO alone were 38.65%, 28.83%, 21.19%, and 22.83%, respectively. However, the degradation rate was significantly improved after adding the compound reagent of MCO and DAO, with degradation rates of at least 49.63%, 38.51%, 38.26%, and 35.40% for histamine, tyramine, cadaverine, and putrescine, respectively, demonstrating that the combined use of the two has a good synergistic degradation effect on biogenic amines. Among them, the reagents described in Examples 3 and 4 showed the best degradation rate for the four monomeric biogenic amines. Although the degradation effect of the reagent described in Example 4 was better than that in Example 3, the higher dosage of DAO resulted in higher cost, while the improvement effect was limited. Therefore, overall, the compound biogenic amine degrading enzyme described in Example 3 had the best effect. In summary, the reagent of this invention can effectively control the content of biogenic amines without affecting the quality of Sichuan sausages, and has important application value and promotion potential in fermented meat products.

[0092] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. Application of polycopper oxidase in reducing the content of biogenic amines in fermented meat products.

2. The application according to claim 1, characterized in that, The fermented meat product is Sichuan sausage.

3. The application according to claim 1, characterized in that, The biogenic amines include at least one of histamine, tyramine, cadaverine, and putrescine.

4. A reagent containing a polycopper oxidase, characterized in that, It contains polycopper oxidase.

5. The reagent containing polycopper oxidase according to claim 4, characterized in that, The reagent contains diamine oxidase.

6. The reagent containing polycopper oxidase according to claim 5, characterized in that, The mass ratio of the polycopper oxidase to the diamine oxidase is 1-4:1-3.

7. The reagent containing polycopper oxidase according to any one of claims 4-6, characterized in that, The amount of the compound reagent added to fermented meat products is (20-50) mg / kg.

8. The reagent containing polycopper oxidase according to any one of claims 4-6, characterized in that, The polycopper oxidase was obtained through heterologous expression and then isolated and purified.

9. The reagent containing polycopper oxidase according to claim 8, characterized in that, The heterologous expression process includes the following steps: First, genomic DNA was extracted from *Lactobacillus plantarum*, and primers were designed to amplify the multi-copper oxidase gene; simultaneously, using... EcoR I and Hind III. The plasmid pET28a was double-digested, and the amplified MCO gene was recombined with the double-digested plasmid. The recombinant gene was then identified and transformed into the expression strain. E. coli BL21 induces the expression of polycopper oxidase.

10. The use of the reagent containing polycopper oxidase according to any one of claims 4-9 in reducing the biogenic amine content of fermented meat products.

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