Recombinant lactobacillus plantarum for degrading biogenic amine and application of recombinant lactobacillus plantarum
By overexpressing the polycopper oxidase gene into Lactobacillus plantarum SH7, recombinant Lactobacillus plantarum was obtained, which solved the problem of bioamine accumulation in fermented foods, achieved a significant bioamine degradation effect, and was suitable for the production of low-salt fermented meat products.
Patent Information
- Application Number
- CN202510216950.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively reduce the accumulation of bioamines in fermented foods, especially under low salt conditions, resulting in increased health risks.
Recombinant Lactobacillus plantarum is obtained by overexpressing the polycopper oxidase gene into Lactobacillus plantarum SH7, which is a microbial preparation that can significantly improve the degradation ability of bioamines.
Recombinant Lactobacillus plantarum can significantly degrade six biological amines, with a total degradation rate of 70.62%, and has the characteristics of amine-lowering, and is suitable for the production of low-salt fermented meat products.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial technology, and particularly relates to a recombinant Lactobacillus plantarum for degrading biogenic amines and its application. Background Art
[0002] Biogenic amines are a class of low-molecular-weight nitrogen-containing basic compounds that widely exist in fermented foods. When the human body ingests foods containing excessive biogenic amines, it will cause poisoning reactions and endanger the health of consumers. High salt content and inoculation with amine-reducing starters can both inhibit the accumulation of biogenic amines in fermented foods. However, high-salt foods can increase the risk of cardiovascular and cerebrovascular diseases such as hypertension. In order to reduce the salt content of fermented foods while ensuring the quality and safety of products, there is an urgent need for a starter with a higher efficiency of inhibiting biogenic amine formation. Summary of the Invention
[0003] The purpose of the present invention is to provide a method for improving the ability of Lactobacillus plantarum to degrade biogenic amines.
[0004] The present invention provides a recombinant Lactobacillus plantarum for degrading biogenic amines, which is obtained by overexpressing a multi-copper oxidase protein using Lactobacillus plantarum as the starting strain.
[0005] Further defined, the starting strain is Lactobacillus plantarum SH7.
[0006] Further defined, the nucleic acid molecule encoding the multi-copper oxidase protein is as shown in SEQ ID NO.3.
[0007] Further defined, the amino acid sequence encoding the multi-copper oxidase protein is as shown in SEQ ID NO.4.
[0008] The present invention provides a microbial preparation containing the above recombinant Lactobacillus plantarum.
[0009] The present invention provides an application of the above recombinant Lactobacillus plantarum or the above microbial preparation in degrading biogenic amines or improving the ability to degrade biogenic amines.
[0010] Further defined, the biogenic amines are tryptamine, phenethylamine, putrescine, cadaverine, histamine and tyramine.
[0011] The present invention provides an application of the above recombinant Lactobacillus plantarum or the above microbial preparation in fermenting low-salt dried sausage.
[0012] The present invention provides a method for degrading biogenic amines, which is to add the above recombinant Lactobacillus plantarum to a solution containing biogenic amines at a concentration of 50 mg / ml and culture at 37°C for 18 h.
[0013] The present invention provides a method for fermenting and producing low-salt dried sausage. The above-mentioned recombinant Lactobacillus plantarum is inoculated into the meat stuffing for fermentation, and the inoculation amount is 10 6 CFU / g of meat stuffing, and cultured at 37 °C for 18 h.
[0014] Beneficial effects: Overexpression of the gene shown in SEQ ID NO.3 can significantly improve the ability of Lactobacillus plantarum to degrade biogenic amines (P<0.05), and has different degrees of degradation effects on six biogenic amines. The degradation rates of tryptamine, phenethylamine, putrescine, cadaverine, histamine, and tyramine reach 24.16%, 46.50%, 77.45%, 71.89%, 78.67%, 56.51% respectively, and the total biogenic amine degradation rate reaches 70.62%. It has the property of amine reduction and can be used as a starter for reducing the content of biogenic amines in the production of low-salt fermented meat products. Description of the Drawings
[0015] Figure 1 It is a graph of the effect data for degrading biogenic amines. Detailed Embodiments
[0016] Example 1. Obtaining recombinant Lactobacillus plantarum
[0017] 1) Extracting the genome of Lactobacillus plantarum
[0018] Lactobacillus plantarum SH7 is a strain with amine-reducing ability screened from dried sausage (Screening and evaluating microorganisms with broad-spectrum biogenic amine-degrading ability from naturally fermented dry sausage collected from Northeast China). After activating the frozen bacterial liquid by inoculating it into MRS medium, the genome of Lactobacillus plantarum is extracted using a bacterial genomic DNA extraction kit.
[0019] 2) Amplifying the target gene (primer sequences, reaction system and procedure)
[0020] Primers F: cccggggatcgatcctctagaATGGCAAAAAAAGTCTATACTGATTATT (SEQ ID NO.1) and R: ttttcagactttgcaaagcttTTACATACCGGGCATCCAAAC (SEQ ID NO.2) were designed according to the base sequence shown in SEQ ID NO.3 contained in the strain and the restriction enzyme sites XbaⅠ and HindⅢ. Using the genomic DNA in the Lactiplantibacillus plantarum SH7 strain as a template, PCR amplification was performed using DNA polymerase and primers F and R to obtain the gene sequence shown in SEQ ID NO.3. Table 1 and Table 2 are the PCR amplification reaction system and procedure respectively. After the reaction was completed, the size of the DNA band was detected by agarose gel electrophoresis, and the amplified SEQ ID NO.3 gene sequence was purified using an agarose gel recovery kit. Subsequently, the DNA concentration was detected using a micro nucleic acid detector, and the product was stored in a -40°C refrigerator.
[0021] (SEQ ID NO.3):
[0022]
[0023] (SEQ ID NO.4):
[0024] MAKKVYTDYFFDEPAYNTHDGGYIPLVTPKVDPQPLAIPPLLKPDRQTDTDDYYTVTAQES
[0025] ETQFLPGKKTKTWGYNAGFLGQTIVFRNGKQTHIDLENKLPELTTFHWHGLNVPGPITD
[0026] GGCHAPVYPGETNHIDFKVHQPAATTWLHAHPCPSTATQVWKGLATMVIIKDDVEDQLPL
[0027] PRNYGVDDIPLVLQDREFHDDNQFDYRADYDPDGVQGHTALVNGTVNPYFDVTTQRVRL
[0028] RILDGSNRREWRLHFNDDLEFAQVASDGGILPAPVYMTKVMMTCAERDEIVVDFGQYQP
[0029] GDEVTLMTDDTPLCRFRIKSFVPDDTKLPEHLVDIPDETPTPDLPVRTITMDGMDDEVALD
[0030] GKKFDMSRIDARQKVGDVAIWEIRNTNSTENGMVHPFHVHGTQFRVLARNDGPVYPNEH
[0031] GLKDTVGVNPGETVRIKVKFELTGVYMYHCHIIEHEDGGMMAQIESYDPQHPQTYHLMDMDTLRNAFAKEQGIKPEDVWMPGM;
[0032] Result: After inoculating the cryopreserved SH7 bacterial liquid into MRS medium for activation, the genomic DNA of Lactiplantibacillus plantarum was extracted using a bacterial genomic DNA extraction kit, and the concentration of the obtained genome was 148.86 ng / μL.
[0033] Using the extracted DNA as a template, PCR amplification was performed using DNA polymerase and primers F and R to obtain the gene shown in SEQ ID NO.3, with concentrations of 40.25 ng / μL and 35.15 ng / μL.
[0034] Table 1 PCR amplification reaction system
[0035]
[0036] Table 2 PCR Reaction Program
[0037]
[0038] 3) Digest the vector plasmid (reaction system)
[0039] Select the PMG-36e plasmid as the vector. Extract the plasmid from Escherichia coli carrying the plasmid according to the kit and detect its concentration. Use the restriction enzymes XbarⅠ and HindⅢ to perform double digestion on the PMG-36e plasmid. The reaction system is shown in Table 3, and the digestion condition is at 37°C for 15 min. Use agarose gel electrophoresis to detect the band size of the digestion product, and use an agarose gel recovery kit to purify the amplified digestion product. Then measure the concentration of the digestion product and store the product in a -40°C refrigerator.
[0040] Result: Extract the plasmid from Escherichia coli carrying the plasmid according to the kit, with a concentration of 159.45 ng / μL. Then use the restriction enzymes XbarⅠ and HindⅢ to perform double digestion on the PMG-36e plasmid, with a concentration of 11.65 ng / μL.
[0041] Table 3 Double Digestion Reaction System
[0042]
[0043] 4) Ligate and transform the digestion product and the target gene
[0044] Use DNA ligase to ligate the digestion product and the gene fragment shown in SEQ ID NO.3. The reaction system is shown in Table 4, and the ligation condition is at 37°C for 30 min to obtain the ligation product, and then immediately place it on ice for cooling. Thaw Escherichia coli competent cells (DH5α) on ice, take 10 μL of the recombinant product and mix it with 100 μL of the competent cells. After standing on ice for 30 min, subject it to heat shock at 42°C for 90 s, and then immediately cool it on ice for 3 min. Put the obtained recombinant product into 900 μL of LB medium, culture it in a shaker at 37°C for 1 h, then centrifuge at 5000 rpm / min for 5 min, discard 900 μL of the supernatant, resuspend the bacterial cells and spread them on LB medium containing 600 mg / ml erythromycin for culturing for 18 h.
[0045] Table 4 Ligation System of the Digestion Product and the Target Gene
[0046]
[0047] 5) Identification and sequencing identification of positive clones
[0048] After the cultivation was completed, single colonies of Escherichia coli grown on the LB plate were picked and placed in a PCR tube. PCR amplification solution was added to the PCR tube, and positive clones were identified by PCR amplification and gel electrophoresis using primers PMG-f and PMG-r to obtain Escherichia coli carrying the correctly ligated recombinant plasmid PMG36e. The correctly identified clones were cultured in LB liquid medium supplemented with 600 μg / ml erythromycin at 37 °C on a shaker for 12 h, preserved, and sent to Sangon Biotech Co., Ltd. in Shanghai for sequencing. After the cultivation was completed, the recombinant plasmid PMG36e in the bacterial liquid was extracted using a plasmid extraction kit, and the DNA concentration was detected using a micro nucleic acid detector. The product was stored in a -40 °C refrigerator. Among them, the colony PCR amplification system is shown in Table 5, and the colony PCR amplification program is shown in Table 6.
[0049] Results: After the cultivation was completed, single colonies of Escherichia coli grown on the LB plate were picked, and positive clones were identified by PCR amplification and gel electrophoresis to obtain Escherichia coli carrying the correctly ligated recombinant plasmid PMG36e. Then the plasmid was extracted, and the concentration was 545.15 ng / μL.
[0050] Table 5 Colony PCR amplification system
[0051]
[0052]
[0053] Table 6 Colony PCR amplification program
[0054]
[0055] 6) Preparation of competent cells of Lactiplantibacillus plantarum SH7
[0056] The cryopreserved SH7 bacterial liquid was inoculated into MRS medium and activated overnight, then inoculated into MRS medium containing 2.5% glycine and cultured until the OD value of the bacterial liquid reached 0.5 - 0.6. Centrifuge at 4 °C, 12000 rpm / min for 10 min to collect the bacterial cells. Add 25 ml of sterile water to the centrifuge tube containing the bacterial cells and wash twice (centrifuge at 4 °C, 12000 rpm / min for 10 min), discard the supernatant. Resuspend the bacterial cells in 0.05 mol / L EDTA solution, incubate on ice for 5 min, then add 25 ml of pre-cooled sterile water, centrifuge at 4 °C, 12000 rpm / min for 10 min, and then wash with 25 ml of pre-cooled sterile water. Discard the supernatant and wash twice with 25 ml of pre-cooled electroporation buffer, centrifuge at 4 °C, 12000 rpm / min for 10 min. Resuspend the bacterial cells in 0.8 ml of electroporation buffer, evenly distribute them into 8 1.5 ml centrifuge tubes, and store them in a -80 °C refrigerator for later use. All the above processes need to be carried out on ice.
[0057] 7) Electrotransformation of the reconstructed strain and positive verification
[0058] Prepare a sterile electroporation cuvette in advance (soak it in absolute ethanol overnight, then soak it in 75% ethanol for 3 h, and expose it to ultraviolet light and ventilation in a laminar flow hood until the alcohol evaporates before use), thaw the competent cells, add 10 μL of the recombinant plasmid PMG36e, mix gently, incubate on ice for 5 min, and transfer the mixture into a pre-cooled electroporation cuvette with a distance of 2 mm between the electrodes. Immediately apply an electric shock of 2.5 V. After the electric shock, quickly add 890 μL of MRS medium to the electroporation cuvette, mix well and transfer it to a 2 mL centrifuge tube. Incubate at 28 °C for 2.5 h for static resuscitation culture. After cell resuscitation, centrifuge the bacterial solution at 8000 rpm for 3 min, discard 900 μL of the supernatant, resuspend the bacterial cells, and spread them on MRS solid medium containing 100 μg / mL erythromycin. Culture on a shaker at 37 °C for 24 h. After the culture, pick a single colony of lactic acid bacteria grown on the MRS plate and place it in a PCR tube. Add PCR amplification solution to the PCR tube. Use primers PMG-f: 5'-ATTCGGTCCTCGGGATATG-3' (SEQ ID NO.5) and PMG-r: 5'-TTCATTCAGTCATCGGCT-3' (SEQ ID NO.6) to identify positive clones by PCR amplification and gel electrophoresis, and obtain the reconstructed strain carrying the correctly ligated plasmid PMG36e. Inoculate the reconstructed strain into MRS liquid medium containing 100 μg / mL erythromycin and culture it on a shaker at 37 °C for 12 h for bacterial solution verification. The systems and PCR procedures for both verifications are the same as those in Tables 6 and 7. Store the correctly verified reconstructed strain at -40 °C in a cryotube containing glycerol.
[0059] Example 2. Detection of the yield of multicopper oxidase in the overexpressing strain
[0060] After activating the original strain L1 (Lactobacillus plantarum SH7) and the overexpressing strain L2 (the recombinant Lactobacillus plantarum obtained in Example 1), they were respectively inoculated into MRS liquid medium without antibiotics and containing 100 μg / ml erythromycin and cultured at 37°C for 18 h. After centrifuging at 4°C and 12,000 rpm for 10 min, the cells were collected, washed twice with 20 mmol / L PBS, and then placed in an ice-water bath for ultrasonic disruption. Ultrasonic disruption conditions: working for 2 s, stopping for 3 s, power of 400 w, disrupting for 20 min. Then it was placed in a high-speed low-temperature centrifuge at 4°C and centrifuged at 12,000 rpm for 10 min to collect the supernatant, obtaining the recombinant enzyme, and detecting the concentration of the recombinant enzyme. The protein content was determined using a BCA protein content determination kit (Shanghai Beyotime Biotechnology Co., Ltd.). The activity of the multicopper oxidase was determined by the ultraviolet-visible light absorption method. Using ABTS as the substrate, the activity of the multicopper oxidase was characterized by the rate of oxidation of ABTS by the multicopper oxidase per unit time. The reaction system was 0.05 mL of enzyme solution, 0.95 mL of 50 mmol / L citric acid-sodium citrate buffer containing 1 mmol / L ABTS and 1 mmol / L CuCl 2 of the buffer. The oxidation rate of ABTS was measured at 420 nm using an ultraviolet-visible light spectrophotometer. The amount of enzyme required to oxidize 1 μmol of ABTS per minute was defined as one enzyme activity unit (U).
[0061] Results: The activity of the multicopper oxidase was determined by the ultraviolet-visible light absorption method. Using ABTS as the substrate, the activity of the multicopper oxidase was characterized by the rate of oxidation of ABTS by the multicopper oxidase per unit time. The protein concentrations of the two groups were L1: 0.094 mg / ml; L2: 0.053 mg / ml. The enzyme activities of the two groups were L1: 20.66 U / mg; L2: 89.20 U / mg.
[0062] Example 3. Detection of the ability of the overexpressing strain to degrade biogenic amines
[0063] The L1 and L2 strains were inoculated into MRS broth containing 6 biogenic amines (tryptamine, phenethylamine, putrescine, cadaverine, histamine, and tyramine; concentration of 50 mg / L) at a concentration of 10 6 CFU / mL. A biogenic amine-containing medium without inoculating microorganisms was set as the blank control group. The three groups of samples were cultured at 37°C for 4 days. The determination of the biogenic amine content in the dried sausage was carried out following the determination method for aquatic products and meats in the first method of liquid chromatography in GB 5009.208—2016 "Determination of Biogenic Amines in Foods".
[0064] Biogenic amine degradation rate calculation formula: X = C 0 - C / C 0 × 100%
[0065] C 0 : Biogenic amine content in the control group
[0066] C: Biogenic amine content in the strain culture solution 4 days after inoculation
[0067] Results:
[0068] Table 7 Degradation rates of biogenic amines by L1 and L2 strains in MRS medium
[0069]
[0070]
[0071] Example 4. Application of overexpressing strains in low-salt dry-cured sausages
[0072] To prepare low-salt dry-cured sausages with a salt content of 2%, the L1 and L2 strains were subcultured twice in MRS medium and then cultured at 37 °C for 18 h. After centrifugation at 4 °C and 10,000 r for 5 min, they were reserved for use. When adding the starter culture, the addition amount of lactic acid bacteria was 10 6 CFU / g meat stuffing. The determination of biogenic amine content in dry-cured sausages was carried out according to the first method of liquid chromatography for the determination of aquatic products and meats in GB 5009.208—2016 "Determination of Biogenic Amines in Foods".
[0073] 3.1 Preparation of the starter culture
[0074] Two strains of lactic acid bacteria (the original strain L1 (Lactobacillus plantarum SH7) and the overexpressing strain L2 (the recombinant Lactobacillus plantarum obtained in Example 1)) were subcultured twice in MRS medium and then cultured at 37 °C for 18 h. After centrifugation at 4 °C and 10,000 g for 5 min, they were reserved for use. When adding the starter culture, the addition amount of lactic acid bacteria was 10 6 CFU / g meat stuffing.
[0075] 3.2 Production of dry-cured sausages
[0076] 2.7 kg of lean meat (pork loin), 0.3 kg of fat (pork back fat), 0.03 kg of Chinese rice wine, 0.06 kg of salt, 0.0027 kg of sodium nitrite, 0.03 kg of granulated sugar, 0.009 kg of monosodium glutamate, 0.024 kg of mixed seasonings (Shiyitang dry sausage seasoning). Process flow: raw meat (removing connective tissues such as lymph, tendons, and blood vessels) → dicing (meat cubes of 1 cm3) → marinating (adding salt and seasonings, etc.) → adding the starter culture (not added in the control group) → mixing the stuffing → stuffing (length 20 cm, diameter 3 cm) → fermenting and drying (temperature 25 ± 2 °C, humidity 65 ± 5%).
[0077] 3.3 Determination of biogenic amine content in dry-cured sausages
[0078] The method of Example 3 was followed to determine the biogenic amine content in dry-cured sausages.
[0079] Figure 1 This is a comparison of the contents of eight biogenic amines in dry-cured sausages inoculated with the original strain L1, the overexpressing strain L2, and the uninoculated dry-cured sausages (control group) on the 8th day of fermentation. The test results show that overexpressing the gene shown in SEQ ID NO.3 can significantly improve the ability of Lactobacillus plantarum to degrade biogenic amines (P<0.05), and has different degrees of degradation effects on six biogenic amines. The degradation rates of tryptamine, phenethylamine, putrescine, cadaverine, histamine, and tyramine reach 24.16%, 46.50%, 77.45%, 71.89%, 78.67%, 56.51% respectively, and the total biogenic amine degradation rate reaches 70.62%, with the characteristics of amine reduction, and is higher than the degradation rate of the original strain inoculation group (10.74%, 20.73%, 61.48%, 62.89%, 65.19%, 43.39%, and the total biogenic amine degradation rate is 58.17%). It can be used as a starter for reducing biogenic amine content in the production of low-salt fermented meat products.
[0080] Formula for the total biogenic amine degradation rate: X = l 0总 -l 2总 / l 0总 ×100%.
[0081] Formula for the degradation rate: X = l 0 -l 2 / l 0 ×100%.
[0082] l 0 : Biogenic amine content in the blank group;
[0083] l 2 : Biogenic amine content in the overexpression group.
Claims
1. A recombinant Lactobacillus plantarum for degrading biogenic amines, characterized in that: It was obtained by overexpressing multi-copper oxidase protein using Lactobacillus plantarum as the starting strain.
2. The recombinant Lactobacillus plantarum according to claim 1, characterized in that The starting strain is Lactobacillus plantarum SH7.
3. The recombinant Lactobacillus plantarum according to claim 1, characterized in that The nucleic acid molecule encoding the multi-copper oxidase protein is shown in SEQ ID NO.
3.
4. The recombinant Lactobacillus plantarum according to claim 1, characterized in that The amino acid sequence encoding the multi-copper oxidase protein is shown in SEQ ID NO.
4.
5. A microbial preparation containing the recombinant Lactobacillus plantarum according to any one of claims 1 to 4.
6. Use of the recombinant Lactobacillus plantarum according to any one of claims 1 to 4 or the microbial preparation according to claim 5 in degrading biogenic amines or improving the ability of degrading organisms.
7. The use according to claim 6, characterized in that: The biogenic amines are tryptamine, phenylethylamine, putrescine, cadaverine, histamine and tyramine.
8. Use of the recombinant Lactobacillus plantarum according to any one of claims 1 to 4 or the microbial preparation according to claim 5 in fermented low-salt air-dried sausage.
9. A method for degrading biogenic amines, characterized in that: The recombinant Lactobacillus plantarum according to any one of claims 1 to 4 is added to a solution containing a biogenic amine at a concentration of 50 mg / ml, and cultured at 37° C. for 18 hours.
10. A method for producing low-salt air-dried sausage by fermentation, characterized in that: The recombinant Lactobacillus plantarum according to any one of claims 1 to 4 is inoculated into the meat filling for fermentation, with an inoculation amount of 10 6 CFU / g minced meat, cultured at 37℃ for 18h.
Citation Information
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