Recombinant lactobacillus sake capable of degrading biogenic amine and application of recombinant lactobacillus sake

Abstract

The invention discloses recombinant lactobacillus sake for degrading biogenic amine and application of the recombinant lactobacillus sake, and belongs to the technical field of microorganisms. The invention aims to provide a method for improving biogenic amine degrading capability of lactobacillus sake. The invention provides recombinant lactobacillus sake capable of degrading biogenic amine, which is obtained by taking lactobacillus sake as a starting strain and overexpressing polyketide oxidase protein. The biological agent prepared from the lactobacillus sake with the gene as shown in the SEQ ID NO.3 through overexpression can reduce product differences caused by different environmental factors in the fermentation process, and the quality, flavor and safety of the air-dried sausages are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a recombinant Lactobacillus sakei for degrading biogenic amines and its application. Background Art

[0002] Air-dried sausage is a traditional fermented meat product in Northeast China. It is highly favored by consumers due to its high nutritional value and pleasant flavor. Its production method usually involves filling a mixture of lean pork, pork back fat, sugar, nitrite, spices, and salt into the casings of pig small intestines. The diameter of the sausage is about 2.5 - 3.0 cm, and then it undergoes rapid natural fermentation under the combined action of raw meat and microorganisms in the surrounding fermentation environment. Proteins in air-dried sausage are degraded by endogenous proteases and microbial proteases to produce a large amount of free amino acids. These free amino acids can promote the formation of the flavor of air-dried sausage, but they are also the precursor substances of biogenic amines. Especially in the presence of microorganisms with decarboxylase positivity, biogenic amines are more likely to form and accumulate.

[0003] Biogenic amines are a class of low-molecular-weight nitrogen-containing organic basic compounds that widely exist in fermented products. They are mainly produced through the decarboxylation of corresponding precursor free amino acids or the interconversion between biogenic amines. Biogenic amines can be roughly divided into three categories according to their structures: aliphatic biogenic amines (putrescine, cadaverine, spermidine, and spermine), aromatic biogenic amines (tyramine and β-phenethylamine), and heterocyclic biogenic amines (histamine and tryptamine). Since monoamine oxidase and diamine oxidase in the human body can rapidly detoxify biogenic amines, a small amount of biogenic amine intake has no toxicity to the human body and can play a positive role in physiological functions, such as participating in brain activities, regulating body temperature, regulating the pH value in the stomach, promoting gastric acid secretion, participating in immune responses, and cell growth and differentiation. On the contrary, too high a content of biogenic amines may cause various toxic effects, such as headache, migraine, oral burning, sweating, and hypertension. In addition, biogenic amines also reduce the sensory properties of food and are considered as a marker of the level of food microbial contamination. Biogenic amines are mainly produced through the decarboxylation of free amino acids by microorganisms with decarboxylase activity or the amination and transamination of aldehydes and ketones.

[0004] At present, some strategies for eliminating biogenic amines in fermented foods mainly include three types: directly degrading biogenic amines using biogenic amine degrading enzymes, inoculating non-biogenic amine-producing or biogenic amine-degrading strains as starters, and adding other antibacterial substances to inhibit the production of biogenic amines. Among them, although using degrading enzymes to degrade biogenic amines is efficient, it cannot guarantee the activity of effective substances during fermentation. In comparison, the effect of reducing amines by inoculating starters is not significant enough. Therefore, achieving the purpose of efficiently reducing amines based on genetic engineering means is a technical problem that urgently needs to be solved at present and has broad application prospects. Summary of the Invention

[0005] The object of the present invention is to provide a method for improving the ability of Lactobacillus sakei to degrade biogenic amines.

[0006] The present invention provides a recombinant Lactobacillus sakei for degrading biogenic amines, which is obtained by overexpressing polyketone oxidase protein using Lactobacillus sakei as the starting strain.

[0007] Further defined, the starting strain is Lactobacillus sakei MDJ6.

[0008] Further defined, the nucleic acid molecule encoding the polyketone oxidase protein is as shown in SEQ ID NO.3.

[0009] Further defined, the amino acid sequence of the polyketone oxidase protein is as shown in SEQ ID NO.4.

[0010] The present invention provides a microbial preparation containing the above recombinant Lactobacillus sakei.

[0011] The present invention provides an application of the above recombinant Lactobacillus sakei or the above microbial preparation in degrading biogenic amines or improving the ability of degradation.

[0012] Further defined, the biogenic amines are tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine.

[0013] The present invention provides an application of the above recombinant Lactobacillus sakei or the above microbial preparation in fermenting to obtain dry-cured sausage.

[0014] The present invention provides a method for degrading biogenic amines, adding the above recombinant Lactobacillus sakei into a solution containing 50 mg / L of biogenic amines, and culturing at 37 °C for 4 days.

[0015] The present invention provides a method for fermenting to obtain low-salt dry-cured sausage, inoculating the above recombinant Lactobacillus sakei into minced meat for fermentation, and the inoculation amount is 10 6 CFU / g of minced meat, and culturing at 37 °C for 18 h.

[0016] Beneficial effects: The sake lactic acid bacterium overexpressing the gene shown in SEQ ID NO.3 has a high expression level of the gene shown in SEQ ID NO.3. The intracellular polyketone oxidase activity is 171.18 U / g protein, and the extracellular polyketone oxidase activity is 349.01 U / g protein. On the 9th day of fermentation of the dry-cured sausage inoculated with the sake lactic acid bacterium overexpressing the gene shown in SEQ ID NO.3, the contents of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine are 3.90 mg / kg, 1.30 mg / kg, 13.98 mg / kg, 33.82 mg / kg, 0.79 mg / kg, 9.01 mg / kg, 1.99 mg / kg and 11.02 mg / kg respectively. The total biogenic amine content is reduced by 69.35% compared with the uninoculated dry-cured sausage, showing a significant effect of reducing the biogenic amine content (P<0.05). Moreover, the biological preparation prepared from the sake lactic acid bacterium overexpressing the gene shown in SEQ ID NO.3 can reduce the differences in products caused by different environmental factors during fermentation, and improve the quality, flavor and safety of dry-cured sausages. Brief Description of the Drawings

[0017] Figure 1 Construction process and verification gel diagram of the sake lactic acid bacterium overexpressing the gene shown in SEQ ID NO.3. Detailed Description of the Invention

[0018] Lactobacillus sakei MDJ6: Screening and evaluating microorganisms with broad-spectrum biogenic amine-degrading ability from naturally fermented dry sausage collected from Northeast China. (Meat Science. 210(2024)109438)

[0019] Example 1. Construction method of the engineering strain overexpressing the gene shown in SEQ ID NO.3

[0020] 1. Obtaining of the recombinant vector pMG36e

[0021] Using the genome of Lactobacillus sakei MDJ6 as a template, the gene shown in SEQ ID NO.3 was obtained by PCR amplification using the primers up and down in Table 1. The sequence is as shown in SEQ ID NO.3. The reaction system and reaction conditions of PCR are as follows:

[0022] Table 1 Primer sequences used

[0023]

[0024] The PCR reaction system is as follows: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mM), 2 μL each of the upstream primer up and the downstream primer down (10 μM), 0.5 μL of template, 1 μL of Phanta Max SuperFidelity DNA Polymerase, and sterile water is added to a final volume of 50 μL.

[0025] The PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 52°C for 12 s, extension at 72°C for 14 s, 35 cycles of reaction, and final extension at 70°C for 5 min.

[0026] Gene (SEQ ID NO.3): atgaaa acctatacgg actatttctt tgatgagcca gcgtttgatc

[0027] tccatgatgg cgggtacgtg ccgcttgagg tcagtgatgc gcctgagaag cccttaaatg

[0028] tgccgccgtt gttgaaaccg gataaagaga cggcgaccga cgtttattac acggtgacag

[0029] cagaggctgg ggaaacgcaa ctgttacctg gggcgaagac caagacgtgg ggctataata

[0030] ccagtctgtt aggtcagacg attgtgtatc gccgtggtca gcatacgcat gtgacactga

[0031] aaaacactct gcctgagttg accacttttc attggcatgg ggctaatgtc agtggccctt

[0032] atgttgatgg cggatgccat gcgccggttt atccgggtga aagtaagcat atcgacttca

[0033] cattggaaca accggcgacg actttgtggc tgcacgcgca tccgtgccca tccacagccg

[0034] agcaggtttg gcatggtttg gctgccatgg tgattgtcaa agacgaccat gaagccagcc

[0035] tgccattgcc aaggaactat ggcgttgacg atattccggt cattttgcaa gaccggcgtt

[0036] ttcatgagaa caaccagtgg gactaccggg ctgattatga tcctgacggt gttgctgggc

[0037] caactgcaat gattaacggt acaatcaatc cctattttga tgtcaccacg caaaaggtcc

[0038] ggttgcgttt tctggatggt gctaatcgcc gtgaatggcg gttgcatttt tccgatgacc

[0039] tgccatttac gcaaattggc ggggatggct cactgttacc ggagccggtc aaatttaccc

[0040] atttgatgct gacttgtgct gagcgtgccg aagtgattgt tgattttggc caataccatg

[0041] aaggcgacga ggtcacctta tatacagatg atgtgccatt gctaaagttc cgcattcatg

[0042] cgttcaaacc ggatcagact accttgcctg ataagttgtt cgatgtgaag gcaccagtgg

[0043] ttgacccggc tttgccagtt cgccacgttg tgatgcaggg gatggacgaa ggtgttgcga

[0044] ttgatggtaa aaagtttgcc atgcagcgga ttgatgccac gcaaccaatt ggcaaagccc

[0045] agtactggga tgttaccaat agcaatgatg cgcctggaat ggttcatcca ttccatgtgc

[0046] atggaaccca attcttagtc ttgtcgcgga atgggcatgc gccgtatcca aatgaacatg

[0047] gtttcaaaga tacaattggc gtgaatcctg gtgaaacggt tcggctgctg gttcgctttg

[0048] atttgccagg ggtttatatg tatcactgcc atatcattga gcacgaagat ggcggcatga

[0049] tggcacagat tgaaacattc gatccagcca agccaaagca agaatataaa ttgatggata

[0050] tggatacgtt aatgatggcc ttggctaaag aacgtggcgt caaaccatct gagatttgga

[0051] tgggtggtat gcagtcttat gaaaaaatgg gaatgaaaat gtaa。

[0052] Amino acid sequence (SEQ ID NO.4):

[0053] mktytdyffd epafdlhdgg yvplevsdap ekplnvppll kpdketatdv yytvtaeage

[0054] tqllpgaktk twgyntsllg qtivyrrgqh thvtlkntlp elttfhwhga nvsgpyvdgg

[0055] chapvypges khidftleqp attlwlhahp cpstaeqvwh glaamvivkd dheaslplpr

[0056] nygvddipvi lqdrrfhenn qwdyradydp dgvagptamingtinpyfdv ttqkvrlrfl

[0057] dganrrewrl hfsddlpftq iggdgsllpe pvkfthlmlt caeraevivd fgqyhegdev

[0058] tlytddvpll kfrihafkpd qttlpdklfd vkapvvdpal pvrhvvmqgm degvaidgkk

[0059] famqridatq pigkaqywdv tnsndapgmv hpfhvhgtqf lvlsrnghap ypnehgfkdt

[0060] igvnpgetvr llvrfdlpgv ymyhchiieh edggmmaqie tfdpakpkqe yklmdmdtlm

[0061] malakergvk pseiwmggmq syekmgmkm。

[0062] The construction scheme of the recombinant plasmid pMG36e is as Figure 1 shown in (A) below. The obtained vector pMG36e (e.g., purchased from Miaoling Biology) was double-digested with XbaI and HindIII (both purchased from TaKaRa). The fragment of the gene shown in SEQ ID NO.3 obtained by amplification was gel-extracted. Then, the recovered gene fragment shown in SEQ ID NO.3 was ligated with the plasmid fragment pMG36e treated with XbaI and HindIII double digestion to obtain a ligation product. The ligation product was transformed into competent Escherichia coli DH5α cells and evenly spread on an LB plate with erythromycin resistance (600 μg / mL). It was cultured overnight at 37 °C. Single colonies were picked and verified by colony PCR and restriction digestion. After correct sequencing alignment, as Figure 1 shown in (B) below, the vector pMG36e containing the gene shown in SEQ ID NO.3, namely the recombinant vector pMG36e, was obtained.

[0063] Among them, the LB medium is: Tryptone: 10.0 g / L, Yeast extract: 5.0 g / L, NaCl: 10.0 g / L. After dissolving with deionized water, it was made up to 1.0 L, and the pH was adjusted to 7.0 - 7.2. 1.5% agar powder was added to the solid medium. It was sterilized at 121 °C for 20 min.

[0064] 2. Construction of the strain overexpressing the gene shown in SEQ ID NO.3

[0065] The competent cells were prepared according to the following method:

[0066] 1. The strain L. sakei MDJ6 was inoculated into 10 mL of MRS medium at 2%, and statically cultured at 37 °C for 16 h;

[0067] 2. 1 mL of the cultured strain was inoculated into 100 mL of MRS medium containing 2.5% glycine concentration. After culturing for 3 - 4 h, the OD 600 value was measured. When it reached 0.5 - 0.6, the culture was stopped. The cells were collected by centrifugation at 4 °C, 12000 rpm / min for 10 min;

[0068] 3. The cells were washed twice with 25 mL of sterilized sterile water. After centrifugation, the supernatant was discarded, and it should be noted that the whole process was operated at low temperature;

[0069] 4. The washed cells were resuspended in 0.05 mol / L EDTA solution and ice - bathed for 5 min. Then 25 mL of pre - cooled sterile water was added, and centrifuged at 4 °C, 12000 rpm / min for 10 min. The cells were washed again with 25 mL of pre - cooled sterile water;

[0070] 5. The cells were centrifuged at 4 °C, 12000 rpm / min for 10 min with 25 mL of pre - cooled electroporation buffer, and this step was repeated once;

[0071] 6. The cells washed with electroporation buffer were resuspended in 0.8 mL of electroporation buffer, and aliquoted into sterilized 1.5 mL centrifuge tubes at a volume of 100 μL each. It should be noted that the aliquoting was performed on ice to obtain competent cells, which were stored at - 80 °C for later use.

[0072] The electroporation of L. sakei was carried out according to the following method:

[0073] 1. 100 μL of competent cells were thawed on ice, and 10 μL of recombinant vector pMG36e DNA was added and gently pipetted to mix evenly. After ice - bathing for 5 min, a recombinant vector pMG36e mixture was obtained; 100 μL of competent cells were thawed on ice, and 10 μL of pMG36e vector DNA was added and gently pipetted to mix evenly. After ice - bathing for 5 min, a pMG36e mixture was obtained;

[0074] 2. The above two 110 μL mixtures were respectively transferred into an electroporation cuvette with a pre - cooled distance of 1 mm on ice, and immediately placed in an electroporator. Electroporation was carried out at a voltage of 1.25 kV. If the distance between the electroporation cuvettes was 2 mm, the voltage of 2.5 kV was used for electroporation;

[0075] 3. After the electrotransformation is completed, quickly add 890 μL of fresh MRS culture medium to the electroporation cup. Gently pipette and mix the mixture with a pipette tip, and then transfer the mixture to a sterile 2 mL centrifuge tube. Incubate the cells at 28 °C for 2.5 h to recover.

[0076] 4. Centrifuge the cultured bacterial solution at 8000 rpm / min for 3 min, discard 900 μL of the supernatant, resuspend the cells with the remaining 100 μL of the supernatant, and spread it on an MRS resistance plate containing 100 μg / mL erythromycin. Incubate the plate at 28 °C statically.

[0077] 5. Pick a single colony and culture the single colony in MRS culture medium containing erythromycin to obtain the strain L. sakei MDJ6-pMG36e containing the empty plasmid and the strain overexpressing the gene shown in SEQ ID NO.3, respectively. Perform colony PCR verification, and the verification results are as Figure 1 shown in (C) of, that is, the correct overexpressing strain is obtained.

[0078] Example 2. Determination of biogenic amine degradation ability in the culture medium system

[0079] I. Polyketone oxidase activity of the overexpressing strain:

[0080] Inoculate two strains of lactic acid bacteria, L. sakei MDJ6 and L. sakei overexpressing the gene shown in SEQ ID NO.3, into MRS medium and activate them for 3 generations to prepare lactic acid bacteria culture solutions. Inoculate the strains into their suitable media at 2% (v / v), culture them at 37 °C for 18 hours, and centrifuge them at 6000×g for 15 min. Take the supernatant and precipitated cells to measure the extracellular biogenic amine degrading enzyme activity and intracellular biogenic amine degrading enzyme activity, respectively. Treat the uninoculated MRS and MSA broths in the same way as controls.

[0081] Preliminary gradient precipitation experiments of proteins in the culture medium were carried out to determine the optimal (NH 4 ) 2 SO 4 concentrations for precipitating biogenic amine degrading enzymes in the MRS supernatant and MSA supernatant were 230.10 and 536.90 g / L, respectively. Add the optimal concentration of (NH 4 ) 2 SO 4 to the culture solution and mix well. Let it stand at 4 °C for 1 h and centrifuge it at 8000×g for 15 min. The precipitate obtained by centrifugation is the crude protein. Dissolve the protein in sterile distilled water to obtain the extracellular biogenic amine degrading enzyme solution.

[0082] Extraction of intracellular biogenic amine degrading enzymes: The cell precipitate of the strain obtained by centrifugation was washed twice with 50 mmol / L phosphate buffered saline (PBS, pH 7.4) and resuspended in PBS, and then 500 μL of 1 mmol / L benzylsulfonyl fluoride was added. The cells were disrupted using an ultrasonic device equipped with a 6.0 mm diameter probe: ultrasonic power 600 W, ultrasonic time 20 min (the ultrasonic working and pause times were 2 s and 2 s, respectively). The ultrasonicated solution was centrifuged at 10000×g for 15 min at 4 °C, and the supernatant was collected as the intracellular biogenic amine degrading enzyme solution.

[0083] The protein content, monoamine oxidase activity, and diamine oxidase activity in the samples were measured using BCA assay kits, MOA assay kits, and DAO assay kits. Using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the reaction substrate, in a reaction system of 50 μL of enzyme solution, 950 μL of 50 mmol / L citrate buffer (containing 1 mmol / L ABTS and 1 mmol / L CuCl 2 ), the amount of ABTS oxidized within 1 min was detected to calculate the polyketone oxidase activity. The amount of enzyme used to oxidize 1 μmol of ABTS per minute was defined as one enzyme activity unit (U), and it was measured using an ultraviolet-visible spectrophotometer at 420 nm. The activities of biogenic amine degrading enzymes (monoamine oxidase, diamine oxidase, and polyketone oxidase) were calculated as follows:

[0084]

[0085] Where A1 represents the biogenic amine degrading enzyme activity (U / L) in the protein solution, and C1 is the protein concentration (mg / L) in the protein solution.

[0086] The results are shown in Table 2. The intracellular and extracellular polyketone oxidase activities of the original strain L. sakei MDJ6 were 83.42 and 110.60 U / g protein, respectively; the intracellular and extracellular polyketone oxidase activities of the overexpressing strain were 171.18 and 349.01 U / g protein, respectively. The extracellular polyketone oxidase activities of both L. sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 were higher than the intracellular ones, indicating that this enzyme mainly exists extracellularly. The intracellular polyketone oxidase activity of Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 was increased by 1.05 times compared with the original strain L. sakei MDJ6, and the extracellular polyketone oxidase activity was increased by 2.16 times, indicating that the expression level of the polyketone oxidase gene in this overexpressing strain was effectively enhanced.

[0087] Table 2 Intracellular and extracellular polyketone oxidase activities (U / g protein) of the original strain Lactobacillus sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3

[0088]

[0089] Different lowercase letters (a - b) in the same column indicate significant differences (P < 0.05)

[0090] II. Biogenic amine degradation ability of the overexpressing strain in the culture medium system:

[0091] Eight biogenic amines (tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) were added to MRS and MSA broths to a final concentration of 50 mg / L for each biogenic amine in the medium, thus obtaining MRS - BA and MSA - BA media. The original strain L. sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 were inoculated into MRS - BA and MSA - BA at an initial cell concentration of approximately 7 lg CFU / mL and cultured at 37 °C for 4 days. The blank medium was used as a control.

[0092] The determination of eight biogenic amines (tryptamine, β-phenethylamine, putrescine, cadaverine, tyramine, histamine, spermidine, and spermine) was carried out according to the methods of extraction, purification, derivatization, and determination in GB 5009.208 - 2016 "National Food Safety Standard - Determination of Biogenic Amines in Foods".

[0093] The content of biogenic amines is expressed as mg / L of the medium, and the calculation formula for the biogenic amine degradation rate is as follows:

[0094]

[0095] where C 0 is the content of biogenic amines before fermentation, and C is the content of biogenic amines in the strain culture solution 4 days after inoculation (mg / L). Table 3 Degradation rates of eight biogenic amines by the original strain Lactobacillus sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 in the culture medium system

[0096]

[0097]

[0098] The results are shown in Table 3. The degradation rate of spermine by Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 was the highest (26.34%), which was 2.41 times higher than that of the control L. sakei MDJ6; followed by the degradation rate of putrescine, which was 12.19%, which was 1.95 times higher than that of the control L. sakei MDJ6. In addition to putrescine and spermine, cadaverine is also a biogenic amine with a high content in air-dried intestines, but the results did not show a significant increase in the degradation rate of cadaverine by the overexpression strain. Although tyramine and histamine are not the main biogenic amines in air-dried intestines, their accumulation in food has also attracted much attention due to their strong toxicity. Among them, the degradation rate of histamine by Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 was not significantly improved; while the degradation rate of tyramine was increased by 0.54 times. The degradation rate of total biogenic amines in the culture medium system by Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 was 8.66%, which was 0.91 times higher than the total biogenic amine degradation rate of 4.53% of the control L. sakei MDJ6.

[0099] Example 3. Preparation of microbial preparation of Lactobacillus sakei

[0100] 1. Activation of sake lactobacillus:

[0101] 100-300 μL of the sake Lactobacillus culture medium overexpressing the gene shown in SEQ ID NO.3 in the glycerol tube was taken and inoculated into 500-1000 mL of MRS broth medium, and incubated at 37°C for 24-48 hours. This step was repeated 2-4 times to obtain the activated sake Lactobacillus culture medium. The activated culture medium was centrifuged, the supernatant was discarded, and then washed with sterile physiological saline and resuspended, centrifuged and the supernatant was discarded. This step was repeated 2-3 times to obtain a bacterial cell concentration of 10 6 ~10 7 CFU / g of active bacterial sludge.

[0102] 2. Preparation of bacterial powder biological preparations:

[0103] Skim milk was selected as a freeze-drying protective agent, and the bacterial sludge obtained in the previous step and the skim milk were mixed at a mass ratio of 1:10 to obtain a bacterial cell concentration of 10 5 ~10 6 CFU / mL of skim milk suspension of sake lactobacillus overexpressing the gene shown in SEQ ID NO.3. It was pre-cultured at 37°C for 2 hours and then placed in a -40°C refrigerator for pre-freezing for about 5 to 7 hours. After the pre-freezing was completed, it was placed in a vacuum freeze dryer for freeze drying. 5 ~10 7 CFU / g of freeze-dried biological preparation of Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3.

[0104] Application of Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3

[0105] I. Production of dry-cured sausage

[0106] (1) Preparation of dry-cured sausage

[0107] Raw materials: 9.0 kg of lean meat (pork loin), 1.0 kg of fat (pork back fat), 0.30 kg of Daqu wine, 0.20 kg of salt, 0.0090 kg of sodium nitrite, 0.5 kg of granulated sugar, 0.15 kg of monosodium glutamate, 0.05 kg of mixed seasonings (dry sausage seasoning of Shiyitang).

[0108] (2) Preparation of starter culture

[0109] Using the dry-cured sausage without inoculating strains as the control, another 2 groups of treatment groups inoculated with L. sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 were set up. After the biological agent Lactobacillus sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 were passaged twice in MRS medium, they were cultured at 37 °C for 18 h. After centrifugation at 4 °C and 10000×g for 5 min, they were reserved. When adding the starter culture, the addition amount of lactic acid bacteria was 10 6 CFU / g of meat stuffing.

[0110] Technological process: Raw meat (removing connective tissues such as lymph, tendons, blood vessels, etc.) → Dicing (meat dices of 1 cm 3 ) → Marinating (adding salt and seasonings, etc.) → Adding starter culture → Mixing the stuffing → Stuffing (length 20 cm, diameter 3 cm) → Fermenting and air-drying (temperature 25 ± 2 °C, humidity 65 ± 5%).

[0111] II. Determination of biogenic amines

[0112] Take 5.0 g of the sample and mix it thoroughly with 500 μL of the internal standard solution 1,7-diaminoheptane with a concentration of 1.0 mg / mL. The determination of 8 biogenic amines (tryptamine, β-phenethylamine, putrescine, cadaverine, tyramine, histamine, spermidine and spermine) was carried out according to the methods of GB 5009.208-2016 "National Food Safety Standard - Determination of Biogenic Amines in Foods" for extraction, purification, derivatization and determination. The content of biogenic amines was expressed as mg / kg of dry-cured sausage, and the calculation method of biogenic amine degradation rate was as follows:

[0113]

[0114] where C 0 is the biogenic amine content of the dry-cured sausage without inoculating strains, and C is the biogenic amine content in the dry-cured sausage after inoculating strains (mg / L).

[0115] The results are shown in Table 4. Compared with inoculation with L. sakei MDJ6, the biogenic amine content of the dry-cured sausage inoculated with Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 is lower. On the 9th day of fermentation, the contents of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine are 3.90 mg / kg, 1.30 mg / kg, 13.98 mg / kg, 33.82 mg / kg, 0.79 mg / kg, 9.01 mg / kg, 1.99 mg / kg and 11.02 mg / kg respectively. The total biogenic amine content is reduced by 69.35% compared with the non-inoculated dry-cured sausage. This result is consistent with the research results of biogenic amine reduction in the culture medium system. Overall, both the strain L. sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 can significantly reduce the biogenic amine content in dry-cured sausage, and the biogenic amine reduction ability of Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 is stronger. The difference in their biogenic amine reduction abilities may be due to the action of the overexpressing strain producing more polyketone oxidase, or the two strains differently changing the microbial community composition in the dry-cured sausage. Obviously, Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3 has a better degradation effect on biogenic amines in dry-cured sausage and has certain application potential in the biogenic amine degradation of fermented meat products.

[0116] Table 4 Contents (mg / kg) and degradation rates of total biogenic amines in dry-cured sausages without inoculation, inoculated with the original strain Lactobacillus sakei MDJ6 and Lactobacillus sakei overexpressing the gene shown in SEQ ID NO.3

[0117]

[0118]

Claims

1. A recombinant Lactobacillus sakei for degrading biogenic amines, characterized in that: It was obtained by overexpressing polyketide oxidase protein using Lactobacillus sakei as the starting strain.

2. The recombinant Lactobacillus sakei according to claim 1, characterized in that The starting strain is Lactobacillus sakei MDJ6.

3. The recombinant Lactobacillus sakei according to claim 1, characterized in that The nucleic acid molecule encoding the polyketide oxidase protein is shown as SEQ ID NO.

3.

4. The recombinant Lactobacillus sakei according to claim 1, characterized in that The amino acid sequence encoding the polyketide oxidase protein is shown in SEQ ID NO.

4.

5. A microbial preparation containing the recombinant Lactobacillus sakei according to any one of claims 1 to 4.

6. Use of the recombinant Lactobacillus sakei 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, β-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine.

8. Use of the recombinant Lactobacillus sakei according to any one of claims 1 to 4 or the microbial preparation according to claim 5 in obtaining air-dried sausage by fermentation.

9. A method for degrading biogenic amines, characterized in that: The recombinant Lactobacillus sakei described in any one of claims 1 to 4 is added to a solution containing 50 mg / L of biogenic amines and cultured at 37° C. for 4 days.

10. A method for obtaining low-salt air-dried sausage by fermentation, characterized in that: The recombinant Lactobacillus sakei described in 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.

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