Lactobacillus plantarum with overexpressed luxS gene, microbial preparation and application of lactobacillus plantarum

Abstract

The invention discloses lactobacillus plantarum for overexpressing a luxS gene, a microbial preparation and application of the lactobacillus plantarum, and belongs to the technical field of microorganisms. In order to solve the technical problem that excessive biogenic amines are accumulated in a fermented meat product in the prior art, the invention provides lactobacillus plantarum for overexpressing a luxS gene and a microbial preparation containing the lactobacillus plantarum. The lactobacillus plantarum for overexpressing the luxS gene, provided by the invention, has the capability of highly producing signal molecules AI-2 and has the capability of remarkably improving the activities of diamine oxidase and polyketide oxidase; the lactobacillus plantarum overexpressing the luxS gene and the microbial preparation containing the lactobacillus plantarum can be used as a leavening agent for reducing biogenic amine to be applied to production of fermented meat products, the content of biogenic amine in the air-dried sausages can be effectively reduced, and the quality 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 Lactobacillus plantarum, a microbial preparation and their applications. Background Art

[0002] Dry-cured sausage is a traditional fermented meat product, which is deeply loved by consumers for its unique taste and flavor. Traditional dry-cured sausage is made by mixing lean pork, pork back fat, salt, nitrite, Daqu and various spices and stuffing them into sausage casings; the fermentation and ripening of traditional dry-cured sausage rely on endogenous enzymes and the microbial community in the natural state to achieve. During the fermentation process, the degradation of proteins provides favorable conditions for flavor formation, but it will also cause the accumulation of harmful factors such as biogenic amines (BAs). Biogenic amines are also the precursor substances of the carcinogen nitrosamine. A small amount of biogenic amines ingested by the human body can regulate normal physiological functions, but excessive intake of biogenic amines will lead to poisoning and even death. Therefore, the problem of reducing biogenic amines in fermented meat products has always been highly concerned, and it is of great significance for improving the safety and quality of traditional fermented meat products.

[0003] Quorum sensing (QS) is a communication mechanism adopted by bacterial communities, in which the release of signal molecules helps the expression of specific genes and regulates community metabolism; this process is of great significance for controlling the metabolic "rhythm" of the entire community and promoting fermentation. Exogenous addition of the signal molecule AI-2 has been proven to significantly enhance the probiotic characteristics of lactic acid bacteria during fermentation, including the production of antibacterial substances, the inhibition of the accumulation of harmful substances, and the production of flavor compounds.

[0004] As the most commonly used bacterial starter in fermented meat products, lactic acid bacteria can actively inhibit the growth of spoilage microorganisms, improve fermentation characteristics, and enhance the quality and safety of products. Research has found that most lactic acid bacteria in fermented foods are "positively regulated" by quorum sensing to increase the safety and quality of fermented foods. Therefore, those skilled in the art are eager to develop a method that can enhance the fermentation characteristics of lactic acid bacteria and reduce the content of biogenic amines in fermented meat products through the quorum sensing system. Summary of the Invention

[0005] In order to solve the technical problem of excessive accumulation of biogenic amines in fermented meat products in the prior art, the present invention provides a Lactobacillus plantarum overexpressing the luxS gene, a microbial preparation and their applications.

[0006] One of the purposes of the present invention is to provide a Lactobacillus plantarum overexpressing the luxS gene, which is obtained by transforming the plasmid pMG36e-luxS overexpressing the luxS gene into Lactobacillus plantarum SH7.

[0007] In a preferred embodiment of the present invention, the preservation number of the Lactobacillus plantarum SH7 is CGMCC NO. 28113.

[0008] The second object of the present invention is to provide the application of the above-mentioned Lactobacillus plantarum overexpressing the luxS gene in the preparation of dried sausage.

[0009] The third object of the present invention is to provide a Lactobacillus plantarum microbial preparation, and the microbial preparation contains the above-mentioned Lactobacillus plantarum overexpressing the luxS gene.

[0010] The fourth object of the present invention is to provide a preparation method of the above-mentioned Lactobacillus plantarum microbial preparation, and the preparation method includes the following steps: activating the above-mentioned Lactobacillus plantarum overexpressing the luxS gene, centrifuging to discard the supernatant to obtain an active bacterial sludge; mixing the above-mentioned active bacterial sludge with skimmed milk and culturing to obtain a mixture; subjecting the above-mentioned mixture to a pre-freezing treatment, and after the pre-freezing is completed, freeze-drying in a vacuum freeze dryer to obtain a microbial preparation.

[0011] In a preferred embodiment of the present invention, the cell concentration of the Lactobacillus plantarum overexpressing the luxS gene in the active bacterial sludge is 10 6 -10 7 CFU / g.

[0012] In a preferred embodiment of the present invention, the mixing mass ratio of the active bacterial sludge to the skimmed milk is 1:10.

[0013] In a preferred embodiment of the present invention, the culturing conditions after mixing the active bacterial sludge with the skimmed milk are: culturing at 37°C for 2 h.

[0014] In a preferred embodiment of the present invention, the conditions of the pre-freezing treatment are: pre-freezing in a -40°C refrigerator for 5 - 7 h.

[0015] The fifth object of the present invention is to provide the application of the above-mentioned Lactobacillus plantarum microbial preparation in the preparation of dried sausage.

[0016] The beneficial effects of the present invention: The present invention provides a Lactobacillus plantarum overexpressing the luxS gene, which is obtained by transforming the plasmid pMG36e-luxS overexpressing the luxS gene into Lactobacillus plantarum SH7, and provides a microbial preparation containing the above-mentioned Lactobacillus plantarum.

[0017] The present invention utilizes Lactobacillus plantarum overexpressing the luxS gene to regulate the LuxS / AI-2 gene through the quorum sensing system, enhancing the "positive regulation" of the quorum sensing system in Lactobacillus plantarum and enhancing the fermentation characteristics of Lactobacillus plantarum, thereby achieving the purpose of degrading the existing biogenic amines in fermented meat products, reducing the content of biogenic amines in fermented meat products, and improving the safety and quality of fermented meat products.

[0018] The Lactobacillus plantarum overexpressing the luxS gene provided by the present invention has a relatively high expression level of the quorum sensing signal molecule AI-2 and has the ability to significantly enhance the activities of diamine oxidase and polyamine oxidase (P<0.05). The extracellular diamine oxidase activity is 976.85 U / g protein, and the extracellular polyamine oxidase activity is 192.73 U / g protein; the degradation rates of putrescine and cadaverine in air-dried sausage are as high as 75.26% and 61.40% respectively, and the total biogenic amine degradation rate reaches 56.50%. It can be seen that the Lactobacillus plantarum overexpressing the luxS gene provided by the present invention, as well as the microbial preparation containing the above-mentioned Lactobacillus plantarum, can be used as a biogenic amine-reducing starter in the production of fermented meat products, effectively reducing the content of biogenic amines in air-dried sausage (including: Harbin air-dried sausage) and improving the quality and safety of air-dried sausage. Brief Description of the Drawings

[0019] Figure 1 It is the construction process and verification gel diagram of Lactobacillus plantarum L.plantarum SH7-pMG36e-luxS; A is the construction flow chart of the recombinant plasmid pMG36e-luxS, B is the verification gel diagram of the pMG36e vector, C is the verification gel diagram of gel recovery, D is the verification gel diagram of enzyme digestion, and E is the verification gel diagram of colony PCR;

[0020] Figure 2 It is the result diagram of the ability of different strains to produce the signal molecule AI-2;

[0021] Figure 3 It is the diagram of the influence of different strains on the activity of diamine oxidase;

[0022] Figure 4 It is the diagram of the influence of different strains on the activity of polyamine oxidase;

[0023] Figure 5 It is the diagram of the influence of different strains on the biogenic amine content and degradation rate at the end of air-dried sausage fermentation. Detailed Description of the Invention

[0024] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes, or appropriate alterations and combinations, to the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0025] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0026] The Lactobacillus plantarum SH7 used in the present invention is deposited under the accession number CGMCC NO. 28113 at the China General Microbiological Culture Collection Center, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date being August 7, 2023.

[0027] Example 1: Construction of Lactobacillus plantarum overexpressing the luxS gene

[0028] 1. Obtaining pMG36e-luxS:

[0029] Using the genome of Lactobacillus plantarum SH7 as a template, PCR amplification was carried out using the primers shown in Table 1 to obtain the luxS gene with the nucleotide sequence shown in SEQ ID NO. 1. The PCR reaction system was: 2×Phanta Max Buffer 25 μL, dNTPMix (10 mM) 1 μL, upstream primer luxS-up and downstream primer luxS-down (10 μM) 2 μL each, template 0.5 μL, PhantaMax Super-Fidelity DNA Polymerase 1 μL, and sterile water was added to a final volume of 50 μL; the PCR reaction conditions were: 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 post-extension at 70 °C for 5 min.

[0030] Table 1

[0031]

[0032]

[0033] The construction scheme of the recombinant plasmid pMG36e-luxS is as follows Figure 1 A. Use XbaI and HindIII (both purchased from TaKaRa) to perform double digestion on the pMG36e vector purchased from Miaoling Biology (as shown in Figure 1 B); Gel extract the fragment of the luxS gene obtained by the above amplification, as shown in Figure 1 C; Ligate the recovered luxS gene fragment with the plasmid fragment pMG36e treated with XbaI and HindIII double digestion to obtain a ligation product; Transform the ligation product into Escherichia coli DH5α competent cells, and evenly coat it on an LB medium (the composition of the LB medium is: tryptone: 10.0 g / L, yeast extract: 5.0 g / L, NaCl: 10.0 g / L, dissolved in deionized water, made up to 1.0 L, pH adjusted to 7.0 - 7.2, 1.5% agar powder added to the solid medium, sterilized at 121°C for 20 min) plate with erythromycin resistance (600 μg / mL), culture overnight at 37°C, pick monoclonal colonies, perform colony PCR verification and restriction enzyme digestion verification, and after correct sequencing comparison, as shown in Figure 1 D, obtain the vector pMG36e containing the luxS gene, abbreviated as: pMG36e-luxS.

[0034] 2. Preparation of Lactobacillus plantarum overexpressing the luxS gene

[0035] S1: Inoculate Lactobacillus plantarum SH7 at an inoculation amount of 2% into 10 mL of MRS medium, and statically culture at 37°C for 16 h;

[0036] S2: Take 1 mL of Lactobacillus plantarum SH7 cultured in S1 and inoculate it into 100 mL of MRS medium containing a glycine concentration of 2.5%. After culturing for 3 - 4 h, detect its OD 600 value. When it reaches 0.5 - 0.6, stop culturing, centrifuge at 4°C and 12000 rpm / min for 10 min, and collect the bacterial cells; Add 25 mL of sterilized sterile water to the above bacterial cells and wash twice, centrifuge, and discard the supernatant, noting that the whole process is operated at low temperature;

[0037] S3: Resuspend the washed bacterial cells in S2 in 0.05 mol / L EDTA solution, ice-bath for 5 min, add 25 mL of pre-cooled sterile water, centrifuge at 4°C and 12000 rpm / min for 10 min, and wash again with 25 mL of pre-cooled sterile water;

[0038] S4: Wash the cells after washing in S3 again with 25 mL of pre-cooled electroporation buffer, centrifuge at 4°C and 12,000 rpm for 10 min, and repeat step S3 once;

[0039] S5: Resuspend the cells after washing in S4 in 0.8 mL of electroporation buffer, aliquot into sterile 1.5 mL centrifuge tubes at a volume of 100 μL each, and operate on ice during aliquoting to obtain competent cells, and store at -80°C for later use;

[0040] S6: Thaw 100 μL of the competent cells obtained in S5 on ice, add 10 μL of pMG36e-luxS vector DNA, gently pipette and mix well, and incubate on ice for 5 min to obtain the pMG36e-luxS mixture;

[0041] S7: Thaw 100 μL of the competent cells obtained in S5 on ice, add 10 μL of pMG36e vector DNA, gently pipette and mix well, and incubate on ice for 5 min to obtain the pMG36e mixture;

[0042] S8: Take 110 μL each of the pMG36e-luxS mixture and the pMG36e mixture obtained in S6 and S7, transfer them into electroporation cups with a spacing of 1 mm pre-cooled on ice respectively, immediately place them in the electroporator, and perform electroporation at a voltage of 1.25 kV. If the spacing of the electroporation cup is 2 mm, use a voltage of 2.5 kV for electroporation; after electroporation, quickly add 890 μL of fresh MRS culture medium to the electroporation cups respectively, gently pipette and mix well with a pipette tip, and then transfer the mixed solutions to sterile 2 mL centrifuge tubes respectively, and incubate statically at 28°C for 2.5 h to recover the cells;

[0043] S9: Centrifuge the bacterial solutions after recovery in S7 at 8,000 rpm for 3 min, discard 900 μL of the supernatant, resuspend the cells with the remaining 100 μL of the supernatant, and spread them on MRS resistance plates containing 100 μg / mL erythromycin respectively, and incubate statically at 28°C;

[0044] S10: Pick single colonies cultured in S9 respectively, and culture the single colonies in MRS culture medium containing erythromycin respectively to obtain strains containing the empty plasmid (abbreviation: L.plantarum SH7-pMG36e) and L.plantarum SH7 lactobacillus plantarum overexpressing the luxS gene (abbreviation: L.plantarum SH7-pMG36e-luxS).

[0045] In this example, the obtained strains were verified by bacterial liquid PCR, and the verification results are as Figure 1As shown in Figure E, it indicates that the correct strains L. plantarum SH7-pMG36e and L. plantarum SH7-pMG36e-luxS were successfully obtained.

[0046] Effect data:

[0047] I. Determination of the production of quorum sensing signal molecule AI-2:

[0048] Inoculate V. harveyi BB170 (purchased from Shanghai Biological Collection Center with the number SHBCC D24812) into AB medium (17.5 g of sodium chloride, 12. g of magnesium sulfate and 2.0 g of acid hydrolysate casein per liter) at 2%, and place it in a shaker at 30 °C and 120 r / min for culturing until the second generation. Culture until the OD 600 nm value is between 0.7 and 1.2. Dilute the V. harveyi BB170 culture solution with fresh AB medium at a ratio of 1:5000 (v / v), mix well and set aside;

[0049] Inoculate the following 3 strains, namely L. plantarum SH7, L. plantarum SH7-pMG36e obtained in Example 1, and L. plantarum SH7-pMG36e-luxS obtained in Example 1, into MRS medium respectively, and prepare strain culture solutions after activating for 3 generations;

[0050] Centrifuge the L. plantarum SH7 strain culture solution, L. plantarum SH7-pMG36e strain culture solution, L. plantarum SH7-pMG36e-luxS strain culture solution, V. harveyi BB170 culture solution, AB medium and MRS liquid medium at 8000 r / min (4 °C) for 5 min respectively, discard the bacterial sludge, and filter the supernatant with a 0.22 μm sterile membrane to remove impurities, and obtain the samples to be tested (L. plantarum SH7 strain culture solution, L. plantarum SH7-pMG36e strain culture solution and L. plantarum SH7-pMG36e-luxS strain culture solution), positive control (sterile supernatant of V. harveyi BB170 culture solution), negative control (sterile supernatant of AB medium), and medium control (sterile supernatant of MRS liquid medium) respectively;

[0051] Mix the above-mentioned samples to be tested, positive, negative, and medium controls with the diluted V. harveyi BB170 bacterial solution at a ratio of 1:100 (v / v), and continue to shake and culture. Measure the fluorescence intensity value every 4 hours within 0 - 24 hours. When the fluorescence intensity value of the negative control reaches the lowest point at 3.0 hours, measure the fluorescence intensity of each sample, and calculate its relative fluorescence intensity to represent the activity of the signal molecule AI-2. The relative fluorescence intensity is as follows: Equation (1): Relative fluorescence intensity of the Vibrio positive control group = Fluorescence intensity value of the positive control / Fluorescence intensity value of the negative control;

[0052] Relative fluorescence intensity of the strain = Fluorescence intensity value of the sample to be tested / Fluorescence intensity value of the medium control.

[0053] The results are as Figure 2 shown. When the culture time exceeds 4 hours, the ability of L. plantarum SH7-pMG36e-luxS strain to produce the signal molecule AI-2 in the medium is significantly higher than that of L. plantarum SH7-pMG36e and L. plantarum SH7 strains inoculated, and there is no significant difference in the ability of L. plantarum SH7-pMG36e and L. plantarum SH7 strains inoculated in the medium to produce the signal molecule AI-2. It can be seen that the Lactobacillus plantarum overexpressing the luxS gene provided by the present invention has a high ability to produce the signal molecule AI-2.

[0054] II. Determination of extracellular diamine oxidase activity:

[0055] Scrape a whole inoculation loop of L. plantarum SH7, L. plantarum SH7-pMG36e, and L. plantarum SH7-pMG36e-luxS strains respectively, and inoculate them into 250 mL conical flasks containing MRS medium, and culture them at 37 °C with a rotation speed of 200 rpm in a shaker for 24 hours. Take the supernatant every 4 hours to extract proteins by 70% ammonium sulfate salting-out, place it at 4 °C and let it stand for 1 hour to fully extract, then discard the supernatant. Dissolve the obtained proteins in an equal volume of phosphate buffer, and use a diamine oxidase DAO kit (purchased from Nanjing Jiancheng Bioengineering Institute) to measure the extracellular protein concentration and diamine oxidase activity, and use a Bradford method protein content detection kit (purchased from Shanghai Jingkang Bioengineering Co., Ltd.) to measure the protein content.

[0056] The results are as Figure 3As shown, the activity of diamine oxidase in the medium inoculated with L. plantarum SH7-pMG36e-luxS strain was significantly higher than that in the medium inoculated with L. plantarum SH7 and L. plantarum SH7-pMG36e strains (P<0.05); it can be seen that the Lactobacillus plantarum overexpressing the luxS gene provided by the present invention has the ability to increase the activity of diamine oxidase.

[0057] III. Determination of the activity of multi-copper oxidase:

[0058] Scrape a whole inoculation loop of L. plantarum SH7, L. plantarum SH7-pMG36e and L. plantarum SH7-pMG36e-luxS strains respectively, and inoculate them into 250 mL conical flasks containing MRS medium, and culture them at 37 °C with a rotation speed of 200 rpm in a shaker for 24 h; take the supernatant every 4 h, and use the ultraviolet-visible light absorption method to determine the activity of multi-copper oxidase. Using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as the substrate, the activity of multi-copper oxidase is characterized by the rate of multi-copper oxidase oxidizing ABTS per unit time; the reaction system is: 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 CuCl2; use an ultraviolet-visible light spectrophotometer to measure the oxidation rate of ABTS at 420 nm, and define the amount of enzyme required to oxidize 1 μmol of ABTS per minute as one enzyme activity unit (U);

[0059] The specific steps are as follows: Take 10 mL of the culture solution, break the cells in an ultrasonic crusher under ice bath, turn on for 3 s and turn off for 3 s, and alternate the crushing cycle for a total of 15 min; centrifuge the broken cells at 13,000 r / min at 4 °C for 10 min, and then centrifuge at 10,000 rpm at 4 °C for 10 min to obtain the crude enzyme solution;

[0060] The determination of lipase activity mainly uses p-nitrophenyl palmitate (p-NPP) as the reaction substrate. Take 600 μL of 2.0 mM p-nitrophenol, add 25 μL of the crude enzyme solution and react at 40 °C for 15 min. After the reaction is completed, add 500 μL of 95% ethanol to terminate the reaction; the control group adds 25 μL of pH 7.0 Tris-HCl buffer, incubate at 40 °C for 15 min and then add 500 μL of 95% ethanol; after mixing, centrifuge at 10,000 rpm at 4 °C for 3 min, and then measure the absorbance at 410 nm; One enzyme activity unit (U) represents the amount of enzyme that produces 1 μM p-nitrophenol (pNP) by catalytic hydrolysis of the substrate p-nitrophenyl palmitate (pNPP) within 1 minute.

[0061] The results are as Figure 4 shown. The activity of multi-copper oxidase in the medium inoculated with L. plantarum SH7-pMG36e-luxS strain was significantly higher than that in the medium inoculated with L. plantarum SH7 and L. plantarum SH7-pMG36e strains (P<0.05). It can be seen that the Lactobacillus plantarum overexpressing the luxS gene provided by the present invention has the ability to increase the activity of multi-copper oxidase.

[0062] Example 2: Preparation of a microbial agent containing Lactobacillus plantarum overexpressing the luxS gene

[0063] S1: Pipette 100 - 300 μL of the bacterial liquid of L. plantarum SH7-pMG36e-luxS in the glycerol tube and inoculate it into 500 - 1000 mL of MRS broth medium. Incubate at 37 °C for 24 - 48 h, and repeat this step 2 - 4 times to obtain the activated L. plantarum SH7-pMG36e-luxS bacterial liquid; centrifuge the activated bacterial liquid, discard the supernatant, wash and resuspend it with sterile physiological saline, centrifuge and discard the supernatant, and repeat this step 2 - 3 times to obtain the L. plantarum SH7-pMG36e-luxS bacterial strain active bacterial mud with a cell concentration of 10 6 -10 7 CFU / g;

[0064] S1: Select skim milk emulsion as the freeze-drying protectant, and mix the L. plantarum SH7-pMG36e-luxS bacterial strain active bacterial mud obtained in S1 and the above skim milk emulsion at a mass ratio of 1:10 to obtain an L. plantarum SH-seven-pMG36e-luxS skim milk suspension with a cell concentration of 10 6 -10 7 CFU / mL; pre-culture it at 37 °C for 2 h, then place it in a -40 °C refrigerator for pre-freezing for about 5 - 7 h. After pre-freezing, place it in a vacuum freeze-dryer for freeze-drying to obtain a microbial agent of freeze-dried Lactobacillus plantarum L. plantarum SH7-pMG36e-luxS with a viable cell concentration of 10 6 -10 7 CFU / g.

[0065] Example 3: Application of a microbial agent containing Lactobacillus plantarum overexpressing the luxS gene in the preparation of dried sausage

[0066] (1) Preparation of dried sausage

[0067] Raw materials: 9.0 kg of lean meat (pork tenderloin), 1.0 kg of fat (pork back fat), 0.30 kg of Daqu liquor, 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 (Shiyitang dry sausage seasoning);

[0068] (2) Preparation of starter

[0069] The microbial preparations (L.plantarum SH7-pMG36e-luxS) and L.plantarum SH7 prepared in Example 2 were respectively placed in MRS medium for two subcultures, cultured at 37 °C for 18 h, centrifuged at 4 °C and 10000×g for 5 min, and then reserved; when adding the microbial preparations, the addition amount of the strains was 10 7 CFU / g of meat stuffing;

[0070] Technological process: Raw meat (removing connective tissues such as lymph, tendons, blood vessels, etc.) → Dicing (meat dices of 1 cm 3 in size) → Marinating (adding salt and seasonings, etc.) → Adding starter → Mixing stuffing → Stuffing (length 20 cm, diameter 3 cm) → Fermenting and air-drying (temperature 25 ± 2 °C, humidity 65% ± 5%).

[0071] Effect experiment:

[0072] 1. Determination of biogenic amine content in air-dried sausage

[0073] The determination of biogenic amine content in air-dried sausage was carried out in accordance with 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".

[0074]

[0075] In the formula: C2 is the concentration of biogenic amines in the control group air-dried sausage (mg / kg); C3 is the concentration of biogenic amines in the air-dried sausage with test strains (mg / kg).

[0076] The results are as Figure 5As shown, the content of biogenic amines in the air-dried sausage inoculated with the microbial preparation containing Lactobacillus plantarum overexpressing the luxS gene (L.plantarum SH7-pMG36e-luxS) is lower, significantly lower than that in the air-dried sausage inoculated with L.plantarum SH7 and the non-inoculated treatment group; and at the end of fermentation, the degradation rates of putrescine and cadaverine in the air-dried sausage by the L.plantarum SH7-pMG36e-luxS strain are as high as 75.26% and 61.40% respectively, and the total biogenic amine degradation rate reaches 56.50%. It can be seen that the Lactobacillus plantarum overexpressing the luxS gene provided by the present invention significantly enhances the amine-reducing characteristics of Lactobacillus plantarum SH7 after overexpressing the luxS gene. Therefore, the Lactobacillus plantarum overexpressing the luxS gene provided by the present invention can be used as a functional microbial preparation for reducing biogenic amines and applied to the production of fermented meat products.

[0077] The content not detailedly described in the specification of the present invention is well-known technology to those skilled in the art. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Lactobacillus plantarum overexpressing the luxS gene, characterized in that, The Lactobacillus plantarum is obtained by transforming the plasmid pMG36e-luxS overexpressing the luxS gene into Lactobacillus plantarum SH7.

2. The Lactobacillus plantarum according to claim 1, characterized in that, The preservation number of Lactobacillus plantarum SH7 is CGMCC NO.28113.

3. Use of the Lactobacillus plantarum overexpressing the luxS gene according to any one of claims 1 to 2 in the preparation of dried sausage.

4. A Lactobacillus plantarum microbial preparation, characterized in that, The microbial preparation contains the Lactobacillus plantarum overexpressing the luxS gene according to any one of claims 1 to 2.

5. The preparation method of the Lactobacillus plantarum microbial preparation according to claim 4, characterized in that, The preparation method includes the following steps: activating the Lactobacillus plantarum overexpressing the luxS gene according to any one of claims 1 to 2, centrifuging to discard the supernatant to obtain an active bacterial sludge; mixing the above active bacterial sludge with skim milk emulsion and culturing to obtain a mixture; performing a pre-freezing treatment on the above mixture, and after the pre-freezing is completed, placing it in a vacuum freeze dryer for freeze-drying to obtain a microbial preparation.

6. The preparation method according to claim 5, characterized in that, The cell concentration of Lactobacillus plantarum overexpressing the luxS gene in the active bacterial sludge is 10 6 -10 7 CFU / g.

7. The preparation method according to claim 5, characterized in that, The mixing mass ratio of the active bacterial sludge to the skim milk emulsion is 1:

10.

8. The preparation method according to claim 5, characterized in that, The conditions for culturing after mixing the active bacterial sludge with the skim milk emulsion are: culturing at 37°C for 2 h.

9. The preparation method according to claim 5, characterized in that, The conditions for the pre-freezing treatment are: pre-freezing in a -40°C refrigerator for 5 - 7 h.

10. Use of the microbial preparation of Lactobacillus plantarum according to claim 4 in the preparation of dried sausage.

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