Lactic acid bacteria strain with low volatile sulfide yield and construction method thereof

Abstract

The invention discloses a lactic acid bacteria strain with low volatile sulfide yield and a construction method of the lactic acid bacteria strain. The lactic acid bacteria strain is obtained by knocking out a metC gene of cystathionine beta-lyase in a lactic acid bacteria starting strain or down-regulating expression of the metC gene of the cystathionine beta-lyase in the lactic acid bacteria starting strain. The lactic acid bacteria strain constructed by the invention can reduce the generation of volatile sulfides, effectively reduce the unpleasant odor in the fermented food, improve the flavor of the product, and maintain the fermentation performance of lactic acid bacteria at the same time.

Description

Technical Field

[0001] The present application relates to the field of microbial genetic engineering, and in particular, to a lactic acid bacteria strain with low volatile sulfide production and a construction method thereof. Background Art

[0002] Lactic acid bacteria is a general term for a class of bacteria that can produce lactic acid from fermentable carbohydrates. Lactic acid bacteria fermented juice is widely popular due to its high nutritional value, unique flavor and good health benefits. However, the disadvantage of the existing technology is that lactic acid bacteria will produce volatile sulfides, such as dimethyl sulfide and dimethyl trisulfide, during the fermentation process. These compounds will give the juice an unpleasant odor and affect the sensory quality of the product. The traditional solution relies on the addition of chemical inhibitors, but this method may change the original flavor of the juice, and consumers are less and less accepting of chemical additives. Therefore, there is an urgent need for a technical solution that does not rely on chemical additions and can improve the flavor quality of fermented juice. Summary of the Invention

[0003] The present application aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0004] In its first aspect, the present application proposes a lactic acid bacteria strain that produces low levels of volatile sulfides. According to an embodiment of the present application, the lactic acid bacteria strain is obtained by knocking out or downregulating the expression of the metC gene for cystathionine β-lyase in a starting lactic acid bacteria strain. Thus, by knocking out or downregulating the metC gene for cystathionine β-lyase in the starting lactic acid bacteria strain, a key step in the metabolic pathway is blocked, thereby reducing the production of volatile sulfides, effectively reducing unpleasant odors in fermented foods, and improving product flavor while maintaining the fermentation performance of the lactic acid bacteria.

[0005] According to an embodiment of the present application, the low volatile sulfide-producing lactic acid bacteria strain of the first aspect of the invention may also have at least one of the following additional technical features:

[0006] According to an embodiment of the present application, the metC gene has a sequence as shown in SEQ ID NO: 1.

[0007] According to an embodiment of the present application, the starting strain of lactic acid bacteria is Lactococcus lactis strain P1, and its preservation number is CGMCCNO.31831.

[0008] According to an embodiment of the present application, the volatile sulfide includes at least one of methyl mercaptan, dimethyl disulfide and dimethyl trisulfide.

[0009] In the second aspect of this application, a method for constructing the aforementioned lactic acid bacteria strain is proposed. According to an embodiment of this application, the method comprises: using the genome of the starting strain as a template, designing primers to amplify the upstream sequence of the metC gene and the downstream sequence of the metC gene, respectively, to obtain the upstream and downstream fragments of the metC gene; inserting the upstream and downstream fragments of the metC gene into a plasmid to obtain a recombinant plasmid carrying an antibiotic resistance gene; transforming the starting strain with the recombinant plasmid, and screening for antibiotic resistance to obtain the lactic acid bacteria strain. Thus, the method of the second aspect of this application can efficiently construct the aforementioned lactic acid bacteria strain.

[0010] According to an embodiment of the present application, the primers for the upstream fragment of the metC gene have sequences shown as SEQ ID NO: 2 and SEQ ID NO: 3.

[0011] According to an embodiment of the present application, the primers for the downstream fragment of the metC gene have sequences shown as SEQ ID NO: 4 and SEQ ID NO: 5.

[0012] According to an embodiment of the present application, the antibiotic resistance gene includes a chloramphenicol resistance gene.

[0013] According to an embodiment of the present application, the plasmid includes a pSET4s1 plasmid.

[0014] In a third aspect of the present application, a microbial agent is provided. According to an embodiment of the present application, the microbial agent includes the aforementioned lactic acid bacteria strain.

[0015] In a fourth aspect of the present application, a food is provided. According to an embodiment of the present application, the food comprises at least one of the aforementioned lactic acid bacteria strains or the aforementioned microbial agents.

[0016] In a fifth aspect of the present application, a method for preparing food is provided. According to an embodiment of the present application, the method comprises: fermenting a food raw material to be fermented with one or more of the aforementioned lactic acid bacteria strains and the aforementioned microbial agents to obtain the food.

[0017] According to an embodiment of the present application, the food raw materials include: melon, watermelon, cucumber, broccoli, radish, apple, pomegranate, date, citrus, cherry, kiwi, wolfberry, asparagus, carrot, tomato, lychee, raspberry, pineapple, grape, blueberry, sugarcane, and beet.

[0018] According to some specific embodiments of the present application, the food raw materials are preferably melon, watermelon, cucumber, broccoli, and radish.

[0019] In a sixth aspect of the present application, a method for reducing volatile sulfide production by lactic acid bacteria is provided. According to an embodiment of the present application, the method comprises: knocking out or downregulating the expression of the metC gene of cystathionine β-lyase in the lactic acid bacteria, wherein the lactic acid bacteria is Lactococcus lactis strain P1, with a deposit number of CGMCC No. 31831.

[0020] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0022] Figure 1 This is a flowchart for constructing the knockout vector in Example 1 of this application;

[0023] Figure 2 This is the electrophoresis diagram of the assembled fragment PCR product in Example 1 of the present application, where lanes 1 and 2 are the metc-up-cmr-metc-down fragments, and M is a DNA marker.

[0024] Figure 3 This is the electrophoresis diagram of the PCR products of the vector construction colonies in Example 1 of the present application, wherein lanes 1 to 11 are the PCR products of E. coli colonies transformed with the knockout vector pSET4s1-metc-loxp-cmr, and M is a DNA marker;

[0025] Figure 4 This is a schematic diagram of the construction of the gene knockout strain in Example 1 of the present application;

[0026] Figure 5 This is an electrophoresis diagram of the PCR products of the knockout strain constructed in Example 1 of the present application, wherein lanes 1 to 8 are the PCR products of the P1 colony transformed with the knockout vector pSET4s1-metc-loxp-cmr, and M is a DNA marker;

[0027] Figure 6 This is the electrophoresis diagram of the plasmid loss colony PCR in Example 1 of the present application, wherein lanes 1 to 8 are P1ΔmetC colony PCR products, and M is a DNA marker. DETAILED DESCRIPTION

[0028] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0030] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0031] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in this application, but not excluding other contents.

[0032] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0033] Lactic acid bacteria strains

[0034] In its first aspect, the present application proposes a lactic acid bacteria strain that produces low levels of volatile sulfides. According to an embodiment of the present application, the lactic acid bacteria strain is obtained by knocking out the metC gene for cystathionine β-lyase in a starting lactic acid bacteria strain or downregulating the expression of the metC gene for cystathionine β-lyase in the starting lactic acid bacteria strain. Thus, by knocking out or downregulating the metC gene for cystathionine β-lyase in the starting lactic acid bacteria strain, a key step in the metabolic pathway is blocked, thereby reducing the production of volatile sulfides, effectively reducing the unpleasant odor in the fermented juice, improving the product flavor, and maintaining the fermentation performance of the lactic acid bacteria.

[0035] According to an embodiment of the present application, the low volatile sulfide-producing lactic acid bacteria strain of the first aspect of the invention may also have at least one of the following additional technical features:

[0036] According to an embodiment of the present application, the metC gene has a sequence as shown in SEQ ID NO: 1. Thus, the metC gene of the present application encodes cystathionine β-lyase, which participates in the methionine metabolic pathway of Lactococcus lactis. In the methionine metabolic pathway, methionine can generate methyl mercaptan through an elimination reaction under the action of cystathionine β-lyase, or generate α-keto-methylthiobutyric acid through a transamination reaction and further generate methyl mercaptan. Subsequently, methyl mercaptan is converted into dimethyl disulfide and dimethyl trisulfide through autooxidation.

[0037] According to an embodiment of the present application, the starting strain of lactic acid bacteria is Lactococcus lactis strain P1, and the deposit number is CGMCC NO. 31831. Thus, the starting strain Lactococcus lactis strain P1 used in the present application was screened for self-fermented peppers, and the whole genome of strain P1 was sequenced. The accession number of its genome sequence in NCBI is CP118738, and it was deposited by the China General Microbiological Culture Collection Center (CGMCC) on September 3, 2024, with the deposit number CGMCC No. 31831, and classified as Lactococcus lactis strain P1 (Lactococcus lactis). By knocking out or downregulating the metC gene of Lactococcus lactis strain P1, a lactic acid bacteria strain with low volatile sulfide production can be obtained.

[0038] According to an embodiment of the present application, the volatile sulfide includes at least one of methyl mercaptan, dimethyl disulfide, and dimethyl trisulfide. Thus, by reducing the generation of these sulfides, the flavor of the fermented food is effectively improved.

[0039] Method for constructing lactic acid bacteria strains

[0040] In the second aspect of this application, a method for constructing the aforementioned lactic acid bacteria strain is proposed. According to an embodiment of this application, the method comprises: using the genome of the starting strain as a template, designing primers to amplify the upstream sequence of the metC gene and the downstream sequence of the metC gene, respectively, to obtain the upstream and downstream fragments of the metC gene; inserting the upstream and downstream fragments of the metC gene into a plasmid to obtain a recombinant plasmid carrying an antibiotic resistance gene; transforming the starting strain with the recombinant plasmid, and screening for antibiotic resistance to obtain the lactic acid bacteria strain. Thus, the method of the second aspect of this application can efficiently construct the aforementioned lactic acid bacteria strain.

[0041] According to an embodiment of the present application, the primers for the upstream fragment of the metC gene have the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. Thus, the upstream region of the cystathionine β-lyase gene metC in the starting strain of lactic acid bacteria is amplified by precisely designed specific primers, ensuring the accuracy and specificity of gene editing and providing the necessary DNA homology arms for subsequent gene knockout or expression downregulation.

[0042] According to an embodiment of the present application, the primers for the downstream fragment of the metC gene have the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5. Thus, the downstream region of the cystathionine β-lyase gene metC in the starting strain of lactic acid bacteria is directed amplified by precisely designed specific primers, ensuring the accuracy and specificity of gene editing and providing the necessary DNA homology arms for subsequent gene knockout or expression downregulation.

[0043] According to an embodiment of the present application, the antibiotic resistance gene includes a chloramphenicol resistance gene. Thus, during gene knockout or downregulation of lactic acid bacteria strains, the chloramphenicol resistance gene is used as a screening marker to help identify and select bacterial cells that have successfully undergone the desired genetic changes, thereby screening strains containing the target gene modification in the transformed bacterial population, ensuring the accuracy and efficiency of the genetic modification process.

[0044] According to an embodiment of the present application, the plasmid includes a pSET4s1 plasmid. Thus, the pSET4s1 plasmid introduces the recombinant DNA fragment into the starting strain for homologous recombination, and its temperature-sensitive suicide property allows the plasmid to be lost, thereby obtaining a strain that has successfully integrated the target gene modification but no longer contains the plasmid, ensuring the efficiency and accuracy of genetic modification while ensuring that the final fermentation product does not contain any foreign plasmid DNA.

[0045] microbial agents

[0046] In a third aspect of the present application, a microbial agent is provided. According to an embodiment of the present application, the microbial agent includes the aforementioned lactic acid bacteria strain.

[0047] Those skilled in the art will appreciate that the characteristics and advantages described above for the lactic acid bacteria strains are also applicable to the microbial agent and will not be elaborated here.

[0048] food

[0049] In a fourth aspect, this application provides a food product. According to embodiments of this application, the food product includes at least one of the aforementioned lactic acid bacteria strains or microbial agents. This significantly improves the flavor quality of the fermented food and reduces unpleasant odors while maintaining the original nutritional value and benefits of the food.

[0050] Methods of preparing food

[0051] In its fifth aspect, the present application provides a method for preparing food. According to embodiments of the present application, the method comprises fermenting a food raw material to be fermented with one or more of the aforementioned lactic acid bacteria strains and microbial agents to obtain the food. Thus, fermentation using the lactic acid bacteria strains or microbial agents of the present application can significantly improve the flavor quality of the fermented food and reduce unpleasant odors while maintaining the original nutritional value and efficacy of the food.

[0052] According to an embodiment of the present application, the food raw materials include: melon, watermelon, cucumber, broccoli, radish, apple, pomegranate, date, citrus, cherry, kiwi, wolfberry, asparagus, carrot, tomato, lychee, raspberry, pineapple, grape, blueberry, sugarcane, and beet.

[0053] According to some specific embodiments of the present application, the food raw materials are preferably melon, watermelon, cucumber, broccoli, and radish.

[0054] Method for reducing the production of volatile sulfides by lactic acid bacteria

[0055] In a sixth aspect of the present application, a method for reducing volatile sulfide production by lactic acid bacteria is provided. According to an embodiment of the present application, the method comprises: knocking out or downregulating the expression of the metC gene of cystathionine β-lyase in the lactic acid bacteria, wherein the lactic acid bacteria is Lactococcus lactis strain P1, with a deposit number of CGMCC No. 31831.

[0056] The sequences involved in the present invention are detailed in Table 1.

[0057] Table 1. Nucleotide sequence description

[0058]

[0059]

[0060]

[0061] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0062] Example 1: Construction of mutant strains

[0063] (1) Using the genome of the starting strain P1 as a template, the upstream and downstream homologous sequences of the metC gene were amplified:

[0064] The starting strain Lactococcus lactis strain P1 used in this application was isolated by the inventors from fermented chili peppers. Its BioProject No. is PRJNA937623, and its BioSample No. is SAMN33411931. The inventors performed whole-genome sequencing on strain P1, and its genome sequence is registered with NCBI as CP118738. It is deposited with the China General Microbiological Culture Collection (CGMCC) under the accession number CGMCC No. 31831.

[0065] The P1 genome was extracted according to the instructions of the bacterial genome extraction kit. Using P1 genomic DNA as a template, the upstream and downstream homology arms of the metC gene were amplified using the primer pairs metc-y1 / metc-y2 and metc-y3 / metc-y4, respectively. The nucleic acid sequences of the primer pairs metc-y1 / metc-y2 and metc-y3 / metc-y4 are shown in SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, and SEQ ID No: 5. The PCR reaction system and procedure are shown in Tables 2 and 3. Product fragment size was determined by 1% agarose gel electrophoresis, and the gel was excised and recovered.

[0066] Table 2. PCR reaction system

[0067]

[0068] Table 3. PCR reaction conditions

[0069]

[0070] (2) Using the plasmid pPK-Ts-Cmr-Cre as a template, PCR amplify the chloramphenicol resistance gene Cmr fragment:

[0071] Plasmid pPK-Ts-Cmr-Cre was extracted from E. coli DH5α according to the instructions of the bacterial plasmid extraction kit. The extracted plasmid DNA was used as a template to amplify the chloramphenicol resistance gene Cmr fragment using primers TY-loxp71 and TY-loxp66. The sequence of primer TY-loxp71 is shown in SEQ ID No: 6, and the sequence of primer TY-loxp66 is shown in SEQ ID No: 7. The product fragment size was determined by 1% agarose gel electrophoresis and the gel was excised and recovered. The PCR reaction system and reaction conditions used were the same as (1).

[0072] (3) Double digest the plasmid pSET4s1 with restriction endonucleases SacI and XhoI:

[0073] Plasmid pSET4s1 was extracted from E. coli DH5α according to the instructions of the bacterial plasmid extraction kit. Double digestion of pSET4s1 with restriction endonucleases SacI and XhoI was performed at 37°C for 1 hour. Product fragment size was determined by 1% agarose gel electrophoresis and the fragments were recovered from the gel. The enzyme digestion system is shown in Table 4.

[0074] Table 4. Double enzyme digestion system

[0075]

[0076] (4) The upstream and downstream homologous sequences of the metC gene obtained in steps (1), (2), and (3), the Cmr fragment amplification product, and the linearized pSET4s1 plasmid were subjected to Gibson assembly and PCR amplification to obtain a recombinant product:

[0077] like Figure 1 As shown in Table 5, the upstream and downstream homology arms of the metC gene, the Cmr fragment amplification product, and the linearized pSET4s1 plasmid were assembled according to the system in Table 5 and reacted at 50°C for 60 minutes. The above three fragments were assembled into the linearized plasmid pSET4s1 after double enzyme digestion by Gibson to obtain the metc-up-cmr-metc-down fragment. The assembled fragment was used as a template and PCR amplification was performed using primers metc-y1 / metc-y4. The PCR reaction system and procedure were the same as (1). The electrophoresis results of the amplified products are shown in Figure 5. Figure 2 As shown, lanes 1 and 2 represent the metc-up-cmr-metc-down fragment, and M represents a DNA marker. The amplified product of the metc-up-cmr-metc-down fragment should be 2236 bp in size. The electrophoresis results are consistent with the expected size, and the sequencing results are correct, indicating successful fragment assembly.

[0078] Table 5. Assembly system

[0079]

[0080] (5) The recombinant product obtained in step (4) was transformed into Escherichia coli by heat shock transformation method, and transformants were screened using LB resistance plates. Transformants grown on LB resistance plates were selected for PCR verification. Positive transformants were screened and expanded in LB resistance liquid medium. Plasmids were extracted to obtain knockout vectors:

[0081] Add 10 μL of the recombinant product to 100 μL of thawed Trans1-T1 competent cells, gently tap the tube wall to mix, let it stand on ice for 30 minutes, heat shock in a 42°C water bath for 45 seconds, and immediately cool it on ice for 2 minutes. Add 900 μL of LB broth medium, culture at 37°C at 220 rpm for 1 hour, centrifuge at 5000 rpm for 5 minutes, discard 900 μL of supernatant, resuspend the cells in the remaining culture medium, spread on LB plate medium containing chloramphenicol, and culture upside down at 37°C for 12 to 16 hours.

[0082] Positive colonies were selected from the chloramphenicol-resistant plates and identified by colony PCR using primers PSET4s1-YZF and CMR-YZR. The colony PCR system and reaction procedures are shown in Tables 6 and 7. The sequence of primer PSET4s1-YZF is shown in SEQ ID No: 8, and the sequence of primer CMR-YZR is shown in SEQ ID No: 9. Product fragment size was determined by 1% agarose gel electrophoresis. Based on the electrophoresis results, corresponding colonies were selected and expanded in LB liquid medium containing chloramphenicol. Plasmids were extracted and sequenced using primers PSET4s1-YZF and M13R. The sequence of primer M13R is shown in SEQ ID No: 11.

[0083] The assembled fragments were transformed and cultured in E. coli, and the positive transformants were identified and extracted to obtain the metC gene knockout vector. Figure 3 The product size of the knockout vector pSET4s1-metc-loxp-cmr should be 876 bp. The electrophoresis results are consistent with the expected size, and the sequencing results are correct, indicating that the knockout vector was successfully constructed.

[0084] Table 6. Colony PCR system

[0085]

[0086] Table 7. Colony PCR program

[0087]

[0088] (6) The knockout vector obtained in step (5) was transformed into the starting strain P1 by electroporation, and transformants were screened using GM17 resistance plates. Transformants grown on GM17 resistance plates were selected for PCR verification, and positive transformants were screened and expanded in GM17 resistance liquid culture medium to obtain recombinant strains:

[0089] Glycerol-preserved P1 strains were streaked onto GM17 solid plates and incubated anaerobically at 30°C for 36-48 hours. Single colonies were picked and transferred to GM17 liquid medium, cultured overnight at 30°C, and then transferred to GM17 medium at a 1% inoculum size and incubated anaerobically at 30°C for approximately 8 hours to an OD600nm of 1. A 10% inoculum size was inoculated into SGM17 medium and cultured overnight at 30°C. Transferred a second time to SGM17 medium and cultured at 30°C to an OD600 of 0.3-0.4. The cells were centrifuged at 6000 rpm for 10 minutes at 4°C. After harvesting, the cells were resuspended once in pre-chilled electroporation buffers I, II, and I, respectively, incubated on ice for 20 minutes, and centrifuged at 6000 rpm for 10 minutes at 4°C. Finally, the cells were resuspended in 0.1 mL of pre-chilled electroporation buffer I to prepare P1 competent cells.

[0090] Add 1 μg of knockout vector to 100 μL of P1 competent cells and mix thoroughly by pipetting. Transfer to a bacterial electroporation cup (0.2 cm) and place on ice for 5 minutes. Assemble the electroporator and place in the electroporation cup for electroporation transformation. The electroporation parameters are set to 5ms for electric shock time and 2.5kV for voltage. After the electric shock, quickly add 0.9mL of recovery solution to the electroporation cup, transfer the solution in the electroporation cup to a sterile EP tube, and culture at 30°C for 2 hours to allow the cells to recover. Take 200μL of the recovered bacterial solution and spread it on a GM17 plate culture medium containing chloramphenicol, and culture it upside down at 30°C for 36 to 48 hours. The transformed bacteria are identified as positive clones.

[0091] Single colonies on the chloramphenicol-resistant plate were picked and added to 50 μL of lysis buffer, and lysed at 80°C for 15 min. After lysis, the supernatant was centrifuged at low speed and 1 μL was collected for colony PCR according to Tables 8 and 9.

[0092] Schematic diagram of gene knockout strain construction Figure 4 The gene knockout vector pSET4s1-metc-loxp-cmr was electroporated into P1 and cultured under resistance conditions for homologous recombination. The colony PCR results of the positive transformants were shown in Figure 2. Figure 5 Among them, 3 colonies of the ΔmetC transformant had the correct band size, and the product size should be 791 bp.

[0093] Table 8. Colony PCR system

[0094]

[0095] Table 9. Colony PCR reaction program

[0096]

[0097] After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis. After the electrophoresis, the PCR product that matched the size of the target gene was sequenced using M13F and CMR-YZR primers. At the same time, a single colony whose PCR product matched the size of the target gene was inoculated into liquid GM17 broth containing chloramphenicol and cultured overnight. The plasmid was extracted and sequenced using M13F and CMR-YZR primers. The sequence of primer M13F is shown in SEQ ID No: 10.

[0098] (7) Using the thermosensitive suicide property of plasmid pSET4s1, the recombinant strain inoculated on the GM17 resistance plate was subjected to plasmid loss. PCR amplification was performed using primers to verify whether the plasmid was lost, and a knockout mutant strain was obtained:

[0099] Single colonies with correct sequencing results were selected and cultured in GM17 broth containing chloramphenicol at 30°C to an OD600nm of 0.6. The colonies were then streaked onto solid GM17 plates containing chloramphenicol. Utilizing the thermosensitive suicide property of plasmid pSET4s1, the plates were incubated at 39°C for plasmid loss and integration. Positive colonies on the chloramphenicol-resistant plates were selected and identified by colony PCR using primers metc-yzF / metc-yzR / CMR-YZR. The colony PCR system and reaction procedures are the same as those in Tables 6 and 7. Product fragment sizes were determined by 1% agarose gel electrophoresis, and bands of correct size were excised and recovered. Sequencing was performed using primer metc-yzF. Strains with correct sequencing results were designated P1ΔmetC, stored in 50% glycerol solution, and frozen at -80°C for subsequent experiments. The sequences of primer metc-yzF are shown in SEQ ID No: 12, and those of primer metc-yzR are shown in SEQ ID No: 13. The PCR results of positive clones were as follows Figure 6 As shown, one positive clone of the knockout strain had a band of the correct size (number 4). The PCR products of P1ΔmetC should be 854 bp and 2469 bp. The bands of the correct size were recovered and sequenced, and the sequencing results aligned correctly, indicating that the P1ΔmetC knockout strain was successfully constructed.

[0100] Example 2: Application of lactic acid bacteria strains in food

[0101] Illustratively, this embodiment uses muskmelon as a food raw material for fermentation treatment.

[0102] (1) Preparation of melon juice fermented by wild-type strain and mutant strain

[0103] Fresh melons were pre-cooled at 4°C for 24 hours, peeled and deseeded, and the pulp was cut into small pieces of about 20 g. The pulp was then pulped using a juicer and homogenized at 5000×g for 8 minutes using a homogenizer. The homogenized melon juice was placed in food-grade PET / PE bags, pasteurized at 85°C for 15 minutes, and then immediately cooled to 25°C on ice. The wild-type P1 and mutant P1ΔmetC were centrifuged at 10,000×g for 10 minutes, washed twice with sterile saline to remove the culture medium, and finally redissolved in sterile saline to prepare a suspension with an OD600nm = 1 (cell concentration 10 9 CFU / g). The cell suspension was added to pasteurized melon juice at a concentration of 1% (v / v) to obtain 10 7 The initial cell concentration was CFU / g and cultured at 37°C under anaerobic conditions for 24 h.

[0104] (2): Fermentation performance analysis of wild-type strains and mutant strains

[0105] Soluble sugars and organic acids in melon pulp were extracted using high-performance liquid chromatography (HPLC). A 5.0 g melon juice sample was placed in a 10 mL centrifuge tube and centrifuged at 10,000 × g for 10 minutes at 4°C. The supernatant was filtered through a 0.22 μm aqueous filter and then analyzed for soluble sugars and organic acids.

[0106] Waters high performance liquid chromatography (equipped with Alliance e2695 separation module and 2414 differential refractive index detector, quaternary pump) and An amino column (250×4.6 mm, 3.5 μm) was used to determine the fructose, glucose, and sucrose content in the samples. The mobile phase was an acetonitrile / water (80 / 20, v / v) solution containing 0.2% (v / v) triethylamine. The injection volume was 10 μL, the column temperature was 40°C, the flow rate was 0.5 mL / min, and the detection time was 30 min. The three soluble sugars were qualitatively and quantitatively determined by comparing the retention time of the standard and the external calibration curve under the same detection conditions.

[0107] The chromatographic analysis was performed using a Waters high performance liquid chromatography (equipped with an Alliance e2695 separation module and a 2996 diode array detector, a quaternary pump) and a Rezex TM Lactic acid content in samples was determined using a ROA organic acid column (300×7.8mm, 8μm). The following conditions were followed according to the column's manufacturer's instructions: a 5 mmol / L aqueous solution of H₂SO₄ as the mobile phase, an injection volume of 10μL, a column temperature of 50°C, a flow rate of 0.5 mL / min, a detection time of 30 minutes, and a detection wavelength of 210 nm. Lactic acid was qualitatively and quantitatively determined using the retention time of the standard and an external calibration curve under the same conditions. pH was measured directly using a pH meter.

[0108] The results are shown in Table 10. Compared with the initial strain, the fermentation performance of the lactic acid bacteria strain obtained in the present application did not change significantly, and metC gene knockout did not affect the fermentation performance of Lactococcus lactis P1.

[0109] Table 10. Fermentation performance of melon juice fermented by mutants and wild strains

[0110] Wild strain P1 Mutant strain P1ΔmetC Glucose (g / kg) 21.69±0.57 22.38±0.92 Fructose (g / kg) 24.08±0.58 25.02±0.38 Sucrose (g / kg) 41.59±0.57 42.70±0.91 pH 4.30±0.10 4.43±0.10 Lactic acid (g / kg) 6.46±0.23 6.10±0.67

[0111] (3): Aroma analysis of melon juice fermented by wild-type strain and mutant strain

[0112] The volatile aroma compounds were extracted and detected using HS-SPME-GC-MS. The four sulfides were qualitatively and quantitatively analyzed using GC-MS in single ion scanning mode (SIM). Standards of varying concentrations were prepared and a five-point calibration curve was drawn for quantification. The mass-to-charge ratios of the four sulfides are as follows:

[0113] The fragment ion of methyl mercaptan has a mass-to-charge ratio of 48 and a retention time of 2.14 minutes; the fragment ions of dimethyl disulfide have mass-to-charge ratios of 94 and 79 and a retention time of 5.09 minutes; the fragment ions of 3-methylthiopropionaldehyde have mass-to-charge ratios of 48 and 104 and a retention time of 11.56 minutes; and the fragment ion of dimethyl trisulfide has mass-to-charge ratios of 126 and 79 and a retention time of 13.72 minutes. All other conditions in SIM mode were the same as those in SCAN mode.

[0114] In order to verify the effect of metC gene deletion on the aroma production performance of P1, the inventors conducted quantitative detection of the aroma of melons fermented by wild strains and mutant strains. The results are shown in Table 10. In terms of dimethyl sulfide content, compared with the dimethyl disulfide (25.35 μg / kg) and dimethyl trisulfide (5.94 μg / kg) contents of melon juice fermented by wild-type strain P1, the contents of dimethyl disulfide and dimethyl trisulfide in melon juice fermented by mutant strain P1ΔmetC in which the cystathionine β-lyase gene metC was knocked out were significantly reduced (p<0.05), and the content of their precursor methyl mercaptan was also significantly reduced (p<0.05), which is consistent with the predicted main production pathway of dimethyl sulfide. Knockout of the metC gene results in the inability to form the cystathionine β-lyase (CBL) it encodes, which in turn blocks the pathway for the formation of methyl mercaptan by β elimination of methionine, ultimately leading to a reduction in the content of dimethyl sulfide derived from methyl mercaptan. However, there was no significant difference in the content of 3-methylthiopropionaldehyde between the two strains after fermentation. This is because in addition to the main elimination pathway mentioned above, there is also a secondary pathway for methionine to be converted into 3-methylthiopropionaldehyde: methionine is converted into α-keto-methylthiobutyric acid (KMBA) under the action of branched-chain amino acid transaminase, and KMBA can be decarboxylated to 3-methylthiopropionaldehyde under the action of α-ketoacid decarboxylase.

[0115] The results showed that the knockout strain P1ΔmetC constructed in the present invention achieved the effect of reducing the volatile sulfide content after fermentation in melon juice without affecting other fermentation properties.

[0116] Table 11. Volatile sulfide concentrations in fermented melon juice from mutants and wild-type strains (μg / kg)

[0117]

[0118] Note: Different lowercase letters in the same row indicate significant differences (p<0.05).

[0119] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0120] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A lactic acid bacteria strain with low volatile sulfide production, characterized in that: The lactic acid bacteria strain is obtained by knocking out the metC gene of cystathionine beta-lyase in the starting lactic acid bacteria strain or down-regulating the expression of the metC gene of cystathionine beta-lyase in the starting lactic acid bacteria strain.

2. The lactic acid bacteria strain according to claim 1, characterized in that The metC gene has a sequence as shown in SEQ ID NO:

1.

3. The lactic acid bacteria strain according to claim 1, characterized in that The lactic acid bacteria starting strain is Lactococcus lactis strain P1, and its preservation number is CGMCC NO.31831.

4. The lactic acid bacteria strain according to claim 1, characterized in that The volatile sulfide includes at least one of methyl mercaptan, dimethyl disulfide and dimethyl trisulfide.

5. A method for constructing the lactic acid bacteria strain according to any one of claims 1 to 4, characterized in that: include: Using the genome of the starting strain as a template, primers are designed to amplify the upstream sequence of the metC gene and the downstream sequence of the metC gene, respectively, to obtain the upstream fragment of the metC gene and the downstream fragment of the metC gene; inserting the upstream fragment of the metC gene and the downstream fragment of the metC gene into a plasmid to obtain a recombinant plasmid carrying an antibiotic resistance gene; The recombinant plasmid is used to transform the starting strain, and the lactic acid bacteria strain is obtained by screening for antibiotic resistance.

6. The method according to claim 5, characterized in that The primers for the upstream fragment of the metC gene have the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; Optionally, the primers for the downstream fragment of the metC gene have the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5; Optionally, the antibiotic resistance gene comprises a chloramphenicol resistance gene; Optionally, the plasmid comprises a pSET4s1 plasmid.

7. A microbial agent, characterized in that: The invention comprises the lactic acid bacteria strain according to claim 1.

8. A food, characterized in that The method comprises at least one of the lactic acid bacteria strain according to claim 1 or the microbial agent according to claim 7.

9. A method for preparing food, characterized in that: include: Fermenting one or more of the lactic acid bacteria strain according to claim 1 and the microbial agent according to claim 7 with a food raw material to be fermented to obtain the food; Optionally, the food ingredients include: melon, watermelon, cucumber, broccoli, radish, apple, pomegranate, jujube, citrus, cherry, kiwi, wolfberry, asparagus, carrot, tomato, lychee, raspberry, pineapple, grape, blueberry, sugarcane, and beet.

10. A method for reducing the production of volatile sulfides by lactic acid bacteria, characterized in that: include: The metC gene of the cystathionine beta-lyase of the lactic acid bacteria is knocked out or down-regulated. The lactic acid bacteria is the Lactococcus lactis strain P1, with a preservation number of CGMCC NO.31831.

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