Screening method for rapidly identifying tetragenococcus halophilus and application of screening method
A primer set for PCR amplification of halophilic Tetragenococcus strains addresses the inefficiencies in traditional screening methods by rapidly identifying strains that do not produce biogenic amines, enhancing the stability and quality of fermented foods.
Patent Information
- Application Number
- CN202411460154.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2024-12-13
AI Technical Summary
Existing methods for identifying and screening halophilic Tetragenococcus strains in fermented foods are time-consuming, labor-intensive, and lack specificity, leading to instability in product quality and flavor due to amino acid degradation by certain strains.
A primer set comprising specific primers for 16S rDNA, histidine decarboxylase, and tyramine decarboxylase genes is used for PCR amplification to rapidly and accurately screen halophilic Tetragenococcus strains that do not produce biogenic amines like histamine and tyramine.
The method significantly reduces screening time and cost while ensuring high accuracy, enabling efficient identification of strains with excellent industrial traits, thereby stabilizing the quality and flavor of fermented products.
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Figure CN119120744A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a screening method for rapid identification of halophilic tetragenococci and its application. Background Art
[0002] Halophilic Tetragenococcus is a moderately salt-tolerant lactic acid bacteria widely found in fermented products such as soy sauce and fish sauce. It is salt-tolerant and resistant to high osmotic pressure. It can effectively improve the quality of fermented products and shorten the fermentation cycle. It is an important fermentation strain. Studies have shown that Halophilic Tetragenococcus can increase the content of various flavor substances such as organic acids, aldehydes, esters, amino acids, etc. in fermented products, providing a soft and rich taste to seasonings such as soy sauce and fish sauce.
[0003] However, some strains of halophilic tetragenous cocci can degrade specific amino acids, such as histidine, tyrosine, arginine, serine, threonine, phenylalanine, etc. The degradation of amino acids will lead to a decrease in the amino acid index of soy sauce, thus adversely affecting the flavor and taste of soy sauce. Although the degradation pathways of these amino acids have not been fully elucidated, some of them become decarboxylated substances through decarboxylation reactions. The natural fermentation environment of soy sauce contains a rich composition of lactic acid bacteria, including strains that produce excellent traits and strains that bring adverse metabolites. The uncertainty of their strain metabolism affects the stability of the performance of fermented products such as soy sauce. In the soy sauce system, artificial intervention to strengthen strains with excellent traits is halophilic tetragenous cocci, which can inhibit the growth of adverse strains and reduce the production of adverse metabolites. In order to obtain fermented products with stable and excellent characteristics and prevent the accumulation of adverse metabolites such as amino acid degradation, strengthening halophilic tetragenous cocci strains that cannot metabolize amino acids is being promoted.
[0004] Therefore, it is urgent to screen industrial halophilic tetragenococcus strains with excellent traits. Among the methods for species identification of the screened strains, the most commonly used is genotype identification, that is, separation and identification through highly conserved nucleic acid sequences of microorganisms, including 16S rRNA gene sequence analysis of bacteria and 18S rRNA gene sequence analysis of fungi and ITS region sequence analysis. Genotype identification is to amplify highly conserved nucleic acid sequences through specific primer pairs, and use a variety of high-throughput sequencing platforms to sequence the amplified sequences and then perform sequence comparison analysis through bioinformatics tools to reveal the relationship between microbial species. This method is relatively mature, but amplicon sequencing takes a certain amount of time, and the online comparison and analysis of the obtained sequences is also time-consuming and labor-intensive, and can only obtain species classification information. On the other hand, with the development of sequencing technology, whole genome sequencing and comparison of microorganisms can not only be used for classification and identification, but also more comprehensive genetic information of microorganisms can be obtained, such as whether they carry some specific genes. However, whole genome sequencing requires the extraction of the whole genome sequence of the strain first, which takes a long time to send to the machine for inspection and is expensive, and additional testing of strains of other species also adds unnecessary costs.
[0005] At present, most soy sauce companies adopt the natural fermentation mode. The entire fermentation system is in an open state, relying on natural sun exposure, without adding strains, and relying on microorganisms in the natural environment to participate in fermentation. The participation of various microorganisms such as Aspergillus, yeast, and lactic acid bacteria promotes the formation of more flavor substances, making the soy sauce flavor more unique, but the complex microbial system also leads to the unstable quality of fermented products. Although the Japanese soy sauce process originated from the Chinese soy sauce process, the traditional Chinese natural fermentation process has been improved by temperature-controlled fermentation. It adopts large-scale closed fermentation tanks for fermentation, brine cooling to form koji, artificial enhancement of functional yeast and halophilic lactic acid bacteria, regular ventilation and stirring, etc., which improves the performance and stability of the product. However, for the isolation, identification and screening of halophilic lactic acid bacteria with excellent characteristics, the traditional strain identification method is not specific, resulting in a large amount of sequencing detection, which is relatively time-consuming and labor-intensive, and has low detection efficiency.
[0006] In order to solve the problem of instability in performance and quality of traditional fermented foods, a method for efficiently screening halophilic Tetragenococcus strains with excellent traits is needed. Summary of the invention
[0007] The purpose of the present application is to overcome the problem of instability in performance and quality of traditional fermented foods, and to provide a screening method for rapid identification of halophilic tetragenococci and its application. The screening method of the present application is simple, efficient and highly accurate.
[0008] To achieve the above objectives, this application adopts the following technical solutions:
[0009] The present application provides a primer set for screening or assisting screening of halophilic tetragenococci that do not produce biogenic amines, the primer set comprising a specific primer 1 for 16S rDNA of halophilic tetragenococci, a specific primer 2 for a histidine decarboxylase gene, and a specific primer 3 for a tyramine decarboxylase gene;
[0010] The nucleotide sequence of the specific primer 1 is shown in SEQ NO: 1-2;
[0011] The nucleotide sequence of the specific primer 2 is shown in SEQ NO: 3-4;
[0012] The nucleotide sequence of the specific primer 3 is shown in SEQ NOs: 5-6.
[0013] In the technical solution of the present application, the comparison and analysis of the nucleic acid sequence of halophilic Tetragenococcus revealed a highly conserved specific region on its 16SrDNA. By designing specific primers 1 (P1 and P2, respectively, with nucleotide sequences such as SEQNO: 1 to 2) for PCR amplification, halophilic Tetragenococcus can be screened out quickly and accurately.
[0014] Since halophilic tetragenococci that produce the amino acid metabolites histamine and tyramine usually carry histidine decarboxylase genes and tyramine decarboxylase genes, the present application designs specific primers 2 (P3 and P4, respectively, with nucleotide sequences such as SEQ NOs: 3 to 4, respectively) for the histidine decarboxylase gene and specific primers 3 (P5 and P6, respectively, with nucleotide sequences such as SEQ NOs: 5 to 6, respectively) for the tyramine decarboxylase gene through PCR amplification to simply and efficiently screen for excellent halophilic tetragenococci that do not produce biogenic amines such as histamine and tyramine.
[0015] Preferably, the biogenic amine comprises histamine or tyramine.
[0016] The present application provides a reagent or a kit for screening or auxiliary screening of halophilic Tetragenococcus, wherein the reagent or the kit comprises the above-mentioned primer set.
[0017] By using the reagent or kit containing the primer set, halophilic tetragenococci can be screened and identified quickly and accurately, and excellent halophilic tetragenococci that do not produce biogenic amines such as histamine and tyramine can be screened out efficiently.
[0018] Preferably, the kit further comprises a PCR reaction solution.
[0019] The kit also includes a high-fidelity Taq enzyme premix.
[0020] The present application provides a method for screening or assisting in screening halophilic tetragenococci that do not produce biogenic amines, the method comprising the following steps:
[0021] 1) Extracting DNA from the sample to be tested;
[0022] 2) Using the DNA of the sample to be tested as a template, performing PCR amplification with the above primer set, identifying and screening the selected monoclonal colonies, and obtaining halophilic tetragenococci that do not produce biogenic amines.
[0023] The screening method of the present application first designs specific primers 1 based on the 16S rDNA of halophilic tetragenococci, and can simultaneously realize the rapid identification of halophilic tetragenococci strains and the rapid screening of strains that do not have the ability to metabolize amino acids through PCR amplification; and further designs specific primers 2 for the histidine decarboxylase gene and specific primers 3 for the tyramine decarboxylase gene, and efficiently screens excellent halophilic tetragenococci that do not produce biogenic amines such as histamine and tyramine through PCR amplification.
[0024] The present application uses PCR amplification to achieve specific screening and identification of halophilic tetragenococci, without the need for genome extraction and machine sequencing of the strains, thereby improving screening efficiency and reducing screening costs; based on the specific gene amplification of key enzymes for the production of biogenic amines, strains that do not produce biogenic amines are screened, which further improves the screening efficiency and achieves efficient screening of halophilic tetragenococci strains with excellent industrial traits.
[0025] The method of the present application can simultaneously identify halophilic tetragenococci and screen halophilic tetragenococci that do not produce histamine and tyramine, and achieve high-throughput detection, effectively reducing the labor cost and time cost of gene extraction and sequencing.
[0026] Preferably, the step 1) comprises: taking a sauce mash sample and resuspending it in sterile water to obtain a mixed bacterial solution, diluting the mixed bacterial solution, spreading it on a solid plate, and performing anaerobic culture, picking monoclonal colonies with transparent circles, separating and purifying them, and then resuspending the separated and purified monoclonal colonies in sterile water, and treating them in a water bath to obtain the sample DNA to be tested.
[0027] More preferably, the solid plate is a MRS plus salt (5%-10%) solid plate containing 0.5% calcium carbonate.
[0028] More preferably, the temperature of the water bath treatment is 95-98° C., and the water bath treatment time is 10-15 min.
[0029] Preferably, in step 2), the PCR amplification reaction procedure is: 98°C denaturation for 30s, 60°C annealing for 20s, 72°C extension for 30s, and 30 cycles.
[0030] Preferably, in the step 2), the sample DNA to be tested is used as a template, and the above-mentioned specific primer 1 is used for PCR amplification to identify and screen the picked monoclonal colonies to obtain halophilic tetragenous cocci, and then specific primer 2 or specific primer 3 is used for PCR amplification to screen the picked monoclonal colonies to obtain non-histamine-producing halophilic tetragenous cocci and non-tyramine-producing halophilic tetragenous cocci, respectively.
[0031] The present application provides application of the above method in fermented food.
[0032] The present application adopts the above method to efficiently screen halophilic tetragenococci with excellent traits, solving the problem of instability in performance and quality of traditional fermented foods.
[0033] The present application provides the use of the above primer set or the reagent or kit in screening or auxiliary screening of halophilic Tetragenococcus.
[0034] The present application provides the use of the above primer set in preparing a product for screening or assisting in screening of halophilic Tetragenococcus.
[0035] Preferably, the Tetragenococcus halophilus is a Tetragenococcus halophilus that does not produce biogenic amines.
[0036] In the method of the present application, each PCR reaction requires 12.5 μL of high-fidelity Taq enzyme premix and 12.5 pmol of each primer. The total cost of consumables is approximately RMB 1.75 + 0.1 = 1.85 per reaction. In the case of high-throughput detection, the cost of using the PCR instrument can be ignored. The 16S rRNA base pair sequencing fee of a conventional third-party testing agency is RMB 0.08 per base pair, and the full-length 16S rRNA sequencing fee is approximately RMB 90 per sample.
[0037] PCR amplification of specific short fragments less than 1kb only takes about 60 minutes, and 96 samples can be amplified simultaneously. Conventional 16SrRNA amplification requires 90-100 minutes because the fragment is larger than 1kb, and a third-party testing agency needs at least one day to sequence the amplified fragment, and a BLAST comparison analysis of the sequencing results is required later.
[0038] The PCR amplification method of the present application can further save PCR reaction costs and time costs. The method is simple, efficient and highly accurate.
[0039] Compared with the prior art, the beneficial effects of this application are:
[0040] The present application provides a simple, efficient and highly accurate screening method for rapid identification of halophilic tetragenous cocci. The present application realizes specific screening and identification of halophilic tetragenous cocci, without the need for genome extraction and machine sequencing of strains, thereby improving screening efficiency and reducing screening costs; based on the specific gene amplification of key enzymes for biogenic amine production, screening of strains that do not produce biogenic amines is performed, further improving screening efficiency, and realizing efficient screening of halophilic tetragenous cocci strains with excellent industrial traits. The method of the present application can simultaneously identify halophilic tetragenous cocci and screen halophilic tetragenous cocci that do not produce histamine and tyramine, and realizes high-throughput detection, effectively reducing the labor cost and time cost of gene extraction and sequencing, and solving the problem of instability of performance and quality of traditional fermented foods. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1 This is the band analysis diagram of the product amplified by 16S rDNA specific primers;
[0042] Figure 2 This is a sequencing comparison analysis of the PCR products of colonies 1, 2 and 4;
[0043] Figure 3 This is a BLAST analysis comparison of the sequencing sequences of the PCR products of colonies 1, 2 and 4;
[0044] Figure 4 It is the band analysis diagram of the amplification products of histidine decarboxylase gene and tyrosine decarboxylase gene with specific primers;
[0045] Figure 5 This is the band analysis diagram of the amplification products of 16S rDNA, histidine decarboxylase gene and tyrosine decarboxylase gene using specific primers. DETAILED DESCRIPTION
[0046] To better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific examples and accompanying drawings, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present application are commercially available.
[0047] Example 1
[0048] The 16S rDNA-specific primers P1 and P2 of halophilic Tetragenococcus were designed (the primer sequences are described in Table 1). PCR amplification was performed using P1 and P2 as primers. 12.5 μL of high-fidelity Taq enzyme premix and 1.25 μL of 10 pmol of each primer were added to each reaction solution. The reaction conditions were: denaturation at 98°C for 30 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, and 30 cycles.
[0049] Table 1 16S rDNA specific primers of Tetragenococcus halophilus
[0050]
[0051] The strains screened were amplified by PCR using 16S rDNA specific primers of Tetragenococcus halophilus, and the results were as follows after agarose gel electrophoresis. Figure 1 As shown, colonies 1, 2 and 4 showed specific bands with a band size of about 300 bp (theoretical size is 287 bp).
[0052] The PCR products of colonies 1, 2 and 4 were recovered and purified and sent to a third-party sequencing company for sequencing. The three sequences obtained were compared. The results are as follows: Figure 2 As shown, the similarity of the three sequences is close to 100%.
[0053] One of the sequences was compared with the sequence in Genbank by BLAST analysis. The results are as follows Figure 3 As shown, the sequence was determined to have 100% homology with the 16S rRNA sequence of Tetragenococcus halophilus, confirming that it was Tetragenococcus halophilus.
[0054] Example 2
[0055] Specific primers P3 and P4 for the histidine decarboxylase gene of halophilic Tetragenococcus, and specific primers P5 and P6 for the tyrosine decarboxylase gene were designed. PCR amplification was performed using P3, P4, P5 and P6 as double primers. 12.5 μL of high-fidelity Taq enzyme premix and 1.25 μL of 10 pmol of each primer were added to each reaction solution. The reaction conditions were: denaturation at 98°C for 30 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, and 30 cycles.
[0056] Table 2 Specific primers
[0057]
[0058] The halophilic Tetragenococcus colonies obtained by screening were amplified by PCR using the histidine decarboxylase gene and tyrosine decarboxylase gene specific primers.
[0059] The amplified products were subjected to agarose gel electrophoresis. Figure 4 As shown, two specific bands appeared in colony 1, and the band sizes were about 500 bp (histidine decarboxylase gene specific fragment) and about 1000 bp (tyrosine decarboxylase gene specific fragment). Colonies 2 and 3 only had a band of about 500 bp, and colony 5 only had a band of about 1000 bp. Only colony 4 did not have any bands, so this colony was a halophilic tetragenococcus that carried neither the decarboxylase gene for producing histamine nor the decarboxylase gene for producing tyramine.
[0060] Example 3
[0061] The strains obtained by the above screening were amplified by PCR using the specific primers P1, P2, P3, P4, P5 and P6 shown in Tables 1 and 2 as three primers. 12.5 μL of high-fidelity Taq enzyme premix and 1.25 μL of 10 pmol of each primer were added to each reaction solution. The reaction conditions were: denaturation at 98°C for 30 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, and 30 cycles.
[0062] The amplified products were subjected to agarose gel electrophoresis. Figure 5 As shown, all five colonies showed 16S rDNA-specific fragments (about 250 bp), colonies 2 and 5 showed histidine decarboxylase gene-specific fragments (about 500 bp), colony 4 showed tyrosine decarboxylase gene-specific fragments (about 1000 bp), and colonies 1 and 3 had only one band, which was determined to be halophilic Tetragenococcus that carried neither the histamine-producing decarboxylase gene nor the tyramine-producing decarboxylase gene.
[0063] This example also verifies the economic cost and time cost of 20 samples to be tested, and the results are shown in Table 3. The economic cost of screening the halophilic tetragenococcus that carries neither the decarboxylase gene for producing histamine nor the decarboxylase gene for producing tyramine by the three-primer amplification method of the present application is 6 yuan / sample, and the screening time cost is 1 to 1.5 hours. The economic cost and time cost of the screening method using 16S rDNA sequencing and genome sequencing are much higher than the screening method using three-primer amplification of the present application, and the results are shown in Table 3.
[0064] Table 3 Comparison of economic cost and time cost based on 20 samples
[0065]
[0066]
[0067] Example 4
[0068] The five colonies identified as halophilic Tetragenococcus in Example 3 were inoculated into a saline (5%-10%) MRS liquid culture medium and cultured anaerobically at 30°C until the logarithmic phase, and then transferred to a fermentation medium at a 1% inoculum level (the fermentation medium composition was: 0.5% histidine and 0.5% tyrosine were added to a saline (5%-10%) MRS medium). After the fermentation medium was cultured anaerobically at 30°C for 4-5 days, samples were taken respectively, and the histidine and tyrosine contents of each sample were determined by amino acid analysis, and the histamine and tyramine contents of each group of samples were determined by HPLC.
[0069] Table 4 Determination of biogenic amine content of strains in culture medium system
[0070] Colony Histidine content Tyrosine content Total amino acid content Histamine content Tyramine content 1 0.49g / 100mL 0.49g / 100mL 1.58g / 100mL 0.00g / kg 0.00g / kg 2 0.01g / 100mL 0.50g / 100mL 1.03g / 100mL 3.48g / kg 0.00g / kg 3 0.50g / 100mL 0.49g / 100mL 1.58g / 100mL 0.00g / kg 0.00g / kg 4 0.49g / 100mL 0.01g / 100mL 0.99g / 100mL 0.00g / kg 3.65g / kg 5 0.01g / 100mL 0.49g / 100mL 1.01g / 100mL 3.36g / kg 0.00g / kg
[0071] The results are shown in Table 4. Figure 5 As shown in the figure, the samples of colonies 2 and 5 do not degrade tyrosine to produce tyramine, and the sample of colony 4 does not degrade histidine to produce histamine. The samples of colonies 1 and 3 do not degrade histidine and tyrosine, so they do not contain histamine and tyramine, indicating that the rapid specific primer PCR method can be used to accurately determine whether the strains screened are halophilic tetragenococci with histamine or tyramine decarboxylase, thereby improving the screening efficiency of strains that produce neither histamine nor tyramine.
[0072] Example 5
[0073] The five strains identified as halophilic Tetragenococcus in Example 3 were inoculated into MRS liquid medium containing salt (5%-10%) and cultured anaerobically at 30°C until the logarithmic phase. 6 The inoculum of CFU / kg was transferred to the fermentation crude oil, and samples were taken after anaerobic culture at 30℃ for 7 days. The histidine and tyrosine contents of each sample were determined by amino acid analysis, and the histamine and tyramine contents of each group of samples were determined by HPLC.
[0074] Table 5 Determination of biogenic amine content of strains in crude oil system
[0075] Colony Histidine content Tyrosine content Total amino acid content Histamine content Tyramine content 1 0.160g / 100mL 0.21g / 100mL 7.94g / 100mL 0.02g / kg 0.07g / kg 2 0.01g / 100mL 0.22g / 100mL 7.76g / 100mL 1.28g / kg 0.07g / kg 3 0.15g / 100mL 0.21g / 100mL 8.18g / 100mL 0.03g / kg 0.08g / kg 4 0.16g / 100mL 0.02g / 100mL 7.69g / 100mL 0.02g / kg 1.73g / kg 5 0.01g / 100mL 0.21g / 100mL 7.75g / 100mL 1.27g / kg 0.07g / kg
[0076] The results are shown in Table 5. Figure 5 The colonies of the halophilic Tetragenococcus shown were applied to ferment crude oil. The histamine and tyramine contents of the products of colonies 1 and 3 were very low, while the histamine or tyramine contents of the products of colonies 2, 4, and 5 were relatively high. This indicates that the application of halophilic Tetragenococcus that carries neither the histamine-producing decarboxylase gene nor the tyramine-producing decarboxylase gene in the crude oil fermentation system will not produce histamine and tyramine, and has fermentation stability.
[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.
Claims
1. A primer set for screening or assisting screening of halophilic tetragenococci that do not produce biogenic amines, characterized in that: The primer set includes a specific primer 1 for 16S rDNA of Tetragenococcus halophilus, a specific primer 2 for a histidine decarboxylase gene, and a specific primer 3 for a tyramine decarboxylase gene; The nucleotide sequence of the specific primer 1 is shown in SEQ NO: 1-2; The nucleotide sequence of the specific primer 2 is shown in SEQ NO: 3-4; The nucleotide sequence of the specific primer 3 is shown in SEQ NOs: 5-6.
2. A reagent or kit for screening or assisting screening of halophilic tetragenococci, characterized in that: The reagent or kit comprises the primer set as claimed in claim 1.
3. The reagent or kit according to claim 2, characterized in that The kit also includes a PCR reaction solution.
4. A method for screening or assisting screening of halophilic tetragenococci that do not produce biogenic amines, characterized in that: The method comprises the following steps: 1) Extracting DNA from the sample to be tested; 2) Using the DNA of the sample to be tested as a template, performing PCR amplification with the primer set as claimed in claim 1, identifying and screening the selected monoclonal colonies, and obtaining halophilic tetragenococci that do not produce biogenic amines.
5. The method according to claim 4, characterized in that The step 1) comprises: taking a sauce mash sample and resuspending it in sterile water to obtain a mixed bacterial solution, diluting the mixed bacterial solution, applying it on a solid plate, performing anaerobic culture, picking a monoclonal colony with a transparent circle, separating and purifying it, and then resuspending the separated and purified monoclonal colony in sterile water, treating it in a water bath, and obtaining the sample DNA to be tested.
6. The method according to claim 4, characterized in that In the step 2), the PCR amplification reaction program is: 98° C. denaturation for 30 s, 60° C. annealing for 20 s, 72° C. extension for 30 s, and 30 cycles.
7. The method according to claim 4, characterized in that In the step 2), the sample DNA to be tested is used as a template, and PCR amplification is performed using the specific primer 1 as described in claim 1 to identify and screen the picked monoclonal colonies to obtain halophilic tetragenous cocci, and then PCR amplification is performed using the specific primer 2 or the specific primer 3 as described in claim 1 to screen the picked monoclonal colonies to obtain halophilic tetragenous cocci that do not produce histamine and halophilic tetragenous cocci that do not produce tyramine, respectively.
8. Use of the method according to any one of claims 4 to 7 in fermented food.
9. Use of the primer set according to claim 1 or the reagent or kit according to claim 2 or 3 in screening or auxiliary screening of halophilic Tetragenococcus.
10. Use of the primer set according to claim 1 in preparing a product for screening or assisting in screening of Tetragenococcus halophilus.