PCR primer combination for detecting mixed microorganisms in food fermentation process and detection method thereof

Abstract

The invention belongs to the technical field of microbiological detection, and discloses a PCR primer combination for detecting mixed microorganisms in a food fermentation process and a detection method thereof. The primer combination comprises a primer pair used for detecting pediococcus pentosaceus, a primer pair used for detecting plant lactobacillus, a primer pair used for detecting lactobacillus mucus fermentation, a primer pair used for detecting bacillus amyloliquefaciens and a primer pair used for detecting lactobacillus acidophilus. The primer pair is at least one of a primer pair for detecting lactobacillus helveticus and a primer pair for detecting acetobacter pasteurianus. The primer combination or the kit containing the primer combination can realize species level specific detection of microorganisms, and can be used for qualitatively / quantitatively detecting food and key functional microorganisms in the food fermentation process, so that the dynamic change of a target flora in the fermentation process is quickly and accurately monitored.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, and specifically discloses a PCR primer combination and its detection method for detecting mixed microorganisms in food fermentation process. Background Technology

[0002] Fermented foods are a type of food processed using beneficial microorganisms. The growth and metabolism of these microorganisms during fermentation play a decisive role in the quality of the fermented foods. Metabolic products such as alcohols, esters, and acids produced by microorganisms during fermentation directly affect the flavor and quality of the product. Therefore, achieving qualitative and quantitative detection of the microbial community structure and quantity during the fermentation process of fermented foods in a short time is crucial for improving the quality and ensuring the stability of fermented products.

[0003] Early researchers primarily relied on methods for quantitatively studying microorganisms during food fermentation. This involved isolating and identifying microorganisms at different stages of fermentation, studying changes in their types and quantities. Alternatively, they fermented isolated purebred strains and analyzed their metabolites to study the function of the isolated strains. With the development of molecular biology techniques, the use of non-culture methods to study the microbial community of fermented foods has attracted considerable interest from researchers.

[0004] Common non-cultured microbial strain detection techniques include PCR-DGGE, phospholipid fatty acid analysis (PLFA), fluorescence in situ hybridization (FISH), flow cytometry, and metagenomics based on high-throughput sequencing. PCR-DGGE has limitations such as multiple copies and the inability to achieve absolute quantification. FISH requires sophisticated probe design, while flow cytometry has high equipment requirements and high detection costs. Various omics methods are increasingly being applied to the study of fermented food microorganisms, such as metagenomics, metagenomics transcriptomics, and metabolomics. Integrating multiple omics methods enables microbial diversity analysis, functional studies, and interaction studies. While these methods offer various advantages, due to the complex structure of microbial communities during food fermentation, high interspecies sequence homology, potential interference from fermentation products, and dynamic changes in the fermentation substrate, there is currently no rapid and accurate detection and monitoring method suitable for mixed microbial communities in fermented foods. Summary of the Invention

[0005] To address these shortcomings, this invention provides a PCR primer combination and detection method for detecting mixed microorganisms in food fermentation processes. This invention targets *Pediococcus pentosaceus*, a mixed microorganism that may need to be detected during food fermentation. Pediococcus pentosaceus Lactobacillus plantarum ( Lactiplantibacillus plantarum ), fermenting lactobacillus (Limosilactobacillus fermentum Bacillus amyloliquefaciens, Bacillus amyloliquefaciens Lactobacillus acidophilus, Lactobacillus acetotolerans Lactobacillus helveticus, Lactobacillus helveticus and Acetobacter pasteurianus. Acetobacter pasteurianus The present application solves the problem of rapid and accurate quantification of mixed microorganisms in food fermentation by designing specific primer combinations and combining PCR technology.

[0006] To achieve the above object, the present application provides the following technical solutions. In a first aspect, the present application provides a PCR primer combination for detecting mixed microorganisms in food fermentation, which includes at least one of a primer pair for detecting Pediococcus pentosaceus, a primer pair for detecting Lactobacillus plantarum, a primer pair for detecting Lactobacillus fermentum, a primer pair for detecting Bacillus amyloliquefaciens, a primer pair for detecting Lactobacillus acidophilus, a primer pair for detecting Lactobacillus helveticus, and a primer pair for detecting Acetobacter pasteurianus. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Pediococcus pentosaceus are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Lactobacillus plantarum are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Lactobacillus fermentum are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Bacillus amyloliquefaciens are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Lactobacillus acidophilus are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Lactobacillus helveticus are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. The specific base sequences of the upstream primer and the downstream primer in the primer pair for detecting Acetobacter pasteurianus are shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

[0007] Preferably, the primer combination consists of a primer pair for detecting Pediococcus pentosaceus, a primer pair for detecting Lactobacillus plantarum, a primer pair for detecting Lactobacillus fermentum, a primer pair for detecting Bacillus amyloliquefaciens, a primer pair for detecting Lactobacillus acidophilus, a primer pair for detecting Lactobacillus helveticus, and a primer pair for detecting Acetobacter pasteurianus.

[0008] In a second aspect, the present application further provides a kit for qualitatively / quantitatively detecting mixed microorganisms in food fermentation, which comprises the primer combination described above.

[0009] Preferably, the kit is a digital PCR kit or a real-time PCR kit.

[0010] Preferably, the kit is a digital PCR kit or a real-time PCR kit.

[0011] Optionally, the real-time PCR kit further comprises consumables required for real-time PCR.

[0012] The consumables include, but are not limited to, Nuclease-free Water, 96-well plates, 96-well plate films, sterile test tubes, and 8-tube racks with lids.

[0013] Optionally, the digital PCR kit further comprises consumables required for digital PCR.

[0014] The consumables include, but are not limited to, droplet generation cards, droplet generation oil, 96-well plates, 96-well plate films, sterile test tubes, and Nuclease-free Water.

[0015] In a third aspect, the present application further provides use of the primer combination described above or the kit described above in qualitative / quantitative detection of at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus, and Acetobacter pasteurianus.

[0016] Preferably, the use is use of the primer combination or the kit in qualitative / quantitative detection of at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus, and Acetobacter pasteurianus in fermented food and food fermentation.

[0017] Preferably, the use is use of the primer combination or the kit in monitoring changes in abundance of at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus, and Acetobacter pasteurianus in food fermentation.

[0018] In a fourth aspect, the present application further provides a method for rapidly detecting key microorganisms in a food fermentation process, the key microorganism being at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus mucosus, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurianus; the method comprising the following steps: S1: collecting microorganisms from a food fermentation process sample or extracting the genome of the microorganisms in the sample; S2: using the microorganisms or the genome of the microorganisms collected in S1 as a template to be tested, performing PCR gene amplification using the above-mentioned primer combination or the above-mentioned kit to obtain a PCR product; S3: characterizing the PCR product to obtain detection information.

[0019] The purpose of the characterization of the PCR product in step 3 is to confirm the specificity, yield or size of the amplification. Agarose gel electrophoresis can be used to characterize the PCR product, and the presence or absence of a 256 bp amplification band in the gel electrophoresis map indicates the presence or absence of Pediococcus pentosaceus in the sample; the presence of a 397 bp amplification band in the gel electrophoresis map indicates the presence of Lactobacillus plantarum in the sample; the presence of a 291 bp amplification band indicates the presence of Lactobacillus mucosus in the sample; the presence of a 168 bp amplification band in the gel electrophoresis map indicates the presence of Bacillus amyloliquefaciens in the sample; the presence of a 303 bp amplification band in the gel electrophoresis map indicates the presence of Lactobacillus acidophilus in the sample; the presence of a 127 bp amplification band in the gel electrophoresis map indicates the presence of Lactobacillus helveticus in the sample; and the presence of a 315 bp amplification band in the gel electrophoresis map indicates the presence of Acetobacter pasteurianus in the sample.

[0020] Preferably, the reaction system of the PCR is 50 μL, and the system comprises 2×Phanta Flash Master Mix 25 μL, nuclease-free water 20 μL, 2 μL of each of the upstream primer and the downstream primer of the same primer pair, and 1 μL of a template; the concentration of each of the primer pair is 10 μmol / L; and the template comprises microorganisms or the genome of the microorganisms in the food fermentation process sample.

[0021] Preferably, the reaction program of the PCR is as follows: 95℃ 5 min; 95℃ 1 min, 55℃ 1 min, 72℃ 15 s, 40 cycles; 72℃ 1 min; 4℃ storage.

[0022] In a fifth aspect, the present application further provides a method for rapidly and quantitatively detecting key microorganisms in a food fermentation process, which comprises using the digital PCR technology or the fluorescent quantitative PCR technology, and using the primer combination or the kit described above to detect and analyze the key microorganisms in the food fermentation process; the key microorganisms are at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus mucosus, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurianus.

[0023] Preferably, the reaction system of the digital PCR is 20 μL, and the system comprises 2×QX200 TM ddPCR TM EvaGreen Supermix for Probes 10 μL, Nuclease-free Water 7 μL, 1 μL of each of the upstream primer and the downstream primer of the same primer pair, and 1 μL of a template; the concentration of each of the primer pair is 10 μmol / L; the template comprises microorganisms or the genome of microorganisms in a food fermentation process sample; The reaction procedure of the digital PCR is as follows: 95℃ for 5 min; 95℃ for 1 min, 55℃ for 1 min, 40 cycles; 4℃ for 5 min; 90℃ for 5 min.

[0024] Preferably, the reaction system of the fluorescent quantitative PCR is 20 μL, and the system comprises SYBR Premix Ex Taq GC (2×) 10 μL, ROX Reference Dye (50×) 0.4 μL, Nuclease-free Water 6.8 μL, 0.4 μL of each of the upstream primer and the downstream primer of the same primer pair, and 2 μL of a template; the concentration of each of the primer pair is 10 μmol / L; the template comprises microorganisms or the genome of microorganisms in a food fermentation process sample; The reaction procedure of the fluorescent quantitative PCR module is as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 45 cycles; 95℃ for 15 s, 60℃ for 1 min; 95℃ for 15 s.

[0025] Preferably, the preparation method of the template is as follows: 1 g of a sample in a food fermentation process is placed in a 10 mL test tube, and then 9 mL of sterile water is added; the test tube is placed in an ultrasonic cleaning instrument for ultrasonic treatment for 10 min, and then the supernatant is taken as the template.

[0026] In a sixth aspect, the present application further provides a method for monitoring the abundance change of key microorganisms in a food fermentation process, wherein samples are taken at different fermentation times in the food fermentation process, the key microorganisms in the samples are quantitatively detected by using the above method, and the abundance change of each key microorganism in the fermentation process is analyzed according to the obtained copy number or relative fluorescence intensity; the key microorganism is at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurianus.

[0027] The present application has the following beneficial effects: The present application provides a PCR primer combination for mixed microorganisms in a food fermentation process, wherein specific primer pairs for Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurianus are designed. The present application designs specific primers by accurately comparing the 16S rDNA sequence difference intervals of the seven strains, ensures the compatibility of the seven specific primer pairs in the same PCR system and meets the high specificity requirements of digital PCR and fluorescent quantitative PCR, can simultaneously realize specific qualitative detection and high-sensitivity absolute quantification of the seven key strains, and has strong anti-interference ability (tolerance to fermentation products and dynamic fermentation substrate interference) and excellent intra-species universality. The present application provides an efficient and accurate multi-strain dynamic monitoring technical method for fermented food production. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only correspond to some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0029] Figure 1 PCR verification results of the specificity of the primers in Example 1; (in the figure: M represents DL500 marker; lanes 1-7 represent Lactobacillus helveticus, Acetobacter pasteurianus, Lactobacillus acidophilus, Bacillus amyloliquefaciens, Lactobacillus plantarum, Pediococcus pentosaceus and Lactobacillus fermentum, respectively) Figure 2 PCR verification results of the specificity and anti-interference of the primers in Example 1; (in the figure: M represents DL500 marker; lanes 1-7 represent Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus plantarum, Pediococcus pentosaceus, Acetobacter pasteurianus and Bacillus amyloliquefaciens, respectively) Figure 3PCR detection results of different primer pairs of Acetobacter pasteurii in Comparative Example 1; (M represents DL1000 marker; lane 1 represents primer pair Ap-F2 / Ap-R2; lane 2 represents primer pair Ap-F / Ap-R) Figure 4 Electrophoretogram of detection of key microorganisms in food fermentation process by different primers in Comparative Example 2; (M represents DL500 marker; lanes 1-7 represent Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus mucosus, Lactobacillus plantarum, Acetobacter pasteurii, Bacillus amyloliquefaciens and Lactobacillus helveticus, respectively) Figure 5 Digital PCR detection results of key microorganisms in food fermentation process in Example 2; Figure 6 Digital PCR detection results of key microorganisms in food fermentation process in Example 4; Figure 7 Fluorescence quantitative PCR amplification curve of key microorganisms in food fermentation process in Example 6; Figure 8 Abundance change of key microorganisms in solid-state fermentation process of vinegar brewing in Application Example 1; Figure 9 Abundance change of key microorganisms in liquid-state fermentation process of vinegar brewing in Application Example 2; Figure 10 Abundance change of key microorganisms in fermentation process of Hongfang sufu in Application Example 3; Figure 11 Abundance change of key microorganisms in fermentation process of soy sauce in Application Example 4; Figure 12 Abundance change of key microorganisms in fermentation process of chili sauce in Application Example 5. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0031] Source of biological materials used in the embodiments of the present application: The Lactobacillus plantarum, Lactobacillus mucosus, Bacillus amyloliquefaciens, Lactobacillus acidophilus and Pediococcus pentosaceus used in the present application are Lactobacillus plantarum (CGMCC 1.2437), Lactobacillus mucosus (CGMCC 1.1880), Bacillus amyloliquefaciens (CGMCC 1.1881), Lactobacillus acidophilus (CGMCC 1.1882) and Pediococcus pentosaceus (CGMCC 1.1883), respectively. Lactiplantibacillus plantarum Limosilactobacillus fermentum Bacillus amyloliquefaciens ​​) CGMCC 1.10901, Lactobacillus acidophilus (ATCC 4356) Lactobacillus acetotolerans ) CICC 10774, Pediococcus pentosaceus (ATCC 14067) Pediococcus pentosaceus ) CICC 24630, All the strains are from the market.

[0032] The Lactobacillus helveticus and the Acetobacter pasteurianus used in the present application are Lactobacillus helveticus CGMCC 12062 and Acetobacter pasteurianus CGMCC 3089 strains screened and preserved by the Systematic Microbiology and Biological Manufacturing Group of Tianjin University of Science and Technology from vinegar grains, and the strains have been disclosed in Sequential bioaugmentation of the dominant microorganisms to improve the fermentation and flavor of cereal vinegar (2024.11.1).

[0033] The culture medium used in the embodiments of the present application is as follows: MRS liquid medium (g / L): glucose 20, proteose peptone 10, beef infusion powder 8, yeast extract powder 4, potassium phosphate dibasic 2, ammonium citrate dibasic 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 80 1, pH 6.0, sterilized at 121°C for 20 min. If a solid culture medium is prepared, 15.0 g / L of agar is added.

[0034] LB liquid medium (g / L): tryptone 10, yeast extract 5, sodium chloride 10, pH 7.0-7.4, sterilized at 121°C for 20 min. If a solid culture medium is prepared, 15.0 g / L of agar is added.

[0035] GY liquid medium (g / L): glucose 20, yeast extract 5, pH 6.0, sterilized at 115°C for 15 min. If a solid culture medium is prepared, 15.0 g / L of agar is added.

[0036] Unless otherwise specified, the technical means used in the embodiments is the conventional means familiar to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used are commercially available.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as familiar to one skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. The preferred implementation methods and materials described herein are only for demonstration.

[0038] Example 1 primer design and specificity verification 1. Sequence analysis and primer design Based on the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, Pediococcus pentosaceus ( Pediococcus pentosaceus Lactobacillus plantarum ( Lactiplantibacillus plantarum ), fermenting lactobacillus ( Limosilactobacillus fermentum ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), acid-resistant lactobacillus ( Lactobacillus acetotolerans Lactobacillus helveticus ( Lactobacillus helveticus ) and Pasteurella multocida ( Acetobacter pasteurianus The 16S rDNA gene sequence information of the strains was obtained, and the specific sequences of the 16S rDNA of different strains were analyzed using the multiple sequence alignment tool in Snapgene7 (Sequence ID: NR_042439.1, Sequence ID: ON116120.1, Sequence ID: ON340628.1, Sequence ID: NR_044699.2, Sequence ID: FN429065.1, Sequence ID: MW564017.1 and Sequence ID: PP236940.1), as detailed in Table 1.

[0039] Using the gene fragments shown in the above specific sequences as template sequences, specific amplification primers for different strains were designed, as shown in Table 1. The primer sequences were synthesized and purified by Genewiz Biotechnology Co., Ltd.

[0040] Table 1 Primer Sequences

[0041] 2. Primer specificity verification Bacterial strains stored at -80℃ were inoculated into corresponding 5 mL liquid culture media for cultivation. Specifically, *Pediococcus pentosaceus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus acidophilus*, and *Lactobacillus helveticus* were inoculated into MRS liquid medium and cultured statically at 37℃ for 24 h after inoculation. *Bacillus amyloliquefaciens* was inoculated into LB liquid medium and cultured with shaking at 37℃ for 24 h after inoculation. *Acetobacter pasteurellium* was inoculated into GY liquid medium and cultured with shaking at 30℃ for 24 h after inoculation. The culture media of each strain were then diluted with physiological saline to adjust the bacterial concentration to 10. 3 After mixing CFU / mL, the culture media of each strain were mixed in equal proportions to prepare a mixed bacterial system.

[0042] Using the specific primer pairs designed in Table 1, colony PCR amplification was performed on the mixed bacterial system, and the gel electrophoresis results are as follows: Figure 1 As shown.

[0043] PCR reaction system (50 μL): 10 μmol / L of the upstream and downstream primers 2 μL each, 2x Phanta Flash MasterMix 25 μL, mixed microbial system 1 μL, Nuclease-free Water 20 μL.

[0044] PCR reaction program: 98℃ 1 min; 98℃ 10 s, 55℃ 5 s, 72℃ 15 s, 35 cycles; 72℃ 1 min.

[0045] From Figure 1 It can be known that under the same PCR condition, the primer pairs of different strains show good strain specificity, and only one specific band is produced. Subsequently, the amplified bands are sequenced by Jinweizhi Biotechnology Co., Ltd., and the sizes of the amplified bands from left to right are 256 bp, 397 bp, 291 bp, 168 bp, 303 bp, 127 bp and 315 bp respectively. Through sequence comparison, it is determined that the sequences of the obtained amplified bands are consistent with the corresponding strain-specific sequences, proving that the primer pairs provided by the application have good specificity.

[0046] 3. Verification of primer specificity and anti-interference 1 g of the sample in the solid-state fermentation of fermented vinegar was taken into a 10 mL test tube, 9 mL of sterile water was then added, ultrasonic cleaning was performed for 10 min in an ultrasonic cleaner, and then the supernatant was taken. The specific primer pairs in Table 1 were used for colony PCR amplification.

[0047] The PCR reaction system was 50 μL, and the system included 2x Phanta Flash Master Mix 25 μL, Nuclease-free Water 20 μL, 10 μmol / L of the upstream and downstream primers 2 μL each (the specific primer pairs in Table 1 were used for different species of microorganisms), and 1 μL of the supernatant.

[0048] PCR reaction program: 98℃ 1 min; 98℃ 10 s, 55℃ 5 s, 72℃ 15 s, 35 cycles; 72℃ 1 min.

[0049] After the PCR amplification, 10 μL of the amplified product was taken for electrophoresis analysis by 1.5% agarose gel, and a photograph was taken in a gel imaging system to verify the size of the PCR product. Figure 2). There are amplified fragments with sizes of 256 bp, 397 bp, 291 bp, 168 bp, 303 bp, 127 bp and 315 bp. Then, the amplified bands are sequenced by Jinweizhi Biotechnology Co., Ltd. Through sequence alignment, it is determined that the amplified bands correspond to Pediococcus pentosaceus (256 bp), Lactobacillus plantarum (397 bp), Lactobacillus fermentum (291 bp), Bacillus amyloliquefaciens (168 bp), Lactobacillus acidophilus (303 bp), Lactobacillus helveticus (127 bp) and Acetobacter pasteurianus (315 bp), proving that the detection primer set provided in the application still maintains good specificity and anti-interference in the complex food fermentation system detection and the complex detection environment of multiple products (such as protease).

[0050] Comparative Example 1 The present comparative example provides a specific primer of Acetobacter pasteurianus, and the primer sequence is shown in the following table.

[0051] Table 2 Acetobacter pasteurianus primer pair

[0052] The strains stored at -80℃ were inoculated into corresponding 5 mL liquid medium for culture, wherein Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus helveticus were inoculated into MRS liquid medium, and the strains were inoculated and cultured at 37℃ for 24 h; Bacillus amyloliquefaciens was inoculated into LB liquid medium, and the strain was inoculated and cultured at 37℃ for 24 h; Acetobacter pasteurianus was inoculated into GY liquid medium, and the strain was inoculated and cultured at 30℃ for 24 h. The cultured strain culture was diluted with normal saline, and the bacterial concentration was adjusted to 10 3 CFU / mL, and the strain culture liquids were mixed in equal proportions to obtain a mixed bacteria system.

[0053] The Acetobacter pasteurianus in the mixed bacteria system was subjected to colony PCR amplification using primer pairs Ap-F / Ap-R (provided in Example 1) and Ap-F2 / Ap-R2, respectively.

[0054] The PCR reaction system (50 μL) was as follows: 2 μL of 10 μmol / L upstream and downstream primers (Ap-F / Ap-R or Ap-F2 / Ap-R2), 25 μL of 2×Phanta Flash Master Mix, 1 μL of mixed bacteria system, and 20 μL of Nuclease-free Water.

[0055] PCR reaction procedure: 98℃ pre-denaturation 1 min; 98℃ denaturation 10 s, 55℃ annealing 5 s, 72℃ extension 15 s, 35 cycles; 72℃ final extension, 1 min.

[0056] After the PCR amplification, 10 μL of the product was taken for agarose gel electrophoresis, and the results of the gel electrophoresis are shown in Fig. 1. It can be seen that under the same conditions, the primer pair Ap-F2 / Ap-R2 has multiple bands (lane 1), indicating that the primer pair has non-specific binding to the homologous sequences in the genomes of some strains in the mixed bacterial system, resulting in non-specific amplification. In contrast, the primer pair Ap-F / Ap-R has only one specific band (lane 2), and the primer pair Ap-F / Ap-R only amplifies the target sequence during PCR, indicating that the primer pair Ap-F / Ap-R has high specificity and can effectively distinguish the target strain from other possible microorganisms. Figure 3 Comparative Example 2

[0057] This comparative example provides a Lactobacillus helveticus specific primer, and the primer sequence is shown in the table below.

[0058] Table 3 Lactobacillus helveticus primer pairs

[0059] 1 g of the sample in the solid-state fermentation of the fermented vinegar was placed in a 10 mL test tube, 9 mL of sterile water was then added, and the mixture was ultrasonically treated in an ultrasonic cleaner for 10 min, after which the supernatant was aspirated. The primers Lp-F / Lp-R, Lf-F / Lf-R, La-F / La-R, Ap-F / Ap-R, Pp-F / Pp-R and Ba-F / Ba-R in Example 1 and the primer pair Lh-F2 / Lh-R2 provided in Comparative Example 2 were used to detect the colonies in the food fermentation process by colony PCR.

[0060] The PCR reaction system was 50 μL, and the system included 2×Phanta Flash Master Mix 25 μL, nuclease-free water 20 μL, 10 μmol / L of the upstream and downstream primers 2 μL each (corresponding to the use of the above specific primer pairs for different species of microorganisms), and 1 μL of the supernatant.

[0061] PCR reaction procedure: 98℃ pre-denaturation 1 min; 98℃ denaturation 10 s, 55℃ annealing 5 s, 72℃ extension 15 s, 35 cycles; 72℃ final extension, 1 min; 4℃ storage.

[0062] ​After PCR amplification, 10 μL of the amplification product was taken for electrophoresis analysis on a 1.5% agarose gel, and a photograph was taken in a gel imaging system, and the results are shown in Figure 4 As can be seen from the figure, the primer pair Lh-F2 / Lh-R2 provided by Comparative Example 2 has multiple bands (lane 7), indicating that the primer pair has poor specificity in the detection of complex food fermentation systems.

[0063] Example 2 Verification of digital PCR primers and dynamic range verification experiment 1 g of sample in the fermentation process of solid-state fermented vinegar was taken into a 10 mL test tube, and then 9 mL of sterile water was added. The test tube was placed in an ultrasonic cleaner for 10 min, and then the supernatant was aspirated. The obtained supernatant was stored in a -20℃ refrigerator for standby. The supernatant was gradient diluted with physiological saline to obtain a series of sample supernatants with bacterial concentrations of 10 0 -10 7 copies / mL. Subsequently, the sample supernatants with different concentrations were subjected to digital PCR reaction.

[0064] The digital PCR reaction system was 20 μL, and the system included 2×QX200 TM ddPCR TM EvaGreen Supermix for Probes 10 μL, 10 μmol / L of each 1 μL of upstream and downstream primers (corresponding to the specific primer pairs provided by Example 1 for different species of microorganisms), 1 μL of sample supernatant, and 7 μL of Nuclease-free Water.

[0065] Digital PCR reaction method: the prepared 20 μL digital PCR reaction system was added to the sample hole of the droplet generation card, 70 μL of droplet generation oil was added to the oil hole of the droplet generation card, and the QX200 droplet generator was used for droplet generation. Then 40 μL of droplets were transferred to a 96-well plate, and the PX1 heat sealer was used for film sealing. After film sealing, PCR reaction was performed.

[0066] Digital PCR reaction program: 95℃ 5 min; 95℃ 1 min, 55℃ 1 min, 40 cycles; 4℃ 5 min; 90℃ 5 min.

[0067] After the reaction is completed, the 96-well plate is placed in a microdroplet analyzer, and sample information is sequentially entered. After the detection starts, the instrument automatically identifies each sample microdroplet in sequence. Under the cooperation of the microdroplet reader, the microdroplets pass through the dual-color detector in sequence. The positive and negative results are determined according to the intensity of the fluorescence signal emitted by the microdroplets, and the number of positive and negative microdroplets of each sample is recorded. After the signal collection is completed, the software Quantasoft calculates the final result to generate a microdroplet scatter plot. Figure 5 ).

[0068] It can be seen from the detection results that all the target bacteria positive signals are successfully detected by the method, and the method has good detection ability and high sensitivity for bacterial concentrations in the range of 10 0 -10 4 copy / mL.

[0069] Example 3: Digital PCR accuracy verification 1. The strains stored at -80°C were inoculated into corresponding 5 mL liquid culture media for culture. Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus helveticus were inoculated into 5 mL MRS liquid culture medium, and the strains were inoculated and cultured at 37°C for 24 h. Bacillus amyloliquefaciens was inoculated into 5 mL LB liquid culture medium, and the strain was inoculated and cultured at 37°C for 24 h. Acetobacter pasteurianus was inoculated into 5 mL GY liquid culture medium, and the strain was inoculated and cultured at 30°C for 24 h. The cultured bacterial liquid was serially diluted by 10 times with physiological saline to obtain 10 -1 -10 -7 dilution gradient of bacterial suspension.

[0070] 2. The bacterial suspension was evenly coated on the corresponding strain solid culture medium. Specifically, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus helveticus used MRS solid culture medium; Bacillus amyloliquefaciens used LB solid culture medium; and Acetobacter pasteurianus used GY solid culture medium. Then, the strains were cultured in an inverted incubator at the optimum temperature for 2 days. After uniform colonies appeared, colony counting was performed, and the bacterial concentrations (CFU / mL) of the bacterial suspensions with different concentrations of each strain were recorded.

[0071] 3. The digital PCR reaction system of the bacterial suspension with different concentrations of each strain was prepared. The digital PCR reaction system was 20 μL, and the system included 2×QX200 TM ddPCR TMEvaGreen Supermix for Probes 10 μL, 10 μmol / L of the upper and lower primers 1 μL (for different species of microorganisms, the specific primer pairs provided in Example 1 are used), 1 μL of bacterial suspension, 7 μL of nuclease-free water.

[0072] 4. Perform digital PCR reaction. Add the prepared 20 μL digital PCR reaction system to the sample well of the droplet generation card, add 70 μL droplet generation oil to the oil well of the droplet generation card, and use the QX200 droplet generator to generate droplets. Then transfer 40 μL droplets to a 96-well plate, seal the film using the PX1 heat sealer, and perform PCR reaction after sealing the film.

[0073] Digital PCR reaction program: 95℃ 5 min; 95℃ 1 min, 55℃ 1 min, 40 cycles; 4℃ 5 min; 90℃ 5 min.

[0074] After the reaction is completed, place the 96-well plate in the droplet analyzer, perform droplet analysis, and record the digital PCR copy number (copy / mL) of each strain at different concentrations of bacterial suspension.

[0075] 5. Perform regression analysis on the bacterial concentration and digital PCR copy number of each strain at different concentrations of bacterial suspension determined. By constructing a standard curve of bacterial concentration and digital PCR copy number, the correlation between the two is evaluated, and the results are shown in Table 4. R 2 All are greater than 0.9, showing a good linear relationship, proving that the use of digital PCR technology for quantitative analysis of key microorganisms in food fermentation process is accurate.

[0076] Table 4 Relationship between bacterial concentration and digital PCR copy number

[0077] Note: x is the number of bacterial cells per unit volume log (CFU / mL), and y is the number of copies per unit volume log (copy / mL).

[0078] Example 4 This example provides a method for rapidly and quantitatively detecting key microorganisms in food fermentation process using digital PCR, and the specific steps are as follows: 1. Take 1 g of sample in the food fermentation process and place it in a 10 mL test tube, then add 9 mL of sterile water. Place the test tube in an ultrasonic cleaner and ultrasonic for 10 min, then aspirate the supernatant.

[0079] 2. Prepare the digital PCR reaction system: the digital PCR reaction system is 20 μL, and the system includes 2×QX200 TM ddPCR TM EvaGreen Supermix for Probes 10 μL, 10 μmol / L of the upstream and downstream primers 1 μL each (use the specific primer pair provided in Example 1 corresponding to different species of microorganisms), supernatant 1 μL, Nuclease-free Water 7 μL.

[0080] 3. Perform digital PCR reaction: add the prepared 20 μL digital PCR reaction system to the sample well of the droplet generation card, add 70 μL droplet generation oil to the oil well of the droplet generation card, and use the QX200 droplet generator to generate droplets. Then transfer 40 μL droplets to a 96-well plate and use the PX1 heat sealer to seal the film.

[0081] 4. After sealing the film, perform PCR reaction: PCR reaction program: 95℃ 5 min; 95℃ 1 min, 55℃ 1 min, 40 cycles; 4℃ 5 min; 90℃ 5 min.

[0082] 5. After the reaction is completed, place the 96-well plate in the droplet analyzer and perform droplet analysis.

[0083] Take the samples during the fermentation process of solid-state fermented vinegar and detect them according to the method described in Example 4, successfully detect positive droplets of 7 key microorganisms, and obtain Figure 6 The digital PCR detection results of key microorganisms in the fermentation process of foods are shown in Table 1, which proves that digital PCR can be used to detect key microorganisms in the fermentation process of foods.

[0084] Example 5 The present example provides a method for detecting the abundance change of key microorganisms in the fermentation process of food.

[0085] Step 1: Take 1 g of samples at different fermentation stages of food in 10 mL test tubes, add 9 mL of sterile water, and ultrasonicate in an ultrasonic cleaner for 10 min, then aspirate the supernatant. The obtained supernatant is stored in a -20℃ refrigerator for standby.

[0086] Step 2: Prepare the digital PCR reaction system of supernatant at different fermentation days. The PCR reaction system is 20 μL, and the system includes 2×QX200 TM ddPCR TMEvaGreen Supermix for Probes 10 μL, 10 μmol / L of the upstream and downstream primers 1 μL (corresponding to the specific primer pairs provided in Example 1 for different species of microorganisms), supernatant 1 μL, Nuclease-free Water 7 μL.

[0087] Step 3: Perform digital PCR reaction. The prepared 20 μL digital PCR reaction system is added to the sample well of the droplet generation card, 70 μL droplet generation oil is added to the oil well of the droplet generation card, and the QX200 droplet generator is used for droplet generation. Then 40 μL droplets are transferred to a 96-well plate, and the PX1 heat sealer is used for sealing. After sealing, the digital PCR reaction is performed.

[0088] PCR reaction program: 95°C 5 min; 95°C 1 min, 55°C 1 min, 40 cycles; 4°C 5 min; 90°C 5 min.

[0089] After the reaction is completed, the 96-well plate is placed in the droplet analyzer, and droplet analysis is performed. The digital PCR copy number of each key microorganism is recorded, and the abundance change of each key microorganism in the fermentation process is analyzed according to the obtained digital PCR copy number.

[0090] Example 6 Verification of specificity of fluorescent quantitative PCR primers Take 1 g of sample in the fermentation process of solid-state fermented vinegar and place it in a 10 mL test tube. Then add 9 mL of sterile water. Place the test tube in an ultrasonic cleaner and ultrasonicate for 10 min. Then, take the supernatant of these different fermentation process samples and perform a fluorescent quantitative PCR reaction.

[0091] The fluorescent quantitative PCR reaction system is 20 μL, which includes SYBR Premix Ex Taq GC (2x) 10 μL, ROX Reference Dye (50x) 0.4 μL, Nuclease-free Water 6.8 μL, upstream and downstream primers 0.4 μL each (corresponding to the specific primer pairs provided in Example 1 for different species of microorganisms), template 2 μL; The concentration of each of the primer pairs is 10 μmol / L. The fluorescent quantitative PCR reaction program is: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, for 45 cycles; 95°C for 15 s, 60°C for 1 min; 95°C for 15 s.

[0092] From the detection results Figure 7It can be seen that each primer pair only produces a typical amplification curve in the corresponding target bacterial template, the amplification curve is standard S-shaped, and the Ct value is within the effective detection window. It shows that the primers can specifically recognize the target microorganism, and the amplification efficiency is good.

[0093] Application Example 1: Detection of the abundance change of key microorganisms in the solid-state fermentation process of vinegar Take 1 g of the 0th, 1st, 3rd, 5th, 7th and 9th day's vinegar dregs samples of Shanxi old vinegar solid-state fermentation in 10 mL test tubes, add 9 mL of sterile water, and ultrasonic for 10 min in the ultrasonic cleaning instrument. Then, the supernatant is obtained. The obtained supernatant is stored in a-20℃ refrigerator for standby. Then, the digital PCR method described in Example 5 is used to detect Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurianus, and analyze the abundance change of key microorganisms in the vinegar brewing process. The results are shown in Figure 8 .

[0094] From Figure 8 It can be seen that the bacterial diversity is high at the initial stage of acetic acid fermentation (0th day), and the dominant bacterial species has not been established. Multiple lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum and Pediococcus pentosaceus etc. together constitute the dominant community, among which Lactobacillus plantarum and Lactobacillus helveticus have high relative abundance, indicating that this stage is mainly in the stage of lactic acid metabolism, which helps to preliminarily establish the fermentation environment. With the progress of fermentation, the relative abundance of Acetobacter pasteurianus significantly increases from the 1st day, and begins to replace the original lactic acid bacteria to become the dominant bacteria, and maintains a stable high abundance (about 40%-50%) from the 3rd to the 9th day, becoming the key bacterial species that continuously dominates the whole fermentation process. Its significant advantage in the middle and late stages indicates that it plays a dominant role in acetic acid synthesis and maintaining acidic environment, and is the core bacterial species that determines the flavor and quality of the fermentation product. At the same time, the relative abundance of microorganisms such as Lactobacillus acidophilus, Bacillus amyloliquefaciens and Lactobacillus fermentum increases to a certain extent from the 3rd to the 7th day, indicating that they may participate in the synthesis of secondary metabolites or form a synergistic effect with the dominant bacteria in the middle and late stages. While bacterial species such as Lactobacillus plantarum and Pediococcus pentosaceus rapidly decay after the 1st day, indicating that they mainly play a role in the early stage of fermentation, and are then replaced by bacteria that are more suitable for acidic environment.

[0095] Application Example 2: Detection of the abundance change of key microorganisms in the liquid-state fermentation process of vinegar Take Longmen vinegar liquid fermentation of the first, 6, 11, 16, 21, 26 and 30 days of vinegar sample 1 mL in 10 mL test tube, add 9 mL sterile water, in ultrasonic cleaning instrument ultrasonic 10 min, after the supernatant. The supernatant obtained into-20℃ refrigerator storage for later use. Then according to the method described in example 5, detection of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurianus, analysis of the key microorganism abundance change of the vinegar brewing process, results as shown in Figure 9 .

[0096] From Figure 9 It can be seen that the initial fermentation of Lactobacillus acidophilus (45.48%) and Acetobacter pasteurianus (53.45%) were enriched together, although the proportion of Lactobacillus plantarum (0.67%), Lactobacillus fermentum (0.27%), Lactobacillus helveticus (0.01%) and Pediococcus pentosaceus (0.12%) was low, but they cooperated to establish an acidic microenvironment. In the middle stage (6-16 days), Acetobacter pasteurianus dominated the synthesis of acetic acid, rising to 71.52% at 6 days and maintaining 66.23% at 16 days, while Lactobacillus acidophilus (28.34%) and Lactobacillus helveticus (13.63%) temporarily increased at 11 days, suggesting that lactic acid bacteria played an auxiliary role in secondary metabolism and flavor precursor generation; In the later stage (21-30 days), the lactic acid bacteria community was again humidified, with Lactobacillus acidophilus abundance increasing to 65.07% at 21 days, Lactobacillus fermentum reaching 34.89% at 26 days, and Lactobacillus acidophilus (55.24%) and Lactobacillus fermentum (34.24%) accounting for nearly 89.48% by 30 days, while Acetobacter pasteurianus decreased to 9.40%, indicating that lactic acid bacteria at the late stage significantly improved the texture stability and flavor complexity of liquid fermented vinegar through extracellular polysaccharides and secondary metabolites regulation.

[0097] Example 3 Detection of the change of key microorganism abundance in the fermentation process of fermented bean curd Take Wang Zhihe Hongfang fermented bean curd fermentation of the 10th, 30th and 60th day of post fermentation sample 1 g in 10 mL test tube, add 9 mL sterile water, in ultrasonic cleaning instrument ultrasonic 10 min, after the supernatant. The supernatant obtained into-20℃ refrigerator storage for later use. Then according to the method described in example 5, detection of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus and Lactobacillus helveticus, analysis of the key microorganism abundance change of the fermented bean curd fermentation process, results as shown in Figure 10 .

[0098] From Figure 10It can be seen that the dynamic succession of the relative abundance of the community from the 10th day to the 60th day can be divided into three stages: on the 10th day, Pediococcus pentosaceus (35.31%) and Lactobacillus helveticus (31.04%) are dominant, followed by Lactobacillus plantarum (20.14%), Lactobacillus scarpens (10.19%), Lactobacillus acidophilus (1.90%) and Bacillus amyloliquefaciens (1.42%) at trace levels, indicating that polysaccharide decomposition and primary acidification are dominant in the early stage; on the 30th day, the abundance of Lactobacillus helveticus increased significantly to 42.96%, Lactobacillus plantarum (29.30%), Lactobacillus scarpens (14.51%) and Lactobacillus acidophilus (3.38%) increased at the same time, while Pediococcus pentosaceus (9.58%) and Bacillus amyloliquefaciens (0.28%) decreased significantly, indicating that the community has been dominated by lactic acid bacteria in the middle stage. By the 60th day, Lactobacillus plantarum increased to 45.26%, Lactobacillus helveticus and Lactobacillus acidophilus were 37.32% and 17.44% respectively, Lactobacillus scarpens (9.94%) remained at a medium level, Pediococcus pentosaceus (7.37%) and Bacillus amyloliquefaciens (0.14%) remained at a low level, indicating that the community has entered a mature and stable stage dominated by lactic acid bacteria in the later stage.

[0099] Application Example 4 Detection of the abundance change of key microorganisms in soy sauce fermentation Take 1 g of Lemin soy sauce fermentation samples at 0, 30, 90, 150 and 180 days in 10 mL test tubes, add 9 mL of sterile water, and ultrasonicate in an ultrasonic cleaner for 10 min, then aspirate the supernatant. The obtained supernatant is stored in a -20°C refrigerator for standby. Then, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus scarpens, Lactobacillus acidophilus and Lactobacillus helveticus are detected according to the method described in Example 5 to analyze the abundance change of key microorganisms in the soy sauce fermentation process, and the results are shown in Table 2. Figure 11

[0100] From Table 2, it can be seen that the relative abundance of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus scarpens, Lactobacillus acidophilus and Lactobacillus helveticus in the Lemin soy sauce fermentation process is shown in Table 2. Figure 11 ​It can be seen that at day 0, the relative abundance of P. pentosaceus was the highest, reaching 46.62%, followed by L. helveticus and L. plantarum, and the contents of L. acidophilus and L. fermentum were lower, indicating that the initial stage was mainly mediated by P. pentosaceus for polysaccharide hydrolysis and primary acidification; by day 30, L. plantarum and L. fermentum were significantly enriched, L. helveticus remained at a high level, L. acidophilus also increased, and P. pentosaceus decreased significantly; by day 90, the community distribution tended to be balanced, L. plantarum (26.74%), L. fermentum (22.95%), L. acidophilus (20.21%), and L. helveticus (23.58%) cooperated with each other, and P. pentosaceus further decreased; by day 150, L. acidophilus increased to 33.33%, L. helveticus, L. plantarum, and L. fermentum were co-enriched, and P. pentosaceus further decreased to 2.67%; by day 180, L. acidophilus accounted for 56.36%, became the dominant bacteria, the contents of L. plantarum, L. helveticus, and L. fermentum decreased, and P. pentosaceus was only 1.04%, indicating that the later community had entered the mature acidification and texture stabilization stage dominated by L. acidophilus.

[0101] Example 5 Detection of the abundance change of key microorganisms in chili sauce fermentation Take 1 g of samples of Tianxiahong chili sauce fermentation at day 0, 20, 40, 60, 90, and 120 in a 10 mL test tube, add 9 mL of sterile water, and ultrasonicate in an ultrasonic cleaner for 10 min, then aspirate the supernatant. The obtained supernatant is stored in a -20°C refrigerator for standby. Then, P. pentosaceus, L. plantarum, L. fermentum, L. acidophilus, and L. helveticus are detected according to the method described in Example 5 to analyze the abundance change of key microorganisms in the fermentation process of the chili sauce, and the results are shown in Table 2. Figure 12

[0102] As shown in Table 2, the relative abundance of P. pentosaceus was the highest at day 0, reaching 46.62%, followed by L. helveticus and L. plantarum, and the contents of L. acidophilus and L. fermentum were lower, indicating that the initial stage was mainly mediated by P. pentosaceus for polysaccharide hydrolysis and primary acidification; by day 30, L. plantarum and L. fermentum were significantly enriched, L. helveticus remained at a high level, L. acidophilus also increased, and P. pentosaceus decreased significantly; by day 90, the community distribution tended to be balanced, L. plantarum (26.74%), L. fermentum (22.95%), L. acidophilus (20.21%), and L. helveticus (23.58%) cooperated with each other, and P. pentosaceus further decreased; by day 150, L. acidophilus increased to 33.33%, L. helveticus, L. plantarum, and L. fermentum were co-enriched, and P. pentosaceus further decreased to 2.67%; by day 180, L. acidophilus accounted for 56.36%, became the dominant bacteria, the contents of L. plantarum, L. helveticus, and L. fermentum decreased, and P. pentosaceus was only 1.04%, indicating that the later community had entered the mature acidification and texture stabilization stage dominated by L. acidophilus. Figure 12 ​It can be seen that Lactobacillus helveticus is dominant in the early fermentation of chili sauce, with a relative abundance of up to 34.62%, followed by Lactobacillus acidophilus, Pediococcus pentosaceus and Lactobacillus fermentum are at a medium level, and Lactobacillus plantarum is the lowest, indicating that the primary decomposition of protein and acidification in the start-up stage is mainly mediated by Lactobacillus helveticus; when the fermentation time is 20 days, Lactobacillus plantarum increases significantly, and starts to drive the fermentation together with Lactobacillus helveticus; when the fermentation time is 40 days, Lactobacillus plantarum has the highest abundance and becomes the dominant bacteria, Lactobacillus helveticus and Lactobacillus acidophilus decrease, and Pediococcus pentosaceus and Lactobacillus fermentum gradually increase, indicating that the diversification of lactic acid bacteria is enhanced in the middle period; when the fermentation time is 60 days, Lactobacillus plantarum still maintains the advantage, and the microbial community structure tends to be stable and synergistic; when the fermentation time is 90 days in the late fermentation, Lactobacillus acidophilus increases significantly to 24.65% and becomes the new dominant bacteria, and the proportions of Lactobacillus plantarum, Lactobacillus fermentum and Pediococcus pentosaceus are close, and Lactobacillus helveticus is the lowest, reflecting that the fermentation system has transitioned to the steady-state acidification stage regulated by Lactobacillus acidophilus; at the end of fermentation, when the fermentation time is 120 days, Lactobacillus acidophilus further increases to 33.82%, and Lactobacillus fermentum rapidly increases to 23.56%, indicating that the fermentation system has entered the mature acidification and texture stabilization stage dominated by Lactobacillus acidophilus, laying a microecological foundation for the final flavor and safety of chili sauce.

[0103] In summary, the primer combination provided by the present application can be used for rapid detection of key microorganisms in the fermentation process of food, and can simultaneously realize species level specific identification and digital PCR absolute quantification of 7 key functional bacteria, has strong anti-interference ability and excellent intra-species universality, and helps precise regulation and standardized production of the fermentation food production process.

[0104] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A PCR primer set for detecting mixed microorganisms in food fermentation processes, characterized in that, The primer combination includes those for detecting Pediococcus pentosaceus (… Pediococcus pentosaceus Primer pairs for detecting *Lactobacillus plantarum* ( Lactiplantibacillusplantarum Primer pairs for detecting fermenting Lactobacillus mucilaginosus ( Limosilactobacillus fermentum Primer pairs for detecting Bacillus amyloliquefaciens (BAM) Bacillusammyloliquefaciens Primer pairs for detecting acid-fast lactobacilli ( Lactobacillusacetotolerans Primer pairs for detecting Lactobacillus helveticus ( Lactobacillushelveticus Primer pairs for detecting *Acetobacter pasteurellosis* ( ) and primers for detecting *Acetobacter paste Acetobacterpasteurianus At least one of the primer pairs; The specific base sequences of the upstream and downstream primers in the primer pair used for detecting Pediococcus pentosaceus are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The specific base sequences of the upstream and downstream primers in the primer pair used for detecting Lactobacillus plantarum are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The specific base sequences of the upstream and downstream primers in the primer pair used for detecting fermenting Lactobacillus mucilaginosus are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The specific base sequences of the upstream and downstream primers in the primer pair used for detecting Bacillus amyloliquefaciens are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The specific base sequences of the upstream and downstream primers in the primer pair used for detecting acid-fast lactobacilli are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. The specific base sequences of the upstream and downstream primers in the primer pair used for detecting Lactobacillus helveticus are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. The specific base sequences of the upstream and downstream primers in the primer pair used for detecting Acetobacter pasteurellosis are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

2. The primer combination according to claim 1, characterized in that, The primer pair consists of primer pairs for detecting Pediococcus pentosaceus, primer pairs for detecting Lactobacillus plantarum, primer pairs for detecting Lactobacillus fermentum, primer pairs for detecting Bacillus amyloliquefaciens, primer pairs for detecting acid-fast Lactobacillus, primer pairs for detecting Lactobacillus helveticus, and primer pairs for detecting Acetobacter pasteurellosis.

3. A kit for qualitative / quantitative detection of mixed microorganisms during food fermentation, characterized in that, The kit comprises the primer combination as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The kit is a digital PCR kit or a real-time PCR kit.

5. The use of the primer combination of claim 1 or 2 or the kit of claim 3 or 4 in the qualitative / quantitative detection of at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus and Acetobacter pasteurellosis.

6. The application according to claim 5, characterized in that, The application refers to the use of the primer combination or the kit in the qualitative / quantitative detection of at least one of the following bacteria during fermentation: *Pediococcus pentosaceus*, *Lactobacillus plantarum*, *Lactobacillus fermentatus*, *Bacillus amyloliquefaciens*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, and *Acetobacter pasteurellosis*; or The application refers to the use of the primer combination or the kit in monitoring the abundance changes of at least one of the following bacteria during food fermentation: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus, and Acetobacter pasteurellosis.

7. A method for rapid detection of key microorganisms during food fermentation, characterized in that, The key microorganism is at least one of the following: *Pediococcus pentosaceus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Bacillus amyloliquefaciens*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, and *Acetobacter pasteurellosis*; the method includes the following steps: S1: Collect microorganisms from food fermentation process samples or extract the genome of microorganisms from the samples; S2: Using the microorganisms or microbial genomes collected in S1 as templates to be tested, PCR gene amplification is performed using the primer combination described in claim 1 or 2 or the kit described in claim 3 to obtain PCR products; S3: Characterize the PCR product to obtain detection information.

8. A method for rapid quantitative detection of key microorganisms during food fermentation, characterized in that, Using digital PCR or quantitative real-time PCR, and employing the primer combination described in claim 1 or 2 or the kit described in claim 4, key microorganisms in the food fermentation process are detected and analyzed; the key microorganisms are at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus, and Acetobacter pasteurellosis.

9. The method according to claim 8, characterized in that, The digital PCR reaction system was 20 μL, containing 2×QX200... TM ddPCR TM 10 μL of EvaGreen Supermix for Probes, 7 μL of Nuclease-free Water, 1 μL each of the upstream and downstream primers of the same primer pair, and 1 μL of template; the concentration of each primer in the primer pair is 10 μmol / L; the template contains microorganisms or the genome of microorganisms in the food fermentation process sample; The digital PCR reaction program is as follows: 95℃ for 5 min; 95℃ for 1 min, 55℃ for 1 min, 40 cycles; 4℃ for 5 min; 90℃ for 5 min; or The reaction system for the quantitative real-time PCR was 20 μL, comprising 10 μL of SYBR Premix Ex Taq GC (2×), 0.4 μL of ROX Reference Dye (50×), 6.8 μL of Nuclease-free Water, 0.4 μL each of the upstream and downstream primers of the same primer pair, and 2 μL of template; the concentration of each primer in the primer pair was 10 μmol / L; the template contained microorganisms or the genome of microorganisms from the food fermentation sample; The reaction procedure for the quantitative real-time PCR was as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 45 cycles; 95℃ for 15 s, 60℃ for 1 min; 95℃ for 15 s.

10. A method for monitoring changes in the abundance of key microorganisms during food fermentation, characterized in that, Samples were taken at different fermentation times during the food fermentation process, and the key microorganisms in the samples were quantitatively detected using the method described in claim 8 or 9. The abundance changes of each key microorganism during the fermentation process were analyzed based on the obtained copy number or relative fluorescence intensity. The key microorganisms are at least one of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus fermentum, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus helveticus, and Acetobacter pasteurellosis.

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