Beer obligate anaerobic pectobacterium and application thereof

Abstract

The invention discloses a strain of beer obligate anaerobic pectobacterium frisinensis, and the preservation number of the beer obligate anaerobic pectobacterium frisinensis is CGMCC (China General Microbiological Culture Collection Center) No. 7.552. The invention further discloses application of the pectobacterium in construction of a pectobacterium and lactic acid bacteria symbiotic system, the pectobacterium and lactic acid bacteria symbiotic system and application of the pectobacterium and lactic acid bacteria symbiotic system. A strain of obligate anaerobic pectobacterium CGMCC No.7.552 is separated and identified, a symbiotic test system is constructed, RT-PCR and GC-MS technologies are adopted, symbiotic behaviors, metabolites and pH changes of pectobacterium and lactic acid bacteria in different environments are qualitatively and quantitatively studied, and the pectobacterium and lactic acid bacteria in different environments can be identified. The method determines the influence of multiple factors on the growth of pectobacterium in the symbiotic environment of pectobacterium and lactic acid bacteria, including the activity of pectobacterium, the amount of initial contaminating bacteria and the pH value of the symbiotic environment, is beneficial to prevention and control of harmful microorganisms in the beer fermentation process, optimization of the beer production process and improvement of the beer quality, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of obligate anaerobic pectinobacterium brevicaulis and its applications. Background Technology

[0002] Beer is a beverage made primarily from malt and hops, fermented with yeast. It contains CO2, low alcohol content, and various nutrients such as amino acids, vitamins, and proteins, and is one of the world's most consumed alcoholic beverages. Beer has long been recognized as a safe food due to its specific physicochemical properties. It contains a certain concentration of alcohol (0.5%~10%, w / w), extremely low oxygen content (<0.1ppm), a high concentration of carbon dioxide (approximately 0.5%, w / w), a low pH (3.8~4.7), and contains hop bitter substances with antibacterial properties. At the same time, it lacks the nutrients (such as monosaccharides and amino acids) necessary for microbial growth and reproduction. Furthermore, beer has a certain antibacterial effect, resisting the invasion of common microorganisms and preventing the emergence of pathogenic bacteria in humans. However, during the beer brewing process, there may still be some obligate or facultative anaerobic microorganisms that are resistant to acid and hop bitter substances. These microorganisms can grow and reproduce using intermediate metabolites and autolysates of yeast, causing microbial contamination of beer and seriously affecting the quality of beer products, such as causing changes in beer flavor and affecting its biological stability.

[0003] Beer contains many types of spoilage bacteria, most commonly lactic acid bacteria (LAC) Lactic Acid Bacteria Lactic acid bacteria (LAB) are the primary source of beer contamination, accounting for approximately 90% of all beer contamination. Most LABs are Gram-positive facultative anaerobic bacteria, capable of growth under both aerobic and anaerobic conditions, and thrive in acidic environments. LABs, due to their strong fermentation and acid-producing capabilities, exhibit high tolerance to hop acids and lower pH levels, increasing the acidity and flavor complexity of beer, and can be present throughout the entire cold-zone brewing process. In industrial beer, if non-acidic beers (such as pale ales and lagers) are contaminated with lactic acid bacteria (such as... Lactobacillus brevis, Lactobacillus plantarum Excessive growth of these organisms can produce byproducts such as volatile acids and aldehydes, which can lead to unpleasant flavors in beer, such as sourness and rottenness. In severe cases, they can also generate turbidity and sediment, affecting the appearance and taste of the beer, causing it to lose its original transparency or clarity, resulting in a decline in beer quality or a shortened shelf life, and bringing huge negative impacts to beer brewing.

[0004] In recent years, with the improvement of beer brewing technology, the decrease in oxygen content in beer has led to contamination by obligate anaerobic putrefactive bacteria, especially Gram-negative bacteria of the genus *Pectobacterium* (…). Pectinatus sp. ) and the genus *Macrococcus* ( Megasphaera sp.The pollution frequency of Pectinatus spp. increases, which seriously affects the quality and safety of beer and brings new challenges to the microbial control of beer enterprises. It is a kind of obligate anaerobe, mainly including Pectinatus frisingensis 、 Pectinatus cerevisiiphilus and Pectinatus haikarae three categories, which was first isolated and identified by Lee et al. in 1978 in a beer factory in the United States, and then found in beer factories in many countries, becoming a common spoilage bacteria in beer brewing process. Pectinatus spp. can survive in low-pH beer environment and exhibit strong acid tolerance and high metabolic activity, which is a typical spoilage bacteria in beer brewing and storage. Pectinatus spp. can produce a variety of volatile compounds, especially hydrogen sulfide, propionic acid, butyric acid and acetic acid, etc., which cause unpleasant sour smell, moldy smell or sulfur smell in beer, making it lose fresh and balanced taste. In addition, Pectinatus spp. can also release polysaccharides, proteins and other substances during reproduction, which are easy to accumulate in beer and produce turbidity and sediment, affecting the transparency and appearance of beer and reducing the shelf life of beer. Therefore, the presence of Pectinatus spp. poses a serious threat to the quality and safety of beer. Studying the characteristics, source and detection technology of Pectinatus spp. is beneficial to the prevention and control of beer microorganisms and has important significance for improving the microbial control level of enterprises and improving beer quality.

[0005] However, the current research on Pectinatus spp. mainly focuses on its biological characteristics, metabolic mechanism and interaction with other microorganisms, and the symbiotic relationship between Pectinatus spp. and lactic acid bacteria in beer fermentation process is relatively less, which is very unfavorable for the prevention and control of Pectinatus spp. The existing technology cannot accurately predict the influence of pH change in the symbiotic environment on the growth of Pectinatus spp., so as to effectively control the growth of Pectinatus spp. and avoid its negative impact on beer quality. These problems limit the understanding of the symbiotic relationship between Pectinatus spp. and lactic acid bacteria, and are not conducive to the progress of harmful microorganism control technology in beer fermentation process.

[0006] Therefore, further study on the symbiotic relationship between Pectinatus spp. and lactic acid bacteria in beer fermentation process is helpful to better understand the synergistic competition effect of the two in metabolism, flavor and growth, and provide scientific basis for the pollution prevention and control of harmful microorganisms in beer. SUMMARY

[0007] The purpose of the present application is to solve the above technical problems, and provide a method for constructing a symbiotic system of Pectinatus spp. and lactic acid bacteria for the prevention and control of obligate anaerobic harmful microorganisms.

[0008] To achieve the above invention purpose, the present application provides the following technical scheme: In a first aspect, the present application provides a beer obligate anaerobic Pectinatus spp. Pectinatus frisingensisand its preservation number is CGMCC No. 7.552.

[0009] In a second aspect, the present application provides an application of the beer-specific anaerobic Pectinatus in constructing a Pectinatus and lactic acid bacteria symbiotic system.

[0010] Preferably, the constructing comprises the following steps: S1, qualitative experiment: separately culturing the beer-specific anaerobic Pectinatus and lactic acid bacteria and symbiotic culturing the two in different concentrations; S2, quantitative experiment: symbiotic culturing the beer-specific anaerobic Pectinatus and lactic acid bacteria in different concentrations, detecting the flavor substance fingerprint of the metabolic product by using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry, and identifying the strains in the symbiotic system by using RT-PCR technology.

[0011] Preferably, the chromatographic conditions are as follows: The carrier gas is helium, a constant flow mode is adopted, the flow rate is 1.0 mL / min, and the injection port temperature is 250℃. Programmed temperature rising: the initial temperature is 60℃, the retention time is 4 min, the temperature is raised to 210℃ at a rate of 5℃ / min, the temperature is raised to 250℃ at a rate of 10℃ / min, and the retention time is 10 min.

[0012] Preferably, the mass spectrometry conditions are as follows: The ion source temperature is 280℃, the transmission line temperature is 250℃, the ion source is an EI source, the electron impact source is 70 eV, and the MS scanning range is 35-350 amu.

[0013] In a third aspect, the present application provides a beer-specific anaerobic Pectinatus and lactic acid bacteria symbiotic system, which comprises the beer-specific anaerobic Pectinatus and lactic acid bacteria of the present application.

[0014] Preferably, the lactic acid bacteria comprise any one or more of Lactobacillus brevis, Lactobacillus brevis Lactobacillus casei, Lactobacillus casei Lactobacillus plantarum, Lactobacillus plantarum and the like.

[0015] In a fourth aspect, the present application further provides an application of the beer-specific anaerobic Pectinatus and lactic acid bacteria symbiotic system in controlling the pollution of harmful bacteria in beer. Preferably, the pH of the beer fermentation liquor is as close to 4 as possible.

[0016] Fifthly, the present invention also provides a method for co-culturing Pectinobacterium and Lactobacillus, comprising: inoculating the Pectinobacterium and Lactobacillus into NBB-B medium for mixed culture. More preferably, the culture conditions are as follows: using NBB-P powder and water:beer in a volume ratio of 1:1 as a solvent, a culture medium with a pH of 5.8-6.0 is prepared, and the inoculation amount is simultaneously added to the culture medium according to the inoculation amount designed for the symbiotic experiment, and cultured under anaerobic conditions at 26°C.

[0017] Sixthly, the present invention also provides a method for reducing the risk of pectinobacterium contamination in beer fermentation broth, which includes bringing the pH of the beer fermentation broth as close to 4 as possible. Further, the method also includes reducing Lactobacillus brevis contamination in the beer fermentation broth, indirectly degrading pectinobacterium contamination.

[0018] Due to the obligate anaerobic nature of pectinobacteria, it is usually difficult to sample and enrich them individually. This invention isolates and identifies a strain of obligate anaerobic pectinobacteria in beer (…). Pectinatus frisingensis According to CGMCC No. 7.552, a symbiotic experimental system was constructed. RT-PCR and GC-MS techniques were used to qualitatively and quantitatively study the symbiotic behavior, metabolites, and pH changes of *Pectinobacterium* and lactic acid bacteria under different environments. The study determined that the growth of *Pectinobacterium* in the symbiotic environment with lactic acid bacteria is influenced by multiple factors, including the viability of *Pectinobacterium* itself, the amount of initial contaminating bacteria, and the pH value of the symbiotic environment. Specifically, when the viability of *Pectinobacterium* in the symbiotic system is high, the concentration of contaminating bacteria is high, and the pH buffering capacity of the symbiotic environment is strong, *Pectinobacterium* can grow and produce the marker metabolite propionic acid before being inhibited by lactic acid bacteria. However, as the acid production of lactic acid bacteria rapidly accumulates, and the pH of the substrate drops below 4, the growth of *Pectinobacterium* is inhibited. The pectinobacterium and lactic acid bacteria symbiotic system constructed by this invention can predict potential pectinobacterium contamination areas by utilizing the detected lactic acid bacteria types and pH changes. This allows for enhanced monitoring and cleaning of key areas, reducing the probability of contamination by Lactobacillus brevis, which coexists with pectinobacterium, and thus reducing the risk of pectinobacterium contamination. Furthermore, by adjusting the pH of beer to be as close as possible to or below 4, the risk of pectinobacterium contamination is significantly reduced.

[0019] This invention overcomes the shortcomings of existing technologies, not only improving our understanding of the symbiotic relationship between pectinobacteria and lactic acid bacteria, but also providing strong technical support for the control of harmful microorganisms during beer fermentation. This invention can not only help optimize beer production processes and improve beer quality, but can also be applied to other food fermentation industries, offering more possibilities for food safety and quality assurance, and has broad application prospects. Attached Figure Description

[0020] Figure 1A flowchart for RT-PCR sample detection is shown.

[0021] Figure 2 The morphology of isolated colonies on solid culture plates is shown. The left image shows MRS medium, and the right image shows NBB-A medium.

[0022] Figure 3 It shows Pectinatus sp. Microscopic images. a: OM 400x; b: EM 10000x; c: EM 18200x.

[0023] Figure 4 The melting curve of the sample detected by RT-PCR in 640 channels is shown.

[0024] Figure 5 The GC-MS flavor profile of Pectinobacterium is shown.

[0025] Figure 6 The GC-MS flavor profile of Lactobacillus brevis is shown.

[0026] Figure 7 The GC-MS flavor profile of Lactobacillus plantarum is shown.

[0027] Figure 8 The GC-MS flavor profile of the mixed sample containing Pectinobacterium is shown. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0030] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0033] I. Methods 1.1 Isolation and Identification of Pectinobacterium Turbid beer samples were streaked onto deoxygenated NBB solid medium (Döhler, Germany; catalog number: 2.04716.462) and MRS solid medium (OXOID, UK; catalog number: CM0361B) and cultured anaerobically for 7-10 days. Single colonies were identified by gene sequencing and RT-PCR to determine the species of the isolated strains.

[0034] 1.2 RT-PCR method for qualitative detection of harmful microorganisms in beer The experimental samples were tested according to the operating instructions for the ROCH RT-PCR instrument, as follows: Figure 1 As shown, qualitative judgments are made based on the characteristic peak values ​​of harmful microorganisms in different types of beer.

[0035] 1.3 Preparation of bacterial suspension for experimental strains Two experimental strains, Pectinobacterium and Lactobacillus, were activated to the logarithmic stationary phase using NBB-B liquid medium (Döhler, Germany). They were then diluted to different gradients in an anaerobic chamber using a 10-fold serial dilution method, and the experimental strains were inoculated according to the experimental design.

[0036] All three experimental lactic acid bacteria strains were commercial strains purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC), including *Lactobacillus brevis*. Lactobacillus brevis Lactobacillus plantarum, catalog number GDMCC NO.1.1496 Lactobacillus plantarum Lactobacillus casei, catalog number GDMCC NO. 1.1516 Lactobacillus casei The serial number is GDMCCNO.1.411.

[0037] 1.4 Symbiotic Experimental Design Scheme Qualitative experiments: Freshly cultured pectinobacteria and lactic acid bacteria were cultured separately and in combination with different concentrations in an anaerobic chamber using NBB-B medium and beer as substrates (see Table 1). 100 μL of bacterial suspensions of different concentrations were inoculated into the medium or beer according to the experimental design. The culture temperature, culture time, and other conditions were kept consistent for each group to maintain the comparability of results between the experimental groups.

[0038] Preferably, the culture conditions are as follows: Döhler NBB-P powder (Germany) and a water-to-beer ratio of 1:1 (volume ratio) are used as solvents to prepare a culture medium with a pH of 5.8-6.0. The inoculum is simultaneously inoculated into the culture medium according to the inoculation amount designed in the following symbiotic experiment, and cultured at 26°C in an ELECROTEK AW500TG anaerobic workstation (5% O2 + 10% CO2 + 85% N2).

[0039] Table 1: Experimental Design for Qualitative Analysis of Pectinobacterium and Lactobacillus Symbiosis

[0040] Quantitative experiment: In an anaerobic chamber, pectinobacterium and experimental lactic acid bacteria with different concentrations were co-cultured in NBB-B medium after half a month of growth following inoculation (Table 2). 100 μL of bacterial suspensions of different concentrations were inoculated into the medium according to the experimental design. The culture temperature, culture time and other conditions of each group were kept consistent to maintain comparability between experimental groups.

[0041] Table 2: Quantitative experimental design for the symbiosis of pectinobacter and lactic acid bacteria

[0042] 1.5 Metabolite Analysis The major metabolites in the experimental samples were analyzed using gas chromatography-mass spectrometry (GC-MS), as follows: (1) Sample preparation: Take 5 mL of sample into a 20 mL headspace vial, and seal it with a sealing gasket and an aluminum cap. Insert the SPME syringe through the sample vial septum and into the vial. Place the fiber tip in the upper space about 20 mm away from the sample surface. Incubate at 40℃ for 30 min, then remove the fiber tip and inject the sample directly. Desorb for 5 min and detect the flavor substances in the sample.

[0043] (2) A method for detecting and analyzing the flavor compounds of common harmful bacteria metabolites in beer was established by using headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS).

[0044] Chromatographic conditions: Helium (He) (purity ≥99.999%) was used as the carrier gas. Constant flow mode was employed at a flow rate of 1.0 mL / min, and the injection port temperature was 250 °C. Splitless injection was used. Temperature program: Initial temperature 60 °C, hold for 4 min, increase to 210 °C at 5 °C / min, increase to 250 °C at 10 °C / min, hold for 10 min.

[0045] Mass spectrometry conditions: ion source temperature 280 ℃, transfer line temperature 250 ℃, ion source is EI source, electron impact source 70 eV, MS scan range is 35-350 amu.

[0046] Detectable substances: Flavor compounds in beer samples, for identification. Pectinatus sp. Characteristic metabolite propionic acid.

[0047] (3) Use the NIST spectral library to find Pectinatus The characteristic metabolite propionic acid produced by *Pectinobacterium* growth is used to qualitatively determine whether growth has occurred, while quantitative results are calculated based on peak area. The detection of propionic acid indicates the presence of *Pectinobacterium* growth and metabolism in the sample; conversely, its absence indicates the absence of growth.

[0048] II. Results 2.1 Isolation and Identification of Pectinobacterium 2.1.1 Isolation of Pectinobacterium Pure colonies were isolated from turbid and spoiled beer using the anaerobic plate culture method on deoxygenated MRS and NBB-A solid plates, respectively. Figure 2 The bacteria appear smooth with slightly raised edges on MRS solid plates, and are white in the middle with green edges due to the indicator bromocresol green. On NBB-A solid plates, the color is light yellow.

[0049] Figure 2 The morphology of isolated colonies on solid culture plates is shown. The left image shows MRS medium, and the right image shows NBB-A medium.

[0050] 2.1.2 Identification of Pectinobacterium There are several methods for identifying Pectinobacter, including macroscopic colony observation, microscopic observation, Gram staining, and RT-PCR identification. 16S rDN A rapid identification method 、 The identification results of the strains isolated in this paper using gas chromatography and GC-MS are as follows: 2.1.2.1 Microscopic observation Due to their unique physiological characteristics, *Pectinobacterium* exhibits different activity states at different growth stages. Cells in earlier growth stages are comb-like in arrangement due to their lateral flagella, and the cells are mobile, allowing their curved morphology to be observed under an optical microscope (OM).Figure 3 The activity of the lateral flagella (a) can also be observed using an electron microscope (EM). Figure 3 The c in the middle varies in length and size. Figure 3 (b) In contrast, pectinobacteria in the senescent stage are inactive due to flagella degeneration, and their cells look like immobile lactobacilli.

[0051] 2.1.2.2 RT-PCR identification Uses German BIOTECONDiagnostics foodproof ® Data analysis of the beer screening kit (S 400 18) and beer testing kit (R 310 02) after running on the RT-PCR instrument is as follows: Figure 4 As shown in Table 3, according to Figure 4 The corresponding melting temperatures were screened from the standard reference table, and the isolated strains were identified as follows: Pectinatus genus 。

[0052] Table 3: Screening results of melting temperature of separated samples using the RT-PCR standard reference table

[0053] 2.1.2.3 16S rDNA sequencing The results of sequencing the isolated samples by BGI Genomics are detailed in Table 4. After BLAST comparison in NCBI, the isolated samples were confirmed to be... Pectinatus frisingensis The strain has been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 7.552 and deposit date of November 25, 2025.

[0054] Table 4: Sequencing alignment results of isolated strains

[0055] 2.2 Results of bacterial suspension counting of symbiotic bacteria experimental strains Table 5: Results of bacterial suspension counting of symbiotic relationship experimental strains

[0056] Table 6: Results of Artificial Counting of Strains in Quantitative Experiments on Symbiotic Relationships

[0057] 2.3 Results of the Symbiosis Experiment Fresh bacterial suspensions activated within one week were inoculated into deoxygenated NBB-B medium and beer according to the "1.4 Symbiotic Experimental Design Scheme". NBB culture was carried out for 15 days. The total inoculum size was calculated based on the bacterial suspension results and dilution factors in Table 5. GC-MS was used to detect metabolites in different experimental groups. The pH of each experimental group was measured and RT-PCR was performed. Detailed results are shown in Tables 7 and 8. Figure 5 - Figure 8 As shown.

[0058] Table 7: Detection results of different experimental groups after growth in NBB-B matrix

[0059] Note: "-" indicates no or negative; "+" indicates the substance was detected.

[0060] Table 8: Detection results of different experimental groups after growth in beer matrix

[0061] Note: "-" indicates no or negative; "+" indicates the substance was detected.

[0062] From the experimental results of groups 1-6 in Tables 7 and 8, it can be concluded that propionic acid is a marker metabolite of Pectinobacterium. Figure 5 Butyric acid is a hallmark metabolite of lactic acid bacteria. Figure 6 , Figure 7 The main metabolic substances of *Pectinobacterium* differ between NBB-B and beer. Acetoin was also detected in NBB-B, possibly due to the different nutrient components in NBB-B and beer, leading to different substances synthesized by *Pectinobacterium* after metabolism. Comparing pH results, the pH decrease after growth of *Pectinobacterium* in NBB-B and beer was smaller than that of lactic acid bacteria. This may be due to its inherent characteristics or its slower growth rate. Comparing pH changes in different substrates, in the nutrient-rich NBB-B medium, the pH of the *Pectinobacterium* sample decreased significantly from 5.68 to approximately 4.4. However, in the beer substrate, due to the buffering capacity of beer, the pH decreased less significantly after growth, from 4.52 to 4.25.

[0063] The symbiotic experiments in groups 7-15 of Tables 7 and 8 can be combined with the metabolites of the experimental strains ( Figure 8The presence of growth was determined by RT-PCR results. From the RT-PCR results, except for group 15 in Table 7 where *Pectinobacterium* was not identified, *Pectinobacterium* was identified in all other groups. However, considering the flavor metabolites, propionic acid, a metabolite of *Pectinobacterium*, was detected in this group, indicating that *Pectinobacterium* was growing. Acetoin was not detected, which, combined with the RT-PCR results, indicates that the growth of *Pectinobacterium* in this group was relatively low. Lactic acid bacteria did not grow in some experimental groups, possibly due to the small inoculum size. From the NBB-B experiments in Table 7, it was found that groups 7, 13, and 15 did not detect acetoin metabolites, indicating that the growth of *Pectinobacterium* may be low. Furthermore, the pH of these groups was below 4, exceeding their growth range, indicating that the symbiotic growth of *Pectinobacterium* is pH-dependent.

[0064] The test was conducted using Pectinobacterium that had grown for half a month after inoculation, with relatively low viability. It was inoculated into NBB-B medium according to the "1.4 Symbiotic Experimental Design Scheme" and cultured for 15 days. The total inoculation amount was calculated based on the bacterial suspension results and dilution factor in Table 6. The experimental results are shown in Table 9.

[0065] Table 9: Changes in major flavor compounds in symbiotic microbiota detected by GC-MS semi-quantitative analysis

[0066] Note: "-" indicates none or negative.

[0067] Within the symbiotic system, when the inoculum size of *Pectinobacterium* was large, it was confirmed by RT-PCR to grow. Lactic acid bacteria, however, failed to grow due to the extremely low inoculum size. *Pectinobacterium* and lactic acid bacteria coexisted, producing small amounts of propionic acid or acetoin, indicating their symbiotic relationship. However, some experimental groups did not show detectable marker metabolites, suggesting that the growth of *Pectinobacterium* in these groups was not significant. The propionic acid content in the symbiotic system was significantly lower than in the groups where *Pectinobacterium* was absolutely dominant (groups 3, 6, and 9), indicating that the test bacteria had a certain inhibitory effect on the growth of *Pectinobacterium*. The pH was observed to be below 4 at these times, consistent with the qualitative experimental results, indicating that when the pH of the symbiotic environment exceeds the growth range of *Pectinobacterium*, its growth will be inhibited or cease. When the inoculum size of *Pectinobacterium* was small, *Pectinobacterium* was not identified in any of the groups, nor was its characteristic metabolite propionic acid detected. Only a few groups showed the presence of acetoin, indicating that the inoculum size affects the symbiotic growth of *Pectinobacterium*. Compared with the qualitative experiment, at the same inoculum size, the symbiotic growth of *Pectinobacterium* experimental strains with shorter growth cycles and higher activity was better when the activity was lower, as the bacteria produced propionic acid more effectively. This indicates that the activity of the strain affects the symbiotic growth.

[0068] Combining qualitative and quantitative experiments with microbial characterization of the symbiotic process, it was found that *Pectinobacterium* requires a strictly anaerobic environment for growth. The symbiotic lactic acid bacteria can grow in a facultative anaerobic environment and consume oxygen, providing anaerobic conditions for *Pectinobacterium* growth. Therefore, *Pectinobacterium* can grow under symbiotic conditions. However, because the growth rate of *Pectinobacterium* is much lower than that of lactic acid bacteria, which grow rapidly and produce acid in a short time, the pH of the symbiotic environment decreases. When the pH drops below 4, it exceeds the pH requirement of >4.4 for *Pectinobacterium* growth, thus inhibiting its growth. Relatively speaking, beer has a better buffering capacity than culture medium, prolonging the pH drop time and providing more favorable conditions for *Pectinobacterium* growth. The final growth amount of *Pectinobacterium* in the symbiotic environment also depends on its viability and initial concentration. When viability decreases or the initial concentration is low, the time required to grow to a detectable concentration level is longer, and growth is prematurely inhibited due to the pH drop.

[0069] This study isolated and identified an obligate anaerobic pectinobacterium. Pectinatus frisingensis (CGMCC No. 7.552) This study qualitatively and quantitatively investigated the symbiotic relationship between *Pectinobacterium* and common lactic acid bacteria using PCR and GC-MS. The results showed that the growth of *Pectinobacterium* in its symbiotic environment with lactic acid bacteria depends on the viability and initial concentration of *Pectinobacterium* cells, as well as the pH of the symbiotic environment. Within the symbiotic system, the stronger the viability and the higher the concentration of *Pectinobacterium* cells, and the stronger the pH buffering capacity of the symbiotic environment, the more readily *Pectinobacterium* can grow and produce propionic acid before being inhibited by lactic acid bacteria. However, as the acid production of lactic acid bacteria rapidly accumulates, the pH of the substrate drops below 4, inhibiting the growth of *Pectinobacterium*. This study provides supporting data for the study of the symbiotic relationship between *Pectinobacterium* and lactic acid bacteria in beer fermentation, and can help understand the synergistic competitive effects between the two in metabolism, flavor, and growth.

[0070] Due to the obligate anaerobic nature of *Pectinobacterium*, it is usually difficult to collect samples alone, and enrichment is also challenging. The *Pectinobacterium*-lactobacter symbiotic system constructed using this invention can predict potential *Pectinobacterium* contamination areas by utilizing the detected lactic acid bacteria types and pH changes, thereby strengthening the monitoring and cleaning of key areas. This is of great significance for the control of this type of bacteria. In actual production, by strengthening the monitoring and equipment cleaning of the main contamination areas where *Lactobacillus brevis* was detected in positive samples, the probability of contamination by *Lactobacillus brevis* symbiotic with *Pectinobacterium* can be reduced, thus reducing the risk of *Pectinobacterium* contamination. Furthermore, by adjusting the pH of beer to below 4, the risk of *Pectinobacterium* contamination is significantly reduced.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A strain of obligate anaerobic pectinobacterium beer Pectinatus frisingensis Its accession number is CGMCC No.7.

552.

2. The application of the beer-specific anaerobic pectinobacterium as described in claim 1 in constructing a symbiotic system of pectinobacterium and lactic acid bacteria.

3. The application according to claim 2, characterized in that, The construction includes the following steps: S1. Qualitative experiments: Beer obligate anaerobic pectinobacter and lactic acid bacteria were cultured separately, as well as in combination with different concentrations of the two in a symbiotic culture. S2. Quantitative experiment: The beer-specific anaerobic pectinobacterium and lactic acid bacteria were co-cultured at different concentrations. The flavor fingerprint of the metabolites was detected by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry, and the strains in the co-culture system were identified by RT-PCR.

4. The application according to claim 3, characterized in that, The chromatographic conditions are as follows: Helium was used as the carrier gas, and a constant flow mode was used with a flow rate of 1.0 mL / min and an injection port temperature of 250℃. Temperature program: Start at 60℃, hold for 4 min, increase to 210℃ at 5℃ / min, increase to 250℃ at 10℃ / min, hold for 10 min.

5. The application according to claim 3, characterized in that, The mass spectrometry conditions are as follows: The ion source temperature was 280 °C, the transfer line temperature was 250 °C, the ion source was an EI source, the electron impact source was 70 eV, and the MS scan range was 35-350 amu.

6. A symbiotic system of beer-specific anaerobic pectinobacterium and lactic acid bacteria, characterized in that, The system includes beer-specific anaerobic pectinobacteria and lactic acid bacteria as described in claim 1.

7. The beer-specific anaerobic pectinobacterium and lactic acid bacteria symbiotic system according to claim 6, characterized in that, The lactic acid bacteria include any one or more of Lactobacillus brevis, Lactobacillus casei, and Lactobacillus plantarum.

8. The application of the beer-specific anaerobic pectinobacterium and lactic acid bacteria symbiotic system as described in claim 6 in controlling harmful bacteria contamination in beer.

9. The application according to claim 8, characterized in that, Keep the pH of the beer fermentation liquid as close to or below 4 as possible.

10. A method for co-culturing obligate anaerobic pectinobacterium in beer with lactic acid bacteria, characterized in that, include: The beer-specific anaerobic pectinobacterium and lactic acid bacteria as described in claim 1 were inoculated into NBB-B medium for culture.

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