Screening method and application of microbial strain with high yield of ergothioneine

Abstract

The invention belongs to the field of synthetic biology and metabolic engineering, and discloses a screening method and application of a microbial strain with high yield of ergothioneine, a promoter (such as PECL1, PBSC5, PEDC2 and the like) responding to ergothioneine is screened through transcriptomics analysis based on saccharomyces cerevisiae, and the promoter can present high sensitivity and linear response under 0-1000 mg / L of ergothioneine. The strain is used for driving fluorescent protein gene expression, a fluorescence report strain is constructed, the strain can rapidly and sensitively detect the concentration of ergothioneine in fermentation liquor by virtue of fluorescence signal change, and high-yield strains are efficiently screened. The method overcomes the problems of low flux, long period, low sensitivity and the like of traditional screening, and the fluorescence signal intensity is in significant linear correlation with the ergothioneine concentration. And the perforated plate and the microplate reader are used for automatic high-throughput screening, so that the efficiency is improved, the resource consumption is reduced, effective support is provided for production optimization of ergothioneine, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of synthetic biology and metabolic engineering, and relates to a screening method of a microbial strain with high ergothioneine production and application, in particular to an efficient screening method for driving the expression of a reporter gene by constructing a promoter responsive to ergothioneine. BACKGROUND

[0002] Ergothioneine (L-Ergothioneine, EGT) is a natural sulfur-containing amino acid with unique antioxidant properties and physiological functions. Since it was first isolated from Claviceps purpureus in 1909, Claviceps purpurea Ergothioneine has been widely studied and shown great application prospects in medicine, cosmetics, functional foods and other fields. Ergothioneine not only has excellent antioxidant capacity, but also has significant physiological effects in cell protection, anti-inflammatory, anti-aging, immune regulation, etc. It can effectively scavenge free radicals, especially has a significant protective effect on oxidative damage in mitochondria and cell nucleus, and is therefore known as "longevity vitamin". Therefore, the production and application of ergothioneine have become the focus of attention in multiple industries.

[0003] However, the industrial production of ergothioneine still faces many challenges. The existing production methods mainly include natural extraction method, chemical synthesis method and biosynthesis method, each of which has certain limitations and deficiencies, which seriously restricts the feasibility of large-scale and low-cost production. First of all, the limitations of natural extraction method are obvious. The natural sources of ergothioneine mainly include edible fungi, cereals and animal tissues, especially in edible fungi. Although some traditional natural extraction methods (such as ethanol reflux extraction method, enzymatic hydrolysis method and ultrasonic extraction method) can efficiently extract ergothioneine, their production efficiency is low and they are limited by the seasonality and yield of raw materials. Edible fungi have a long cultivation period, low yield, and the extraction process is complex and costly, which makes it difficult for natural extraction method to meet the needs of industrialization. In addition, with the increasing demand for ergothioneine, the production method relying on natural raw materials is unsustainable, so it is urgent to find a more efficient and sustainable production approach. Secondly, although chemical synthesis method has made certain progress in laboratory conditions and can achieve the synthesis of ergothioneine, its process is complex, involving multiple reactions, and often accompanied by the generation of toxic by-products, which poses certain environmental risks. In addition, chemical synthesis requires precise control of stereoselectivity and purity, and the production cost is high, and it is difficult to achieve large-scale production. Therefore, although chemical synthesis method can provide a synthesis route for ergothioneine, its application prospect in industrial production is still limited.

[0004] Biosynthesis has become the main research direction of ergothioneine production due to its green environmental protection, low cost, simple operation and other advantages. Microbial fermentation method realizes the synthesis of ergothioneine by genetically engineering microorganisms (such as Escherichia coli, Saccharomyces cerevisiae, Aspergillus etc.). Compared with traditional extraction and chemical synthesis method, microbial synthesis method has many advantages, including short culture period, sufficient raw materials, environmentally friendly production process and easy to scale up, so it is considered as the most promising production method.

[0005] However, the biosynthesis of ergothioneine still faces many bottlenecks. First, the biosynthesis pathway of ergothioneine is not completely clear, although a variety of model microorganisms (such as Escherichia coli, Saccharomyces cerevisiae and Neurospora crassa) have been used to construct the synthesis path of ergothioneine, but its synthesis efficiency is still limited. In order to improve the production efficiency, researchers have modified microbial strains through genomics, metabolic engineering, synthetic biology and other means, such as through multi-copy integration of key enzyme genes, optimization of precursor supply pathway and other strategies, but these modifications have not been able to significantly improve the yield of ergothioneine. Second, the existing high-yield ergothioneine yeast strains are still in the preliminary research stage, and lack efficient screening methods. Traditional screening methods require a large amount of time and resources, and cannot screen the best high-yield strain in a short time. Therefore, developing an efficient and rapid screening method is crucial to improve the production level of ergothioneine.

[0006] Currently, the methods for screening high-yield ergothioneine yeast strains mainly include metabolic phenotype-based screening and genomics method. Metabolic phenotype screening method usually relies on the accumulation of ergothioneine on the culture medium, however, this method is low in efficiency and is easily disturbed by external environment. Genomics screening method relies on the regulation of the expression level of synthesis pathway related genes, although this method can improve the screening accuracy, but still requires a large amount of genomic data and relatively complex screening steps. Therefore, the existing screening methods have many shortcomings in high-throughput, rapidity, sensitivity and other aspects, and a new screening technology is needed to make up for these defects. SUMMARY

[0007] In order to make up for the shortcomings of the prior art, the present application relates to a screening method for high-yield ergothioneine microbial strains and its application. Specifically, the present application screens a specific response ergothioneine promoter based on Saccharomyces cerevisiae transcriptomics analysis, and uses the promoter to drive the expression of fluorescent protein coding gene to construct a fluorescent reporter strain. The reporter strain can be used for high-throughput screening of fermentation products containing ergothioneine, and the content of ergothioneine in the fermentation product is reflected by the intensity of the fluorescent signal, and then the high-yield ergothioneine microbial strain (test strain) is screened.

[0008] The core idea of the present application is to provide an efficient and rapid screening method by constructing a biosensor system (reporter strain) specifically responsive to ergothioneine, overcoming the limitations of traditional screening methods (high performance liquid chromatography, etc.), such as low throughput and long cycle.

[0009] The specific technical solutions of the present application include: A screening method for a microbial strain with high ergothioneine yield, comprising the following steps: S1. Screening and application of a promoter specifically responsive to ergothioneine; A promoter specifically responsive to ergothioneine is screened through transcriptomic analysis. Specifically, a model microorganism with a clear genetic background is selected as the research object, and it is treated with different concentrations of ergothioneine. Total RNA is extracted and subjected to transcriptomic analysis. By comparing the gene expression differences between the treatment group and the control group, genes with significantly up-regulated expression after ergothioneine stimulation are screened, and by analyzing the promoter region, a promoter with high sensitivity and linear response is selected. These promoters can drive gene expression in the presence of different concentrations of ergothioneine, providing a promoter element for subsequent construction of an ergothioneine biosensor system (reporter strain).

[0010] S2. Construction of a fluorescent reporter strain; A fluorescent reporter strain is constructed by combining the selected promoter specifically responsive to ergothioneine with a fluorescent protein gene. Specifically, the ergothioneine-responsive promoter, fluorescent protein gene, and terminator are linked to construct a recombinant expression plasmid expressing fluorescent protein, and the recombinant plasmid is transformed into a model microorganism with a clear genetic background through genetic transformation technology. The successfully transformed microbial strain can be used as a reporter strain for detecting ergothioneine content.

[0011] S3. High-throughput screening; Based on the reporter strain constructed in S2, the present application designs a high-throughput screening method for microbial strains producing ergothioneine based on a fluorescent reporter system (see Figure 1 for the specific screening process). Specifically, the test strains are cultured in a multi-well microbioreactor, and the culture time is determined according to different microorganisms. After the culture is completed, a certain concentration of reporter strain is added, and the culture is continued to induce the expression of fluorescent protein. The fluorescence signal is detected using a multifunctional enzyme marker instrument. By analyzing the relationship between the fluorescence signal and the ergothioneine content, the test strains with high ergothioneine yield are quickly identified.

[0012] In embodiment S1, the model microorganism with a clear genetic background includes Saccharomyces cerevisiae, Escherichia coli, Aspergillus niger, and other model microorganisms.

[0013] In embodiment S1, the different concentrations of ergothioneine are 0-1000 mg / L.

[0014] In implementation scheme S1, when the model fungus Saccharomyces cerevisiae S Saccharomyces cerevisiae S288C cells were treated with 0-1000 mg / L ergothioneine, and transcriptomic analysis revealed that the promoter specifically responding to ergothioneine was P. ECL1 P BSC5 P EDC2 .

[0015] In implementation scheme S2, the fluorescent protein includes green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.

[0016] In implementation scheme S2, the genetic transformation technology includes chemical transformation, electrotransformation, protoplast transformation, Agrobacterium-mediated genetic transformation, etc.

[0017] In implementation scheme S2, when based on the model fungus Saccharomyces cerevisiae S . cerevisiae S288C was used to construct a reporter strain, and the promoter (P) was ligated via seamless cloning or restriction enzyme ligation technology. ECL1 P BSC5 or P EDC2 The green fluorescent protein (GFP) gene and the CYC1 terminator were combined to construct a GFP expression cassette. Using the free expression plasmid pYES2-KanMX as a backbone, recombinant expression plasmids pYES2-KanMX-P were constructed. ECL1 / P BSC5 / P EDC2 -GFP-T CYC1 .

[0018] In implementation scheme S2, the reporter strain is a model microbial strain successfully introduced with a free recombinant expression plasmid. When the reporter strain is placed in a liquid system containing ergothioneine, the promoter specifically responding to ergothioneine will be induced by exogenous ergothioneine to synthesize fluorescent protein, and the change in fluorescence intensity is well correlated with the ergothioneine content. By detecting the fluorescence intensity, the ergothioneine yield in the liquid system can be determined.

[0019] In implementation scheme S3, the porous microbioreactor includes 24-well plates, 48-well plates, 96-well plates, 384-well plates, etc.

[0020] In implementation scheme S3, the test strains include wild-type strains or genetically engineered strains of Saccharomyces cerevisiae, Escherichia coli, filamentous fungi, actinomycetes, etc., that can produce ergothioneine.

[0021] In implementation scheme S3, when the added reporter strain is a Saccharomyces cerevisiae strain, the added cell concentration is 10. 6 -10 8CFU / mL, and the incubation time is 2-6 h.

[0022] Compared with the prior art, the present application has the beneficial effects that: Although ergothioneine as a natural product with broad application prospects, there are many methods for research and production, but the current production method still has the problems of high cost, low efficiency and the like. Therefore, developing a high-efficiency, low-cost and green production process, especially through a biosynthetic pathway for production, has become a technical problem to be solved. The introduction of the biosensor system provides a new idea for solving this problem. Through the expression of the reporter gene driven by the ergothioneine-responsive promoter, the screening efficiency can be improved in high-throughput screening, and a new tool is provided for screening of high-ergothioneine-producing strains.

[0023] The biosensor system of the present application can be widely applied to the screening, optimization and modification of high-ergothioneine-producing microbial strains. Compared with the traditional metabolic phenotype screening method (HPLC, etc.), the biosensor system of the present application can significantly improve the screening efficiency, save time and resources, and quickly identify excellent strains with high ergothioneine production in genetically edited or metabolically engineered microbial strains.

[0024] The present application screens the ergothioneine-responsive promoter based on the transcriptomic analysis of Saccharomyces cerevisiae, and uses the promoter to drive the expression of the fluorescent protein gene to construct a fluorescent reporter strain. The reporter strain can quickly and sensitively detect the ergothioneine concentration in the fermentation broth through the change of the fluorescent signal, thereby realizing efficient screening of high-ergothioneine-producing microbial strains. The biosensor system of the present application selects promoters that can exhibit high sensitivity and linear response under different concentrations of ergothioneine (0-1000 mg / L), such as P ECL1 , P BSC5 , P EDC2 , etc., to overcome the problems of low throughput, long cycle and low sensitivity of traditional screening methods. There is a significant linear relationship between the intensity of the fluorescent signal and the concentration of ergothioneine, which can efficiently screen high-ergothioneine-producing strains. The method uses multi-well plates and an enzyme label instrument to realize automated high-throughput screening, significantly improving the screening efficiency and reducing the experimental time and resource consumption. The technology of the present application not only provides a new tool for screening of high-ergothioneine-producing strains, but also provides effective support for the production optimization of ergothioneine in microbial fermentation process, and can be widely applied in the fields of synthetic biology, metabolic engineering and green biological manufacturing. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a flow chart for screening of high-ergothioneine-producing microbial strains; Figure 2is volcano plot of differentially expressed genes (DEGs) between 2 h and 0 h ergothioneine-treated S. cerevisiae; Figure 3 is GO enrichment results of molecular function, cellular component and biological process categories between 2 h and 0 h ergothioneine-treated S. cerevisiae; Figure 4 is KEGG pathway enrichment of DEGs between 2 h and 0 h ergothioneine-treated S. cerevisiae; Figure 5 is a Venn diagram showing genes up-regulated in response to ergothioneine between 2 h and 0 h ergothioneine-treated S. cerevisiae; Figure 6 is promoter P ASH1 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 7 is promoter P BSC5 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 8 is promoter P CHA1 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 9 is promoter P ECL1 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 10 is promoter P ECM13 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 11 is promoter P EDC2 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 12 is promoter P PDR3 Construction process of recombinant plasmid for driving expression of green fluorescent protein (GFP); Figure 13 is comparison of induction effects of ergothioneine on 7 promoters; Figure 14 is promoter P ECL1 , P BSC5 , P EDC2 response to ergothioneine. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0027] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] like Figure 1 As shown, the screening process for high-ergothioneine-producing microbial strains includes: liquid culture of the test strains using a porous microbioreactor, with the culture time determined according to the specific microorganism; after culture, a certain concentration of reporter strain is added, and culture continues to induce fluorescent protein expression; the fluorescence signal is detected using a multifunctional microplate reader. By analyzing the relationship between the fluorescence signal and ergothioneine content, high-ergothioneine-producing test strains can be rapidly identified.

[0029] Example 1: Transcriptome analysis of gene expression in Saccharomyces cerevisiae in response to ergothionein This embodiment aims to study *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) using transcriptome analysis technology. S. cerevisiae Gene expression changes induced by ergothioneine (EGT) were investigated. Saccharomyces cerevisiae strain S288C was selected as the experimental subject and cultured to the logarithmic growth phase. The strains were then divided into a control group (0 mg / L ergothioneine) and experimental groups (treated with 50 mg / L, 100 mg / L, and 200 mg / L ergothioneine, respectively). Three biological replicates were set up for each group. Yeast samples were collected 2 hours after treatment, and total RNA was extracted and sequenced using high-throughput RNA sequencing. Differential expression analysis (DEGs) identified 2618 differentially expressed genes, of which 1103 were upregulated and 1515 were downregulated. Figure 2 Further GO enrichment analysis showed that the upregulated genes are mainly involved in biological processes such as redox reactions, energy metabolism, and cellular respiration. Figure 3 The upregulation of these genes may be due to yeast cells responding to ergothioneine-induced oxidative stress. KEGG pathway analysis further revealed that most of the upregulated genes are involved in mitochondrial function, energy metabolism, and redox processes, suggesting that the antioxidant effect of ergothioneine may exert its effect by regulating mitochondrial oxidative stress responses. Figure 4 Furthermore, it was found that ergothionein-induced gene expression is closely related to mitochondrial energy metabolism, providing theoretical support for subsequent screening of high-yield ergothionein-producing yeast strains using biosensors, and providing important molecular mechanism data for further research on the physiological effects of ergothionein.

[0030] Example 2: Screening and identification of promoters in Saccharomyces cerevisiae that respond to ergothioneine This embodiment screened for promoters responding to ergothioneine based on transcriptome analysis results and constructed a fluorescent reporter system to verify their function. First, through transcriptome analysis, 190 significantly upregulated genes were screened from gene expression data of *Saccharomyces cerevisiae* treated with ergothioneine.Figure 5 ). Seven candidate promoters were designed according to the promoter regions of these genes (e.g. P CHA1 , P ECM13 , P ECL1 , P BSC5 , P EDC2 , P ASH1 , P PDR3 , etc.) (Table 1). Then, using the pYES2-URA3 vector, the promoters were linked to the green fluorescent protein gene (GFP) to construct GFP reporter plasmids, and the constructed plasmids (pYES2-URA3-P Figure 6-12 ) were introduced into the S. cerevisiae BY4741 strain by yeast transformation technology. The transformed strain was cultured in different concentrations of ergothioneine (0, 50, 100 mg / L), and the changes in fluorescence signals were monitored in real time by fluorescence microscopy and a microplate reader. The results showed that the P ECL1 , P BSC5 , P EDC2 promoters exhibited significant fluorescence enhancement under ergothioneine treatment, and the fluorescence intensity increased with increasing ergothioneine concentration. Under a concentration of 100 mg / L ergothioneine, the P ECL1 , P BSC5 , P EDC2 promoters drove significant enhancement of GFP fluorescence signals, indicating that these promoters had good responsiveness to ergothioneine ( Figure 13 ). In addition, these promoters exhibited good linear relationships under different concentrations of ergothioneine, and could be used as ideal elements for constructing ergothioneine-responsive biosensors. This example not only screened some efficient ergothioneine-responsive promoters, but also provided a basis for the optimization and application of subsequent biosensors.

[0031] Table 1 Seven candidate genes with the most obvious up-regulation

[0032] Example 3: Linear analysis of the concentration gradient response of promoters P ECL1 , P BSC5 , P EDC2 to ergothioneine This example mainly discusses the response characteristics of P ECL1 , P BSC5 , P EDC2 promoters under different concentrations of ergothioneine, and analyzes the linear relationship between the fluorescence signals and the concentration of ergothioneine. First, the pYES2-URA3 vector containing P ECL1 , P BSC5 , P EDC2The GFP reporter plasmid with different promoters was transformed into the BY4741 strain of Saccharomyces cerevisiae, and then the transformed strains were inoculated into the medium containing different concentrations of ergothioneine (0, 50, 100, and 200 mg / L) for culture. The OD values and fluorescence intensities of each strain were measured at 2 h and 6 h, respectively. 600 The results showed that the GFP fluorescence signal driven by the P EDC2 promoter gradually increased with the increase of the concentration of ergothioneine, and showed a good linear correlation in the whole concentration range. Specifically, in the concentration range of 50 mg / L to 200 mg / L, the P EDC2 promoter showed a more significant fluorescence enhancement, and the linear correlation between the fluorescence signal and the concentration of ergothioneine was stronger (R² > 0.95). In contrast, the P ECL1 and P BSC5 promoters showed more obvious differences in fluorescence signals in the low concentration range of 0-50 mg / L, but the increase in fluorescence signals gradually slowed down in the concentration range of 50 mg / L to 150 mg / L. Through further linear regression analysis, it was confirmed that there was a certain linear relationship between the fluorescence signals of P ECL1 and P BSC5 and the concentration of ergothioneine, although the responsiveness was lower than that of the P EDC2 promoter, but still had good application potential ( Figure 14 ). This result provided a basis for the characteristic analysis of different promoters in the concentration range of ergothioneine response. The Saccharomyces cerevisiae strain into which the free GFP expression plasmid was transformed in this example was a reporter strain.

[0033] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can make equivalent substitutions or changes to the technical solutions and inventive concepts of the present application within the scope of the disclosure, and all such substitutions or changes should be covered within the protection scope of the present application.

Claims

1. A method for screening a microbial strain having high ergothioneine productivity, characterized by, Comprising the following steps: S1. Screening and application of specific response to ergothioneine promoter: Selecting a model microorganism with clear genetic background as the research object, stimulating it with different concentrations of ergothioneine, extracting total RNA and performing transcriptome analysis, comparing the gene expression differences between the treatment group and the control group, selecting the genes with significantly up-regulated expression after response to ergothioneine stimulation, analyzing the promoter region, and selecting the promoter with high sensitivity and linear response; S2. Constructing a fluorescent reporter strain: Using the selected specific response to ergothioneine promoter combined with a fluorescent protein gene to construct a fluorescent reporter strain; S3. High-throughput screening: Based on the reporter strain constructed in step S2, using a multi-well micro bioreactor to culture the test strain, adding a certain concentration of reporter strain after the culture is completed, continuing to culture to induce the expression of fluorescent protein, using a multifunctional enzyme marker to detect the fluorescence signal, and quickly identifying the high-yield ergothioneine test strain through the relationship between the fluorescence signal and the ergothioneine content.

2. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S1, the model microorganism with clear genetic background includes at least one of Saccharomyces cerevisiae, Escherichia coli, and Aspergillus niger.

3. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S1, the different concentrations of ergothioneine are 0-1000 mg / L.

4. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S1, when the model fungus Saccharomyces cerevisiae S Saccharomyces cerevisiae S288C is treated with 0-1000 mg / L ergothioneine, transcriptional profiling analysis screens for promoters P ECL1 , P BSC5 , and P EDC2 that are specifically responsive to ergothioneine.

5. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S2, the fluorescent protein includes at least one of green fluorescent protein, red fluorescent protein, and yellow fluorescent protein.

6. The method for screening of high-ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S2, the genetic transformation technology includes at least one of chemical transformation, electroporation, protoplast transformation, and Agrobacterium-mediated genetic transformation.

7. The method for screening of high-ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S2, when the model fungus S. cerevisiae Saccharomyces cerevisiae S288C construction report strain, by seamless cloning or enzyme cutting connection technology connection promoter (P ECL1 , P BSC5 or P EDC2 ), green fluorescent protein (GFP) gene and CYC1 terminator, construct green fluorescent protein expression cassette, with free expression plasmid pYES2-KanMX as the skeleton to construct recombinant expression plasmid pYES2-KanMX-P ECL1 -GFP-T CYC1 , pYES2-KanMX-P BSC5 -GFP-T CYC1 , pYES2-KanMX-P EDC2 -GFP-T CYC1 .

8. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S2, the reporter strain is a model microorganism strain successfully introduced with a free recombinant expression plasmid. When the reporter strain is placed in a liquid system containing ergothioneine, the specific response to ergothioneine promoter will be induced to synthesize fluorescent protein, and the change in fluorescence intensity has a good correlation with the ergothioneine content. By detecting the fluorescence intensity, the ergothioneine production in the liquid system can be determined.

9. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S3, the multi-well micro bioreactor includes at least one of a 24-well plate, a 48-well plate, a 96-well plate, and a 384-well plate.

10. The method for screening of high ergothioneine-producing microbial strain according to claim 1, characterized in that, In step S3, the to-be-tested strains include wild strains or genetically engineered strains capable of producing ergothioneine in Saccharomyces cerevisiae, Escherichia coli, filamentous fungi, and actinomycetes; when the added reporter strain is a Saccharomyces cerevisiae strain, the added cell concentration is 10 6 -10 8 CFU / mL, and the continued culture time is 2-6 h.

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