The ldh Gene Plays a Crucial Role in Mediating the Pathogen Control of Lactiplantibacillus plantarum AR113

Abstract

Effectively managing foodborne pathogens is imperative in food processing, where probiotics play a crucial role in pathogen control. This study focuses on the Lactiplantibacillus plantarum AR113 and its gene knockout strains, exploring their antimicrobial properties against Escherichia coli O157:H7 and Staphylococcus aureus. Antimicrobial assays revealed that the inhibitory effect of AR113 increases with its growth and the potential bacteriostatic substance is acidic. AR113Δldh, surpassed AR113Δ0273&2024, exhibited a complete absence of bacteriostatic properties, which indicates that lactic acid is more essential than acetic acid in the bacteriostatic effect of AR113. However, the exogenous acid validation test affirmed the equivalent superior bacteriostatic effect of lactic acid and acetic acid. Notably, AR113 has high lactate production and deletion of the ldh gene not only lacks lactate production but also affects acetic production. This underscores the ldh gene’s pivotal role in the antimicrobial activity of AR113. In addition, among all the selected knockout strains, AR113ΔtagO and ΔccpA also had lower antimicrobial effects, suggesting the importance of tagO and ccpA genes of AR113 in pathogen control. This study contributes insights into the antimicrobial potential of AR113 and stands as the pioneering effort to use knockout strains for comprehensive bacteriostatic investigations.

Introduction

Bacterial pathogens constitute a group of highly contagious and pathogenic microorganisms capable of inducing diverse intestinal infectious diseases (Siebers and Finlay, 1995; Fernandez-Lopez et al., 2019). According to the World Health Organization, one in ten individuals is affected by bacterial pathogens annually worldwide (WHO, 2019). Inhibiting the growth and development of bacterial pathogens is crucial in food quality and safety control (Bazargani and Rohloff, 2016). Antibiotics are traditional suppressants of the growth of bacterial pathogens. However, the continued emergence of antibiotic-resistant strains limits our ability to control pathogens (Mir and Kudva, 2019). Furthermore, antibiotics also exhibit profound side effects on the intestinal microbiota that alter the nutrient-absorbing landscape of the gut and can lead to the expansion of pathogenic populations (Bäumler and Sperandio, 2016). Due to the lack of new classes of antibiotics, alternative therapies represent a promising area of investigation (Fleitas Martinez et al., 2019; Kim, 2019).

The use of probiotics as a novel alternative to antibiotics represents a novel research direction. Lactic acid bacteria (LAB), which are common probiotics found in fermented foods, have garnered significant interest owing to their antimicrobial properties (Hu et al., 2019). Lactiplantibacillus plantarum is an optional heterofermentative LAB belonging to the genus Lactiplantibacillus. L. plantarum has obtained Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA) and Generally Recognized as Safe (GRAS) status from the U.S. Food and Drug Administration (US FDA) (Li et al., 2023). Our team previously isolated L. plantarum AR113 from traditional Chinese kimchi. According to our research, AR113 can survive in low pH, high concentrations of bile salts, and high osmotic pressure (Lin et al., 2020). In addition, AR113 possesses optimal probiotic and health-promoting properties such as acceleration of liver regeneration (Xie et al., 2022), antioxidation under in vitro (Lin et al., 2020) and in vivo conditions (Xia et al., 2023), and alleviation of colitis (Xia et al., 2020). In this study, the pathogen control of AR113 in vitro was systematically investigated.

Inactivating the production of substances by manipulating their genes is an effective method for studying the functions of substances (Zhang et al., 2021). The knockout strains showed unfavorable effects, indicating the critical role of this gene. Previously, upon examining gene knockout strains of AR113, we confirmed that bile salt hydrolase (bsh) 1 and bsh 3 genes on AR113 are crucial in ameliorating colitis (Feng et al., 2023; Shao et al., 2023). In this study, we conducted antimicrobial experiments using AR113 Δldh and Δ0273&2024, which disrupt common organic acid kinase, to elucidate its primary bacteriostatic substances. In addition, strains with knockout mutations in the exopolysaccharide (EPS) synthesis cluster, as well as structure- and growth-related genes, were also examined to explore the impact of these genes on AR113 bacterial inhibition. Our research has laid the theoretical foundation for understanding the antimicrobial effect of L. plantarum AR113. Moreover, using knockout strains in antimicrobial experiments has provided a novel avenue for further elucidating the underlying mechanisms.

Materials and Methods

Strains and cultural conditions

L. plantarum AR113 (CGMCC No. 13909), identified by Shanghai Miki Biological Co., Ltd., was maintained at the China General Microbiological Culture Collection Center. Knockout strains of AR113 (Table 1) were obtained from our collection. These strains were cultured in the deMan, Rogosa, and Sharpe (MRS) medium. Escherichia coli O157:H7 (CICC NO. 10907) and Staphylococcus aureus (CICC NO. 10384) were selected as pathogenic indicator strains and were grown in Luria-Bertani broth (LB) and nutrient broth (NB) basal medium, respectively. Both strains were maintained at the China Center of Industrial Culture Collection. All strains were incubated at 37°C.

Table 1.

AR113 and Corresponding Gene Knockout Strains

Strains	Descriptions
L. plantarum AR113	Lactiplantibacillus plantarum AR113
AR113 Δltas	Lipoteichoic acid synthesis (ltas) gene knockout strain
AR113 ΔtagO	Teichoic acid synthesis (tagO) gene knockout strain
AR113 ΔEPS	Exopolysaccharide synthesis cluster (EPS) gene knockout strain
AR113 ΔsrlD	Sorbitol synthesis (srlD) gene knockout strain
AR113 Δ0273&2024	Acetate kinase (0273&2024) gene knockout strain
AR113 Δldh	D-type and L-type lactate dehydrogenase (ldh) gene knockout strain
AR113 ΔccpA	Catabolite control protein A (ccpA) gene knockout strain

Δ represents a knockout, followed by a letter or number code representing the AR113 gene.

Preparation of fermentation broth and cell-free supernatant

The bacterial solution was used as the bacterial fermentation broth at the end of the extended incubation in an MRS liquid medium. The cell-free supernatant (CFS) was obtained after centrifugation (10 min at 3000×g at 4°C) and sterilized filtration of fermentation broth liquid supernatant.

Antimicrobial activity assay

The cylinder plate method determined the antimicrobial activity (Beadle et al., 1945). Briefly, 300 μL of the test sample was inserted into the wells (8 mm in diameter) in agar medium that mixed with the indicator bacterial suspension (0.1% v/v). After incubation for 24 h, the antimicrobial activity was measured as represented by inhibition halos (mm), calculated as inhibition zone diameter minus 8 mm.

Measurement of antimicrobial activity of AR113 under conditions involving different culture concentrations

Antimicrobial experiments were conducted using fermentation broths of AR113 cultured to four concentration levels corresponding with optical density readings at 600 nm (OD₆₀₀) of 1.0, 1.5, 2.0, and 2.5. The number of live colony-forming units (CFUs) of AR113 was counted by the plate enumeration method at each OD₆₀₀ value. Meanwhile, the pH of the bacterial suspension was measured.

Influence of pH, pasteurization, and enzyme on the antimicrobial effects of AR113 against indicator strains

To investigate the characteristics of the potential antimicrobial effects of AR113, the fermentation broth or CFS, after incubation to an OD₆₀₀ of 2.5, was subjected to different treatments according to Hu et al., with modifications (Hu et al., 2019).

Antimicrobial effect of fermentation broth and CFS under conditions involving neutral pH

The fermentation broth and CFS of AR113 were adjusted to neutral pH (7.0) with 1 mol/L NaOH or calcium carbonate, respectively. Antimicrobial effects of these solutions were compared with those of liquid without pH adjustment.

Antimicrobial effect of pasteurization in fermentation broth

AR113 fermentation broth was processed by pasteurization (conducted in 65°C water bath for 30 min) and then cooling to room temperature to examine the bacteriostatic properties.

Antimicrobial effects of the additive enzyme in CFS

The CFS of AR113 was digested at 37°C with proteinase K (Solarbio, Solarbio Science & Technology Co., Ltd., Beijing, China) for 2 h or catalase (Sigma, Aldrich Corporation, Luxembourg) for 1 h, respectively, to determine whether strains can produce antimicrobial peptides or hydrogen peroxide. Following this, the treated CFS was heated at 100°C for 5 min. The enzymatic solution was subjected to an antimicrobial assay.

Utilization of knockout strains for antimicrobial activity

Antimicrobial activity of knockout strains

The gene knockout strains of AR113 were tested for antimicrobial activity when cultured until the OD₆₀₀ reached a consistent value of 1.8. AR113 was used as a control. Furthermore, the pH of the fermentation broth of each strain was tested.

Determination of ATP content

ATP content was determined by the CFS of AR113 or AR113Δldh. After coculture with indicator strains for 24 h, the ATP content of the fermentation mixture was determined according to the ATP content detection kit (Beyotime Biotech, S0026). A blank MRS medium was used as a control.

Growth inhibition test

AR113 or AR113Δldh was mixed and cocultured with indicator strains. Each mixture was prepared at a ratio of 1:1, and the inoculation involved adding 50 μL in 5 mL of mixed medium (MRS: LB = 1:1), followed by incubation at 200 rpm in a shaker. The number of CFUs of each indicator strain was counted by the plate enumeration method at 0 h, 6 h, 12 h, and 24 h, respectively, of coculture.

Exogenous acid validation test

Hydrochloric acid, sulfuric acid, lactic acid, and acetic acid were used to test bacteriostatic properties. Since the pH of the L. plantarum AR113 was stabilized at approximately 4.0 at an OD₆₀₀ of 2.5, the pH of the exogenous acid was adjusted to 4.0. The four exogenous acids were validated in a bacteriostatic test.

Determination of lactic and acetic acid content

Comparing the yield of lactic and acetic acids, the CFS of broth fermented for 24 h was used as the sample to be tested. Further purification was conducted using HPLC (Waters Symmetry C18 column, 250 × 4.6 mm I.D., 5 μm, Dublin, Ireland). Isocratic elution: 97% solvent A (H₂O with 11.5% ammonium dihydrogen phosphate) and 3% solvent B (methanol). The flow rate was set to 0.7 mL/min, the temperature was set to 25°C, and a volume of 10 μL was injected.

Statistical analysis

The Statistical Package for Social Sciences (SPSS) version 22.0 (SPSS Inc.) was used to perform statistical analysis. p < 0.05 were considered significant. Experimental results were plotted using GraphPad Prism 6.0 (GraphPad Software) software.

Results

Antimicrobial effects of AR113 under conditions involving different culture concentrations

Figure 1A illustrates the pH dynamics and live bacterial count of AR113 fermentation broth. As the concentration of AR113 increased, the pH of the fermentation broth decreased simultaneously. Specifically, when the OD₆₀₀ reached 2.5, the pH of the fermentation broth stabilized at 3.65 ± 0.31. Figures 1B depicts the width and morphology of the inhibition halo generated by AR113. At an OD₆₀₀ value of 1.0, a noticeable bacterial inhibition was observed. As AR113 continued to grow, the fermentation broth exhibited enhanced pathogen control, as evident by more pronounced zones of inhibition (Fig. 1C to D). The inhibitory halos against E. coli O157:H7 and S. aureus were 9.38 ± 0.20 mm and 9.26 ± 0.21 mm, respectively, at an OD₆₀₀ value of 2.5, where the live bacterial count of AR113 in the fermentation broth was approximately 10¹¹ CFU/mL.

FIG. 1.

AR113 parameters under conditions involving different culture concentrations. (A) pH value and live bacterial count of AR113 fermentation broth; (B) width of the inhibition halos; (C, D) Inhibition zone of AR113 against E. coli O157:H7 and S. aureus.

Antimicrobial activity of AR113 against two indicators after different treatments

As shown in Figure 2, the inhibitory halos of AR113 fermentation broth against E. coli O157:H7 and S. aureus were significantly higher than other groups (p < 0.05). However, in comparison with the untreated CFS group, the antimicrobial activity remained unchanged after treatment with fermentation broth, inactivated by pasteurization and with proteinase K and catalase of CFS. Notably, the fermentation broth and CFS had lower pH, but upon neutralization with NaOH or CaCO₃, the antimicrobial activity against both indicator strains was completely abolished. This phenomenon suggests that the probable bacteriostatic substance of AR113 is acidic.

FIG. 2.

Changes in antimicrobial activity against two pathogens of AR113 after various treatments. (A) E. coli O157:H7 as an indicator; (B) S. aureus as an indicator.

Antimicrobial activity of AR113 knockout strains

Table 2 presents the pH values and antimicrobial characteristics of AR113 fermentation broth and its knockout strain when cultured to an OD₆₀₀ of 1.8. At this concentration, the pH values of AR113ΔEPS and AR113ΔsrlD remained significantly unchanged compared with those of AR113 (p < 0.05). The pH values of AR113Δ0273&2024, AR113ΔtagO, AR113Δldh, AR113Δltas, and AR113ΔccpA were all higher than 5.0. Among these, AR113Δldh exhibited the highest pH (nearly neutral). Regarding the width of inhibition halos against indicator strains for each gene knockout strain, all strains, except AR113ΔEPS, showed significantly lower inhibitory activity than AR113 (p < 0.05). Notably, the inhibition halos of AR113Δldh disappeared completely against both indicators. AR113ΔtagO and AR113ΔccpA demonstrated similar antimicrobial effects against both indicators with minimal inhibitory zones.

Table 2.

Characterization of the Knockout Strains

Knockout strains	pH of fermentation broth of the knockout strains (OD₆₀₀ = 1.8)	Inhibition halos^a (mm) against different indicator pathogens
Knockout strains		Escherichia coli O157:H7	Staphylococcus aureus
AR113	4.50 ± 0.14^b	6.88 ± 0.25^e	6.29 ± 0.39^e
AR113ΔEPS	4.60 ± 0.08^b	6.29 ± 0.31^de	5.34 ± 0.53^de
AR113 Δ0273&2024	5.13 ± 0.15^c	5.70 ± 0.44^d	4.85 ± 0.19^d
AR113 Δldh	6.90 ± 0.08^e	0.00 ± 0.00^b	0.00 ± 0.00^b
AR113 ΔtagO	5.18 ± 0.10^c	5.11 ± 0.53^c	4.41 ± 0.18^c
AR113 Δltas	5.07 ± 0.09^c	5.92 ± 0.27^d	5.13 ± 0.35^d
AR113 ΔsrlD	4.58 ± 0.10^b	6.06 ± 0.43^d	5.29 ± 0.15^d
AR113 ΔccpA	5.36 ± 0.13^d	4.69 ± 0.55^c	4.19 ± 0.17^c

Value represents the mean ± SD.

b–e

Means in columns with different superscripts represent significant differences (p < 0.05).

ATP concentration and growth inhibition of indicator pathogens

Figure 3A and B shows the effects of AR113 CFS and AR113Δldh CFS on the intracellular ATP concentration in E. Coli O157:H7 (A) and S. aureus (B). With the addition of AR113 CFS, the ATP levels of the indicator pathogens were significantly decreased by various multiples (p < 0.05). However, this phenomenon was not recorded in the groups comprising AR113Δldh CFS.

FIG. 3.

Effects of AR113 and AR113Δldh in ATP content and growth inhibition for indicator pathogens. (A) ATP levels of E. coli O157:H7; (B) ATP levels of S. aureus; (C) the number of E. coli O157:H7; (D) the number of S. aureus; C(a): inhibition zone against E. coli O157:H7; D-(b): inhibition zone against S. aureus.

According to Figure 3C and D, indicator bacteria grew faster in the first 12 h of cocultivation. However, AR113 significantly inhibited the growth of the indicator bacteria during this period (p < 0.05). Compared with the control group, the number of live E. coli O157:H7 and S. aureus in the AR113 coculture was continuously and significantly reduced over the entire culture duration (p < 0.05), and an order of magnitude was noted between the numbers of live indicator bacteria in each group. However, in contrast to AR113, AR113Δldh showed no inhibition halos against E. coli O157:H7 and S. aureus (Fig. 3C-(a) and Figure 3D-(b)), and the growth of the indicator bacteria was not affected.

Exogenous acid validation test

To further validate the bacteriostatic mechanism of lactic acid, other exogenous acids were used to compare the antimicrobial activity. The results are shown in Table 3. Lactic acid and acetic acid produced significantly larger inhibition zones than hydrochloric acid and sulfuric acid at the same pH value (p < 0.05). However, no significant difference in bacterial inhibition was observed between lactic acid and acetic acid against each indicator strain (p < 0.05).

Table 3.

Antimicrobial Activity of Different Exogenous Acids at pH 4.0

Exogenous acid	Inhibition halos^a (mm) against different indicator pathogens
Exogenous acid	Escherichia coli O157:H7	Staphylococcus aureus
Hydrochloric acid	7.54 ± 0.20^b	7.30 ± 0.08^b
Sulfuric acid	8.20 ± 0.11^c	8.09 ± 0.10^c
Lactic acid	9.29 ± 0.10^d	9.21 ± 0.06^d
Acetic acid	9.14 ± 0.09^d	9.01 ± 0.05^d

Value represents the mean ± SD.

b–e

Means in columns with different superscripts indicate significant differences (p < 0.05).

Lactic and acetic acid content assay

The ability of the strain to produce lactic and acetic acid is demonstrated in Figure 4. After 24 h of incubation, AR113 produced 336.18 ± 8.94 μM of lactic acid, which was nearly five times more than that of acetic acid (51.91 ± 4.76 μM). AR113Δ1dh lactate content was extremely low and almost undetectable. Notably, there was no significant difference of lactate production in AR113Δ0273&2024 compared with AR113, but AR113Δldh showed a decrease in acetic yield, which was significantly lower than that of AR113 (p < 0.05).

FIG. 4.

Determination of lactic and acetic acid content.

Discussion

The preliminary study examining the antibacterial activity of AR113 revealed that L. plantarum AR113 had the potential to eliminate pathogens E. coli O157:H7 and S. aureus, and the antimicrobial effect improved with the increase in the amount of AR113. Exploration of the antimicrobial potential of L. plantarum strains has been documented in existing literature, including studies on L. plantarum NTU 102 (Lin and Pan, 2019) and L. plantarum N20 (Jomehzadeh et al., 2020). In conclusion, L. plantarum is widely used in the food industry for pathogen control, with AR113 contributing to this group category.

Some proteinaceous substances (Rocchetti et al., 2021) and H₂O₂-producing microbial metabolic processes (Gilliland and Speck, 1974; Ito et al., 2003) have been characterized as the potential bacteriostatic substances to inhibit bacteria. However, our investigation revealed that proteins and H₂O₂ are not the likely inhibitory substances of AR113. Furthermore, pasteurization of the bacterial suspension showed a sterilizing effect similar to that of the CFS, indicating that the antimicrobial substance can withstand pasteurization temperatures used in food industry processing. Similar findings have been reported for antimicrobial substances isolated from other probiotics that remained stable after exposure to temperatures ranging from 60°C to 100°C for 20 min (Pringsulaka et al., 2012).

The decrease in pH mediated by acids can remarkably inhibit the growth of other bacteria (Guimarães et al., 2018). In our previous study, the bacteriostatic effects improved concomitantly with the progressively increased culture concentration and decreased pH value in the AR113 fermentation broth. In addition, the fermentation broth and CFS of AR113 at pH 7.0 did not exhibit complete antibacterial properties. Findings from similar studies have shown that the antimicrobial properties of supernatants from most LAB strains were eliminated when the pH was adjusted to 6.5 (Arrioja-Bretón et al., 2020). L. plantarum strains can produce a variety of organic acids, mediating a decrease in the pH of the fermentation broth (Li et al., 2023). The optimal pH value for the growth of pathogenic bacteria ranges from 6.0 to 7.0, but acid substances could lower pH than values within this range and destroy the membrane of pathogens, creating unfavorable conditions for the growth of potentially pathogenic microorganisms (Ammor et al., 2006; Huang et al., 2022; Toushik et al., 2023). Hence, this is the primary method for inhibiting the growth of the causative agent. To neutralize imbalanced acidity in acid environments, bacteria expend considerable ATP, resulting in reduced cellular ATP levels representing eventual pathogen demise due to irreversible denaturation (Cui et al., 2015). According to our study, AR113Δldh has no antimicrobial activity completely, and AR113Δldh did not affect the ATP levels of indicator strains. Then we proposed the hypothesis that lactic acid, responsible for the most significant pH alteration, might be the primary antimicrobial substance of AR113, which corroborated that the ldh gene on AR113 plays a crucial role in the inhibitory effect of the indicator bacteria.

Some experiments have been conducted to determine the changes in the bacteriostatic effects over time based on the number of pathogenic bacteria (Yang et al., 2009). Generally, a rapid onset of the antimicrobial effect is associated with a lower likelihood of inducing bacterial resistance (Luo et al., 2022). According to the growth inhibition test in our present study, AR113 significantly inhibited the growth of the indicator bacteria in the first 12 h and eliminated pathogens E. coli O157:H7 and S. aureus by a tenfold reduction for 24 h of coculturing. Conclusively, the pathogen control of AR113 is relatively rapid and stable. However, the inhibition test of AR113Δldh exhibited the same bacteriostatic effect as the blank MRS group, further illustrating the key role of ldh gene in the antimicrobial effect of AR113.

Significantly, the low acidity in the cytoplasm of pathogens hinders the entry of dissociative acids, whereas undissociated acids, carrying hydroxyl, easily penetrate the pathogenic cytoplasm, leading to dissociation and the subsequent destruction of pathogenic bacteria (Cui et al., 2015; Guimarães et al., 2018). The primary undissociated organic acids with antimicrobial effects include acetic and lactic acids (Zalán et al., 2010; Guimarães et al., 2018). They demonstrated significant efficacy in inhibiting bacteria compared with hydrochloric and sulfuric acids at equivalent pH levels, as confirmed by our exogenous acid validation test. Our findings align with a previous study reporting that lactic acid exhibits superior antipathogenic activity compared with HCl (Wang et al., 2014). Notably, there is no significant difference in the bacteriostatic properties of lactic and acetic acids under the same pH conditions. However, the knockout strain of AR113Δ0273&2024, despite lowering the pH of the fermentation broth, exhibited reduced inhibitory activity, suggesting that acetic acid secretion by AR113 had a relatively minor impact on the inhibitory effect, deviating from other findings (Sohail and Hume, 2019; Shin et al., 2020). According to our further study, this discrepancy might be attributed to the lower acetate yield in AR113 after 24 h of incubation, and AR113 produced almost fivefold more lactic acid than acetic acid. Notably, AR113Δldh produced almost no lactic acid and affected the production of acetic acid, whereas AR113Δ0273&2024 still produced a small amount of acetic acid and had no effect on the production of lactic acid. This result further confirms the essential role of the ldh gene in the bacterial suppression of AR113.

In addition, our study on EPS produced conflicting outcomes. EPS, considered a secondary metabolite of Lactobacillus microorganisms, is known to impede the formation of pathogenic biofilms and eradicate pathogenic bacteria (Ma'unatin et al., 2020; Xu et al., 2020). Nevertheless, in our investigation, the AR113ΔEPS demonstrated comparable inhibitory effects against E. Coli O157:H7 and S. aureus when compared with AR113, suggesting that the EPS produced by AR113 might not be the primary factor contributing to its antimicrobial effects. Moreover, we also tested genes associated with microbial structure and growth, such as ltas, tagO, srlD, and ccpA genes. The strains with the knockout genes encoding the aforementioned factors are significantly different in bacteriostatic properties compared with the AR113 strain (p < 0.05). However, within these strains, the antimicrobial effect against both indicators was consistently weaker for AR113ΔtagO and AR113ΔccpA, positioning them just below AR113Δldh. This diminished antimicrobial impact implies that tagO and ccpA genes, lower than ldh gene, also serve as important factors contributing to the antimicrobial effect of AR113.

Conclusion

L. plantarum AR113 demonstrates excellent pathogen exclusion properties against E. coli O157:H7 and S. aureus. The ldh gene is the crucial factor impacting the pathogen control of AR113. AR113Δldh did not demonstrate any bacteriostatic properties and did not mediate a change in ATP levels within the indicator bacteria and inhibited their growth. Lactic acid is the primary bacteriostatic substance produced by AR113. Deletion of the ldh gene not only eliminates lactic acid production, but also interferes with the production of acetic acid for AR113. In addition, the absence of the tagO and ccpA genes also diminishes the inhibitory effect of AR113, suggesting these genes also serve as important factors devoted to the antimicrobial effect of AR113.

Footnotes

Authors’ Contributions

X.L.: conception and design, data collection and analysis, and article writing. G.W.: project administration and data mapping. J.W.: data collecting and analysis. X.S. and Z.X.: collected references. Y.X.: revised, formatted, and edited the article. L.A.: supervision and project administration.

Disclosure Statement

No competing financial interests exist.

Funding Information

The work was supported by the National Science Fund for Distinguished Young Scholars (Grant NO. 32025029), the CIFST-Abbott Foundation of Food Nutrition and Safety (Grant NO. 2022-F04), the Shanghai Engineering Research Center of Food Microbiology Program (19DZ2281100), and the Shanghai Education Committee Scientific Research Innovation Projects (2101070007800120).

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