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Pectobacterium carotovorum is a Gram-negative plant-specific pathogen that causes severe rot of postharvest vegetables. Given the safety consideration of pesticides, we developed a peptide approach...

This study aimed to investigate the effect of Pichia galeiformis on disease resistance and elucidate the changes in phenylpropane biosynthesis treated by P. galeiformis in postharvest citrus. The results showed that P. galeiformis reduced the disease incidence and lesion diameters without direct contact with the pathogen Penicillium digitatum. Transcriptome analysis revealed that phenylpropanoid biosynthesis was triggered by P. galeiformis. Genes encoding phenylpropanoid biosynthesis were upregulated, including phenylalanine ammonia-lyase (PAL), 4-coumaroyl-CoA ligase (4CL), cinnamate-4-hydroxylase (C4H), peroxidase (POD), cinnamyl alcohol dehydrogenase (CAD), O-methyltransferase, and hydroxyl cinnamoyl transferase. Moreover, P. galeiformis increased the activity of PAL, 4CL, C4H, POD, polyphenol oxidase, and CAD in citrus pericarp. In addition, P. galeiformis treated citrus displayed higher levels of total phenolic compounds, flavonoid, and lignin and higher amounts of ferulic and sinapic acid. In conclusion, these results suggested that P. galeiformis could induce resistance through modulating the pathway of phenylpropanoid biosynthesis in postharvest citrus.

Background:Antimicrobial resistance is found in all microorganisms and has become one of the biggest threats to global health. New antimicrobials with different action mechanisms are effective weapons to fight against antibiotic-resistance.Objective:This review aims to find potential drugs which can be further developed into clinic practice and provide clues for developing more effective antimicrobials.Methods:DNA replication universally exists in all living organisms and is a complicated process in which multiple enzymes are involved in. Enzymes in bacterial DNA replication of initiation and elongation phases bring abundant targets for antimicrobial development as they are conserved and indispensable. In this review, enzyme inhibitors of DNA helicase, DNA primase, topoisomerases, DNA polymerase and DNA ligase were discussed. Special attentions were paid to structures, activities and action modes of these enzyme inhibitors.Results:Among these enzymes, type II topoisomerase is the most validated target with abundant inhibitors. For type II topoisomerase inhibitors (excluding quinolones), NBTIs and benzimidazole urea derivatives are the most promising inhibitors because of their good antimicrobial activity and physicochemical properties. Simultaneously, DNA gyrase targeted drugs are particularly attractive in the treatment of tuberculosis as DNA gyrase is the sole type II topoisomerase in Mycobacterium tuberculosis. Relatively, exploitation of antimicrobial inhibitors of the other DNA replication enzymes are primeval, in which inhibitors of topo III are even blank so far.Conclusion:This review demonstrates that inhibitors of DNA replication enzymes are abundant, diverse and promising, many of which can be developed into antimicrobials to deal with antibioticresistance.

Loss and waste of postharvest vegetables are the main challenges facing the world’s vegetable supply. In this study, an innovative method of value-added transformation was provided: production of bacteriocin from vegetable waste, and then its application to preservation of vegetables. Antibacterial activity to soft rot pathogen Pectobacterium cartovorum (Pcb BZA12) indicated that tomato performed best in the nutrition supply for bacteriocin production among 12 tested vegetables. Moreover, the antibacterial activity was from Lactobacillus paracasei WX322, not components of vegetables. During a fermentation period of 10 days in tomato juice, L. paracasei WX322 grew well and antibacterial activity reached the maximum on the tenth day. Thermostability and proteinase sensitivity of the bacteriocin from tomato juice were the same with that from Man-Rogosa-Sharpe broth. Scanning electron microscope images indicated that the bacteriocin from tomato juice caused great damage to Pcb BZA12. At the same time, the bacteriocin from tomato juice significantly reduced the rotten rate of Chinese cabbage from 100% ± 0% to 20% ± 8.16% on the third day during storage. The rotten rate decrease of cucumber, tomato, and green bean was 100% ± 0% to 0% ± 0%, 70% ± 14.14% to 13.33% ± 9.43%, and 76.67% ± 4.71% to 26.67% ± 4.71%, respectively. Bacteriocin treatment did not reduce the rotten rate of balsam pear, but alleviated its symptoms.

Food-borne pathogenic bacteria are dispersed throughout the entire chain of the food industry. However, many food preservatives are limited by poor biocompatibility such as cumulative poisoning. The antimicrobial peptide is increasingly regarded as a promising preservative in food research due to its high bioactivity and low cytotoxicity. In this study, thirteen peptides were designed, synthesized, and screened for application as food preservatives. One of them, termed zp65, whose sequence is GIOAOIIIOIOO-NH2, demonstrated potent bactericidal effect against common Gram-negative strains including enterohemorrhagic Escherichia coli, Salmonella, and Citrobacter freundii. Encouragingly, zp65 showed negligible cytotoxicity to both mammalian cells and Galleria mellonella larvae. Peptide zp65 was prone to form α-helix structure in amphiphilic environments, facilitating its affinity with bacterial membrane. Furthermore, the proteolytic stability of zp65 was much higher than its derivatives consisting of totally natural amino acids. Isothermal titration calorimetry indicated that zp65 has a strong binding affinity to lipopolysaccharide with Kd = 1.3 μM, suggesting its possible action target on the bacterial envelope. Mechanistic studies revealed that this peptide also influenced the membrane potential of E.coli O157:H7 (O157) in a dose-dependent manner. Surprisingly, peptide zp65 did not induce disruption of membrane permeability even at a higher concentration of 4-fold minimal inhibitory concentration. By employing confocal microscopy, peptide zp65 labeled by fluorescein isothiocyanate mainly aggregated on the bacterial membrane. These results suggested that the bactericidal mode of action of zp65 is likely attributed to depolarization of the cell membrane. The minced lean beef experiment indicated that the maximum reduction of O157 reached 1.46 log colony-forming unit (CFU) per gram on day 1 after zp65 treatment at the dosage of 40 μg/g. Compared with the untreated cooked beef sample, the CFU of the zp65-treated group remained at a much lower level after 10-day storage. Subsequently, treatment with zp65 at concentrations above 32 μM also significantly reduced O157 viable counts in fresh tomato juice. And the zp65 treatment could rescue about 40% of Galleria mellonella larvae injected with O157-contaminated tomato juice. The peptide zp65 exhibits great potential and deserves further study as a candidate for food preservative.

Biofilm-producing pathogens, such as Acinetobacter baumannii, have aroused escalating attention. Because these bacteria could secrete mixture with close-knit architecture and complicated components to resist traditional antibiotics. Here, we reported an amphiphilic peptide denoted as zp3 (GIIAGIIIKIKK-NH2), which showed favorable bioactivity against Acinetobacter baumannii ATCC 19606 (minimal inhibitory concentration, MIC = 4 μM) and low cytotoxicity to mammalian cells Vero (half maximal inhibitory concentration, IC50 > 100 μM). Importantly, zp3 could inhibit the formation of biofilm at micromole level and eliminate around 50% preformed biofilm at 32 μM after 6 h treatment. This peptide was able to bind with biofilm while maintaining a helical structure in a mimic biofilm-rich environment. In vivo test demonstrated that zp3 rescued 33.3% of larvae after 48 h infection and reduced 1 log live bacteria inside the animal body after 6 h treatment. The bactericidal mode for zp3 was attributed to the combination of influencing ions balance at low concentration and inducing permeability alteration and pore formation on the Acinetobacter baumannii membrane at high concentration. Application on medical textiles also proved that zp3 could perform a good antibacterial activity in practice.

In this study, the transcriptional profiling of Penicillium digitatum after C12O3TR treatment was analyzed by RNA-Seq technology. A total of 2562 and 667 genes in P. digitatum were differentially expressed after 2 and 12 h treatment, respectively. These genes were respectively mapped to 91 and 79 KEGG pathways. The expression patterns of differentially expressed genes (DEGs) at 2 and 12 h were similar, mainly were the metabolic processes in cell wall, cell membrane, genetic information and energy. Particularly, the main metabolic process which was affected by C12O3TR stress for 2 and 12 h was cell integrity, including cell wall and cell membrane. The changes of chitin in cell wall was observed by Calcofluor White (CFW) staining assay. The weaker blue fluorescence in the cell wall septa, the decrease of β-1, 3-glucan synthase activity and the increase of chitinase and AKP activity showed that C12O3TR could damage the cell wall integrity. In conclusion, these results suggested that C12O3TR could inhibit the growth of P. digitatum through various mechanisms at transcriptional level, and could influence the cell wall permeability and integrity.

Multidrug-resistant (MDR) bacterial infection still poses a serious threat to public health, therefore, effective and safe antimicrobial agents are urgently needed. In this study, recombinant bacteriocin BMP32 (BMP32r) prepared by the Escherichia coli expression system had a broad-spectrum antibacterial activity even against some MDR bacteria and its minimum inhibitory concentration ranged from 9.2 to 36.8 mg/L. Furthermore, BMP32r showed good stable performance in heat, pH and storage. Moreover, the scanning electron microscope and transmission electron microscope revealed that BMP32r killed indicator strains through cell wall destruction, pore formation, and the membrane permeability increasing which was proved by propidium iodide uptake investigation. The wound healing of an animal MDR S. aureus infected model was promoted by BMP32r, and the safety was verified by the cytotoxicity assay that the viability of HFF cells remained 87.3% in even when the concentration of BMP32r was as high as 147.2 mg/L. In addition, no abnormalities or damages to major organs was found in vivo assessments after treatment with BMP32r. In conclusion, BMP32r has great potential to be developed as a safe antimicrobial agent to treat MDR bacterial infections.

This study investigated the effect of arginine (Arg) on the antagonistic activity of Metschnikowia citriensis against sour rot caused by Geotrichum citri-aurantii in postharvest citrus, and evaluated the possible mechanism therein. Arg treatment up-regulated the PUL genes expression, and significantly induced the pulcherriminic acid (PA) production of M. citriensis, which related to the capability of iron depletion of M. citriensis. By comparing the biocontrol effects of Arg-treated and untreated yeast cells, it was found that Arg treatment significantly enhanced the biocontrol efficacy of M. citriensis, and 5 mmol L−1 Arg exerted the best effect. Additionally, the biofilm formation ability of M. citriensis was greatly enhanced by Arg, and the higher population density of yeast cells in citrus wounds was also observed in Arg treatment groups stored both at 25 °C and 4 °C. Moreover, Arg was shown to function as a cell protectant to elevate antioxidant enzyme activity [including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX)] and intracellular trehalose content to resist oxidative stress damage, that directly helped to enhance colonization ability of yeasts in fruit wounds. These results suggest the application of Arg is a useful approach to improve the biocontrol performance of M. citriensis.

Listeria monocytogenes and Cronobacter sakazakii are notorious pathogens involved in numerous foodborne outbreaks after ingested contaminated food. Bacteriocins are natural food preservatives, some of which have antimicrobial activity comparable with antibiotics. In this study, a plasmid encoded novel bacteriocin BMP11 produced by Lactobacillus crustorum MN047 was innovatively identified by combining complete genome and LC-MS/MS. The BMP11 was found to have rich α-helix conformation after prediction. Moreover, the antimicrobial activity of BMP11 was verified after its heterologous expression in E. coli with 1280 and 640 AU/mL against L. monocytogenes and C. sakazakii, respectively. After purification by anion-exchange chromatography and HPLC, BMP11 had MIC values of 0.3–38.4 μg/mL against tested foodborne pathogens. Further, it was found that BMP11 had bactericidal action mode with concomitant cell lysis to pathogens by growth curve and time-kill kinetics. The results of scanning electron microscope (SEM) and transmission electron microscope (TEM) indicated that BMP11 destroyed the integrity of cell envelope of pathogens with cell wall perforation and cell membrane permeabilization. The destruction of cell envelope integrity was further verified by propidium iodide (PI) uptake and lactic dehydrogenase (LDH) release. BMP11 increased inner-membrane permeability of C. sakazakii in a concentration-dependent manner. Meanwhile, BMP11 exhibited antibiofilm formation activity. In addition, BMP11 inhibited the growth of L. monocytogenes in milk. Therefore, BMP11 had promising potential as antimicrobial to control foodborne pathogens in dairy products.

Foodborne pathogens cause diseases by food chain transmission, therefore preservatives or antimicrobials are essential for food products, among which bacteriocins are considered as promising alternatives of chemical preservatives. Moreover, bacteriocins can potentially be used as antibiotic alternatives. In this study, two genes of novel bacteriocins (BM1157 and BM1300) from probiotic Lactobacillus crustorum MN047 were identified, cloned, and then expressed in Escherichia coli expression system. The genes were inserted into expression vector pET-28a and transformed into competent E. coli BL21 (DE3) pLysS, after which the two bacteriocins were successfully heterologously expressed. Further, it showed that the BM1157 and the BM1300 had broad spectrum activity against gram-positive and gram-negative bacteria, including multidrug-resistant strains. The characteristics and action mode of the BM1157 were further investigated because of its higher antimicrobial activity. It was found that the BM1157 had MIC value of 5.2 μg/mL against both S. aureus and E. coli. Moreover, it was stable at high temperature and resistant to proteinases. The BM1157 had bactericidal action mode according to time-kill curve. The results of scanning electron microscope and transmission electron microscope demonstrated that the BM1157 killed Listeria monocytogenes by biofilm destruction and pore formation. The antibiofilm activity and pore formation were further verified by crystal violet dye and lactic dehydrogenase release. In addition, the BM1157 inhibited the growth of L. monocytogenes in milk.

Metschnikowia citriensis FL01 has great potential for biocontrol applications for its excellent biocontrol efficacy on postharvest diseases of citrus fruit, and the iron depletion by pulcherriminic acid (PA) and then formation of insoluble pigment pulcherrimin had been speculated as an important action mechanism. To identify the genes involved in pulcherrimin synthesis and reutilization in M. citriensis FL01, we de novo assembled the genome of M. citriensis FL01 based on long-read PacBio sequencing. The final assembled genome consisted of 12 contigs with a genome size of 25.74 Mb, G + C content of 49.16% and 9310 protein-coding genes. The genome-wide BLAST of the PUL genes of M. pulcherrima APC 1.2 showed that the four PUL genes were clustered and located on Contig 4 of M. citriensis FL01. In order to further clarify the role of pulcherrimin pigment on biocontrol of M. citriensis FL01, CRISPR/cas9 technology was used to knock out PUL2 gene that was responsible for PA synthesis and the pigmentless mutants with stable phenotype were obtained. The mutant strains of M. citriensis FL01 lost the ability to produce pulcherrimin pigment, and simultaneously lost the ability to inhibit the growth of Geotrichum citri-aurantii in vitro. Moreover, the biocontrol efficacy of pigmentless mutant strains against sour rot was about 80% lower than that of wild-type M. citriensis FL01. These results directly proved that the iron depletion was an important mechanism of M. citriensis FL01.

Bacterial soft rot caused by Pectobacterium carotovorum has brought huge economic losses to post-harvest vegetables. In this study, vegetables were used as raw materials to produce food-grade antibacterial products, fermented by lactic acid bacteria, to control P. carotovorum. A total of 32 kinds of vegetables were fermented by Lactobacillus paracasei WX322, results showed that 31 vegetable fermentation products had antibacterial activities at different levels. The green pepper fermentation antibacterial product (GPFA) had the highest activity and was selected for further analysis. On the 7th day of fermentation, the antibacterial activity of GPFA reached the maximum, with 640 AU/mL. Two bacteriocins, GP-19 and EP-20, were identified by LC-MS/MS. Growth curve and time-kill curve showed that the GPFA could effectively inhibit and kill P. carotovorum at a dose-dependent manner. SYTO9TM/PI staining observation further confirmed its bactericidal ability. Meanwhile, GPFA treatment led to irregular deformation of cells with rough surface observed using scanning electron microscopy (SEM). Moreover, in vivo assay showed that GPFA treatment significantly reduced the disease incidence and decay degree of pepper soft rot. In addition, GPFA treatment could prolong the shelf life of fresh-cut bell pepper. This study indicates that antibacterial products based on vegetable fermentation can provide a new method for the control of vegetable soft rot.

Superbugs or multi-drug resistant pathogens are not only clinical issue but also highlighted in food safety. However, development of new antibiotics and safe preservatives is declining. A novel workflow was used to purify antimicrobial substance produced by Lactobacillus rhamnosus LS-8, in which ethyl acetate extraction, polypeptide column and HPLC were involved in the purification process. Subsequently, a novel antimicrobial substance was purified and identified as 4,6-dimethyl-1,2,5-triazepane-3,7-dione (C6H11N3O2) by mass spectrum, 1H NMR and 13C NMR analysis. Fortunately, it has significant inhibitory effect on both gram-positive and gram negative strains including multi-drug resistance pathogens. Also it had good heat stability and resistant to proteolytic enzymes, however, it was only active in acidic condition. The results of scanning electron microscopy (SEM) and transmission electron microscope (TEM) showed that LS-8-25 destroyed the ultrastructure of S. aureus and E. coli. The discovery is adding a new variety of antimicrobial substance produced by lactic acid bacteria.

Background Penicillium digitatum is one of the most important pathogens causing citrus green mold, leading to significant economic losses. Traditionally, synthetic fungicides are used to control diseases. However, the side effects of fungicides should not be ignored. Thus, antagonistic yeasts were proposed to be safe and effective alternatives for managing diseases. Orchards are excellent sources of naturally occurring antagonists against pathogens. Therefore, in the present study, antagonistic yeasts obtained from orchards were screened, and the possible biocontrol mechanisms of the most promising yeast were investigated. Results Seventy-eight isolates of yeasts (15 species of 10 genera) were obtained from citrus orchards. In in vitro assays, 16 strains showed antifungal activity against Pichia digitatum and 15 strains showed biocontrol potential against green mold on Olinda oranges. Pichia galeiformis (BAF03) exhibited the best antagonistic activity against P. digitatum during 6 days storage at 25 °C and a good antagonistic activity during 29 days at 4 °C. Pichia galeiformis (BAF03) could colonize and amplify quickly in wounded citrus. Scanning electron microscopy results showed that the citrus wound was colonised by the yeast. A total of eight volatile organic compounds (VOCs) were identified by gas chromatography-mass spectrometry The VOCs produced by P. galeiformis (BAF03) efficiently inhibited P. digitatum. Conclusion Pichia galeiformis (BAF03) isolated from a citrus orchard showed potential to control postharvest green mold of citrus. The possible mechanisms of action likely include competition for space and nutrients as well as production of VOCs.

Utilizing antagonistic yeasts is a promising approach for managing postharvest decay of fruits. However, it is well established that various severe stresses encountered in the environment and production process cause the intracellular reactive oxygen species (ROS) accumulation in yeast cells, resulting in cell damage and loss of vitality. Here, proline has been shown to function as a cell protectant and inducer of biofilm formation able to increase the oxidative stress tolerance and the biocontrol ability of the antagonistic yeast Metschnikowia citriensis. Addition of proline to M. citriensis cells induced a significant rise in superoxide dismutase (SOD) and catalase (CAT) activity in the early and late stages of oxidative stress, respectively, and increased the maroon pigment production that directly reduced intracellular iron content and indirectly diminished intracellular ROS levels and thus inhibited ROS- and iron-induced apoptosis. Treating cells with iron chelator tropolone yielded similar results. Pigment production induced by proline also enhanced the capability of biofilm formation of M. citriensis. These results suggested an important role for pigment of M. citriensis in response to oxidative stress. The abilities of proline to scavenge intracellular ROS and inhibit apoptosis, increase pigment production, and promote biofilm formation contribute to the improvements in oxidative stress tolerance and biocontrol efficacy of M. citriensis.

Many citrus farmers are deprived of their profits as a result of post-harvest losses in citrus production. Diverse microbial environments including fermented foods are necessary to be explored in the quest to get more novel biocontrol yeasts to control post-harvest pathogens in citrus. 90 yeasts were isolated from kimchi in this investigation. Inhibition tests in vitro showed that 10 of the 90 yeasts reduced the development of fungal mycelia by the formation of an inhibition zone. Molecular methods were used to identify the yeast isolates, and they were Pichia sp., Kluyveromyces marxianus, Yarrowia lipolytica, and Issatchenkia orientalis. Three isolates tested in vivo in citrus were able to reduce disease incidence within the range from 18 % to 57 %. The action mechanisms of the three yeasts were studied subsequently by determining the biofilm formation ability of the yeast isolates, screening of yeast isolates for extracellular lytic enzyme activity, evaluating the effect of yeast isolates’ volatile organic compounds (VOCs) on P. digitatum, investigating the competition for nutrients between P. digitatum and yeast isolates, etc. For Pichia kudriavzevii, the most effective one, reduced the incidence of green mold caused by Penicillium digitatum (Pd1 / CECT 20795) by biofilm formation, competition for nutrients, and emitting volatile organic compounds. K. marxianus showed antifungal activity by inducing resistance, and colonization on wound sites. Whereas Y. lipolytica inhibited P. digitatum by adhesion to pathogen mycelia and production of extracellular lytic enzymes. Evidence from this study suggests that some yeast strains isolated from kimchi have the potentials to inhibit green mold in citrus.

E. coli and C. sakazakii are both opportunistic pathogenic bacteria that widely found in food. Emerging of antibiotic resistance makes the treatment of Gram-negative bacteria infection become ever more difficult. Therefore, it is urgent to find new antibacterial compounds against Gram-negative bacteria. Our previous study showed that bacteriocin BM1157 had broad antibacterial activity against both Gram-positive and Gram-negative bacteria. However, its antimicrobial mechanism against Gram-negative bacteria is still unknown. In this study, propidium iodide uptake and lactate dehydrogenase release experiments showed that the membrane integrity and permeability of the two indicator strains were not affected by BM1157 treatment. Observation of the ultrastructure of both bacteria by SEM and TEM also indicated that BM1157 did not damage the cell membrane. However, it significantly inhibited the biofilm formation of both bacteria. Furthermore, the lable-free proteome showed that BM1157 disturbed the carbohydrate metabolism, amino acid metabolism, lipid metabolism and nucleic acid metabolism, and then bioactive molecule synthesis and intracellular metabolism were blocked, resulting in cell death. Down-regulation of gene expression was further confirmed by real-time quantitative PCR. This study indicates that bacteriocin BM1157 inhibited Gram-negative bacteria by non-pore mechanisms.

Bacterial soft rot is a devastating disease that harms vegetables. Lactic acid bacteria (LAB) can be effective antagonist against soft rot pathogens. In this study, Lactobacillus paracasei WX322 and its produced bacteriocin had antibacterial activities against a variety of foodborne pathogens and spoilage bacteria, including both Gram-positive and Gram-negative bacteria, especially the soft rot pathogen Pectobacterium carotovorum subsp. brasiliense BZA12 (Pcb BZA12). The in vivo antibacterial test proved that the bacteriocin produced by the L. paracasei WX322 could control soft rot disease of peppers caused by Pcb BZA12. The L. paracasei WX322 produced bacteriocin after fermentation for 36 h, which had broad antimicrobial activity. In addition, the bacteriocin produced by L. paracasei WX322 had good thermal stability and broad pH adaptation. Then, the complete genome of L. paracasei WX322 was sequenced with one circular chromosome and eight plasmids. Three bacteriocin gene clusters were identified in the genome by antiSMASH. Furthermore, seven genes in the three gene clusters were identified to be bacteriocins. Moreover, three of the seven bacteriocins were novel. The complete genome sequences of the L. paracasei WX322 can provide a better molecular basis of its antibacterial activity and biocontrol effect on bacterial soft rot disease.

The multidrug-resistant strains of food spoilage bacteria and foodborne pathogenic bacteria in food are badly in need of being controlled by effective bio-preservatives. In this study, the XN8 strain was isolated from Jiangshui and identified as Lactobacillus coryniformis according to 16S rRNA gene sequence. One of the bacteriocins produced by XN8 was purified by ammonium sulfate precipitation and a series of chromatographic column, and designated as lactocin XN8-A (LXA). The molecular mass of LXA was 3100.0242 Da by MALDI-TOF MS. The LXA showed good heat, pH and storage stabilities. However, it was sensitive to proteases. The LXA was found to have a broad antimicrobial spectrum on both Gram-positive and Gram-negative bacteria including multidrug-resistant strains and Listeria monocytogenes. Its minimum inhibitory concentration (MIC) for both Escherichia coli and Staphylococcus aureus was 6.85 μg/mL. The LXA had a bactericidal mode without cell lysis by the growth curve and time-kill assay. The results of electron microscope showed that the LXA destroyed membrane permeability and induced pore-formation of target cells. Furthermore, the LXA induced cell cycle arrest at both G1 and G2/M phase by cell cycle analysis. This research suggests that the LXA has promising potential as bio-preservative in food industry.

The presence of pathogens in fresh-cut fruits represents a risk for the public health since these products generally do not receive any further treatment before consumption. In this study, a Lactobacillus pentosus MS031 was isolated from Sichuan Paocai with broad antibacterial activity against foodborne pathogens. Antimicrobial peptides produced by the L. pentosus MS031 had broad activity and resistance to heat, but they were sensitive to proteinases and their activity vanished under alkaline condition. Bacteriocin-like substance obtained by pH-absorption related methods showed activity only against Staphylococcus aureus. However, cell-absorbed substance and cell-secreted into culture substance had activity against both S. aureus and Escherichia coli. Foodborne pathogens in fresh-cut fruit mixture were controlled by the metabolites of the L. pentosus MS031 that Listeria monocytogenes was reduced by 96.3 %, Salmonella typhi and E. coli were decreased to an undetectable level. The complete genome of the L. pentosus MS031 was sequenced using Illumina and MinION nanopore platform with a size of 3,805,216 bp, consisting of one chromosome and eight plasmids. After mining using BAGEL4, two novel bacteriocins, pentocin MS1 and pentocin MS2, were identified. Furthermore, fragments of 5 antimicrobial peptides were identified by LC–MS/MS in the fermentation supernatant. In addition, antimicrobial cyclic dipeptides and small compounds were identified in the antimicrobial metabolites. The results indicate antimicrobial metabolites of the L. pentosus MS031 can control foodborne pathogens in fresh-cut product during subsequent cold storage, which is significant for food industry.

This study investigated the biocontrol efficiency of Metschnikowia citriensis strain FL01 against Geotrichum citri-aurantii, and evaluated possible mechanisms. The results showed that M. citriensis could effectively control the development of sour rot, and significantly inhibit the mycelial growth and spore germination of G. citri-aurantii. The population dynamics results and Scanning electron microscopy (SEM) analysis indicated that M. citriensis could rapidly colonize wounds and tightly adhere to the surface of the wounds to compete with G. citri-aurantii for nutrition and space. M. citriensis also showed the biofilm formation action in vitro. The response of G. citri-aurantii to different components of M. citriensis culture showed that only the yeast cells but not the extracellular metabolites and the volatile organic compounds (VOCs) exhibited inhibitory effect on the growth of G. citri-aurantii. M. citriensis adhered to the hyphae of G. citri-aurantii loosely and sparsely, and the production of lytic enzymes β-1, 3-glucanase (GLU) and Chitinase (CHI) could not be induced by G. citri-auranti. Iron affected the pulcherrimin pigment production and antagonism of M. citriensis indicating iron depletion as the most important antagonistic mechanism. Besides, M. citriensis also induced resistance of fruit against sour rot. These results suggested that M. citriensis could be used as the potential alternative of fungicides to control postharvest pathogens on citrus fruit.

The control of foodborne pathogens in raw/processed foods is urgently needed. Lactic acid bacteria (LAB) bacteriocins are promising natural food preservatives. In this study, 81 antibacterial LAB strains were isolated from pickles and dry-cured meats, among which 38 LAB isolates inhibited both Staphylococcus aureus and Escherichia coli. Broad antibacterial LAB strains consisted of Lactobacillus, Pediococcus, Staphylococcus, and Weissella after identification according to 16S rDNA sequences. Antimicrobial activity of crude bacteriocins of the 38 strains was tested against multiple foodborne pathogens and L. pentosus DZ35 was selected to investigate further. L. pentosus DZ35 started to produce bacteriocin at 36 h during fermentation. The bacteriocin of L. pentosus DZ35 had good thermostability and broad pH adaption ranging from pH 2 to pH 11. Its antibacterial activity was partially lost after treated by proteinases. Genome of L. pentosus DZ35 was sequenced using Illumina sequencing and MinION nanopore sequencing platform with a size of 3,962,622 bp, including nine circular plasmids. Two novel bacteriocins, pentocin DZ1 and pentocin DZ2, were identified using BAGEL4. The present study indicates that pickle has abundant bacteriocin-producing LAB and the probiotic L. pentosus DZ35 has promising potentials to control pathogens in food.

Backgroud: The growing emergence of antibiotic-resistant pathogens including the most dangerous superbugs requires quick discovery of novel antibiotics/biopreservatives for human health and food safety. Bacteriocins, a subgroup of antimicrobial peptides, have been considered as promising alternatives to antibiotics. Abundant novel bacteriocins are stored in genome sequences of lactic acid bacteria. However, discovery of novel bacteriocins still mainly relies on dubious traditional purification with low efficiency. Moreover, sequence alignment is invalid for novel bacteriocins which have no homology to known bacteriocins in databases. Therefore, an efficient, simple, universal, and time-saving method was needed to discover novel bacteriocins. Methods and Results: Crude bacteriocins from both cell-related and culture supernatant of Lactobacillus crustorum MN047 fermentation were applied to LC-MS/MS for peptidome assay, by which 131 extracellular peptides or proteins were identified in the complete genome sequence of L. crustorum MN047. Further, the genes of suspected bacteriocins were verified by expressed in Escherichia coli BL21 (DE3) pLysS. Thereafter, eight novel bacteriocins and two nonribosomal antimicrobial peptides were identified to be broad-spectrum activity against both Gram-positive and Gram-negative bacteria, including some multidrug-resistant strains. Among them, BM1556 located within predicted bacteriocin gene cluster. The most active bacteriocin BM1122 had low MIC values of 13.7 mg/L against both Staphylococcus aureus ATCC29213 and E. coli ATCC25922. The BM1122 had bactericidal action mode by biofilm-destruction, pore-formation, and membrane permeability change. Conclusions: The combination of complete genome and peptidome is a valid approach for quick discovery of novel bacteriocins without/with-low homology to known ones. This method will contribute to deep exploitation of novel bacteriocins in genome of bacteria submitted to GenBank.

The complete genome sequence of Lactobacillus crustorum deciphered by PacBio RS II and Illumina HiSeq 4000 sequencing was first reported with one chromosome and two plasmids. Sequence analysis of L. crustorum MN047 showed probiotic characteristics.

Bacteriocins produced by LAB comprise a variety of antimicrobial proteins or peptides which can be used as food biopreservatives and/or possible antibiotic alternatives. Bacteriocin-producing Lactobacillus coryniformis MXJ 32, which was identified based on its 16S rRNA gene sequence, was isolated from a traditional fermented vegetable ( Jiangshui Cai ) of Xixiang county, Shaanxi province, China. Thereafter bacteriocin (designated as lactocin MXJ 32A) produced by L. coryniformis MXJ 32 was purified by ammonium sulfate precipitation, dialysis and ion exchange chromatography. The molecular mass of lactocin MXJ 32A was 3520 Da determined by LC-ESI/MS and the N-terminal sequence of lactocin MXJ 32A was NH 2 -GEPGPMGPAGAD. Lactocin MXJ 32A had a broad antimicrobial spectrum including many Gram-positive and Gram-negative foodborne pathogens, in which even some antibiotic-resistant foodborne pathogenic strains could also be inhibited. The MIC of lactocin MXJ 32A for Staphylococcus aureus and Escherichia coli was 10 mg/mL. Lactocin MXJ 32A showed good pH-stability and heat stability and was sensitive to proteolytic enzymes. It was shown by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) that the possible mode of action of lactocin MXJ 32A was pore formation of the cytoplasmic membrane of target cells.

The present study evaluated the antifungal properties of tetrapeptide H-OOWW-NH2 (O3TR) and its derivative lipopeptide C12-OOWW-NH2 (C12O3TR) against Penicillium digitatum, one of the main postharvest pathogens in citrus, and the possible mechanisms of their antifungal action. The results showed that the peptides O3TR and C12O3TR could inhibit conidial germination, induce conidia death and reduce the survival of mycelia of P. digitatum in vitro. The antifungal properties of O3TR and C12O3TR against P. digitatum were thermostable (40 °C–80 °C), insensitive to the change of pH (3–10) and varying sensitive to the presence of cations (Na+, Ca2+). In addition, the two peptides could effectively control green mold on citrus in vivo study. In terms of safety evaluation, the hemolytic activity of O3TR was neglectable, and significantly lower than that of C12O3TR, both of which were much lower than that of commercial prochloraz. The signals and intensity of fluorescent dye SYTOX Green (SG) and Propidium Iodide (PI) showed that O3TR and C12O3TR could enhance the mycelial and conidial membrane permeabilization. The antifungal action of O3TR and C12O3TR was further demonstrated by the release of cellular constituents and extracellular conductivity. In conclusion, the two peptides have a promising prospect to be applied as antifungal agents for the control of the green mold of citrus postharvest diseases.

With the appearance of superbug and environment pollution, it is urgent to find a viable alternative to synthetic fungicide for controlling postharvest disease on citrus, especially for the control of the main pathogen Penicillium digitatum. The aim of this study was to isolate lactic acid bacteria (LAB) with antifungal activity against P. digitatum and investigate their antifungal characteristics. Sixty-eight LAB were isolated from surface of citrus fruit, leaves of citrus and peanuts, and soil in citrus orchards. Ten strains which belonging to three species (Lactobacillus sucicola, Weissella paramesenteroides, Pediococcus acidilactici) had strong inhibitory effects in vitro. After neutralization and catalase treatment, the cell-free supernatants of the L. sucicola JT03, W. paramesenteroides JT13 and P. acidilactici JY03 still exhibited partial inhibitory effect. However, their antifungal activities were partially lost after treated by proteinases. Moreover, they were especially sensitive to trypsin that antifungal activity of cell-free supernatants of the three LAB strains was totally lost. This result indicated that proteinaceous compounds might play an important role in antifungal activity for the LAB strains.

The growing emergence of antibiotic-resistant bacteria in the food industry needs to be controlled with effective antimicrobials. In this study, bacteriocin MN047 A (BMA) was found to have antibacterial activity against multidrug-resistant bacteria. It was produced by Lactobacillus crustorum MN047, which was first isolated from koumiss, a traditional fermented dairy product from Xinjiang Autonomous Region, China. It was purified by ammonium sulfate precipitation, ion-exchange chromatography, and reversed-phase chromatography. It had a low molecular mass of 1,770.89 Da according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the sequence was identified as QLPWQILGIVAGMFQA by liquid chromatography-tandem mass spectrometry analysis and MASCOT searching. It was proteinaceous in nature: the bacteriocin was digested by protease but not by α-amylase or lipase. It showed broad pH toleration (pH 2-11), good thermostability, and good storage stability. It had a broad inhibitory spectrum, including both gram-positive and gram-negative bacteria. Growth curve and time-kill kinetics indicated that it was bactericidal to the indicator strains, and this finding was verified by scanning electron microscope and transmission electron microscope after treatment with BMA. As well, BMA halted the growth of Staphylococcus aureus and Escherichia coli in the G1 and G2/M phases according to cell-cycle analysis by flow cytometry, indicating that BMA had comprehensive inhibitory effects against foodborne pathogens.