Characterization of a Vibrio parahaemolyticus-targeting lytic bacteriophage SSJ01 and its application in artificial seawater.

Vibrio parahaemolyticus is a major foodborne pathogen causing serious illnesses. In this study, a new lytic bacteriophage SSJ01 that infects V. parahaemolyticus was isolated and characterized. It had a short non-contractile tail and belonged to the Caudoviricetes class. It rapidly adsorbed onto host cells, exhibited a short latent period, and has a large burst size. It showed lytic activities under a broad range of temperature (- 18 to 60 °C), pH (5 to 11), and salinity (0 to 6%). It contained 35 open reading frames with a G + C content of 49.16% without toxic or lysogen-forming genes. The MOI of 105 phage-treated group in vitro reduced the target cells up to 3.49-log CFU/mL at 6 °C and 3.47-log CFU/mL at 25 °C, respectively. In aquatic environments (6 and 25 °C), bactericidal activities showed a significant decrease within 2 h. Therefore, the bacteriophage SSJ01 has potential as a biocontrol agent to control V. parahaemolyticus in marine culture.

Evaluation of the biocontrol potential of a collagen peptide/trehalose-based Cronobacter sakazakii phage powder in rehydrated powdered infant formula.

Cronobacter sakazakii is a major foodborne pathogen that is mainly transmitted through powdered infant formula (PIF) and has a high mortality rate of up to 80%, particularly in fetuses and neonates. Bacteriophages have emerged as an effective biocontrol agent for antibiotic-resistant bacteria. In this study, lytic phage SG01 was newly characterized and loaded into collagen peptide/trehalose-based powders to develop an antibacterial agent against C. sakazakii contamination in PIF. The phage belongs to the Siphoviridae family, has an icosahedral head and a flexible tail, and showed rapid and persistent antibacterial activity up to 17 h. It was specifically active against C. sakazakii and also exhibited effective anti-biofilm properties. The phage was freeze-dried to a collagen peptide/trehalose-based powder and the phage was tested for viability, storage stability, and antibacterial activity. The optimal composition was 5% (w/v) collagen peptides and 1% (w/v) trehalose, which demonstrated the highest phage viability after freeze-drying. The phage remained stable in the collagen peptide/trehalose-based powder for up to four weeks at 4 °C and 25 °C, indicating that this is a desirable formulation for phage protection. Furthermore, the phage powder showed significant antibacterial efficacy in PIF, with a 4-log CFU/mL reduction within 6 h. Overall, the tested phage powder has the potential to be used as an antimicrobial agent in the food industry, particularly in powdered foods such as PIF.

Complete Genome Sequence of the Bacillus cereus Temperate Bacteriophage BSG01.

The Bacillus cereus phage BSG01 has a siphovirus morphology that can belong to the order Caudovirales. It consists of 81,366 bp, with a GC content of 34.6%, and contains 70 predicted open reading frames. BSG01 includes lysogeny-related genes (tyrosine recombinase and antirepressor protein), indicating that it is a temperate phage.

Complete Genome Sequence of Bacteriophage EO1, Which Infects Both Escherichia coli O157:H7 and Shigella.

The lytic bacteriophage EO1 has been newly isolated. This phage infects Escherichia coli O157:H7 and has a broad antibacterial spectrum, including against Shigella. The complete genome sequence of phage EO1 was determined; its full length is 166,941 bp, and it has a G+C content of 35.46%.

Comparison of chitosan and gelatin-based films and application to antimicrobial coatings enriched with grapefruit seed extract for cherry tomato preservation.

Bio-based single, composite, and bilayer edible films were developed based on chitosan and gelatin, including grapefruit seed extract (GSE) as an antimicrobial agent. The physicochemical and antimicrobial properties of films were analyzed, and it was found that compounding and laminating two polymers could enhance their physicochemical properties. The composite film was strong, endurable, and flexible compared with the single ones. In addition, the composite and bilayer films had lower water vapor permeability than the single ones. Edible films and coatings with GSE presented a greater bactericidal effect than the inactive ones. In addition, the hardness, weight, and color changes of the coated cherry tomatoes during 7-day storage did not differ, whereas a bacterial reduction against Salmonella Typhimurium was revealed. Taken together, composite and bilayer films with CH and GL and enriched with GSE were developed for food packaging applications, and it showed improved mechanical, water barrier, and antimicrobial properties. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01254-9.

Synergistic antimicrobial effect and mode of action of palmarosa oil-loaded nanoemulsion and citric acid against Pectobacterium carotovorum.

The synergistic antimicrobial activity of palmarosa oil (Cymbopogon martini, PO)-loaded nanoemulsion (PO-NE) and citric acid (CA) against Pectobacterium, the major pathogen for soft-rot disease, was evaluated. The combination of PO-NE and CA (PO-NE + CA) significantly improved the storage stability of PO-NE at 30 °C. Compared to the anti-Pectobacterium activity of alone, PO-NE + CA reduced the minimum inhibitory concentration (MIC) by 1/4 and 1/2, respectively. Bactericidal efficacy of PO-NE + CA against P. carotovorum PCC3 was similar of PO-NE alone in the MIC in time-kill kinetic assay. PO-NE treatment mainly influenced membrane integrity, while CA treatment strongly stimulated intracellular ATP depletion. This synergistic combination effectively reduced the use of PO-NE, imparting a strong flavor note without sacrificing the antimicrobial efficacy against Pectobacterium. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01217-6.

Anti-Salmonella polyvinyl alcohol coating containing a virulent phage PBSE191 and its application on chicken eggshell.

The use of antibiotics in the food industry is avoided due to the increase of antibiotic-resistant bacteria. Therefore, the bacteriophage is emerging as an alternative agent. Here, we characterized the Salmonella Enteritidis phage PBSE191 and applied it to a polyvinyl alcohol (PVA) film. Transmission electron microscopic analysis revealed that it belonged to the Caudoviricetes class, with an icosahedral head and flexible tails. The phage showed rapid and strong lytic activity within 1 h. It was active against a broad range of Salmonella isolates, including six serotypes. In 25 min, 99 % of the initial population was adsorbed to the bacterial cell surface. The phage was also applied to 10 % (w/v) PVA films and coatings, which were then characterized in terms of phage stability and antibacterial performance, both in vitro and in foods. The phage remained stable in the 10 % (w/v) PVA solution containing 20 % (w/w, based on PVA weight) sorbitol (PVAS20), indicating that the phage was stable under dry conditions and strongly released in the polymer. Furthermore, significant bacterial cell reduction (2.0 × 105 CFU/film within 2 h) was observed in the phage-containing PVAS20 films. In addition, the PBSE191-containing PVAS20 coating on the chicken eggshell surface showed significant anti-Salmonella efficiency (about 2 log CFU reduction) within 24 h. Overall, the PBSE191 phage possesses a high potential as a biocontrol agent for use as an additive, or as an active antibacterial packaging to improve food safety against Salmonella contamination.

Nocardioides panacis sp. nov., isolated from soil of a ginseng field.

A bacterial strain designated as G188T was isolated from ginseng field soil in the Republic of Korea. Phylogenetic analysis of 16S rRNA gene sequences showed that strain G188T formed a distinct lineage within the genus Nocardioides, family Nocardioidaceae, order Propionibacteriales. Sequence similarity revealed that strain G188T was most closely related to Nocardioides iriomotensis IR27-S3T (97.7 % 16S rRNA similarity). The genome size of strain G188T was 4 901 775 bp, and the genomic DNA G+C content was 72.3 mol%. The average nucleotide identity and DNA-DNA hybridization values with other Nocardioides species were less than 75.6 and 20.1 %, respectively. The main fatty acids of strain G188T were C17 : 0, C17 : 1 ω8c and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol, and the major respiratory quinone was menaquinone 8, supporting that strain G188T was affiliated with the genus Nocardioides. Based on biochemical, chemotaxonomic and phylogenetic analyses, the novel species Nocardioides panacis G188T (KACC 21695T=LMG 31733T) is proposed.

Development of a multilayer insect-repelling film with trans-anethole for the storage of almond flake cereal.

Trans-anethole (AN), which exhibits strong insect-repellent activity against Plodia interpunctella larvae, was applied on a polyethylene terephthalate (PET) film as an active packaging coating layer. All developed films at different concentrations (25%, 30%, 40%, and 50%) exhibited significant insect repellent activities. However, these films did not significantly differ from the control film in terms of color and transparency. In addition, the developed polypropylene (PP) and PET laminated films containing 25% AN (PP/AN25/PET) exhibited strong and continuous insect-repellent activity for up to 42 days. Finally, the developed film showed 2.86-fold stronger repellent activity than that of the control film when applied to the almond flake cereals packaging. These results suggest that PP/AN25/PET could be used as a potent insect-repelling packaging film in a realistic grain-packaging system. PRACTICAL APPLICATION: A PP/trans-anethole/PET film that exhibited good insect-repellent activity for 42 days was newly developed in this study. As it showed strong insect repellency, especially in almond flake cereals packaging, it is expected to have high potential as an insect-repelling grain-packaging film.

Analysis of the insect-repelling mechanism of star anise extract and its major active compounds against Plodia interpunctella.

One of the major stored product pests, Indian meal moth causes the loss on the agriculture and food industries. This study was conducted to screen the insecticidal activity of ethanolic extracts and fractions partitioned by four different solvents [(1) n-hexane; (2) ether; (3) ethyl acetate; (4) water] from star anise (Illicium verum Hook. f.) against Plodia interpunctella larvae. Among all solvent-partitioned fractions, the strongest repellency was found for the n-hexane fraction of star anise extract. Solvent-solvent partitioning and chromatographic methods were further used to isolate and identify major anti-insect compounds from star anise extract. The results showed that trans-anethole (94.24%) was the major active compound showing an insect-repelling activity against P. interpunctella. Consequently, trans-anethole can be utilized as a main natural insect-repelling agent for controlling the P. interpunctella infestation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01053-8.

Hymenobacter armeniacus sp. nov. and Hymenobacter montanus sp. nov., two radiation-resistant bacteria from soil.

Two bacterial strains, designated BT189T and BT664T, were isolated from soil sampled in the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strains BT189T and BT664T belonged to the genus Hymenobacter, family Hymenobacteraceae (order Cytophagales). The 16S rRNA genes of the two strains shared a sequence similarity of 93.7 %. The closely related species of strain BT189T were Hymenobacter rubidus DG7BT (97.1 % 16S rRNA similarity) and Hymenobacter terrae DG7AT (96.7 %). The closest related species to strain BT664T were Hymenobacter sedentarius DG5BT (95.3 %) and Hymenobacter terrenus MIMtkLc17T (95.2 %). The genome sizes of strains BT189T and BT664T were 5 285 287 and 5 475 357 bp, respectively. The genomic DNA G+C contents of strains BT189T and BT664T were 63.2 and 59.3 mol%, respectively. The main fatty acids of strain BT189T were iso-C15 : 0, anteiso-C15 : 0 and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c), and those of strain BT664T were iso-C15 : 0, C16 : 1 ω5c and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c). The main polar lipid in both strains was phosphatidylethanolamine and the predominant respiratory quinone was MK-7, supporting the affiliation of these strains with the genus Hymenobacter. Based on the results of biochemical, chemotaxonomic and phylogenetic analyses, two novel species, Hymenobacter armeniacus BT189T (=KCTC 72341T=NBRC 114843T) and Hymenobacter montanus BT664T (KACC 21967T=NBRC 114856T), are proposed.

Isolation, screening and identification of key components having intense insect repellent activity against Plodia interpunctella from four different medicinal plant materials.

BACKGROUND: Global warming and the indiscriminate use of pesticides have increased the propagation of the stored-product insect pests, leading to enormous losses in the agriculture and food industries. The most used insect repellents are synthetic derivatives; however, these have an adverse effect on human health as well as on the environment. Therefore, we attempted to find materials with insect repellent activity in natural products. The present study aimed to identify the single chemical component with intense insect repellent activity in extracts from four different Oriental medicinal plant materials: (i) Anethum graveolens L. (dill) seeds; (ii) Artemisia capillaris Thunb. (capillary wormwood) leaves; (iii) smoked Prunus mume Siebold & Zucc. (mume) fruits; and (iv) Rhus javanica L. (galls). RESULTS: As a result of the bioassay-guided fractionation of each extract against the Plodia interpunctella, stored-product insect, the n-hexane fraction of dill seeds extract was confirmed as the optimal fraction between all of the fractions. In total, 32 chemical components were identified from the n-hexane fraction of dill seeds by gas chromatography-mass spectrometry analysis, and the two main components were dillapiole (47.51%) and carvone (26.76%). Of the two components, dillapiole was confirmed as the key component playing an essential role in insect repellent activity. CONCLUSION: Our study suggests that dillapiole has the potential to be used as a natural insect repellent for the control of P. interpunctella infestation in agricultural and food products during distribution and storage. © 2021 Society of Chemical Industry.

Synergistic inactivation of Listeria and E. coli using a combination of erythorbyl laurate and mild heating and its application in decontamination of peas as a model fresh produce.

We investigated the synergistic antimicrobial activity of erythorbyl laurate (EL) and mild heating co-treatment on the Gram-positive Listeria innocua and Gram-negative Escherichia coli O157:H7 bacteria. EL (2 mM) and mild heating (55 °C for 3 min) resulted in 3.1 and 0.5 log colony forming units (CFU)/mL reductions in the number of L. innocua, respectively, compared to a 6.4 log CFU/mL reduction induced by the combined treatment of EL and mild heating in saline. EL (10 mM) and mild heating (55 °C for 3 min) resulted in 1.3 and 0.7 log CFU/mL reductions in the number of E. coli O157:H7, respectively, compared to a 6.2 log CFU/mL reduction with the combined treatment in saline. EL, a membrane-active compound, showed a strong synergistic effect with mild heating, possibly due to enhanced disruption of the bacterial cell membrane. The synergistic antibacterial effect was evaluated using inoculated English peas (Pisum sativum) and this combined treatment (2 mM EL and mild heating against L. innocua and 10 mM EL and mild heating against E. coli O157:H7) resulted in more than 7 log reductions in the numbers of L. innocua and E. coli O157:H7, inoculated on the surface of fresh peas. The treatments did not show significant difference in the color or texture of treated peas compared to the non-treated controls. This is the first report illustrating synergistic activity of EL and mild heating for both the gram positive (L. innocua) and the gram negative (E. coli O157:H7) bacteria on food. Overall, this research will illustrate the development of more effective and rapid antibacterial surface disinfection method for application in the processing of minimally processed foods.

Synergistic Inactivation of Bacteria Using a Combination of Erythorbyl Laurate and UV Type-A Light Treatment.

This study evaluated the synergistic antimicrobial activity of erythorbyl laurate (EL) and UV type-A (UVA). To investigate the mode of synergism, changes in gene expression and bacterial inactivation activity were examined. Individual treatments with EL (10 mM) or UVA caused a 1.9- or 0.5-log CFU/ml reduction respectively, whereas EL/UVA co-treatment resulted in a 5.5-log CFU/ml reduction in Escherichia coli viable cell numbers. Similarly, treatment with either EL (2 mM) or UVA for 30 min resulted in a 2.8- or 0.1-log CFU/ml reduction in Listeria innocua, respectively, whereas combined treatment with both EL and UVA resulted in a 5.4-log CFU/ml reduction. Measurements of gene expression levels showed that EL and UVA treatment synergistically altered the gene expression of genes related to bacterial membrane synthesis/stress response. However, addition of 10-50-fold excess concentration of exogenous antioxidant compared to EL reduced the synergistic effect of EL and UVA by approximately 1 log. In summary, the results illustrate that synergistic combination of EL and UVA enhanced membrane damage independent of the oxidative stress damage induced by UVA and thus illustrate a novel photo-activated synergistic antimicrobial approach for the inactivation of both the Gram-positive and Gram-negative bacteria. Overall, this study illustrates mechanistic evaluation of a novel photochemical approach for food and environmental applications.

Hymenobacter negativus sp. nov., bacteria isolated from mountain soil collected in South Korea.

Two novel Gram-negative bacterial strains BT442T and BT584 were isolated from dry soil collected in mountains Busan and Guri, Korea during wintertime. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains BT442T and BT584 both belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia). Strain BT442T was closely related to Hymenobacter soli PB17T (98.0% 16S rRNA gene similarity) and Hymenobacter terrae POA9T (97.6%). No other recognized bacterial species showed more than 97% 16S rRNA gene sequence similarity to strains BT442T. The genome size of strain BT442T was 5,143,362 bp. Bacterial growth was observed at 10-30 °C (optimum 25 °C), pH 6.0-8.0 (optimum pH 6.0) in R2A agar and in the presence up to 1% NaCl. The major cellular fatty acids of strains BT442T and BT584 were iso-C15:0, anteiso-C15:0 and summed feature 3 (C16:1 ω6c / C16:1 ω7c). In addition, their predominant respiratory quinone was MK-7. The major polar lipids of strains BT442T and BT584 were identified to be phosphatidylethanolamine, aminophospholipid, and aminolipid. Based on the biochemical, chemotaxonomic, and phylogenetic analyses, strains BT442T and BT584 are novel bacterial species within the genus Hymenobacter, and the proposed name is Hymenobacter negativus. The strain type of Hymenobacter negativus is BT442T (= KCTC 72902T = NBRC XXXXT).

Hymenobacter citatus sp. nov., isolated from soil in South Korea.

Two novel Gram-negative bacterial strains, BT507T and BT506, were isolated from soil collected in Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains BT507T and BT506 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae; order Cytophagales; class Cytophagia). The 16S rRNA genes of the two strains shared a sequence similarity of 100.0%. Strains BT507T and BT506 are closely related to Hymenobacter profundi M2T (97.2% 16S rRNA gene similarity), Hymenobacter defluvii POA9T (97.1%), and Hymenobacter tenuis POB6T (95.3%). The genome size of strain BT507T is 5,078,289 base pairs. Bacterial growth was observed at 10-37 °C (optimum 25 °C) and pH 6.0-8.0 (optimum pH 6.0). The primary cellular fatty acids of strain BT507T are iso-C15:0, C16:1ω5c, and summed feature 3 (C16:1ω6c/C16:1ω7c). Its predominant respiratory quinone is MK-7. The primary polar lipids of strain BT507T are lipid, aminophospholipid, and phosphatidylethanolamine. Based on the biochemical, chemotaxonomic, and phylogenetic analysis, strains BT507T and BT506 can be described a novel bacterial species within the genus Hymenobacter, and the proposed name is Hymenobacter citatus. The type strain of H. citatus is BT507T is KCTC 82115T and NBRC 114850T.

Phyllobacterium pellucidum sp. nov., isolated from soil.

A bacterial strain, BT25T, was isolated from soil in Korea. The bacterial cells were Gram-negative and rod-shaped. Phylogenetic analysis using 16S rRNA gene sequences showed that the BT25T strain was related to the genus Phyllobacterium. BT25T was 96.6 and 96.5% similar to Phyllobacterium brassicacearum STM 196T and Phyllobacterium myrsinacearum DSM 5892T, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between BT25T and the two closest phylogenetic neighbors were calculated to be 78.5 and 77.7, 21.1 and 21.2%, respectively. The major cellular fatty acids were summed feature 8 (C18:1 ω7c/C18:1 ω6c) (29.3%), cyclo-C19:0 ω8c (27.5%), and C16:0 (16.5%). The BT25T strain had menaquinone Q-10 as the predominant quinone, as well as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylcholine as the major polar lipids. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the BT25T strain was classified as a novel Phyllobacterium species. The name Phyllobacterium pellucidum sp. nov. was proposed. The type strain is BT25T (= KCTC 62765T = NBRC 114381T).

Polycaprolactone film functionalized with bacteriophage T4 promotes antibacterial activity of food packaging toward Escherichia coli.

Bacteriophages (phages) have been extensively utilized as antibacterial agents in the food industry because of their host-specificity. However, their application in polymer films has been limited because of the lack of a strong attachment method for phage to the surface. We developed an antibacterial film by covalently immobilizing Escherichia coli (E. coli)-specific phage T4 on a polycaprolactone (PCL) film. The chemical bond formation was confirmed by XPS analysis, and the covalent attachment of phage T4 effectively inhibited E. coli growth even after external stimulation of the film by sonication. When applied as a packaging film for raw beef inoculated with E. coli O157:H7, the chemically functionalized PCL film showed approximately 30-fold higher bacterial inhibitory effects than the film with physically adsorbed phage T4. These results indicate the promising application potential of chemically functionalized PCL film with phage T4 as an antibacterial food packaging material against the foodborne pathogen E. coli.

Pontibacter fetidus sp. nov. and Pontibacter burrus sp. nov., isolated from the soil.

Two novel strains, BT213T and BT327T, were isolated from the soil collected in Uijeongbu city, Korea. Cells of strains were Gram negative, aerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains BT213T and BT327T formed two distinct lineages within the family Hymenobacteraceae (order Cytophagales, class Cytophagia). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BT213T and BT327T belonged to the genus Pontibacter. Strain BT213T showed the highest similarities of 97.8% with Pontibacter deserti JC215T. Strain BT327T showed the highest sequence similarity of 96.5% with Pontibacter mucosus PB3T. Optimal growth occurred at 25 °C, pH 7, and in the absence of NaCl. The major cellular fatty acid of strains BT213T and BT327T were iso-C15:0 and summed feature 4 (iso-C17:1 I/anteiso-C17:1 B). Strains BT213T and BT327T had MK-7 as major respiratory quinone and phosphatidylethanolamine as major polar lipids. The genome size of strains BT213T and BT327T were 4,072,018 bp and 4,314,171 bp, respectively. The genomic G + C mol% of strains BT213T and BT327T are 45.6% and 46.1%, respectively. Based on biochemical, chemotaxonomic, and phylogenetic analysis, two novel species Pontibacter fetidus BT213T (KCTC 72345T = NBRC 114379T) and Pontibacter burrus BT327T (KCTC 72412T = NBRC 114376T) are proposed as type strains.