Simple Purification and Antimicrobial Properties of Bacteriocin-like Inhibitory Substance from Bacillus Species for the Biopreservation of Cheese.

Bacteriocins may be used as natural preservatives and antibiotic substitutes in various foods. However, the multistep purification process of bacteriocins results in high production costs, which is an obstacle to their commercial use and consumer accessibility. In this study, a bacteriocin-like inhibitory substance (BLIS) from Bacillus spp. isolated from Korean fermented foods was partially purified using the aqueous two-phase system (ATPS). The maximum activity of the BLIS was achieved for ATPS composed of PEG 1000 (15% [w/w])/ammonium sulfate (20% [w/w])/sodium chloride (2% [w/w]), which caused BLIS activity to increase by 3 times with a 99% recovery rate. In particular, B. amyloliquefaciens Y138-6 BLIS exhibited broad antibacterial activity, high resistance to acid-base stress, and excellent thermal stability. This antibacterial substance inhibited the growth of aerobic bacteria and fungi on the walls of cheese and ripening rooms. These antibacterial properties have been shown to increase food safety and have the potential for use as biopreservatives. Moreover, considering that the execution of the ATPS requires only salts and PEG, it is a simple, environmentally friendly, and cost-effective process and may have industrial applications in the recovery of BLIS from fermentation broth.

A highly sensitive lateral flow immunoassay for the rapid and on-site detection of enrofloxacin in milk.

Enrofloxacin (ENR) is a veterinary antibiotic used to treat bacterial infections in livestock. It chiefly persists in foods and dairy products, which in turn pose severe risks to human health. Hence it is very important to detect the ENR in foods and dairy products to safeguard human health. Herein, we attempted to develop a single-step detection lateral flow immunochromatographic assay (LFIA) using gold nanoparticles (AuNPs) for the rapid and on-site detection of ENR in milk samples. An anti-enrofloxacin monoclonal antibody (ENR-Ab) was conjugated with AuNPs for the specific detection of ENR in milk samples. For sensitivity improvement, many optimization steps were conducted on LFIA test strips. The visual limit of detection (vLOD) was found to be 20 ng/ml with a cut-off value of 50 ng/ml in the milk samples. The obtained LOD and cut-off value were within the safety limit guidelines of the Ministry of food and drug safety, South Korea. The test strip showed negligible cross-reactivity with ENR analogs, and other components of antibiotics, this indicates the high specificity of the LFIA test strip towards ENR. The designed test strip showed good reliability. The visual test results can be seen within 10 min without the need for special equipment. Therefore, the test strip can be employed as a potential detection strategy for the qualitative on-site detection of enrofloxacin in milk samples.

Highly Specific Peptide-Mediated Cuvette-Form Localized Surface Plasmon Resonance (LSPR)-Based Fipronil Detection in Egg.

Herein, we have developed peptide-coated gold nanoparticles (AuNPs) based on localized surface plasmon resonance (LSPR) sensor chips that can detect fipronil with high sensitivity and selectivity. The phage display technique has been exploited for the screening of highly specific fipronil-binding peptides for the selective detection of the molecule. LSPR sensor chips are fabricated initially by attaching uniformly synthesized AuNPs on the glass substrate, followed by the addition of screened peptides. The parameters, such as the peptide concentration of 20 µg mL-1 and the reaction time of 30 min, are further optimized to maximize the efficacy of the fabricated LSPR sensor chips. The sensing analysis is performed systematically under standard fipronil solutions and spike samples from eggs. The developed sensor has shown excellent sensitivity towards both standard solutions and spike samples with limit of detection (LOD) values of 0.01 ppb, respectively. Significantly, the developed LSPR sensor chips offer distinct features, such as a facile fabrication approach, on-site sensing, rapid analysis, cost-effectiveness, and the possibility of mass production, in which the chips can be effectively used as a promising and potential on-site detection tool for the estimation of fipronil.

Probiogenomic In-Silico Analysis and Safety Assessment of Lactiplantibacillus plantarum DJF10 Strain Isolated from Korean Raw Milk.

The whole genome sequence of Lactiplantibacillus plantarum DJF10, isolated from Korean raw milk, is reported, along with its genomic analysis of probiotics and safety features. The genome consists of 29 contigs with a total length of 3,385,113 bp and a GC content of 44.3%. The average nucleotide identity and whole genome phylogenetic analysis showed the strain belongs to Lactiplantibacillus plantarum with 99% identity. Genome annotation using Prokka predicted a total of 3235 genes, including 3168 protein-coding sequences (CDS), 59 tRNAs, 7 rRNAs and 1 tmRNA. The functional annotation results by EggNOG and KEGG showed a high number of genes associated with genetic information and processing, transport and metabolism, suggesting the strain's ability to adapt to several environments. Various genes conferring probiotic characteristics, including genes related to stress adaptation to the gastrointestinal tract, biosynthesis of vitamins, cell adhesion and production of bacteriocins, were identified. The CAZyme analysis detected 98 genes distributed under five CAZymes classes. In addition, several genes encoding carbohydrate transport and metabolism were identified. The genome also revealed the presence of insertion sequences, genomic islands, phage regions, CRISPR-cas regions, and the absence of virulence and toxin genes. However, the presence of hemolysin and antibiotic-resistance-related genes detected in the KEGG search needs further experimental validation to confirm the safety of the strain. The presence of two bacteriocin clusters, sactipeptide and plantaricin J, as detected by the BAGEL 4 webserver, confer the higher antimicrobial potential of DJF10. Altogether, the analyses in this study performed highlight this strain's functional characteristics. However, further in vitro and in vivo studies are required on the safety assurance and potential application of L. plantarum DJF10 as a probiotic agent.

Identification and Characterization of a Bacteriocin from the Newly Isolated Bacillus subtilis HD15 with Inhibitory Effects against Bacillus cereus.

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70°C and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

Inhibitory effects of ultraviolet-C light and thermal treatment on four fungi isolated from pig slaughterhouses in Korea.

Pig slaughterhouses harbor high humidity because of the necessary cleaning that takes place simultaneously with slaughter, which facilitates the existence of mold. Due to the enclosed space, there are several limitations to the control of mold growth with respect to cleaning, ventilation, and drying. In this study, the prevalence of fungi was investigated in four pig slaughterhouses in Korea. Four fungi (Aspergillus niger, Penicillium commune, Penicillium oxalicum, and Cladosporium cladosporioides) were detected with the highest frequency. These four strains were subjected to various treatments to reduce their growth. The fungi were inoculated onto stainless steel (SS) chips and treated with ultraviolet (UV)-C irradiation and hot water. Individual treatments with UV-C (15, 30, 90, 150, 300, and 600 mJ/cm2), and hot water (60, 65, 70, and 83°C) were performed to sanitize the SS chips. Simultaneous cleaning with 60°C hot water and more than 150 mJ/cm2 of UV-C reduced the fungal incidence by > 6.5 Log from 6.6-7.0 Log CFU/cm2 (initial count). Our results demonstrate that a combined treatment of UV-C and hot water is the most economical and convenient way to prevent microbiological contamination of small tools (such as knives and sharpeners) and steel surfaces in slaughterhouses.

Development of Desiccation-Tolerant Probiotic Biofilms Inhibitory for Growth of Foodborne Pathogens on Stainless Steel Surfaces.

Lactic acid bacteria biofilms can be used to reduce foodborne pathogen contamination in the food industry. However, studies on growth inhibition of foodborne pathogens by inducing biofilm formation of antagonistic microorganisms on abiotic surfaces are rare. We developed a desiccation-tolerant antimicrobial probiotic biofilm. Lactobacillus sakei M129-1 and Pediococcus pentosaceus M132-2 isolated from fermented Korean foods were found to exhibit broad-spectrum antibacterial activity against Bacillus cereus, Escherichia coli O157:H7, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. Their biofilm levels were significantly (p < 0.05) higher on stainless steel than on polyethylene or ceramic. Biofilms of both isolates showed significantly (p < 0.05) enhanced resistance against desiccation (exposure to 43% atmospheric relative humidity) as compared with the isolates not in the biofilm form. The antimicrobial activity of the isolates was sustained in dried biofilms on stainless steel surface; the initial number of foodborne pathogens (average 7.0 log CFU/mL), inoculated on stainless steel chips containing L. sakei M129-1 or P. pentosaceus M132-2 biofilm decreased to less than 1.0 log CFU within 48 h. The lactic acid bacteria antibacterial biofilms developed in this study may be applied to desiccated environmental surfaces in food-related environments to improve microbiological food safety.

Potential Correlation between Microbial Diversity and Volatile Flavor Compounds in Different Types of Korean Dry-Fermented Sausages.

The microbial community in fermented sausages plays an important role in determining their quality characteristics. The objective of this study was to investigate the correlation between microbial diversity and volatile compounds in dry-fermented sausages procured from different regions of Korea. Results from metagenomics analysis showed that Lactobacillus and Staphylococcus were the predominant bacterial genera, and Penicillium, Debaryomyces, and Candida were the predominant fungal genera. Twelve volatile compounds were detected using an electronic nose. Leuconostoc exhibited a positive correlation with esters and volatile flavor, whereas Debaryomyces, Aspergillus, Mucor, and Rhodotorula exhibited a negative correlation with methanethiol, thus revealing the involvement of the microorganisms in flavor formation. The results of this study may help in understanding the microbial diversity of dry-fermented sausages in Korea and provide a rationale and quality control guideline through potential correlation with volatile flavor analysis.

Broad-spectrum antimicrobial activity of cinnamoyl esterase-producing Lactobacilli and their application in fermented rice bran.

BACKGROUND: Cinnamoyl esterase (CE) can release antioxidant phenolic acids from its non-digestible ester-linked form. Fermentation using CE-producing lactic acid bacteria (LAB) can be useful in the food industry because of its ability to produce bioactive compounds and antibacterial metabolites. The purpose of this study was to confirm the food applicability of LAB with CE-producing ability and broad-spectrum antibacterial activity. RESULTS: Among the 219 bacterial strains identified in infant feces, five Lactobacillus gasseri and six Limosilactobacillus fermentum with a high CE activity were isolated. The survival rate of all selected LABs was > 95% at pH 2.5 for 3 h and > 70% when treated with 0.3% bile salt for 4 h. Moreover, cell-free supernatants of all strains strongly inhibited five food-borne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, Escherichia coli O157:H7, Bacillus cereus, and Staphylococcus aureus) and three toxin-producing fungal pathogens (Aspergillus niger, Penicillium sp., and Fusarium oxysporum). To improve phenolic acid content and rice bran preservation, Limosilactobacillus fermentum J2 with the strongest CE activity and Lactobacillus gasseri N2 with the strongest antibacterial activity were used in rice bran fermentation, respectively. FRB-J2 (fermented rice bran with Limosilactobacillus fermentum J2) and FRB-N2 (fermented rice bran with Lactobacillus gasseri N2) significantly increased caffeic acid and ferulic acid (P < 0.01). FRB-J2 and FRB-N2 artificially inoculated with F. oxysporum showed no visible fungal growth during the test period (21 days). CONCLUSION: Fermentation by Limosilactobacillus fermentum J2 and Lactobacillus gasseri N2 can help extend the shelf life of rice bran-based products and produce bioactive compounds. © 2021 Society of Chemical Industry.

Simple and rapid detection of ractopamine in pork with comparison of LSPR and LFIA sensors.

This study developed a simple and rapid strategic technique to detect ractopamine (chemical growth-promoting agent) in pork. Two highly sensitive and specific gold nanoparticle-based portable sensors, i.e., localized surface plasmon resonance (LSPR) sensors, and lateral flow immunoassay (LFIA) strips were developed to detect veterinary drug residues in food products, that have detrimental effects on humans. Optimization studies were conducted on several sensor devices to improve sensitivity. Each sensor comprised functionalized gold nanoparticles conjugated with ractopamine antibodies. The LSPR sensor chip achieved excellent detection sensitivity = 1.19 fg/mL and was advantageous for quantitative analysis due to its wide dynamic range. On the other hand, LFIA strips provided visual test confirmation and achieved 2.27 ng/mL detection sensitivity, significantly less sensitive than LSPR. The complementary sensors help overcome each other's shortcomings with both the techniques offering ease of use, affordability and rapid diagnosis. Thus, these sensors can be applied on-site for routine screening of harmful drug residues in pork meat. They also provide useful direction for advanced technologies to enhance assay performance for detecting various other food drug contaminants.

A Response Surface Methodology (RSM) Approach for Optimizing the Attenuation of Human IgE-Reactivity to ß-Lactoglobulin (ß-Lg) by Hydrostatic High Pressure Processing.

The response surface methodology (RSM) and central composite design (CCD) technique were used to optimize the three key process parameters (i.e., pressure, temperature and holding time) of the high-hydrostatic-pressure (HHP) processing either standalone or combined with moderate thermal processing to modulate molecular structures of ß-lactoglobulin (ß-Lg) and α-lactalbumin (α-La) with reduced human IgE-reactivity. The RSM model derived for HHP-induced molecular changes of ß-Lg determined immunochemically showed that temperature (temp), pressure (p2) and the interaction between temperature and time (t) had statistically significant effects (p < 0.05). The optimal condition defined as minimum (ß-Lg specific) IgG-binding derived from the model was 505 MPa at 56 °C with a holding time of 102 min (R2 of 0.81 and p-value of 0.01). The validation carried at the optimal condition and its surrounding region showed that the model to be underestimating the ß-Lg structure modification. The molecular change of ß-Lg was directly correlated with HHP-induced dimerization in this study, which followed a quadratic equation. The ß-Lg dimers also resulted in the undetectable human IgE-binding.

Aptamer-Conjugated Polydiacetylene Colorimetric Paper Chip for the Detection of Bacillus thuringiensis Spores.

A colorimetric polydiacetylene (PDA) paper strip sensor that can specifically recognize Bacillus thuringiensis (BT) HD-73 spores is described in this work. The target-specific aptamer was combined with PDA, and the aptamer-conjugated PDA vesicles were then coated on polyvinylidene fluoride (PVDF) paper strips by a simple solvent evaporation method. The PDA-aptamer paper strips can be used to detect the target without any pre-treatment. Using the paper strip, the presence of BT spores is directly observable by the naked eye based on the unique blue-to-red color transition of the PDA. Quantitative studies using the paper strip were also carried out by analyzing the color transitions of the PDA. The specificity of this PDA sensor was verified with a high concentration of Escherichia coli, and no discernable change was observed. The observable color change in the paper strip occurs in less than 1 h, and the limit of detection is 3 × 107 CFU/mL, much below the level harmful to humans. The PDA-based paper sensor, developed in this work, does not require a separate power or detection device, making the sensor strip highly transportable and suitable for spore analysis anytime and anywhere. Moreover, this paper sensor platform is easily fabricated, can be adapted to other targets, is highly portable, and is highly specific for the detection of BT spores.

Inhibitory Effects of Combinations of Chemicals on Escherichia coli, Bacillus cereus, and Staphylococcus aureus Biofilms during the Clean-in-Place Process at an Experimental Dairy Plant.

ABSTRACT: In dairy plants, clean-in-place (CIP) equipment cannot be disassembled, making it difficult to clean the inner surface of pipes. In this study, the inhibitory effects of chemical agents on biofilms formed by three foodborne pathogens, Bacillus cereus, Escherichia coli, and Staphylococcus aureus, was evaluated in a dairy CIP system. The experiment was conducted on a laboratory scale. Each of the three bacteria (200 µL) was inoculated onto stainless steel (SS) chips (25 by 25 mm), and the effect of single cleaning agents was evaluated. Individual treatments with NaClO (30, 50, 100, and 200 ppm), NaOH (0.005, 0.01, 0.05, and 0.1%), citric acid (1, 3, 5, and 7%), and nisin (5, 10, 25, 50, 100, and 200 ppm) were used to clean the SS chip for 10 min. The most effective concentration of each solution was selected for further testing in a commercial plant. Simultaneous cleaning with 200 ppm of NaClO (10 min) and 7% citric acid (10 min) reduced the biofilms of B. cereus, E. coli, and S. aureus by 6.9, 7.0, and 8.0 log CFU/cm2, respectively. Both 7% citric acid and 0.1% NaOH were optimal treatments for E. coli. NaClO and citric acid are approved for use as food additives in the Republic of Korea. Our results revealed that a combined treatment with NaClO and citric acid is the most effective approach for reducing biofilms formed by common foodborne pathogens on CIP equipment. These findings can contribute to the production of safe dairy products.

Gold Nanoparticle-Coated Starch Magnetic Beads for the Separation, Concentration, and SERS-Based Detection of E. coli O157:H7.

Here, we report gold nanoparticle-coated starch magnetic beads (AuNP@SMBs) that were prepared by in situ synthesis of AuNPs on the surface of SMBs. Upon functionalization of the surface with a specific antibody, the immuno-AuNP@SMBs were found to be effective in separating and concentrating the target pathogenic bacteria, Escherichia coli O157:H7, from an aqueous sample as well as providing a hotspot for surface-enhanced Raman scattering (SERS)-based detection. We employed a bifunctional linker protein, 4× gold-binding peptide-tagged Streptococcal protein G (4GS), to immobilize antibodies on AuNP@SMBs and AuNPs in an oriented form. The linker protein also served as a Raman reporter, exhibiting a strong and unique fingerprint signal during the SERS measurement. The amplitude of the SERS signal was shown to have a good correlation with the concentration of target bacteria ranging from 100 to 105 CFU/mL. The detection limit was determined to be as low as a single cell, and the background signals derived from nontarget bacteria were negligible due to the excellent specificity and colloidal stability of the immuno-AuNP@SMBs and SERS tags. The highly sensitive nature of the SERS-based detection system will provide a promising means to detect the pathogenic microorganisms in food or clinical specimen.

Characterisation of fungal contamination sources for use in quality management of cheese production farms in Korea.

OBJECTIVE: This study was conducted to determine the composition and diversity of the fungal flora at various control points in cheese ripening rooms of 10 dairy farms from six different provinces in the Republic of Korea. METHODS: Floor, wall, cheese board, room air, cheese rind and core were sampled from cheese ripening rooms of ten different dairy farms. The molds were enumerated using YM petrifilm, while isolation was done on yeast extract glucose chloramphenicol agar plates. Morphologically distinct isolates were identified using sequencing of internal transcribed spacer region. RESULTS: The fungal counts in 8 out of 10 dairy farms were out of acceptable range, as per hazard analysis critical control point regulation. A total of 986 fungal isolates identified and assigned to the phyla Ascomycota (14 genera) and Basidiomycota (3 genera). Of these Penicillium, Aspergillus, and Cladosporium were the most diverse and predominant. The cheese ripening rooms was overrepresented in 9 farms by Penicillium (76%), while Aspergillusin a single farm. Among 39 species, the prominent members were Penicillium commune, P. oxalicum, P. echinulatum, and Aspergillus versicolor. Most of the mold species detected on surfaces were the same found in the indoor air of cheese ripening rooms. CONCLUSION: The environment of cheese ripening rooms persuades a favourable niche for mold growth. The fungal diversity in the dairy farms were greatly influenced by several factors (exterior atmosphere, working personnel etc.,) and their proportion varied from one to another. Proper management of hygienic and production practices and air filtration system would be effective to eradicate contamination in cheese processing industries.

Paper-based lateral flow strip assay for the detection of foodborne pathogens: principles, applications, technological challenges and opportunities.

As a representative colorimetic biosnesor, paper-based LFSA have emerged as a promising and robust tool that can easily and instansly detect the presence of target biological components in food sample. Recently, LFSAs have gained a considerable attention as an alternative method for rapid diagnosis of foodborne pathogens to the conventional culture-based assays such as plate counting and PCR. One major drawback of the current LFSAs for the detection of pathogenic bacteria is the low sensitivity, limiting its practical applications in POCT. Not like many other protein-based biomarkers that are present in nM or pM range, the number of pathogenic bacteria that cause disease can be as low as few CFU/ml. Here, we review current advances in LFSAs for the detection of pathogenic bacteria in terms of chromatic agents and analyte types. Furthermore, recent approaches for signal enhancement and modifications of the LFSA architecture for multiplex detection of pathogenic bacteria are included in this review, together with the advantages and limitations of each techniques. Finally, the technological challenges and future prospect of LFSA-based POCT for the detection of pathogenic bacteria are discussed.

Expression and Purification of Extracellular Solute-Binding Protein (ESBP) in Escherichia coli, the Extracellular Protein Derived from Bifidobacterium longum KACC 91563.

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

Shape-controlled assemblies of graphitic carbon nitride polymer for efficient sterilization therapies of water microbial contamination via 2D g-C3N4 under visible light illumination.

Bacterial pathogens of water origin have potential public threats thus suggesting the need of developing efficient and sustainable water disinfection strategies from waterborne pathogens. We set out to synthesize different controlled morphologies of graphitic carbon nitride (g-C3N4) polymer, evaluate their comparative effects on the generation of reactive oxygen species (ROS), and investigate potential applications in water purification systems. Characterization of the synthesized microstructures of g-C3N4, such as melamine-cyanuric acid (MCA)-based rosette-type, rod-type, 2D hexagonal, and 3D cubic mesoporous silica was accomplished using Fourier transform infrared (FT-IR), energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM), X-ray diffractometry (XRD), and transmission electron microscopy (TEM). The microbial inhibitory potential of 2D g-C3N4 photocatalyst against waterborne Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium was evaluated based on the effective activity of 2D g-C3N4 upon visible light excitations. The microbicidal efficiency of 2D g-C3N4 was evident within 30 min of visible light exposure via direct interaction, while other microstructures of g-C3N4 demonstrated only slight antimicrobial effects after 120 min, with insufficient ROS generation. The antimicrobial and ROS-generating effects of 2D g-C3N4 depended on the type and surface area of the synthesized 2D g-C3N4 material. Considering its availability and excellent disinfection activity, 2D g-C3N4 obtained from simple and convenient facile synthesis is a promising solar-driven photocatalyst for clearing microbial contamination from water.

Genotypic Classification of Staphylococcus aureus and Bacillus cereus from Korean Slaughterhouses Using Semiautomated Repetitive Sequence-Based Polymerase Chain Reaction.

A repetitive sequence-based polymerase chain reaction (rep-PCR) technique utilizing a semiautomated system, namely DiversiLab, was applied to determine the genotypes of Staphylococcus aureus and Bacillus cereus obtained from slaughterhouses. Twenty-four S. aureus and 16 B. cereus isolates from pigs and Hanwoo cattle from three slaughterhouses were used to create a DNA fingerprint library with the system software. Scatterplots demonstrated that rep-PCR groupings of S. aureus isolates were in good agreement with their origins. Specifically, linked rep-PCR profiles were observed for S. aureus isolates recovered from the same slaughterhouse, and higher genetic similarities were found among strains isolated from adjacent regions. All S. aureus isolates except one (ID: A-Hanwoo-9) from slaughterhouse "A" clustered with the three S. aureus reference strains, Korea Culture Center of Microorganisms (KCCM) 41291, KCCM 12214, and Culture Collection of Antimicrobial Resistant Microbes (CCARM) 3A007 (similarity values >95%). Moreover, most isolates obtained from slaughterhouse "B" clustered with S. aureus KCCM 11335 and KCCM 41331, and two isolates from slaughterhouse "C" clustered with CCARM 0027. Therefore, for this species, genotypic characteristics of regional isolates can be used to track the pathway of contamination. In contrast, B. cereus isolates showed high genetic diversity and could not be clustered with any specific group. Collectively, this system is useful for analyzing genetic diversity and is a rapid and reproducible typing method; however, there is a need to develop rep-PCR libraries for its use as a rapid identification method.

Carvacrol encapsulated nanocarrier/ nanoemulsion abrogates angiogenesis by downregulating COX-2, VEGF and CD31 in vitro and in vivo in a lung adenocarcinoma model.

Nanoemulsion-based synthesis has been introduced to enhance the bioavailability of natural compounds at target sites for their various biomedical applications. In this study, we synthesized carvacrol nanoemulsion (CN) an oil-in-water (O/W) as a nano-emulsion vehicle system by using ultrasonication emulsification for anti-angiogenesis therapy formulated by combining MCT, lecithin, and polysorbate 80 at the O/W interface called carvacrol encapsulated nanoemulsion (CEN). The diameter of CEN determined by TEM analysis was 105.32 nm. The hydrodynamic droplet size was 101.0 nm with a -39.38-mV zeta potential. The stability of the synthesized CEN was approved till 100 days without any change in diameter size distribution and encapsulation efficiency. We evaluated the role of CEN on angiogenesis in lung adenocarcinoma A549 cells both in vitro and in vivo and observed that it reduced the growth and MMP levels of A549 cells in a dose-dependent manner. Exposure to CEN decreased the activation of MAPK p38 as well as ERK. Moreover, we found that CEN reduced the expression of VEGF and CD31 in A549 cells both in vitro and in vivo. Our in-silico study also indicated the binding of carvacrol to COX-2 and VEGF at the active and allosteric sites of CD31 with low binding energy. Overall, CEN induced anti-angiogenic effects in A549 cells in vitro, in silico, and in vivo, thereby establishing its potential as targeted drug delivery vehicle against angiogenesis.