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.

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.

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.

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.

A protamine-conjugated gold decorated graphene oxide composite as an electrochemical platform for heparin detection.

In this study, an effective electrochemical sensor was developed for heparin detection using a protamine-conjugated graphene oxide/gold (GO/Au) composite. Protamine is an antidote that can act as an affinity ligand for heparin. The GO was used as support for signal amplification, and Au nanoparticles (NPs) were employed to immobilize the protamine. This Au NPs also increasing the electron transfer rate and enhancing the signal response during protamine-heparin integration. The proposed affinity sensor had a simple fabrication process, a low detection limit (0.9 nM), a wide linear range (1.9 × 10-7 M to 1.5 × 10-9 M), high stability, and high selectivity in the detection of heparin.

Effect of UV-C light on the microbial and sensory quality of seasoned dried seafood.

This study investigated the effects of different doses of UV-C light at 253.7 nm (0-18 kJ/m(2)) on the reduction of Escherichia coli,Staphylococcus aureus and Bacillus cereus in contaminated seasoned dried filefish (Thamnaconus modestus) and sliced squid (Todarodes pacificus) surfaces and sensory quality. The counts of all three bacteria were significantly (P < 0.05) reduced by the increase of UV-C dosage.E. coli,S. aureus and B. cereus on filefish with 18 kJ/m(2)of UV-C maximally reduced by 2.70, 2.55 and 2.57 log CFU/g, respectively; however, on the sliced squid using the same UV dose reduced the same bacteria by 1.35, 0.54 and 1.05 log CFU/g, respectively. However, the results suggest that 6 to 9 kJ/m(2)of UV-C could be used for the inactivation of E. coli and B. cereus in these dried fishery products without any changes in sensory quality. However, S. aureus levels on sliced squid will require a combination of UV-C light and chemical treatment.