Development of Stabilizing Solution for Long-Term Storage of Bacteriophages at Room Temperature and Application to Control Foodborne Pathogens.

Bacteriophages (phages) have gained considerable attention as effective antimicrobial agents that infect and kill pathogenic bacteria. Based on this feature, phages have been increasingly used to achieve food safety. They are stored in a medium or buffer to ensure stability; however, they cannot be directly applied to food under these conditions due to reasons such as regulatory considerations and concerns about marketability. This study developed a stabilizing solution that allowed the maintenance of phage activity for extended periods at room temperature while being directly applicable to food. The stability of phages stored in distilled water was relatively low. However, adding a stabilizer composed of sugars and salts improved the survival rates of phages significantly, resulting in stability for up to 48 weeks at room temperature. When Escherichia coli O157:H7-contaminated vegetables were washed with tap water containing phages, the phages reduced the pathogenic E. coli count by over 90% compared with washing with tap water alone. Additionally, when pathogenic E. coli-contaminated vegetables were placed in a phage-coated container and exposed to water, the coating of the container dissolved, releasing phages and lysing the pathogenic E. coli. This led to a significant 90% reduction in pathogenic E. coli contamination compared to that after water rinsing. These results suggest an effective and economical method for maintaining phage activity and establishing the potential for commercialization through application in the food industry.

Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain.

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to ß-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.

Characterization of a unique pH-dependent amylosucrase from Deinococcus cellulosilyticus.

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health.

Modified Impedance Sensing System Determination of Virulence Characteristics of Pathogenic Bacteria Klebsiella Species.

An impedance sensing system is a family of biosensors that measure changes in electrical impedance to perform their functions. Physical and chemical changes in the impedance of the sensing element, such as changes in the concentration of a target analyte or changes in the physical properties of the sensing element, can result in changes in the impedance of the sensing element. Many impedance biosensors have been developed for the detection of pathogens in the past few decades. Several types of biosensors have been developed for the detection of infections, including transduction elements, biorecognition components, and electrochemical approaches. In this review, we discuss the characteristics and pathogenic factors associated with 2,3-butanediol-producing Klebsiella pneumoniae collected using impedance sensors. An impedance sensing system was introduced as a great method for monitoring the virulence factors of Klebsiella spp. in situ. Klebsiella pneumoniae produces virulence factors, including capsules, lipopolysaccharides, fimbriae, and siderophores, as part of its pathogenesis. It is possible to examine virulence factors' pathogenic characteristics in vitro and in vivo using real tissues or mouse models in order to conduct experiments. For the monitoring of virulence factors in situ, a novel alternative method has been developed to mimic the environment of real tissues. For the purpose of developing tissue-mimicking models, mucin and mannose were used to modify the surface of gold electrodes. These components are known to contribute to the adhesion of pathogens to epithelial cells in mammals.

A review of nucleic acid-based detection methods for foodborne viruses: Sample pretreatment and detection techniques.

Viruses are major pathogens that cause food poisoning when ingested via contaminated food and water. Therefore, the development of foodborne virus detection technologies that can be applied throughout the food distribution chain is essential for food safety. A common nucleic acid-based detection method is polymerase chain reaction (PCR), which has become the gold standard for monitoring food contamination by viruses due to its high sensitivity, and availability of commercial kits. However, PCR-based methods are labor intensive and time consuming, and are vulnerable to inhibitors that may be present in food samples. In addition, the methods are restricted with regard to site of analysis due to the requirement of expensive and large equipment for sophisticated temperature regulation and signal analysis procedures. To overcome these limitations, optical and electrical readout biosensors based on nucleic acid isothermal amplification technology and nanomaterials have emerged as alternatives for nucleic acid-based detection of foodborne viruses. Biosensors are promising portable detection tools owing to their easy integration into compact platforms and ability to be operated on-site. However, the complexity of food components necessitates the inclusion of tedious preprocessing steps, and the lack of stability studies on residual food components further restricts the practical application of biosensors as a universal detection method. Here, we summarize the latest advances in nucleic acid-based strategies for the detection of foodborne viruses, including PCR-based and isothermal amplification-based methods, gene amplification-free methods, as well as food pretreatment methods. The principles, strengths/disadvantages, and performance of each method, problems to be solved, and future prospects for the development of a universal detection method are discussed.

Extracellular matrix-degrading enzymes as a biofilm control strategy for food-related microorganisms.

Biofilm is one of the major problems in food industries and is difficult to be removed or prevented by conventional sanitizers. In this review, we discussed the extracellular matrix-degrading enzymes as a strategy to control biofilms of foodborne pathogenic and food-contaminating bacteria. The biofilms can be degraded by using the enzymes targeting proteins, polysaccharides, extracellular DNA, or lipids which mainly constitute the extracellular polymeric substances of biofilms. However, the efficacy of enzymes varies by the growth medium, bacterial species, strains, or counterpart microorganisms due to a high variation in the composition of extracellular polymeric substances. Several studies demonstrated that the combined treatment using conventional sanitizers or multiple enzymes can synergistically enhance the biofilm removal efficacies. In this review, the application of the immobilized enzymes on solid substrates is also discussed as a potential strategy to prevent biofilm formation on food contact surfaces.

Recent Advances in Biosensor Development for the Detection of Viral Particles in Foods: A Comprehensive Review.

Viral foodborne diseases cause serious harm to human health and the economy. Rapid, accurate, and convenient approaches for detecting foodborne viruses are crucial for preventing diseases. Biosensors integrating electrochemical and optical properties of nanomaterials have emerged as effective tools for the detection of viruses in foods. However, they still face several challenges, including substantial sample preparation and relatively poor sensitivity due to complex food matrices, which limit their field applications. Hence, the purpose of this review is to provide an overview of recent advances in biosensing techniques, including electrochemical, SERS-based, and colorimetric biosensors, for detecting viral particles in food samples, with emerging techniques for extraction/concentration of virus particles from food samples. Moreover, the principle, design, and advantages/disadvantages of each biosensing method are comprehensively described. This review covers the recent development of rapid and sensitive biosensors that can be used as new standards for monitoring food safety and food quality in the food industry.

Biosynthesis of Silver Nanoparticles from Duchesnea indica Extracts Using Different Solvents and Their Antibacterial Activity.

Silver nanoparticles (AgNPs) were synthesized using the whole plant of Duchesnea indica (DI) which was extracted in different solvents; the antimicrobial effects of the extract were investigated in this study. The extraction of DI was performed using three different solvents: water, pure ethanol (EtOH), and pure dimethyl sulfoxide (DMSO). AgNP formation was monitored by measuring the UV-Vis spectrum of each reaction solution. After synthesis for 48 h, the AgNPs were collected and the negative surface charge and size distribution of the synthesized AgNPs were measured using dynamic light scattering (DLS). The AgNP structure was determined by high-resolution powder X-ray diffraction (XRD) and the AgNP morphology was investigated using transmission electron microscopy (TEM). AgNP antibacterial activities were evaluated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa using the disc diffusion method. Additionally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were also determined. Biosynthesized AgNPs showed enhanced antibacterial activity against B. cereus, S. aureus, E. coli, S. enteritidis, and P. aeruginosa compared with that of pristine solvent extract. These results suggest that AgNPs synthesized from extracts of DI are promising antibacterial agents against pathogenic bacteria and can be further applied in the food industry.

Effects of Blanching Methods on Nutritional Properties and Physicochemical Characteristics of Hot-Air Dried Edible Insect Larvae.

Global meat consumption is increasing worldwide, however, supply remains lacking. Several alternative protein sources, such as cultured meat, plant-based protein production, and edible insects, have been proposed to overcome this shortage. Interestingly, edible insects are characterized by superior digestive and absorptive qualities that make them the ideal replacement for traditional protein production. This study aims to further the processing ability of insect protein by investigating the effects of various pre-treatment methods, such as blanching (HB), roasting (HR), and superheated steam (HS), on the nutritional properties and physicochemical characteristics of proteins extracted from Hermetia illucens larvae. The drying rate, pH value, color analysis, amino and fatty acid profile, as well as bulk density, shear force, and rehydration ratios of the above pre-treatment methods, were explored. HS was found to have the highest drying rate and pH value analysis showed that HB and HS samples have significantly higher values compared to the other modalities. Raw edible insects had the highest value in the sum of essential amino acid (EAA) and EAA index when compared to EAAs. HB and HS showed significantly lower bulk density results, and HS showed the highest shear force and the highest value in rehydration ratio, regardless of immersion time. Therefore, taking the above results together, it was found that blanching and superheated steam blanching pre-treatment were the most effective methods to improve the processing properties of H. illucens after hot-air drying.

Whole-cell bioconversion using non-Leloir transglycosylation reactions: a review.

Microbial biocatalysts are evolving technological tools for glycosylation research in food, feed and pharmaceuticals. Advances in bioengineered Leloir and non-Leloir carbohydrate-active enzymes allow for whole-cell biocatalysts to curtail production costs of purified enzymes while enhancing glucan synthesis through continued enzyme expression. Unlike sugar nucleotide-dependent Leloir glycosyltransferases, non-Leloir enzymes require inexpensive sugar donors and can be designed to match the high value, yield and selectivity of the former. This review addresses the current state of bacterial cell-based production of glucans and glycoconjugates via transglycosylation, and describes how alterations made to microbial hosts to surpass purified enzymes as the preferred mode of catalysis are steadily being acquired through genetic engineering, rational design and process optimization. A comprehensive exploration of relevant literature has been summarized to describe whole-cell biocatalysis in non-Leloir glycosylation reactions with various donors and acceptors, and the characterization, application and latest developments in the optimization of their use.

Synthesis of a New Glycoconjugate with Di-á´ -Psicose Anhydride Structure.

Demand for healthy diets has led researchers to explore new saccharide as sucrose alternatives. ᴅ-Psicose, the C-3 epimer of ᴅ-fructose, has a similar sweetness intensity to sucrose but contributes fewer calories. This study proposes a disaccharide with a stable structure derived from ᴅ-psicose. The compound with a spiro-tricyclic core was generated at 32% conversion via caramelization of ᴅ-psicose under acidic anhydrous conditions. The compound was identified by high-resolution mass spectrometry and multi-dimensional nuclear magnetic resonance (NMR). The molecular formula was established as C12H20O10 from the molecular weight of m/z 324.1055. Twelve signals were observed by the 13C NMR spectrum. This compound, denoted di-ᴅ-psicose anhydride (DPA), exhibited a lower water solubility (40 g/L) and higher thermal stability (peak temperature = 194.7 °C) than that of ᴅ-psicose (peak temperature = 126.5 °C). The quantitatively evaluated metal ion scavenging ability of DPA was the best in magnesium (average 98.6 ± 1.1%). This synthesis methodology can provide disaccharides with high stability-reducing heavy metals.

Efficient Screening of Pesticide Diazinon-Binding Aptamers Using the Sol-Gel-Coated Nanoporous Membrane-Assisted SELEX Process and Next-Generation Sequencing.

Aptamer-based methods for detecting pesticides are more efficient than antibody-based methods by high thermal stability, low molecular weight, easy modification, and low cost. In this study, the systematic evolution of ligands by exponential enrichment (SELEX) process, combined with next-generation sequencing (NGS), was performed to select aptamers specific to the pesticide, diazinon, which was fixed on a sol-gel-coated nanoporous-anodized aluminum oxide membrane to overcome the immobilization effect of general method and simplify the elution step. The frequency of specific nucleotide sequences obtained after SELEX rounds was directly analyzed using NGS to eliminate the time-consuming cloning process used in the general SELEX methods. Nine sequences with the highest frequency after SELEX round 10 followed by NGS were selected and tested to derive their binding affinity with the target, diazinon, through circular dichroism (CD) spectrophotometry. The CD signal difference of the aptamer candidates ranged from 0.13 to 2.242 mdeg between diazinon-only treated and diazinon-aptamer-treated samples at a wavelength near 270 nm. Aptamer D-4, which had the highest binding affinity from CD spectrophotometry analysis, showed no cross-reactivity with non-target pesticides, such as baycarb, bifenthrin, and pyridaben, but interacted with the other pesticides, fipronil and 2-phenylphenol. Therefore, an aptamer was effectively screened by selection of high-frequency candidates after SELEX-NGS followed by CD analysis with the highest difference signal. A follow-up study is needed to confirm whether the proposed SELEX process combined with NGS for the discovery of aptamers for new targets can further shorten the SELEX cycle by reducing the number of SELEX rounds to 10 or less.

Rolling Circle Amplification-based Copper Nanoparticle Synthesis on Cyclic Olefin Copolymer Substrate and Its Application in Aptasensor.

This study aimed to develop a label-free fluorescent aptasensor for the detection of diazinon (DZN) on a cyclic olefin copolymer (COC) substrate. The aptasensor design was based on rolling circle amplification (RCA) technology and the use of self-assembled copper nanoparticles (CuNPs). A dual-function (DF) probe, capable of binding to circular DNA and an aptamer, was designed and immobilized on a COC-bottom 96-well plate. An aptamer was used for selective recognition of DZN, and the specific site of the aptamer that strongly reacted with DZN was successfully identified using circular dichroism (CD) analysis. In presence of DZN, the aptamer and DZN formed a strong complex, thus providing an opportunity for hybridization of the DF probe and circular DNA, thereby initiating an RCA reaction. Repetitive poly thymine (T) sequence with a length of 30-mer, generated in the RCA reaction, served as a template for the synthesis of fluorescent copper nanoparticles, emitting an orange fluorescence signal (at approximately 620 nm) proportional to the amount of RCA product, within 10 min under UV irradiation. The CuNP fluorescence was imaged and quantified using an image analysis software. A linear correlation of the fluorescence signal was confirmed in the DZN concentration range of 0.1-3 ppm, with a detection limit of 0.15 ppm. Adoption of a label-free detection method, utilizing RCA and fluorescent CuNPs on COC substrates, reduced the need for complex equipment and requirements for DZN analysis, thereby representing a simple and rapid sensing method circumventing the limitations of current complex and labor-intensive methods. Electronic Supplementary Material ESM: The online version of this article (doi:10.1007/s12257-021-0220-0) contains supplementary material, which is available to authorized users.

Autoinducer-2 Could Affect Biofilm Formation by Food-Derived Bacillus cereus.

Bacillus cereus is a foodborne pathogen and cause a frequent problem due to the biofilms forming in equipment of food production plants. Autoinducer-2 (AI-2) involved in interspecies communication, plays a role in the biofilm formation of B. cereus. In this study, biofilm formation by thirty-nine B. cereus strains isolated from foods produced in Korea was determined. To investigate the effect of AI-2 on biofilm formation by B. cereus SBC27, which had the highest biofilm-forming ability, biofilm densities formed after addition of the AI-2 from Staphylococcus aureus and Escherichia coli were analysed. As a result, it was found that the quorum sensing molecule AI-2 could induce biofilm formation by B. cereus within 24 h, but it may also inhibit biofilm formation when more AI-2 is added after 24 h. Thus, these results improve our understanding of biofilm formation by food-derived B. cereus and provide clues that could help to reduce the impact of biofilms, the biggest problem in food processing environments, which has an impact on public health as well as the economy.

Selective Binding and Elution of Aptamers for Pesticides Based on Sol-Gel-Coated Nanoporous Anodized Aluminum Oxide Membrane.

Sol-gel-based mesopores allow the entry of target small molecules retained in their cavity and aptamers to bind to target molecules. Herein, sol-gel-based materials are applied to screen-selective aptamers for small molecules, such as pesticides. To enhance the efficiency of aptamer screening using a sol-gel, it is necessary to increase the binding surface. In this study, we applied the sol-gel to an anodized aluminum oxide (AAO) membrane, and the morphological features were observed via electron microscopy after spin coating. The binding and elution processes were conducted and confirmed by fluorescence microscopy and polymerase chain reaction. The sol-gel coating on the AAO membrane formed a hollow nanocolumn structure. A diazinon-binding aptamer was bound to the diazinon-containing sol-gel-coated AAO membrane, and the bound aptamer was effectively retrieved from the sol-gel matrix by thermal elution. As a proof of concept, a sol-gel-coated AAO disc was mounted on the edge of a pipette tip, and the feasibility of the prepared platform for the systematic evolution of ligands by exponential enrichment (SELEX) of the aptamer binding was also confirmed. The proposed approach will be applied to an automated SELEX cycle using an automated dispenser, such as a pipetting robot, in the near future.

Magnetic solid-phase extraction based on magnetic carbon particles from coffee grounds for determining phthalic acid esters in plastic bottled water.

Newly developed magnetic carbon particles prepared from coffee grounds were used as the sorbent for the magnetic solid-phase extraction of eight phthalic acid esters (PAEs) from plastic bottled water prior to their analysis by GC-MS. The method, which uses coffee-ground particles coated with iron oxide, was validated, and exhibited linearities for the eight PAEs, with coefficients of determination above 0.998 in the 0.005 to 0.1 mg/L concentration range. Limits of detection and limits of quantification of 0.00003 to 0.002 mg/L and 0.0001 to 0.005 mg/L, respectively, were achieved, with recoveries (%) ranging between 77% and 120%, and relative standard deviations for intra- and interday precisions below 16.3% at three fortification levels. No PAE residues were detected when the developed and validated method was applied to 10 real plastic bottled water samples. Taken together, the developed magnetic solid-phase extraction method is a useful tool for monitoring phthalate esters in aqueous samples. PRACTICAL APPLICATION: The development of a new, inexpensive, and efficient magnetic sorption material derived from spent coffee grounds, and its ability to determine phthalate esters in aqueous solutions was described by GC-MS/MS. The developed magnetic solid-phase extraction method is a useful tool for monitoring phthalate esters in aqueous samples.

Detection of Escherichia coli O157:H7 Using Automated Immunomagnetic Separation and Enzyme-Based Colorimetric Assay.

The food industry requires rapid and simple detection methods for preventing harm from pathogenic bacteria. Until now, various technologies used to detect foodborne bacteria were time-consuming and laborious. Therefore, we have developed an automated immunomagnetic separation combined with a colorimetric assay for the rapid detection of E. coli O157:H7 in food samples. The colorimetric detection method using enzymatic reaction is fascinating because of its simplicity and rapidity and does not need sophisticated devices. Moreover, the proposed procedures for the detection of bacteria in food take less than 3 h including pre-enrichment, separation and detection steps. First, target-specific immunomagnetic beads were introduced to contaminated milk in a pre-enrichment step. Second, the pre-enriched sample solution containing target bacteria bound on immunomagnetic beads was injected into an automated pretreatment system. Subsequently, the immunomagnetic beads along with target bacteria were separated and concentrated into a recovery tube. Finally, released ß-galactosidase from E. coli O157:H7 after lysis was reacted with chlorophenol red ß-galactopyranoside (CPRG) used as a substrate and the colorimetric change of CPRG was determined by absorbance measuring or the naked eye. By the proposed approach in this study, we could detect 3 × 102 CFU/mL of E. coli O157:H7 from a milk sample within 3 h.

Determination of 60 pesticides in hen eggs using the QuEChERS procedure followed by LC-MS/MS and GC-MS/MS.

An analytical method involving QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation, followed by LC-MS/MS and GC-MS/MS was developed and validated for the determination of 60 pesticides in eggs. Recoveries of 70-120% were achieved for selected pesticides and relative standard deviations <20% were obtained for most analytes at three concentrations. The limit of quantification was <10 µg kg-1 for 83% of the total pesticides. This method was used to analyze 58 egg samples and the residues of seven pesticides (disulfoton, fipronil sulfone, cyromazine, o,p-DDT, p,p-DDD, p,p-DDT, and permethrin) were quantified in 16 egg samples at levels of 5-10 µg kg-1, which was below the corresponding the maximum residue levels, as established by Korean Ministry of Food and Drug Safety. We demonstrated that LC-MS/MS and GC-MS/MS in combination with QuEChERS can be used to routinely monitor multiple pesticide residues in egg samples.

Alpha-Hederin Nanopore for Single Nucleotide Discrimination.

Various types of biological and synthetic nanopores have been developed and utilized for the high-throughput investigation of individual biomolecules. Biological nanopores made with channel proteins are so far superior to solid-state ones in terms of sensitivity and reproducibility. However, the performance of a biological nanopore is dependent on the protein in the channel structure its dimensions are predetermined and are difficult to modify for broader applications. Here inspired by the cytotoxic mechanisms of a saponin derivative, alpha-hederin, we report a nonproteinaceous nanopore that can be formed spontaneously in a lipid membrane. We propose the pore-forming mechanism of alpha-hederin in a cholesterol-rich lipid membrane and a strategy to control the pore-forming rate by a lipid partitioning method. The small diameter and effective thickness of alpha-hederin nanopores enabled us to discriminate ssDNA homopolymers as well as four types of nucleotides, showing its potential as a DNA sequencing tool.

Reduction of DNA Folding by Ionic Liquids and Its Effects on the Analysis of DNA-Protein Interaction Using Solid-State Nanopore.

DNA folding is not desirable for solid-state nanopore techniques when analyzing the interaction of a biomolecule with its specific binding sites on DNA since the signal derived from the binding site could be buried by a large signal from the folding of DNA nearby. To resolve the problems associated with DNA folding, ionic liquids (ILs), which are known to interact with DNA through charge-charge and hydrophobic interactions are employed. 1-n-butyl-3-methylimidazolium chloride (C4 mim) is found to be the most effective in lowering the incident of DNA folding during its translocation through solid-state nanopores (4-5 nm diameter). The rate of folding signals from the translocation of DNA-C4 mim is decreased by half in comparison to that from the control bare DNA. The conformational changes of DNA upon complexation with C4 mim are further examined using atomic force microscopy, showing that the entanglement of DNA which is common in bare DNA is not observed when treated with C4 mim. The stretching effect of C4 mim on DNA strands improves the detection accuracy of nanopore for identifying the location of zinc finger protein bound to its specific binding site in DNA by lowering the incident of DNA folding.