Protocol to reveal the binding partner of secreted housekeeping enzymes in Listeria monocytogenes via in silico prediction to in vivo validation.

Numerous interacting protein partners exist without recognized interactive domains, necessitating a standardized methodology to decipher more in-depth interaction profiles. Here, we present a protocol to reveal the binding partner of a secreted housekeeping enzyme, alcohol acetaldehyde dehydrogenase (Listeria adhesion protein), in Listeria monocytogenes through in silico modeling and in vivo experiments. We describe steps for target protein modeling, biophysical profiling, ClusPro docking optimization, protein variant modeling, and docking comparison. We then provide detailed procedures for in vitro and in vivo protein binding validation. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

A critical study of the nonlinear rheological properties in major classes of foods using the Sequence of Physical Processes (SPP) method and the Fourier Transform Coupled with Chebyshev Decomposition (FTC) method.

The nonlinear rheological behaviors of three different classes of foods (emulsion, suspension, and elastic network) were studied and analyzed using the Rogers Sequence of Physical Processes (SPP) method and the Ewoldt-McKinley method of coupling Fourier Transform with Chebyshev Decomposition (FTC). SPP analysis led to instantaneous rheological parameters G't and G″t at any point in time, providing a more accurate picture of the linear viscoelastic region and crossover points by the 3D amplitude sweep. When G't is plotted against G″t, the resulting graph is a deltoid which offers a detailed and distinctive intracycle behavior of each class of food. Analyzing the revolution of deltoids with increasing strain allows for the determination of a critical strain, beyond which irreversible network breakdown occurs. The strain range between the linear viscoelastic limit and the critical strain found in SPP is comparable to the MAOS region as determined with FTC. Under increasing amplitude, predominantly elastic networks showed a gradual structural rearrangement, while more erratic and abrupt changes were observed in the suspension and emulsion we studied. Under increasing frequency, elastic responses dominate viscous responses in all samples due to the shorter experimental time, allowing less relaxation.

Comparison of the specific mechanical energy, specific thermal energy, and functional properties of cold and hot extruded pea protein isolate.

Pea protein is a popular source of plant-based protein, though its application in meat and dairy analog products is still lacking. This is particularly true in the development of products with fatty and creamy textures. Cold denaturation may be a way to induce these types of textures in food since this is a universal phenomenon in protein that occurs due to a weakening of hydrophobic interactions at cold temperatures. This work utilizes a single screw extruder to systematically study the impacts of moisture content (50-65 %) and pH (2,4.5,8) on the outlet temperatures, specific mechanical energy, specific thermal energy, and texture of cold-extruded pea protein. It was found that at pH 2 and moistures of 60 % and greater, the temperature of the product exiting the extruder is <5.5 °C, and also produced 13.7 %-36.5 % more specific thermal energy, indicating the occurrence of cold denaturation in these products. Based on these findings, a comparison of hot and cold extrusion was conducted as a function of pH and oil content. It was found that cold extrusion imparts 43.0 %-56.2 % more mechanical energy into the protein than hot extrusion, and the cold extruded protein had higher values of Young's modulus and breaking stress. The protein extruded at low temperatures was also able to bind 32.93 % more oil than hot extruded proteins when extruded with 10 % added oil, which may aid in the formation of protein-based fat memetics for the food industry.

Physicochemical characterization of changes in pea protein as the result of cold extrusion.

Pea protein is a popular plant-based protein for mimicking textures in meat and dairy analogues which are more sustainable than their animal-based counterparts. However, precise mechanisms for generating specific textures through different processing methods are still being evaluated. This work utilizes a novel low-temperature extrusion process to selectively alter the chemical structure of pea protein. Changes in secondary structure, surface hydrophobicity, electrostatic interactions, and disulfide bonding are characterized through FTIR, ANS- probes, zeta potential, and SDS-PAGE. Extrudates are further characterized using texture parameter analysis. It was found that a linear combination of physicochemical data, generated with multiple linear regression modelling, led to reasonable estimates of the specific mechanical energy and textural properties. This work offers a new method of reactive extrusion to selectively modify interactions in pea protein using low temperature extrusion, and applications may include fatty textures, since the extrudates are found to be largely stabilized through hydrophobic interactions evaluated with surface hydrophobicity measurements.

Complexation with Polysaccharides Enhances the Stability of Isolated Anthocyanins.

Isolated anthocyanins have limited colonic bioavailability due to their instability as free forms. Thus, many methods have been fabricated to increase the stability of anthocyanins. Complexation, encapsulation, and co-pigmentation with other pigments, proteins, metal ions, and carbohydrates have been reported to improve the stability and bioavailability of anthocyanins. In this study, anthocyanins extracted from purple potatoes were complexed with four different polysaccharides and their mixture. The anthocyanin-polysaccharide complexes were characterized using a zeta potential analyzer, particle size analyzer, scanning electron microscopy, and Fourier-transform infrared spectroscopy. Complexes were subjected to simulated digestion for assessing the stability of anthocyanins. Furthermore, complexes were subjected to different pH conditions and incubated at high temperatures to monitor color changes. A Caco-2 cell monolayer was used to evaluate the colonic concentrations of anthocyanins. In addition, the bioactivity of complexes was assessed using LPS-treated Caco-2 cell monolayer. Results show that pectin had the best complexation capacity with anthocyanins. The surface morphology of the anthocyanin-pectin complex (APC) was changed after complexation. APC was more resistant to the simulated upper gastrointestinal digestion, and high pH and temperature conditions for a longer duration. Furthermore, APC restored the lipopolysaccharide (LPS)-induced high cell permeability compared to isolated anthocyanins. In conclusion, complexation with pectin increased the stability and colonic bioavailability and the activity of anthocyanins.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Bioinformatic Approaches for Characterizing Molecular Structure and Function of Food Proteins.

Structural bioinformatics analyzes protein structural models with the goal of uncovering molecular drivers of food functionality. This field aims to develop tools that can rapidly extract relevant information from protein databases as well as organize this information for researchers interested in studying protein functionality. Food bioinformaticians take advantage of millions of protein amino acid sequences and structures contained within these databases, extracting features such as surface hydrophobicity that are then used to model functionality, including solubility, thermostability, and emulsification. This work is aided by a protein structure-function relationship framework, in which bioinformatic properties are linked to physicochemical experimentation. Strong bioinformatic correlations exist for protein secondary structure, electrostatic potential, and surface hydrophobicity. Modeling changes in protein structures through molecular mechanics is an increasingly accessible field that will continue to propel food science research.

Encapsulation of tannins and tannin-rich plant extracts by complex coacervation to improve their physicochemical properties and biological activities: A review.

As a major class of dietary polyphenols, tannins are demonstrated to have various health-promoting properties. Although tannins have been widely utilized in food, pharmaceutical and many other industries, the applications of tannins are quite limited due to their poor stability, sensory attributes and bioavailability. Encapsulation helps improve all of these properties. Complex coacervation, one of the most effective encapsulation techniques, is known for its simplicity, low cost, scalability and reproducibility in encapsulation of functional components. In recent years, complex coacervation has been successfully used for encapsulation of tannins and tannin-rich plant extracts. In this article, the research progress in encapsulating tannins and tannin-rich plant extracts by complex coacervation to improve their physicochemical properties and biological activities is critically reviewed for the first time. Encapsulation of tannins and tannin-rich plant extracts can effectively improve their sensory characteristics, stabilities, bioavailability, anti-hypercholesterolemia, anti-diabetic, antioxidant, anticancer and antimicrobial activities. In particular, the enhancement of biological activities of tannins and tannin-rich plant extracts is usually correlated to their improved physicochemical properties imparted by the encapsulation technique. Moreover, we introduce the issues that need to be further resolved in future studies on encapsulation of tannins and tannin-rich plant extracts by complex coacervation.

Effect of endogenous wheat gluten lipids on the non-linear rheological properties of the gluten network.

The impact of endogenous wheat lipids on thermal characteristics, mixing behavior, non-linear rheological properties of gluten was studied to explore the contribution of wheat lipids to viscoelastic behavior of gluten under large processing deformations. Thermal analysis indicated higher denaturation temperature for vital wheat gluten (VWG) (69.2 ± 1.2 °C) due to reduced water affinity compared to lipid-removed vital wheat gluten (LRVWG) (63.6 ± 0.2 °C). Development time was reached 4 minutes earlier and consistency increased constantly for LRVWG as Farinograph mixing proceeded, suggesting higher affinity to water for gluten in the absence of lipids. Large Amplitude Oscillatory Shear (LAOS) tests showed a mixture of type III and IV non-linear behavior for gluten. Higher tendency to type III behavior for VWG indicated more extensibility in the presence of lipids. Higher elasticity and strain stiffening obtained for LRVWG under LAOS deformations accentuated the stabilizing effect of lipids on the viscoelastic nature of gluten network during processing.

Applying text mining to identify relevant literature in food science: Cold denaturation as a case study.

In a research environment characterized by the five V's of big data, volume, velocity, variety, value, and veracity, the need to develop tools that quickly screen a large number of publications into relevant work is an increasing area of concern, and the data-rich food industry is no exception. Here, a combination of latent Dirichlet allocation and food keyword searches were employed to analyze and filter a dataset of 6102 publications about cold denaturation. After using the Python toolkit generated in this work, the approach yielded 22 topics that provide background and insight on the direction of research in this field, as well as identified the publications in this dataset which are most pertinent to the food industry with precision and recall of 0.419 and 0.949, respectively. Precision is related to the relevance of a paper in the filtered dataset and the recall represents papers which were not identified in the screening method. Lastly, gaps in the literature based on keyword trends are identified to improve the knowledge base of cold denaturation as it relates to the food industry. This approach is generalizable to any similarly organized dataset, and the code is available upon request. Practical Application: A common problem in research is that when you are an expert in one field, learning about another field is difficult, because you may lack the vocabulary and background needed to read cutting edge literature from a new discipline. The Python toolkit developed in this research can be applied by any researcher that is new to a field to identify what the key literature is, what topics they should familiarize themselves with, and what the current trends are in the field. Using this structure, researchers can greatly speed up how they identify new areas to research and find new projects.

A Low-Field Magnetic Resonance Imaging Aptasensor for the Rapid and Visual Sensing of Pseudomonas aeruginosa in Food, Juice, and Water.

In this work, we present a low-field magnetic resonance imaging (LF-MRI) aptasensor based on the difference in magnetic behavior of two magnetic nanoparticles with diameters of 10 (MN10) and 400 nm (MN400) for the rapid detection of Pseudomonas aeruginosa (P. aeruginosa). First, specific anti-P. aeruginosa aptamers were covalently immobilized onto magnetic nanoparticles via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide chemistry for the capture of the target bacteria. In the presence of P. aeruginosa, an MN10-bacteria-MN400 (MBM) complex was formed after binding between the aptamers on magnetic nanoparticles and P. aeruginosa cells. When a magnetic field was applied, the MBM complex and free MN400 were rapidly magnetically separated, and free MN10 left in the solution worked as a T2 (transverse relaxation time) single readout in MRI measurement. Under optimum conditions, the LF-MRI platform provides both image analysis and quantitative detection of P. aeruginosa, with a detection limit of 100 cfu/mL. The feasibility and specificity of the aptasensor were demonstrated in detecting real food, orange juice, and drinking water samples and validated using plate counting methods.

Cold Denaturation of Proteins: Where Bioinformatics Meets Thermodynamics to Offer a Mechanistic Understanding: Pea Protein As a Case Study.

Protein structure can be altered with heat, but models which predict denaturation show that globular proteins also spontaneously unfold at low temperatures through cold denaturation. By an analysis of the primary structure of pea protein using bioinformatic modeling, a mechanism of pea protein cold denaturation is proposed. Pea protein is then fractionated into partially purified legumin and vicilin components, suspended in ethanol, and subjected to low temperatures (-10 to -20 °C). The structural characterizations of the purified fractions are conducted through FTIR, ζ potential, dynamic light scattering, and oil binding, and these are compared to the results of commercial protein isolates. The observed structural changes suggest that pea protein undergoes changes in structure as the result of low-temperature treatments, which could lead to innovative industrial processing techniques for functionalization by low-temperature processing.

Novel Nondestructive Biosensors for the Food Industry.

An increasing number of foodborne outbreaks, growing consumer desire for healthier products, and surging numbers of food allergy cases necessitate strict handling and screening of foods at every step of the food supply chain. Current standard procedures for detecting food toxins, contaminants, allergens, and pathogens require costly analytical devices, skilled technicians, and long sample preparation times. These challenges can be overcome with the use of biosensors because they provide accurate, rapid, selective, qualitative, and quantitative detection of analytes. Their ease of use, low-cost production, portability, and nondestructive measurement techniques also enable on-site detection of analytes. For this reason, biosensors find many applications in food safety and quality assessments. The detection mechanisms of biosensors can be varied with the use of different transducers, such as optical, electrochemical, or mechanical. These options provide a more appropriate selection of the biosensors for the intended use. In this review, recent studies focusing on the fabrication of biosensors for food safety or food quality purposes are summarized. To differentiate the detection mechanisms, the review is divided into sections based on the transducer type used.

Building a Resilient, Sustainable, and Healthier Food Supply Through Innovation and Technology.

The modern food supply faces many challenges. The global population continues to grow and people are becoming wealthier, so the food production system must respond by creating enough high-quality food to feed everyone with minimal damage to our environment. The number of people suffering or dying from diet-related chronic diseases, such as obesity, diabetes, heart disease, stroke, and cancer, continues to rise, which is partly linked to overconsumption of highly processed foods, especially high-calorie or rapidly digestible foods. After falling for many years, the number of people suffering from starvation or malnutrition is rising, and thishas been exacerbated by the global COVID-19 pandemic. The highly integrated food supply chains that spread around the world are susceptible to disruptions due to policy changes, economic stresses, and natural disasters, as highlighted by the recent pandemic. In this perspective article, written by members of the Editorial Committee of the Annual Review of Food Science and Technology, we highlight some of the major challenges confronting the modern food supply chain as well as how innovations in policy and technology can be used to address them. Pertinent technological innovations include robotics, machine learning, artificial intelligence, advanced diagnostics, nanotechnology, biotechnology, gene editing, vertical farming, and soft matter physics. Many of these technologies are already being employed across the food chain by farmers, distributors, manufacturers, and consumers to improve the quality, nutrition, safety, and sustainability of the food supply. These innovations are required to stimulate the development and implementation of new technologies to ensure a more equitable, resilient, and efficient food production system. Where appropriate, these technologies should be carefully tested before widespread implementation so that proper risk-benefit analyses can be carried out. They can then be employed without causing unforeseen adverse consequences. Finally, it is important to actively engage all stakeholders involved in the food supply chain throughout the development and testing of these new technologies to support their adoption if proven safe and effective.

Detection of Pyocyanin Using a New Biodegradable SERS Biosensor Fabricated Using Gold Coated Zein Nanostructures Further Decorated with Gold Nanoparticles.

In this paper, a biodegradable gold coated zein film surface enhanced Raman spectroscopy (SERS) platform, with gold nanoparticles (AuNPs) deposited on the surface to further enhance the Raman signal, was used to detect pyocyanin (PYO), the toxin secreted by Pseudomonas aeruginosa. An inverted pyramid structure imprinted on a zein film and gold coated during the transfer process was further improved with the deposition and fixing of gold nanoparticles, which resulted in enhancement of the SERS signal by approximately a decade. This new platform served as a lab-on-a-chip sensor to enable the sensitive and rapid detection of PYO in drinking water. The size, distribution, and morphology of the zein film nanostructures including the presence and distribution of gold nanoparticles were characterized by scanning electron microscopy (SEM). The new zein-based platform has the advantage of being largely biodegradable compared with commercial silicon- or glass-based platforms. The limit of detection for PYO using the newly developed zein-based SERS sensor platform was calculated as 25 µM, considerably lower than the concentration of PYO in the blood of people with cystic fibrosis which has been reported to be 70 µM.

Simultaneous immunofluorescent imaging of gliadins, low molecular weight glutenins, and high molecular weight glutenins in wheat flour dough with antibody-quantum dot complexes.

Gluten proteins and their impact in the quality of wheat based food products is well known. In order visualize the 'in situ' distribution of low molecular weight glutenins, high molecular weight glutenins, and gliadins simultaneously in wheat doughs we needed to overcome and eliminate dough auto-fluorescence, and to develop a reliable immunostaining procedure for their simultaneous detection in wheat doughs. We are studying different auto-fluorescence quenchers used in biological fluorescent imaging and their effect on dough auto-fluorescence removal, and the effect of different fixative mediums on the adhesion of wheat flours doughs onto microscope slides. We found that the best method to remove dough auto-fluorescence is removing it as background in the microscope detection system. We also found methanol to be the best fixative medium for dough samples. In this research, we are showing the first 'in situ' localization of these gluten subunits simultaneously in wheat flour dough.

Mechanical and spectroscopic characterization of crosslinked zein films cast from solutions of acetic acid leading to a new mechanism for the crosslinking of oleic acid plasticized zein films.

This study discovered through FTIR, FT-Raman and rheological measurements that glutaraldehyde binds to zein through the amine groups of glutamine turns by replacing the already-bonded oleic acid molecules and forming imine structures. As a secondary crosslinking mechanism, glutaraldehyde unfolds some of the α-helices and turns them into ß-sheets. While crosslinking resulted in stiffer and less ductile zein films, it made the surface of the films rougher, measured using AFM, and more hydrophilic, measured using WCA. In the crosslinking conditions in this study, the number of crosslinks estimated from rubber elasticity theory were not enough to change the water vapor permeability of the films significantly. Improving the understanding of crosslinking mechanism and its effects on physical and chemical properties of zein films can be useful to develop stiffer, stronger and more durable platforms for biodegradable biosensors, microfluidic devices or scaffolds.

Conjugation of Specifically Developed Antibodies for High- and Low-Molecular-Weight Glutenins with Fluorescent Quantum Dots as a Tool for Their Detection in Wheat Flour Dough.

The importance of gluten proteins, gliadins and glutenins, is well-known in the quality of wheat products. To gain more specific information about the role of glutenins in wheat dough, the two major subunits of glutenin, high- and low-molecular-weight (HMW and LMW) glutenins, were extracted, isolated, and identified by mass spectrometry. Antibodies for HMW and LMW glutenins were developed using the proteomic information on the characterized glutenin subunits. The antibodies were found to be specific to each subunit by western immunoblots and were then conjugated to quantum dots (QDs) using site-click conjugation, a new method to keep antibody integrity. A fluorescence-link immunosorbent assay tested the successful QD conjugation. The QD-conjugated antibodies were applied to dough samples, where they recognized glutenin subunits and were visualized using a confocal laser scanning microscope.

Advances in Nanotechnology as They Pertain to Food and Agriculture: Benefits and Risks.

Nanotechnology is an emerging and rapidly developing toolbox that has novel and unique applications to food science and agriculture. Fast and impressive developments in nanotechnology for food and agriculture have led to new experimental prototype technologies and products. Developing various types of nanodelivery systems, detection tools, nanoscale modifications of bulk or surface properties, fabrication of wide-range bionanosensors, and biodegradable nanoplatforms can potentially improve consumer health and safety, product shelf life and stability, bioavailability, environmental sustainability, efficiency of processing and packaging, and real-time monitoring. Some recently developed nanotechnology techniques and potential product applications of nanotechnology are summarized in this review. Exposure to nanomaterials may be harmful to the consumer and the environment and might increase the potential of risk. For this reason, evaluation of the potential risks resulting from the interaction of nanomaterials with biological systems, humans, and the environment is also reviewed.

Advances in Understanding the Molecular Structures and Functionalities of Biodegradable Zein-Based Materials Using Spectroscopic Techniques: A Review.

Zein's amphiphilic properties, film forming capability, and biodegradability make it a highly demanded polymer for fabrication of packaging materials, production of drug carrier nanoparticles, scaffolds in tissue engineering, and formation of biodegradable platforms for biosensors including microfluidic devices. Zein properties can be improved by chemical modifications, which are often analyzed with spectroscopic techniques. However, there is not a consensus on the structure of zein. For this reason, in this Review the aim is to compile the recent studies conducted on zein-based products and compare them under five main spectroscopic techniques: Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, circular dichroism (CD), X-ray diffraction (XRD) and atomic force microscopy (AFM). This Review serves as a library of recent zein studies and helps readers to have a better perception of contradictions in the literature to take their studies one step further.