Essential oil-nanoemulsion based edible coating: Innovative sustainable preservation method for fresh/fresh-cut fruits and vegetables.

Utilizing plant-based sources for the preservation of fresh and fresh-cut fruits and vegetables offers a natural and chemical-free method. However, the inherent instability of plant bioactive compounds underscores the necessity for encapsulation techniques. Essential oil-based nanoemulsions (EO-NEs) stand out among food additives due to their distinctive antibacterial and antioxidant properties. This review delves into recent advancements in the application of EO-NEs as edible coatings for fresh and fresh-cut produce. It examines the efficacy of EO-NEs in enhancing the preservation of fruits and vegetables by harnessing their bioactive compounds for antibacterial, antifungal, and antioxidant activities. Additionally, the review accentuates the efficacy of EO-NEs in inhibiting biofilm formation on fruits and vegetables. It reveals that coatings derived from plant-source nanoemulsions exhibit exceptional mechanical, optical, and microstructural qualities, as well as superior water barrier properties. In contrast to conventional emulsions, nanocoatings facilitate the gradual and controlled release of antimicrobial and antioxidant compounds during food storage. This feature enhances bioactivity, extends shelf life, and enhances the nutritional profile of products. By preserving and protecting shelf stability, EO-NEs contribute to the maintenance of vegetable freshness. Nonetheless, ensuring their commercial viability necessitates additional research into the toxicity of EO-based nanoemulsions.

Bioactive polysaccharides from Aegle marmelos fruit: Recent trends on extraction, bio-techno functionality, and food applications.

Polysaccharides from non-conventional sources, such as fruits, have gained significant attention recently. Aegle marmelos (Bael), a non-conventional fruit, is an excellent source of biologically active components with potential indigenous therapeutic and food applications. Apart from polyphenolic components, this is an excellent source of mucilaginous polysaccharides. Polysaccharides are one the major components of bael fruit, having a high amount of galactose and glucuronic acid, which contributes to its potential therapeutic properties. Therefore, this review emphasizes the conventional and emerging techniques of polysaccharide extraction from bael fruit. Insight into the attributes of polysaccharide components, their techno-functional properties, characterization of bael fruit polysaccharide, emulsifying properties, binding properties, reduction of hazardous dyes, application of polysaccharides in film formation, application of polysaccharide as a nanocomposite, and biological activities of bael fruit polysaccharides are discussed. This review also systematically overviews the relationship between extraction techniques, structural characteristics, and biological activities. Additionally, recommendations, future perspectives, and new valuable insight towards better utilization of bael fruit polysaccharide have been given importance, which can be promoted in the long term.

Plant and animal protein mixed systems as wall material for microencapsulation of Manuka essential Oil: Characterization and in vitro release kinetics.

Combination of plant and animal protein diet is becoming a valuable source of nutrition in the modern diet due to the synergistic functional properties inherent in these protein complexes. Moreover, the synergy between animal and plant proteins can contribute to the high stability and improved solubility of the encapsulated bioactive ingredients (e.g., essential oils). Therefore, the study was designed to evaluate the plant (pea protein (PP) and lupine protein (LP)) and animal protein (whey protein, WP) mixed systems as a wall material for microencapsulation of manuka essential oil, as an example of bioactive compound. Moreover, physicochemical properties and in vitro release profile of encapsulated manuka essential oil were studied. Manuka essential oil microcapsules exhibited low moisture content (5.3-7.1 %) and low water activity (0.33-0.37) with a solubility of 53.7-68.1 %. Change in wall material ratio significantly affected the color of microcapsules, while microcapsules prepared with 1:1 protein/oil ratio demonstrated a high encapsulation efficiency (90.4 % and 89.4 %) for protein mixed systems (PP + WP and LP + WP), respectively. Microcapsules further showed low values for lipid oxidation with a high oxidative stability and antioxidant activity (62.1-87.0 %). The zero order and Korsmeyer-Peppas models clearly explained the release mechanism of encapsulated oil, which was dependent on the type and concentration of the protein mixed used. The findings demonstrated that the protein mixed systems successfully encapsulated the manuka essential oil with controlled release and high oxidative stability, indicating the suitability of the protein mixed systems as a carrier in encapsulation and application potential in development of encapsulated functional foods.

Bioactive Compounds and Health-Promoting Properties of Elephant Apple (Dillenia indica L.): A Comprehensive Review.

Elephant apple (Dillenia indica L.) grows wild in Southeast Asia's forests, including in China, India, Nepal, Bangladesh, and Sri Lanka. Elephant apples are considered essential fruit crops because of their high nutritional value, which includes high levels of vitamin C, carbohydrates, fats, fibre, protein, minerals, and fatty acids. It is important to understand the nutritional value and health benefits of elephant apples in order to increase fruit intake in people's daily diets. The present review paper focuses on elephant apple's phytochemistry, bioactive compounds, therapeutic value, and medicinal capabilities for designing and developing a wide range of food formulations. Proteins, minerals, fats, crude fibre, carbohydrates, vitamin C, tannins, malic acid, and glucose are abundant in the leaves, bark, and fruit of the elephant apple. In addition to nutritional components, many phytochemicals found in elephant apples have been identified as bioactive compounds with a broad range of biological activities, the most prominent of which are antioxidant, anticancer, antidiabetic, and anti-inflammatory properties. Overall, elephant apple is a rich, natural source of bioactive compounds with potential applications in the production of value-added foods and nutraceuticals for disease prevention and management.

Interactions of legume phenols-rice protein concentrate towards improving vegan food quality: Development of a protein-phenols enriched fruit smoothie.

Phenol-protein interaction is considered an effective tool to improve the functional properties of vegan proteins. The present work aimed to evaluate the covalent interaction between kidney bean polyphenols with rice protein concentrate and studied their characteristics for quality improvement in vegan-based foods. The impact of interaction on the techno-functional properties of protein was evaluated and the nutritional composition revealed that kidney bean was rich in carbohydrates. Furthermore, a noticeable antioxidant activity (58.11 ± 1.075 %) due to the presence of phenols (5.5 mg GAE/g) was observed for the kidney bean extract. Moreover, caffeic acid and p-Coumaric acid were confirmed using ultra-pressure liquid chromatography and the amount was 194.43 and 0.9272 mg/kg, respectively. A range of rice protein- phenols complexes (PPC0.025, PPC0.050, PPC0.075, PPC0.1, PPC0.2, PPC 0.5, PPC1) were examined and PPC0.2 and PPC0.5 showed significantly (p < 0.05) higher binding efficiency with proteins via covalent interaction. The conjugation reveals changes in physicochemical properties of rice protein, including, reduced size (178.4 nm) and imparted negative charges (-19.5 mV) of the native protein. The presence of amide Ⅰ, Ⅱ, Ⅲ, was confirmed in native protein and protein-phenol complex with vibration bands, particularly at 3784.92, 1631.07, and 1234 cm-1, respectively. The X-ray diffraction pattern depicted a slight decrease in crystallinity after the complexation and scanning electron microscopy revealed the alteration in morphology from less to improved smoothness and continuous surface characteristics for the complex. Thermo gravimetric analysis revealed high thermal stability of the complex with a maximum weight loss at a temperature range of 400-500 °C. Protein-phenol complex added fruit-based smoothie was developed and it was found to be acceptable in terms of various sensory attributes including color & appearance, textural consistency, and mouthfeel as compared to the control smoothie. Overall, this study provided novel insights to understand the phenol-protein interactions and the possible use of the phenol-rice protein complex in the development of vegan-based food products.

Enzyme-assisted polysaccharides extraction from Calocybe indica: Synergistic antibiofilm and oxidative stability of essential oil nanoemulsion.

Recently, mushroom polysaccharides have been explored to attribute to vital biologically important functions, and several extraction techniques can be employed, therefore, polysaccharides were extracted from the edible mushroom Calocybe indica to explore its functionality. Multiple enzymes viz., cellulase, pectinase, and protease (1:1:1) at temperature 47 °C and pH 4.64 with an extraction time of 2 h yielded 7.24 % polysaccharide content. The thermograph curve of polysaccharides showed two-stage decomposition at a different temperature range and decomposition of polysaccharides initiated with an onset temperature of 226.77 °C and a maximum peak at 248.90 °C. Hydrodistillation processed Eucalyptus globulus leaf oil was characterized using the chromatography technique and eucalyptol, p-cymene, Γ-terpinene, 4-epi-cubebol, spathulenol, viridiflorol, and p-mentha-1,5-dien-8-ol was observed as major components. As well, we formulated nanoemulsion using mushroom polysaccharide and eucalyptus leaf oil with 140.8 nm and evaluated synergistic antimicrobial and antibiofilm activity. MIC and MBC values for Pseudomonas aeruginosa, E. coli, and S. typhi were 12.50-3.12 and 6.25-1.56, and for S. aureus were 6.25, 6.25, 3.12, and 3.12, 3.12, 1.56 and for C. albicans the values were 12.50, 12.50, 6.250 and 6.25, 6.25, and 3.12 µL/mL respectively. The polysaccharides, essential oil, and nanoemulsion showed remarkable antibiofilm activity against S. aureus with inhibition of 57.42 ± 0.19, 59.62 ± 0.15, and 69.34 ± 0.19 %, while E. coli showed the least antibiofilm activity. However, all three tested samples showed significant (p < 0.05) differences against tested pathogenic microorganisms with inhibition of biofilm formation. Therefore, it could be inferred that the synergistic properties of essential oils with mushroom polysaccharides are a promising strategy to enhance antimicrobial efficacy and control foodborne pathogens.

Hazardous impacts of glyphosate on human and environment health: Occurrence and detection in food.

With the ever-increasing human population, farming lands are decreasing every year, therefore, for effective crop management; agricultural scientists are continually developing new strategies. However, small plants and herbs always impart a much loss in the yields of the crop and farmers are using tons of herbicides to eradicate that problem. Across the world, several herbicides are available in the market for effective crop management, however, scientists observed various environmental and health effects of the herbicides. Over the past 40 years, the herbicide glyphosate has been used extensively with the assumption of negligible effects on the environment and human health. However, in recent years, concerns have increased globally about the potential direct and indirect effects on human health due to the excessive use of glyphosate. As well, the toxicity on ecosystems and the possible effects on all living creatures have long been at the center of a complex discrepancy about the authorization for its use. The World Health Organization also further classified glyphosate as a carcinogenic toxic component and it was banned in 2017 due to numerous life-threatening side effects on human health. In the present era, the residues of banned glyphosate are more prevalent in agricultural and environmental samples which are directly affecting human health. Various reports revealed the detailed extraction process of glyphosate from different categories of the food matrix. Therefore, in the present review, to reveal the importance of glyphosate monitoring in the food matrix, we discussed the environmental and health effects of glyphosate with acute toxicity levels. Also, the effect of glyphosate on aquatic life is discussed in detail and various detection methods such as fluorescence, chromatography, and colorimetric techniques from different food samples with a limit of detection values are revealed. Overall, this review will give an in-depth insight into the various toxicological aspects and detection of glyphosate from food matrix using various advanced analytical techniques.

Impact of Different Drying Methods on the Phenolic Composition, In Vitro Antioxidant Activity, and Quality Attributes of Dragon Fruit Slices and Pulp.

The aim of this study was to compare the refractance window drying method (RWD) with the hot air oven drying (HD), vacuum drying (VD), and freeze-drying (FD) methods in order to analyze the outcomes of the qualitative properties of dragon fruit slices and pulp. Moreover, the impact of temperature on the phenolic content, antioxidant activity, color, and texture of the dragon fruit slices and pulp were studied. The results showed that the RWD samples exhibited a high nutritional quality in contrast to the other drying methods. The RWD method had a short drying time of 960 min to reach the final moisture content of 6.50% (dry basis), while the FD, VD, and HD methods had significantly higher drying times of 1320, 1200, and 1080 min, respectively, to reach the final moisture content. Higher values of TPC (182 mg GAE/100 g) and crude fiber (0.98%) were obtained in the RWD dragon fruit samples, indicating the potential of RWD to preserve the quality of dried samples. In conclusion, this study revealed that RWD provides an appropriate drying temperature as an alternative to freeze-drying. RWD may improve dragon fruit drying, adding value to the food industry.

Gum arabic/guar gum stabilized Hydnocarpus wightiana oil nanohydrogel: Characterization, antimicrobial, anti-inflammatory, and anti-biofilm activities.

Hydnocarpus wightiana oil has proven to inhibit the growth of pathogenic microorganisms; however, the raw form is highly susceptible to oxidation, and thus it becomes toxic when uptake is in high amounts. Therefore, to minimize the deterioration, we formulated Hydnocarpus wightiana oil-based nanohydrogel and studied its characteristics as well biological activity. The low energy-assisted hydrogel was formulated by including gelling agent, connective linker, and cross-linker and it resulted in internal micellar polymerization of the milky white emulsion. The oil showed the presence of octanoic acid, n-tetradecane, methyl 11-(2-cyclopenten-1-yl) undecanoate (methyl hydnocarpate), 13-(2-cyclopenten-1-yl) tridecanoic acid (methyl chaulmoograte), and 10,13-eicosadienoic acid. The amount of caffeic acid was 0.0636 mg/g, which was higher than the amount of gallic acid (0.0076 mg/g) in the samples. The formulated nanohydrogel showed an average droplet size of 103.6 nm with a surface charge of -17.6 mV. The minimal inhibitory bactericidal, and fungicidal concentrations of nanohydrogel against pathogenic bacteria and fungi were ranging from 0.78 to 1.56 µl/mL with 70.29-83.62 % antibiofilm activity. Also, nanohydrogel showed a significantly (p < 0.05) higher killing rate for Escherichia coli (7.89 log CFU/mL) than Staphylococcus aureus (7.81 log CFU/mL) with comparable anti-inflammatory activity than commercial standard (49.28-84.56 %). Therefore, it can be concluded that being hydrophobic, and having the capability of target-specific drug absorption as well as biocompatibility nanohydrogels can be utilized to cure various pathogenic microbial infections.

Attenuation of Hyperlipidemia by Medicinal Formulations of Emblica officinalis Synergized with Nanotechnological Approaches.

The ayurvedic herb Emblica officinalis (E. officinalis) is a gift to mankind to acquire a healthy lifestyle. It has great therapeutic and nutritional importance. Emblica officinalis, also known as Indian gooseberry or Amla, is a member of the Euphorbiaceae family. Amla is beneficial for treating illnesses in all its forms. The most crucial component is a fruit, which is also the most common. It is used frequently in Indian medicine as a restorative, diuretic, liver tonic, refrigerant, stomachic, laxative, antipyretic, hair tonic, ulcer preventive, and for the common cold and fever. Hyperlipidemia is also known as high cholesterol or an increase in one or more lipid-containing blood proteins. Various phytocompounds, including polyphenols, vitamins, amino acids, fixed oils, and flavonoids, are present in the various parts of E. officinalis. E. officinalis has been linked to a variety of pharmacological effects in earlier studies, including hepatoprotective, immunomodulatory, antimicrobial, radioprotective, and hyperlipidemic effects. The amla-derived active ingredients and food products nevertheless encounter challenges such as instability and interactions with other food matrices. Considering the issue from this perspective, food component nanoencapsulation is a young and cutting-edge field for controlled and targeted delivery with a range of preventative activities. The nanoformulation of E. officinalis facilitates the release of active components or food ingredients, increased bioaccessibility, enhanced therapeutic activities, and digestion in the human body. Accordingly, the current review provides a summary of the phytoconstituents of E. officinalis, pharmacological actions detailing the plant E. officinalis's traditional uses, and especially hyperlipidemic activity. Correspondingly, the article describes the uses of nanotechnology in amla therapeutics and functional ingredients.

Effect of microfluidization on quality characteristics of sapodilla (Manilkara achras L.) juice.

Various oxidative enzymes account for the quality degradation of sapodilla (Manilkara achras L.) juice and need to be inactivated through emerging and continuous green pressure processing technology. In this study, pressurization of sapodilla juice was attempted via microfluidization (MF) at pressure range of 10,000-30,000 pound per square inch (psi) with 1-3 passes or cycles. The impact of microfluidization on the activity of polyphenol oxidase (PPO), peroxidase (POD), color, total soluble solid (TSS), viscosity, serum cloudiness along with particle size, and microbial load of sapodilla juice was assessed. Results showed that microfluidization (MF) decreased the residual PPO activity from 100 to 80.78 % and POD activity from 100 to 40.57%. However, TSS (18.81-19.01 %), viscosity (2.64-2.06 cP), serum cloudiness (2.19-1.22 %) and total color change (3.19-18.54) was also significantly affected. Most of these changes were observed due to particle size (PS) reduction that varied from 65.19 to 8.13 µm. Microfluidized juice revealed color improvement at particular MF pressure and pass due to enzyme inactivation. Moreover, lowest microbial load (2.89 Log CFU/ mL) was found at 30,000 psi/3 pass of MF as compared to control sample (unprocessed juice) (7.57 Log CFU/ mL). Consequently, MF can be potential candidate in processing of juices against spoilage.

Advances in Nanofabrication Technology for Nutraceuticals: New Insights and Future Trends.

Bioactive components such as polyphenolics, flavonoids, bioactive peptides, pigments, and essential fatty acids were known to ward off some deadliest diseases. Nutraceuticals are those beneficial compounds that may be food or part of food that has come up with medical or health benefits. Nanoencapsulation and nanofabricated delivery systems are an imminent approach in the field of food sciences. The sustainable fabrication of nutraceuticals and biocompatible active components indisputably enhances the food grade and promotes good health. Nanofabricated delivery systems include carbohydrates-based, lipids (solid and liquid), and proteins-based delivery systems. Solid nano-delivery systems include lipid nanoparticles. Liquid nano-delivery systems include nanoliposomes and nanoemulsions. Physicochemical properties of nanoparticles such as size, charge, hydrophobicity, and targeting molecules affect the absorption, distribution, metabolism, and excretion of nano delivery systems. Advance research in toxicity studies is necessary to ensure the safety of the nanofabricated delivery systems, as the safety of nano delivery systems for use in food applications is unknown. Therefore, improved nanotechnology could play a pivotal role in developing functional foods, a contemporary concept assuring the consumers to provide programmed, high-priced, and high-quality research toward nanofabricated delivery systems.

Antimicrobial and Anti-Inflammatory Activity of Low-Energy Assisted Nanohydrogel of Azadirachta indica Oil.

Plant-based bioactive compounds have been utilized to cure diseases caused by pathogenic microorganisms and as a substitute to reduce the side effects of chemically synthesized drugs. Therefore, in the present study, Azadirachta indica oil nanohydrogel was prepared to be utilized as an alternate source of the antimicrobial compound. The total phenolic compound in Azadirachta indica oil was quantified by chromatography analysis and revealed gallic acid (0.0076 ppm), caffeic acid (0.077 ppm), and syringic acid (0.0129 ppm). Gas chromatography−mass spectrometry analysis of Azadirachta indica oil revealed the presence of bioactive components, namely hexadecenoic acid, heptadecanoic acid, ç-linolenic acid, 9-octadecanoic acid (Z)-methyl ester, methyl-8-methyl-nonanoate, eicosanoic acid, methyl ester, and 8-octadecane3-ethyl-5-(2 ethylbutyl). The nanohydrogel showed droplet size of 104.1 nm and −19.3 mV zeta potential. The nanohydrogel showed potential antimicrobial activity against S. aureus, E. coli, and C. albicans with minimum inhibitory, bactericidal, and fungicidal concentrations ranging from 6.25 to 3.125 (µg/mL). The nanohydrogel showed a significantly (p < 0.05) higher (8.40 log CFU/mL) value for Gram-negative bacteria E. coli compared to Gram-positive S. aureus (8.34 log CFU/mL), and in the case of pathogenic fungal strain C. albicans, there was a significant (p < 0.05) reduction in log CFU/mL value (7.79−6.94). The nanohydrogel showed 50.23−82.57% inhibition in comparison to standard diclofenac sodium (59.47−92.32%). In conclusion, Azadirachta indica oil nanohydrogel possesses great potential for antimicrobial and anti-inflammatory activities and therefore can be used as an effective agent.

Effect of stress on germination of djulis (Chenopodium formosanum Koidz.) sprouts: a natural alternative to enhance the betacyanin and phenolic compounds.

BACKGROUND: Germination is regarded as a natural method for improving the bioavailability of seed nutrients against stress, which enhances the accumulation of bioactive compounds. The present study aimed to determine the effect of stress (H2 O2 , catechin, gallic acid, tyrosine, and NaCl) during germination of djulis (Chenopodium formosanum Koidz.) sprouts on betacyanin, phytochemicals, and antioxidant capacities. RESULTS: The betacyanin and antioxidant activities of the djulis sprouts increased significantly compared to seeds. The lowest betacyanin was found in NaCl-stressed sprouts. The djulis sprouts reported the presence of celosianins I and II (50.72%), which was absent in seeds. Hydroxycinnamic acids accounted for > 60% of the total phenolic compounds in sprouts, whereas rutin predominated in the seeds. CONCLUSION: Germination under stress may represent an effective natural method for improving the bioactive potential of sprouts, an alternative to use seeds, in the development of bioactive compounds-enriched healthy foods that are good for public health. © 2022 Society of Chemical Industry.

Recent developments on production, purification and biological activity of marine peptides.

Marine peptides are one of the richest sources of structurally diverse bioactive compounds and a considerable attention has been drawn towards their production and bioactivity. However, there is a paucity in consolidation of emerging trends encompassing both production techniques and biological application. Herein, we intend to review the recent advancements on different production, purification and identification technologies used for marine peptides along with presenting their potential health benefits. Bibliometric analysis revealed a growing number of scientific publications on marine peptides (268 documents per year) with both Asia (37.2%) and Europe (33.1%) being the major contributors. Extraction and purification by ultrafiltration and enzymatic hydrolysis, followed by identification by chromatographic techniques coupled with an appropriate detector could yield a high content of peptides with improved bioactivity. Moreover, the multifunctional health benefits exerted by marine peptides including anti-microbial, antioxidant, anti-hypertension, anti-diabetes and anti-cancer along with their structure-activity relationship were presented. The future perspective on marine peptide research should focus on finding improved separation and purification technologies with enhanced selectivity and resolution for obtaining more novel peptides with high yield and low cost. In addition, by employing encapsulation strategies such as nanoemulsion and nanoliposome, oral bioavailability and bioactivity of peptides can be greatly enhanced. Also, the potential health benefits that are demonstrated by in vitro and in vivo models should be validated by conducting human clinical trials for a technology transfer from bench to bedside.

Recent Advances on Nanoparticle Based Strategies for Improving Carotenoid Stability and Biological Activity.

Carotenoids are natural pigments widely used in food industries due to their health-promoting properties. However, the presence of long-chain conjugated double bonds are responsible for chemical instability, poor water solubility, low bioavailability and high susceptibility to oxidation. The application of a nanoencapsulation technique has thus become a vital means to enhance stability of carotenoids under physiological conditions due to their small particle size, high aqueous solubility and improved bioavailability. This review intends to overview the advances in preparation, characterization, biocompatibility and application of nanocarotenoids reported in research/review papers published in peer-reviewed journals over the last five years. More specifically, nanocarotenoids were prepared from both carotenoid extracts and standards by employing various preparation techniques to yield different nanostructures including nanoemulsions, nanoliposomes, polymeric/biopolymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid nanoparticles, supercritical fluid-based nanoparticles and metal/metal oxide nanoparticles. Stability studies involved evaluation of physical stability and/or chemical stability under different storage conditions and heating temperatures for varied lengths of time, while the release behavior and bioaccessibility were determined by various in vitro digestion and absorption models as well as bioavailability through elucidating pharmacokinetics in an animal model. Moreover, application of nanocarotenoids for various biological applications including antioxidant, anticancer, antibacterial, antiaging, cosmetics, diabetic wound healing and hepatic steatosis were summarized.

In vitro antioxidant activity of Kyoho grape extracts in DPPH and ABTS assays: Estimation methods for EC50 using advanced statistical programs.

The antioxidant capacities of Kyoho skin, seed, and flesh extracts were determined using DPPH and ABTS assays and a suitable statistical program was tested for the prediction of EC50 values of Kyoho skin, seed, and flesh extracts obtained by DPPH and ABTS assays. Statistical programs: OriginPro® version 8, GraphPad Prism® version 7, GraphPad Prism® version 7 five-parameter (5P) logistic model, SigmaPlot® version 14.0, Microsoft Excel® version 2016, interpolation log, and direct interpolation methods were employed to estimate the EC50 values. Linear regression and direct interpolation results showed deviations for non-linear models in the prediction of EC50 values of grape extracts. The correlation results of grape flesh extracts demonstrated a positive correlation (r = ≥ 0.95) between EC50 values of antioxidant assays. Therefore, non-linear methods were appropriate for EC50 calculations. These findings have significant implications for selecting an efficient statistical tool to calculate EC50 values for DPPH and ABTS assays.