Molecular modeling of lactoferrin for food and nutraceutical applications: insights from in silico techniques.

Lactoferrin is a protein, primarily found in milk that has attracted the interest of the food industries due to its health properties. Nevertheless, the instability of lactoferrin has limited its commercial application. Recent studies have focused on encapsulation to enhance the stability of lactoferrin. However, the molecular insights underlying the changes of structural properties of lactoferrin and the interaction with protectants remain poorly understood. Computational approaches have proven useful in understanding the structural properties of molecules and the key binding with other constituents. In this review, comprehensive information on the structure and function of lactoferrin and the binding with various molecules for food purposes are reviewed, with a special emphasis on the use of molecular dynamics simulations. The results demonstrate the application of modeling and simulations to determine key residues of lactoferrin responsible for its stability and interactions with other biomolecular components under various conditions, which are also associated with its functional benefits. These have also been extended into the potential creation of enhanced lactoferrin for commercial purposes. This review provides valuable strategies in designing novel nutraceuticals for food science practitioners and those who have interests in acquiring familiarity with the application of computational modeling for food and health purposes.

Molecular insights into the interaction of apo-lactoferrin with the receptor binding domain of the SARS-CoV-2 spike protein: a molecular dynamics simulation study.

LF is a bioactive protein, derived from colostrum and milk that has been found to possess various immunomodulatory, iron chelating, and antimicrobial properties, especially in its apo-form. Recent studies have demonstrated the functionality of LF in attaching to the S proteins of SARS-CoV-2, thereby preventing it from interacting with the ACE-2 receptor. However, the molecular mechanism mediating the process is poorly understood. In this study, molecular docking and MD simulations coupled with free energy calculations were applied to elucidate the key interaction of apo-LF and its N-lobe and C-lobe derivative forms with the RBD of coronavirus S proteins. This has also been extended into evaluating the L452R mutant, which is associated with the delta variant of SARS-CoV-2. The results demonstrate the efficacy of the apo-LF C-lobe in binding to the RBD of both variants, primarily through electrostatic attractions between the acidic residues of the former and the basic residues of each RBD. Furthermore, due to the additional arginine in the L452R variant, the interaction between the C-lobe and the latter is stronger, resulting in a more favourable binding and tightly bound structure. The simulations highlight that the C-lobe, followed by full-length apo-LF can form a multimeric complex with the RBD of SARS-CoV-2, indicating their potential use as novel therapeutics, particularly the cleaved C-lobe of apo-LF to disrupt the S proteins from binding to the host ACE-2 receptor.Communicated by Ramaswamy H. Sarma.

The effects of temperature on the crystalline properties and resistant starch during storage of white bread.

Resistant starch (RS) can form during storage of foods, thereby bestowing a variety of potential health benefits. The purpose of the current study has been to determine the influence of storage temperature and time on the crystallinity and RS content of bread. Loaves of white bread were baked and stored at refrigeration, frozen and room temperatures with analysis over a period of zero to seven days. RS determination and X-ray diffraction (XRD) were used to evaluate the influence of storage temperature and time on total crystallinity and RS content. The rate of starch recrystallisation was affected by storage temperature and time, where refrigeration temperatures accelerated RS formation and total crystallinity more than storage time at both frozen and room temperature. A strong statistical model has been established between RS formation in bread and XRD patterns, having a 96.7% fit indicating the potential of XRD to measure RS concentrations.

Effect of the glass transition temperature on alpha-amylase activity in a starch matrix.

This study optimises a protocol for the estimation of α-amylase activity in a condensed starch matrix in the vicinity of the glass transition region. Enzymatic activity on the vitrified starch system was compared with that of a reference substrate, maltodextrin. The activity was assayed as the rate of release of reducing sugar using a dinitrosalicylic acid procedure. The condensed carbohydrate matrices served the dual purpose of acting as a substrate as well as producing a pronounced effect on the ability to enzymatic hydrolysis. Activation energies were estimated throughout the glass transition region of condensed carbohydrate preparations based on the concept of the spectroscopic shift factor. Results were used to demonstrate a considerable moderation by the mechanical glass transition temperature, beyond the expected linear effect of the temperature dependence, on the reaction rate of starch hydrolysis by α-amylase in comparison with the low-molecular weight chain of maltodextrin.

Application of the rat liver lysosome assay to determining the reduction of toxic gliadin content during breadmaking.

Enriched caricain was able to detoxify a major proportion of the gliadin in wholemeal wheat dough by allowing it to react for 5h at 37 °C during the fermentation stage. A reduction of 82% in toxicity, as determined by the rat-liver lysosome assay, was achieved using 0.03% enzyme on weight of dough. Without enzyme, only 26% reduction occurred. The difference in reduction of toxicity achieved is statistically significant (p < 0.01). The results are very similar to those obtained in our previous work using an immuno assay and the same enzyme preparation. They confirm the value of caricain as a means of reducing the toxicity of gliadin and open the way for enzyme therapy as an adjunct to the gluten free diet. This approach should lead to better control over the elimination of dietary gluten intake in conditions such as coeliac disease and dermatitis herpetiformis.

The behaviour of whey protein isolate in protecting Lactobacillus plantarum.

There is increasing evidence that whey protein isolates (WPI), can be utilised to encapsulate and protect bioactive substances, including lactic acid bacteria, due to their physicochemical properties. However, little is known about what happens in the immediate vicinity of the cells. This study examined the protective behaviour of WPI for two strains of Lactobacillus plantarum, A17 and B21, during spray drying. B21 was found to be more hydrophobic than A17 and required 50% of the amount of WPI to provide comparably high survival (∼ 90%). We hypothesise that WPI protects the hydrophobic bacteria by initial attachment to the unfolded whey protein due to hydrophobic interactions followed by adhesion to the proteins, resulting in cells being embedded within the walls of the capsules. The encapsulated strains had a moisture content of approximately 5.5% and during storage trials at 20 °C retained viability for at least eight weeks.

The effect of fermentation and addition of vegetable oil on resistant starch formation in wholegrain breads.

Resistant starch has potential health benefits but the factors affecting its formation in bread and baked products are not well studied. Here, the formation of resistant starch in wholemeal bread products was evaluated in relation to the processing conditions including fermentation time, temperature and the inclusion of palm oil as a vitamin source. The effects of each the factor were assessed using a full factorial design. The impact on final starch content of traditional sourdough fermentation of wholemeal rye bread, as well as the bulk fermentation process of wheat and wheat/oat blends of wholemeal bread, was also assessed by enzyme assay. Palm oil content was found to have a significant effect on the formation of resistant starch in all of the breads while fermentation time and temperature had no significant impact. Sourdough fermentation of rye bread was found to have a greater impact on resistant starch formation than bulk fermentation of wheat and wheat blend breads, most likely due the increased organic acid content of the sourdough process.

Assessment of arsenic in Australian grown and imported rice varieties on sale in Australia and potential links with irrigation practises and soil geochemistry.

Chronic dietary exposure to arsenic, particularly the inorganic forms (defined as elemental arsenic, predominantly As(3+) and As(5+), and all its inorganic compounds except arsine), is a matter of concern for human health. Ingestion of arsenic usually occurs via contaminated water but recent studies show there is also a risk of exposure from food, particularly Asian rice (Oryza sativa). Australia is a rice growing country, contributing around 2% of the world rice trade, and a large proportion of the population consumes rice regularly. In the present study we investigated concentrations of arsenic in both Australian grown and imported rice on sale in Australia and examined the potential links with irrigation practises and soil geochemistry. The results indicated a wide spread of arsenic levels of 0.09-0.33 mg kg(-1), with Australian grown Arborio and sushi varieties of O. sativa containing the highest mean value of ∼0.22 mg kg(-1). Arsenic levels in all samples were below the 1 mg kg(-1) limit set by Food Standards Australia New Zealand.

Agent selection and protective effects during single droplet drying of bacteria.

The protective mechanisms of whey protein isolate (WPI), trehalose, lactose, and skim milk on Lactobacillus plantarum A17 during convective droplet drying has been explored. A single droplet drying technique was used to monitor cell survival, droplet temperature and corresponding changes in mass. WPI and skim milk provided the highest protection amongst the materials tested. In situ analysis of the intermediate stage of drying revealed that for WPI and skim milk, crust formation reduces the rate of sudden temperature increase thereby imparting less stress on the cells. Irreversible denaturation of the WPI components might have also contributed to the protection of the cells. Skim milk, however, 'loses' the protective behaviour towards the latter stages of drying. This indicates that the concentration of the WPI components could be another possible factor determining the sustained protective behaviour during the later stages of drying when the moisture content is low.

Controlled release of thiamin in a glassy κ-carrageenan/glucose syrup matrix.

The work dealt with the diffusional mobility of thiamin embedded in a high-solid matrix of κ-carrageenan with glucose syrup. It utilized thermomechanical analysis in the form of modulated differential scanning calorimetry and small-deformation dynamic oscillation in shear, Fourier transform infrared spectroscopy, wide angle X-ray diffraction, scanning electron microscopy and UV-vis spectrophotometry. The structural properties of the matrix were assessed in a temperature induced rubber-to-glass transformation. A thiamin-dye binding assay was employed to monitor the diffusion process of the vitamin from the high-solid preparation to ethylene glycol. The relationship between mechanical properties of the carbohydrate matrix and vitamin mobility was assessed via the application of the combined framework of the free volume theory and the predictions of the reaction rate theory. Results argue that the transport of the micronutrient is governed by the structural relaxation of the high-solid matrix. These were further treated with the concept of Fickian diffusion coefficient to provide the rate of the bioactive compound mobility within the present experimental settings.

Reduction of toxic gliadin content of wholegrain bread by the enzyme caricain.

Increasingly the number of individuals being diagnosed with some form of sensitivity to the proteins in wheat grains represents a cause for concern. Currently, the treatment is dietary withdrawal of gluten, but commercial gluten-free bread presents some undesirable properties. The objective of this study has been to assess the ability of the enzyme caricain (from papaya latex) to detoxify gliadin in whole wheat flour and develop bread suitable for coeliacs and gluten intolerant individuals. Ion exchange chromatography was used to enrich the caricain in papaya latex and an enzyme-linked immunosorbent assay test kit was used for the analysis of gliadin residues in the baked bread. The partially purified enzyme was found to be more effective in reducing gliadin content than the crude papain and the resultant loaves had acceptable crumb and crust characteristics. Caricain appears to be capable of detoxifying gliadin and has the potential to mitigate the problems confronting coeliacs.

Effect of a glassy gellan/polydextrose matrix on the activity of α-D-glucosidase.

An investigation of the ability of the enzyme α-D-glucosidase to act on the substrate 4-nitrophenyl α-D-glucopyranoside (pNPG) while embedded in glassy carbohydrate matrices (deacylated gellan with polydextrose and polydextrose alone) is presented. Physicochemical characterisation of the matrices was achieved using the techniques of modulated differential scanning calorimetry, small deformation dynamic oscillation on shear, Fourier transform infra-red spectroscopy, wide angle X-ray diffraction and scanning electron microscopy. A UV-vis spectrophotometric procedure was adapted for the analysis of the activity of α-D-glucosidase in hydrolysing pNPG in the condensed carbohydrate systems. In order to derive a relationship between the structural properties of the matrix and the enzymatic activity, mechanical spectra were recorded using the combined framework of the Williams, Landel and Ferry equation with the time-temperature superposition principle. Theoretical modelling and experimental observations strongly argue for a pronounced effect of the gelling polysaccharide/co-solute mixture on enzymatic activity near the mechanical Tg of the matrix.

Structural studies on matrices of deacylated gellan with polydextrose.

The effect of varying concentrations of co-solute (polydextrose) on thermomechanical and physicochemical properties of deacylated gellan matrices is presented. Modulated differential scanning calorimetry, micro differential scanning calorimetry, small deformation dynamic oscillation in shear, Fourier transform infrared spectroscopy, wide angle X-ray diffraction and environmental scanning electron microscopy have been used to investigate the structural transformations in aqueous, low-solid and condensed systems. There was a rise in values of storage modulus as the level of co-solute was increased, followed by a significant decline at intermediate concentrations, with high modulus values being regained as more of the co-solute was incorporated. These results confirm the hypothesis of a structural transformation from a highly enthalpic aggregated assembly in the aqueous/low-solid environment to a lightly cross linked polysaccharide network in the high solids regime. Time-temperature superposition (TTS) phenomena observed for amorphous synthetic polymers have been utilised to generate master curves of viscoelasticity, which afforded rationalisation of results on the basis of the free volume theory.

Development of a switchable multidimensional/comprehensive two-dimensional gas chromatographic analytical system.

In this study, a new system for analysis using a dual comprehensive two-dimensional gas chromatography/targeted multidimensional gas chromatography (switchable GC x GC/targeted MDGC) analysis was developed. The configuration of this system not only permits the independent operation of GC, GC x GC and targeted MDGC analyses in separate analyses, but also allows the mode to be switched from GC x GC to targeted MDGC any number of times through a single analysis. By incorporating a Deans switch microfluidics transfer module prior to a cryotrapping device, the flow stream from the first dimension column can be directed to either one of two second dimension columns in a classical heart-cutting operation. Both second columns pass through the cryotrap to allow solute bands to be focused and then rapidly remobilized to the respective second columns. A short second column enables GC x GC operation, whilst a longer column is used for targeted MDGC. Validation of the system was performed using a standard mixture of compounds relevant to essential oil analysis, and then using compounds present at different abundances in lavender essential oil. Reproducibility of retention times and peak area responses demonstrated that there was negligible variation in the system over the course of multiple heart-cuts, and proved the reliable operation of the system. An application of the system to lavender oil, as a more complex sample, was carried out to affirm system feasibility, and demonstrate the ability of the system to target multiple components in the oil. The system was proposed to be useful for study of aroma-impact compounds where GC x GC can be incorporated with MDGC to permit precise identification of aroma-active compounds, where heart-cut multidimensional GC-olfactometry detection (MDGC-O) is a more appropriate technology for odour assessment.

Investigation of folic acid stability in fortified instant noodles by use of capillary electrophoresis and reversed-phase high performance liquid chromatography.

A single enzyme treatment with alpha-amylase, prior to the quantification of added folic acid (FA) in fortified instant fried Asian noodles with analysis performed by capillary zone electrophoresis (CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection, is described. The method was validated and optimized for capillary electrophoresis (CE) with separation achieved using a 8 mM phosphate-12 mM borate run buffer with 5% MeOH at pH 9.5. FA was well separated from matrix components with nicotinic acid (NA) employed as an internal standard. In a comparative study, separation of FA was performed using HPLC with a mobile phase consisting of 27% MeOH (v/v) in aqueous potassium phosphate buffer (3.5 mM KH(2)PO(4) and 3.2 mM K(2)HPO(4)), pH 8.5, and containing 5 mM tetrabutylammonium dihydrogen phosphate as an ion-pairing agent. For both methods, excellent results were obtained for various analytical parameters including linearity, accuracy and precision. The limit of detection was calculated to be 2.2 mg/L for CE without sample stacking and 0.10 mg/L with high performance liquid chromatography (HPLC). Sample extraction involved homogenization and enzymatic extraction with alpha-amylase. Results indicated that FA was stable during four main stages of instant fried noodle manufacturing (dough crumbs, cut sheets, steaming and frying).

Lactulose production from milk concentration permeate using calcium carbonate-based catalysts.

Milk concentration permeate (MCP), a low-value by-product of ultrafiltration plants and calcium carbonate-based catalysts were used for lactulose production. The results obtained show the effectiveness of oyster shell powder and limestone for lactose isomerisation as a replacement for egg shell powder. With the reaction conditions of 12mg/ml catalyst loading, reflux time of 120min at 96°C, a maximum yield of 18-21% lactulose was achievable at a cost of <50% of original lactose degradation (measured by HPLC). De-proteination of MCP by acidification prior to isomerisation helped lactulose formation in the earlier stages, but did not significantly increase the yield. The resulting lactulose MCP (40°B) incorporated at the rate of 3-4% was effective in enhancing the growth rate and acid production of Lactobacillus acidophilus (LA-5) in probiotic products.

Folates in Asian noodles: I. Microbiological analysis and the use of enzyme treatments.

Asian noodles are a major end use of common wheat flour globally. A microbiological assay protocol for the folate contents of flour and Asian noodles has been developed. Different approaches were compared for enzymatic extraction and deconjugation of folate following the establishment of optimum inoculum concentration for analysis. The folate results obtained varied when samples were subjected to different preparation and extraction procedures. The use of trienzyme treatments gave significantly higher content values. The pH and specific conditions of enzyme treatment also influenced the results. In applying well-established methods for folate analysis, it is essential to consider extraction procedures effective for the specific food products being analyzed. A suitable, validated method was developed and is recommended for use in further studies of fortification of wheat flour for Asian noodle processing.

Folates in Asian noodles: II. A comparison of commercial samples and the impact of cooking.

The folate contents of 26 commercial noodle samples were investigated. The impact of ingredients, pH, and cooking on folate content was studied for the 3 predominant styles of noodles: white salted, yellow alkaline, and instant. Some variability was found in the proportion of folate present in the free form and the noodles generally had low total folate contents. The pH values of the samples covered a wide range, varying from 3.7 to 10.3; however, the results did not provide strong evidence for a relationship between pH and folate content for any of the noodle styles studied. Higher folate levels were typically found in yellow alkaline samples compared to white salted and instant noodles. The storage of noodles in dry or moist forms did not appear to influence total folate contents, and subsequent losses during cooking depended upon the time of exposure to elevated temperatures. The enzymatic treatment of samples was particularly important for cooked noodles, indicating that folates were bound or entrapped during this process.

Folates in Asian noodles: III. Fortification, impact of processing, and enhancement of folate intakes.

Asian noodles, a widely consumed staple food, were evaluated as potential vehicles for fortification with folic acid. Samples of white salted, yellow alkaline, and instant noodles, prepared under controlled laboratory conditions, were fortified and folates were measured at each stage of processing using a microbiological assay. Although the 3 styles showed differing patterns of retention, overall losses were slightly more than 40% and were similar for all styles. White salted and yellow alkaline noodles showed no significant decrease in total folate content during production. In contrast, significant losses occurred for instant noodles during steaming and deep-frying of the noodle strands. In all cases, substantial losses occurred during subsequent cooking of the dried noodles. Fortification at a rate of 50% of the reference value per serving resulted in retention of folate at levels corresponding to 30% following cooking, whereas unfortified noodles contributed less than 4% per serving. It is concluded that fortifying Asian noodles provides an effective means for enhancing folate intake.

CE methods applied to the analysis of micronutrients in foods.

This article reviews the applications of CE that are relevant to the analysis of small molecules in foods. CE has been applied to a wide range of important areas of food analysis and is rapidly being established as an alternative technique to chromatographic methods including HPLC and GC within analytical food and research laboratories. In recent years the analysis of food by CE has become more frequent and important and as such a variety of compounds have been separated and quantified. Although many other analytes have been detected by CE, this review will highlight areas relating primarily to the rather broad chemical classes of free amino acids, carbohydrates, organic acids, vitamins and a variety of antioxidants. In addition, information relating to the analyte, sample matrix, mode of CE employed, scope of the methodology and the detection and derivatization of the small molecules are considered and discussed.