Copper-Chelated Chitosan Microgels for the Selective Enrichment of Small Cationic Peptides.

Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan beads were shown to strongly interact with a range of amino acids, in a wide range of pH and saline conditions. The beads exhibited an affinity that seemed to depend on the isoelectric point of the amino acid, with the extent of uptake increasing with decreasing isoelectric point. This selective interaction with anionic amino acids resulted in a significant relative enrichment of the supernatant solution in cationic amino acids. The beads were then studied as a novel fractionation system for complex milk hydrolysates. The copper chitosan beads selectively removed larger peptides from the hydrolysate aqueous solution, yielding a solution relatively enriched in medium and smaller peptides, which was characterized both quantitatively and qualitatively by size exclusion chromatography (SEC). Liquid chromatography-mass spectrometry (LCMS) work provided comprehensive data on a peptide sequence level and showed that a depletion of the anionic peptides by the beads resulted in a relative enrichment of the cationic peptides in the supernatant solution. It could be concluded that after fractionation a dramatic relative enrichment in respect to small- and medium-sized cationic peptides in the solution, characteristics that have been linked to bioactivities, such as anti-microbial and cell-penetrating properties. The results demonstrate the use of the chitosan copper gel bead system in lab scale fractionation of complex hydrolysate mixtures, with the potential to enhance milk hydrolysate bioactivity.

Effect of different botanically-diverse diets on the fatty acid profile, tocopherol content and oxidative stability of beef.

BACKGROUND: Beef from pasture-fed animals is viewed as a healthier and more welfare-friendly alternative to concentrate-fed beef. Botanically-diverse pastures consisting of numerous plant species may alter the fatty acid (FA) profile and the tocopherol content of beef, as well as the oxidative stability of the meat. In the present study, steers were assigned to one of three botanically-diverse diets: perennial ryegrass (PRG), perennial ryegrass + white clover (PRG + WC) or multi-species (MS), all with a finishing diet of the respective botanically-diverse silages plus a cereal-based concentrate, consistent with production systems in Ireland. The FA profile, tocopherol content, oxidative stability and colour of meat during storage were measured. RESULTS: Compared to the other diets, the MS diet resulted in higher proportions of linolenic acid (C18:3n-3c), linoleic acid (C18:2n-6c) and total polyunsaturated fatty acids (PUFA), with higher PUFA:saturated fatty acid and n-6:n-3 ratios in the meat. α-Tocopherol concentrations were lowest in the meat of animals from the MS diet. In uncooked meat, lipid oxidation and colour values were affected by storage time across all diets, whereas the MS diet led to higher hue values only on day 14 of storage. When cooked, meat from animals on PRG + WC and MS diets had higher lipid oxidation on days 1 and 2 of storage than meat from animals on the PRG diet. CONCLUSION: Feeding steers on a botanically-diverse diet consisting of six plant species can improve the n-3 FA and PUFA concentration of beef, affecting the susceptibility of cooked, but not uncooked, beef to oxidation. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Can botanically-diverse pastures positively impact the nutritional and antioxidant composition of ruminant meat? - Invited review.

A desire for more sustainable pasture-based ruminant feeding systems has led to growing interest in utilising botanically-diverse pastures (BDP) over monoculture pastures. Research suggests that, from a human consumption viewpoint, grass-based ruminant feeding leads to more nutritionally desirable fatty acid (FA) and antioxidant concentrations in meat compared with concentrate feeding, which can affect meat quality. The FA, antioxidant and secondary metabolite content of plants differ, depending on species, maturity and seasonality, offering the potential through targeted feeding of BDP to produce meat with superior nutritional and antioxidant profiles. This review explores the effect, if any, that grazing ruminants on BDP has on the FA profile, fat-soluble vitamin, and antioxidant content of meat. The input-output relationship between forage and red meat constituents is complex and is likely affected by species diversity, forage consumption patterns and modulation of rumen fermentation processes. Further investigation is required to fully understand the effect that BDP may have on the composition and quality of ruminant meat.

Chemical and antioxidant characterization of Dovyalis caffra and Dovyalis abyssinica fruits in Kenya.

This study aimed at chemical characterization of Dovyalis caffra (Hook.f. & Harv.) Sim. and Dovyalis abyssinica (A. Rich.) Warb. fruits from Kinamba Town (KT) in Laikipia county and Gitoro Forest (GF) in Meru county of Kenya. All analysed fresh fruit samples had low pH values averaging at 2.67. Other tests showed D. abyssinica-GF to be significantly inferior to D. caffra-GF and D. caffra-KT in terms of TSS, TSS:TTA ratio, and ascorbic acid content. Based on these parameters, D. caffra-KT presents itself with a higher potential for direct consumption as compared to D. caffra-GF. Proximate analysis of dried fruit pulps demonstrated D. abyssinica-GF to be significantly higher in ash content and significantly lower in protein and fat contents compared to the other two samples. There were insignificant difference in the fibre and carbohydrate contents of all the fruit samples. In phytochemical analysis, D. caffra-GF recorded the highest total polyphenol content of 1845 mg Gallic acid equivalent (GAE)/100 g while D. abyssinica-GF reported the lowest figure of 1128 mg GAE/100 g. Flavonoid and simple phenols fractions were in the range of 18.15-26.85% and 73.15-81.85% respectively in all fruit samples. As for antioxidant activity, D. caffra-GF recorded significantly high scores in both DPPH and CUPRAC assays, and D. abyssinica-GF the lowest. The range of DPPH and CUPRAC scores for all samples was 1995-4993 mg l-ascorbic acid/100 g and 1384-2303 mg l-ascorbic acid/100 g respectively. The current study presents the nutritional and health potential of D. caffra and D. abyssinica fruits. This forms a good basis for future adoption and exploitation of these fruits.

Enriching antimicrobial peptides from milk hydrolysates using pectin/alginate food-gels.

Cationic antimicrobial peptides have raised interest as attractive alternatives to classical antibiotics, and also have utility in preventing food spoilage. We set out to enrich cationic antimicrobial peptides from milk hydrolysates using gels containing various ratios of anionic pectin/alginate. All processes were carried out with food-grade materials in order to suggest food-safe methods suited for producing food ingredients or supplements. Hydrolysed caseinate peptides retained in the gel fraction, identified by mass spectrometry, were enriched for potential antimicrobial peptides, as judged by a computational predictor of antimicrobial activity. Peptides retained in a 60:40 pectin:alginate gel fraction had a strong antimicrobial effect against 8 tested bacterial strains with a minimal inhibitory concentration of 1.5-5 mg/mL, while the unfractionated hydrolysate only had a detectable effect in one of the eight strains. Among 110 predicted antimicrobial peptides in the gel fraction, four are known antimicrobial peptides, HKEMPFPK, TTMPLW, YYQQKPVA and AVPYPQR. These results highlight the potential of pectin/alginate food-gels based processes as safe, fast, cost-effective methods to separate and enrich for antimicrobial peptides from complex food protein hydrolysates.

Monitoring the effect of different microwave extraction parameters on the recovery of polyphenols from shiitake mushrooms: Comparison with hot-water and organic-solvent extractions.

The current study aimed to investigate the effect of different microwave-assisted extraction (MAE) parameters (i.e., particle size of the sample, solid-to-liquid ratio, microwave power, and extraction time) on the total phenolic content (TPC), antioxidant capacity (DPPH and CUPRAC), chlorogenic acid and caffeic acid contents of shiitake mushrooms. All the independent variables affected TPC and antioxidant capacity values. Only the sample particle size had no significant effect on phenolic acid contents. The highest TPC, DPPH, and CUPRAC values were obtained when a particle size of 1.75 mm, solid-to-liquid ratio of 1/40, microwave power of 600 W, and extraction time of 15 min were used. The extracts obtained by MAE were compared with those obtained after hot-water extraction (HWE) and organic-solvent extraction (OSE). Scanning electron microscopy (SEM) confirmed that MAE resulted in cell wall disruption which might be due to an increase in the pressure of the inner part of the cells.

Understanding and Controlling Food Protein Structure and Function in Foods: Perspectives from Experiments and Computer Simulations.

The structure and interactions of proteins play a critical role in determining the quality attributes of many foods, beverages, and pharmaceutical products. Incorporating a multiscale understanding of the structure-function relationships of proteins can provide greater insight into, and control of, the relevant processes at play. Combining data from experimental measurements, human sensory panels, and computer simulations through machine learning allows the construction of statistical models relating nanoscale properties of proteins to the physicochemical properties, physiological outcomes, and tastes of foods. This review highlights several examples of advanced computer simulations at molecular, mesoscale, and multiscale levels that shed light on the mechanisms at play in foods, thereby facilitating their control. It includes a practical simulation toolbox for those new to in silico modeling.

Development of a first order derivative spectrophotometry method to rapidly quantify protein in the presence of chitosan and its application in protein encapsulation systems.

A new protein quantification method based on first order derivative spectrophotometry was established to eliminate various interferences, mainly chitosan, to the utmost using bovine serum albumin (BSA) as a model food protein. Absorbance spectra of BSA solutions were recorded and their first order derivative calculated. The values of derivative absorbance at 288 nm were used to generate linear calibration curve of BSA. The new method was applied in entrapping BSA into chitosan-tripolyphosphate beads. A general calibration curve was established with a CI (width of 95% confidence interval of three repeat measurements of unknown samples) less than 0.0262 g/L and a LOQ (limit of quantification) of 0.11 g/L, showing excellent tolerance to various interferences, which was further verified by the good mass balance of BSA during encapsulation. Overall, the method successfully eliminated the interferences from chitosan and other factors to facilitate the measurement of protein in complicated environments.

Incorporation of bioactive dairy hydrolysate influences the stability and digestion behaviour of milk protein stabilised emulsions.

The physical stability of emulsions containing bioactive ingredients is an important aspect of functional food development. This research investigated the effects of a bioactive dairy hydrolysate with anti-inflammatory effects on the properties of oil-in-water emulsions (23% rapeseed oil and 1.5% w/w protein). This was determined by monitoring the effects of various combinations of sodium caseinate (NaCas) and NaCas hydrolysate (NaCasH) (NaCas : NaCasH; 100 : 0, 40 : 60, 30 : 70, 20 : 80 and 0 : 100) on the physico-chemical characteristics (particle size distribution, microstructure, adsorption of protein to the interface, viscosity and creaming stability) of emulsions. Currently, there is growing interest in designing functional foods that modulate lipid digestion. Therefore, emulsion breakdown and subsequent release of free fatty acids (FFAs) from selected NaCasH stabilised emulsions (40 : 60 and 0 : 100) was monitored during in vitro gastrointestinal digestion and compared to the behaviour of emulsions stabilised by NaCas alone. Inclusion of NaCasH generally decreased the stability of the emulsions except when added at a NaCas : NaCasH ratio of 40 : 60 which resulted in emulsions with equivalent stability to the NaCas stabilised emulsions. Although the 40 : 60 combination provided an emulsion system as stable as NaCas, during simulated digestion, these emulsions demonstrated a slower rate of FFA release. This was attributed to the 40 : 60 stabilised emulsions having much larger flocculated lipid droplets than NaCas emulsions, which resulted in reduced surface area and fewer binding sites for lipase adsorption. Accordingly, the 40 : 60 emulsions were hydrolysed more slowly. Emulsions containing only NaCasH exhibited extensive coalescence prior to and during digestion and thus displayed the slowest release of FFA. The results suggest that including NaCasH in the emulsifier blend yields emulsions with modified digestibility and may form the basis of controlling the digestion and release of fat-soluble nutrients in formulated foods. However, further studies are required to optimise the stability of these emulsions before inclusion in such applications.

Casein Hydrolysate with Glycemic Control Properties: Evidence from Cells, Animal Models, and Humans.

Evidence exists to support the role of dairy derived proteins whey and casein in glycemic management. The objective of the present study was to use a cell screening method to identify a suitable casein hydrolysate and to examine its ability to impact glycemia related parameters in an animal model and in humans. Following screening for the ability to stimulate insulin secretion in pancreatic beta cells, a casein hydrolysate was selected and further studied in the ob/ob mouse model. An acute postprandial study was performed in 62 overweight and obese adults. Acute and long-term supplementation with the casein hydrolysate in in vivo studies in mice revealed a glucose lowering effect and a lipid reducing effect of the hydrolysate (43% reduction in overall liver fat). The postprandial human study revealed a significant increase in insulin secretion ( p = 0.04) concomitant with a reduction in glucose ( p = 0.03). The area under the curve for the change in glucose decreased from 181.84 ± 14.6 to 153.87 ± 13.02 ( p = 0.009). Overall, the data supports further work on the hydrolysate to develop into a functional food product.

In vitro evaluation of chitosan copper chelate gels as a multimicronutrient feed additive for cattle.

BACKGROUND: Effective micronutrient supplementation strategies are critical to ensure optimal health and productivity in livestock. The objective of this study was to develop a copper and vitamin (multimicronutrient) delivery system based on chitosan gel beads, and test its suitability, in vitro, for use as a cattle feed additive. RESULTS: Chitosan was chelated with copper sulfate to produce millimetre-scale gel matrices (∼2 mm). The copper content was significantly increased (from 61 to 95 mg g by adjusting pH to alkaline conditions post bead formation. The beads could subsequently be loaded with the model vitamin riboflavin to levels as high as 324 µg g-1 beads. Restricted rehydration of the dried gel matrices in simulated rumen fluid led to a sustained release of riboflavin with no copper released in these neutral conditions for up to 24 h, demonstrating copper rumen bypass. Moreover, sustained release of the mineral was observed in abomasal conditions of pH 2 over a 3 h period. CONCLUSIONS: The matrices showed rumen bypass for copper yet supplied nutritionally relevant levels of the free mineral in abomasal conditions, as required for effective supplementation in cattle. The controlled-release properties demonstrated by the matrices indicate their potential as a multimicronutrient functional feed additive to enhance cattle nutrition and productivity. © 2018 Society of Chemical Industry.

Peptidomic screening of bitter and nonbitter casein hydrolysate fractions for insulinogenic peptides.

Sodium caseinate hydrolysates (NaCaH) contain biologically active peptides that can positively influence human health. However, their intense bitterness hinders their inclusion in food products. To our knowledge, no studies have investigated whether a correlation between bitterness and bioactivity exists in NaCaH, so it is not yet known what effect selective removal of bitterness has on NaCaH bioactivity. A deeper understanding of the physicochemical characteristics affecting both bitterness and bioactivity is therefore needed. The aim of this study was to use in silico analysis to elucidate the relationship between bitterness and bioactivity of the insulinogenic NaCaH. The NaCaH fractions were generated by membrane filtration and flash chromatography and were subsequently evaluated for bitterness by a sensory panel. In this present study, peptidomic and bioinformatic processing of these NaCaH fractions allowed for the identification of insulinogenic peptides as well as other literature-identified peptides in each of the fractions. The results showed that the most bitter fraction contained the highest abundance of insulinogenic peptides, whereas another bitter fraction contained the highest abundance of other literature-identified bioactive peptides exhibiting angiotensin-converting enzyme-inhibition activity. Although some bioactive peptides were identified in the least bitter fractions, the abundance of these peptides was very low. These observations show a correlation between bitter taste and bioactivity, highlighting potential complications in removing bitterness while maintaining bioactivity. However, as the most bitter fraction contained the highest abundance of insulinogenic peptides, there is potential for using a lower dose of this enriched bioactive fraction to exert health benefits. The second most bitter fraction contained a very low abundance of insulinogenic peptides and other bioactive peptides. Therefore, removal of this fraction could reduce the NaCaH product's bitterness without significantly altering overall bioactive potential.

Entrapment of proteins and peptides in chitosan-polyphosphoric acid hydrogel beads: A new approach to achieve both high entrapment efficiency and controlled in vitro release.

Bovine serum albumin (BSA), whey protein isolate (WPI), insulin and a casein hydrolysate were entrapped in chitosan-polyphosphoric acid (PPA) beads. The in vitro release of protein from the beads in simulated gastric fluid (SGF, pH 3) and simulated intestinal fluid (SIF, pH 7) was evaluated. High entrapment efficiencies were achieved for intact proteins (>95% in all cases) but entrapment was lower for the casein hydrolysate (circa 50%), possibly indicating a physical or steric entrapment of the proteins in these chitosan-PPA beads. Inhibited release of BSA, in both SGF and SIF, was achieved with low PPA concentration. Insulin and WPI were effectively retained in SGF and gradually released in SIF. Peptides from casein hydrolysate were partially (circa 35%) but quickly released in SGF with no further release in SIF. Overall, these results indicate that chitosan-PPA beads show potential for lower gastrointestinal delivery of bioactive protein material.

Validation of a paper-disk approach to facilitate the sensory evaluation of bitterness in dairy protein hydrolysates from a newly developed food-grade fractionation system.

Casein-hydrolysates (NaCaH) are desirable functional ingredients, but their bitterness impedes usage in foods. This study sought to validate a paper-disk approach to help evaluate bitterness in NaCaHs and to develop a food-grade approach to separate a NaCaH into distinct fractions, which could be evaluated by a sensory panel. Membrane filtration generated <0.2-µm and <3-kDa permeates. Further fractionation of the <3-kDa permeate by flash-chromatography generated four fractions using ethanol (EtOH) concentrations of 5, 10, 30 and 50%. As some fractions were poorly soluble in water, the fractions were resolubilzed in EtOH and impregnated into paper-disks for sensory evaluation. Bitterness differences observed in the membrane fractions using this sensory evaluation approach reflected those observed for the same fractions presented as a liquid. The flash-chromatography fractions increased in bitterness with an increase in hydrophobicity, except for the 50% EtOH fraction which had little bitterness. Amino acid analysis of the fractions showed enrichment of different essential amino acids in both the bitter and less bitter fractions. Practical Applications: The developed food-grade fractionation system, allowed for a simple and reasonably scaled approach to separating a NaCaH, into physicochemically different fractions that could be evaluated by a sensory panel. The method of sensory evaluation used in this study, in which NaCaH samples are impregnated into paper-disks, provided potential solutions for issues such as sample insolubility and limited quantities of sample. As the impregnated paper-disk samples were dehydrated, their long storage life could also be suitable for sensory evaluations distributed by mail for large consumer studies. The research, in this study, allowed for a greater understanding of the physicochemical basis for bitterness in this NaCaH. As some essential amino acids were enriched in the less bitter fractions, selective removal of bitter fractions could allow for the incorporation of the less bitter NaCaH fractions into food products for added nutritional value, without negatively impacting sensory properties. There is potential for this approach to be applied to other food ingredients with undesirable tastes, such as polyphenols.

Entrapment of protein in chitosan-tripolyphosphate beads and its release in an in vitro digestive model.

This research sought to evaluate the entrapment and in vitro release behaviour of bovine serum albumin (BSA) in chitosan-tripolyphosphate (TPP) hydrogel beads. Beads were manufactured by extruding gel forming solutions containing varying concentrations of chitosan (1-2.5%w/w) and BSA (0.25-10%w/w) into TPP solutions ranging in concentration from 0.1 to 10%w/w and in ionic strength from 0.16 to 0.67M at pH values of 4, 5 and 9.4. Beads produced at a low TPP concentration of 0.4% w/w had the highest BSA entrapment efficiency (71.6±0.7%) and inhibited BSA release in simulated gastric fluid (SGF) to a greater extent. Increasing chitosan concentration resulted in a higher protein entrapment efficiency, but lowered the overall release. Increasing TPP concentration or the BSA concentration loaded, led to early release in SGF. The results indicate that the utilization of lower concentrations of TPP is a good approach to improve the protein retention ability of chitosan-TPP beads in a simulated gastric environment.

Kinetics of immobilisation and release of tryptophan, riboflavin and peptides from whey protein microbeads.

This study investigated the kinetics of immobilisation and release of riboflavin, amino acids and peptides from whey microbeads. Blank whey microbeads were placed in solutions of the compounds. As the volume of microbeads added to the solution was increased, the uptake of the compounds increased, to a maximum of 95% for the pentapeptide and 56%, 57% and 45% for the dipeptide, riboflavin and tryptophan respectively, however, the rate of uptake remained constant. The rate of uptake increased with increasing molecule hydrophobicity. The opposite was observed in the release studies, the more hydrophobic compounds had lower release rate constants (kr). When whey microbeads are used as sorbents, they show excellent potential to immobilise small hydrophobic molecules and minimise subsequent diffusion, even in high moisture environments.

Whey microbeads as a matrix for the encapsulation and immobilisation of riboflavin and peptides.

Whey microbeads manufactured using a cold-set gelation process, have been used to encapsulate bioactives. In this study whey microbeads were used to encapsulate riboflavin using 2 methods. Riboflavin was added to the microbead forming solution however diffusional losses of riboflavin occurred during the subsequent bead preparation. To overcome riboflavin loss, a second approach to 'load' whey microbeads by soaking in riboflavin was assessed. Significantly (p⩽0.05) higher concentrations of riboflavin were obtained in 'loaded' microbeads (361 mg/L) compared to riboflavin added to the microbead forming solution (48 mg/L). Riboflavin uptake by the microbeads was shown to be via a partition process. As partitioning is often driven by hydrophobic interactions the uptake of amino acids and peptides of varying hydrophobicities by the microbeads was examined. The % encapsulation increased with increasing molecule hydrophobicity with a maximum of 89% encapsulation. Whey microbeads are well suited to act as sorbents for encapsulation.

Cold-set whey protein microgels as pH modulated immobilisation matrices for charged bioactives.

The ability of cold-set whey protein microgels to function as pH-sensitive immobilisation matrices for bioactives was investigated. A pH dependent interaction was confirmed between the microgels and charged bioactives and this binding was impeded by the presence of competing ions in the solution, suggesting an electrostatic interaction. The use of a computer generated prediction model for the pH-dependent association of the microgels and further bioactives (including cationic and anionic peptides) was validated. The prediction model was efficient at determining the pH at which the maximum microgel-bioactive interaction occurred. This study highlights the capabilities of these food-grade whey based microgels as matrices that enable the immobilisation of a variety of bioactives by a charge interaction, and shows the potential for these matrices to function as smart delivery systems, in which uptake and release of bioactives is facilitated by environmental pH change.

Cold-set whey protein microgels containing immobilised lipid phases to modulate matrix digestion and release of a water-soluble bioactive.

This study investigated the in-vitro digestibility of cold-set whey protein (WP) microgels prepared by two gelation methods (external and internal) containing lipids (0%, 10% or 20% w/w). The incorporation of lipids into these matrices achieved higher entrapment of the bioactive vitamin riboflavin, as well as significant reductions in rates of both the digestion of the protein matrix, and the subsequent diffusion of the water-soluble bioactive. A biexponential model accounted for the contribution of digestion- and diffusion-driven mechanisms in describing the release of riboflavin into enzyme containing simulated gastrointestinal fluids. In particular, for external gelation microgels, as the lipid load within the matrices increased, the contribution of a faster diffusion-driven release was almost completely negated by a slower digestion-assisted release. Lipid loads provided a composite matrix capable of alternating from a burst to a sustained release of bioactive.

Oxidative stability of water/oil mixtures as influenced by the addition of free Cu2+ or Cu-alginate gel beads.

The formation of Cu-alginate complexes and the impact of free or bound copper on the oxidative stability of model water/oil mixtures containing edible sunflower or corn oil were examined. Equilibrium dialysis showed that copper binding capacity of alginate increased proportionally with copper concentration and the binding was rapid. The results indicated that 25mM CuCl2 was necessary in obtaining beads of spherical shape and adequate mechanical strength (0.45N at 80% compression) to avoid rupture during mixing, whereas lower CuCl2 concentrations resulted in weak gel beads (0.34N) of irregular shape. When Cu-alginate beads were dispersed in the aqueous phase to give 0.5mM copper, the peroxide value of water/corn oil mixtures was 3.7mEq peroxide/kg oil after 7days. Corn oil-containing mixtures with 0.5mM free Cu2+ in the aqueous phase had a peroxide value ∼3 times higher (P<0.001) after the same storage time. However, copper binding by alginate did not significantly reduce the peroxide values of the water/sunflower oil mixtures. Results indicated that the binding of pro-oxidant minerals, such as copper, in alginate beads can reduce the levels of oxidation in water/oil mixtures.