Protein digestion and absorption: the influence of food processing.

The rates of dietary protein digestion and absorption can be significantly increased or decreased by food processing treatments such as heating, gelling and enzymatic hydrolysis, with subsequent metabolic impacts, e.g. on muscle synthesis and glucose homeostasis.This review examines in vivo evidence that industrial and domestic food processing modify the kinetics of amino acid release and absorption following a protein-rich meal. It focuses on studies that used compositionally-matched test meals processed in different ways.Food processing at extremely high temperature at alkaline pH and/or in the presence of reducing sugars can modify amino acid sidechains, leading to loss of bioavailability. Some protein-rich food ingredients are deliberately aggregated, gelled or hydrolysed during manufacture. Hydrolysis accelerates protein digestion/absorption and increases splanchnic utilisation. Aggregation and gelation may slow or accelerate proteolysis in the gut, depending on the aggregate/gel microstructure.Milk, beef and eggs are heat processed prior to consumption to eliminate pathogens and improve palatability. The temperature and time of heating affect protein digestion and absorption rates, and effects are sometimes non-linear. In light of a dietary transition away from animal proteins, more research is needed on how food processing affects digestion and absorption of non-animal proteins.Food processing modifies the microstructure of protein-rich foods, and thereby alters protein digestion and absorption kinetics in the stomach and small intestine. Exploiting this principle to optimise metabolic outcomes requires more human clinical trials in which amino acid absorption rates are measured and food microstructure is explicitly considered, measured and manipulated.

The plasma amino acid response to blended protein beverages: a randomised crossover trial.

Soya-dairy protein blends can extend post-exercise muscle synthesis in young people more than whey protein control. Older adults differ metabolically from young people, and their ability to absorb amino acids from dietary protein is important for muscle function. The objective was to determine how protein source affects postprandial plasma amino acid response and/or metabolomic profile in older adults via a single-blind randomised crossover trial (n 16, males 50-70 years), using three nutritionally equivalent meal replacement drinks containing 30 g protein, from a 1:1 (mass ratio) soya:dairy blend, a 1:2 soya:dairy blend or whey protein. The outcome measures were plasma amino acid concentrations at 0-300 min postprandially and urine metabolomic fingerprint. Soya:dairy drinks gave similar amino acid response in plasma over time and similar urinary metabolite fingerprints. However, there were significant differences in plasma amino acid concentrations and AUC values for the soya:dairy drinks v. the whey protein drink. AUC for Leu, Trp and Lys was lower and AUC for Phe and Pro was higher for the soya:dairy drinks. Differences partly reflected the amino acid profiles of the drinks, but overall plasma amino acid response patterns were qualitatively unchanged. Plasma amino acid differences between the whey protein drink and the soya:dairy blends were reflected in urine metabolite patterns. In conclusion, postprandial plasma amino acid responses were broadly similar, irrespective of protein source (and soya:dairy ratio). There were significant differences for some plasma amino acid concentrations, reflecting different amino acid profiles of the protein source and influencing urine metabolite fingerprints.

Type A and B bovine milks: Heat stability is driven by different physicochemical parameters.

The value of milk hinges on its physicochemical functionality under processing conditions. We examined composition-functionality relationships with individual milks from 24 New Zealand dairy cows, sampled at 3 times over the season. Milks were classified into type A or B, according to the shape of 3-point heat coagulation time versus pH profiles. Milk type changed over the season for half of the cows in the study. Best subsets regression suggested that different factors controlled heat stability in the 2 milk types. Urea concentration was key for both types, but for type A milks, osmotic pressure and milk solids were the most important predictors of heat stability, whereas casein micelle size and ionic calcium predicted heat stability for type B milks. This study revealed that milk type is prone to change over the season, and the findings suggest that optimizing heat stability could be achieved by different means for type A versus type B milks.

Applications of novel processing technologies to enhance the safety and bioactivity of milk.

Bioactive compounds in food can have high impacts on human health, such as antioxidant, antithrombotic, antitumor, and anti-inflammatory activities. However, many of them are sensitive to thermal treatments incurred during processing, which can reduce their availability and activity. Milk, including ovine, caprine, bovine, and human is a rich source of bioactive compounds, including immunoglobulins, vitamins, and amino acids. However, processing by various novel thermal and non-thermal technologies has different levels of impacts on these compounds, according to the studies reported in the literature, predominantly in the last 10 years. The reported effect of these technologies either covers microbial inactivation or the bioactive composition; however, there is a lack of comprehensive compilation of studies that compare the effect of these technologies on bioactive compounds in milk (especially, caprine and ovine) to microbial inactivation at similar settings. This research gap makes it challenging to conclude on the specific processing parameters that could be optimized to achieve targets of microbial safety and nutritional quality at the same time. This review covers the effect of a wide range of thermal and non-thermal processing technologies including high-pressure processing, pressure-assisted thermal sterilization, pulsed-electric field treatment, cold plasma, microwave-assisted thermal sterilization, ultra-high-pressure homogenization, ultrasonication, irradiation on the bioactive compounds as well as on microbial inactivation in milk. Although a combination of more than one technology could improve the reduction of bacterial contaminants to meet the required food safety standards and retain bioactive compounds, there is still scope for research on these hurdle approaches to simultaneously achieve food safety and bioactivity targets.

Nanomechanics of Pectin-Linked ß-Lactoglobulin Nanofibril Bundles.

Nanofibrils of ß-lactoglobulin can be assembled into bundles by site-specific noncovalent cross-linking with high-methoxyl pectin (Hettiarachchi et al. Soft Matter 2016, 12, 756). Here we characterized the nanomechanical properties of bundles using atomic force microscopy and force spectroscopy. Bundles had Gaussian cross sections and a mean height of 17.4 ± 1.4 nm. Persistence lengths were calculated using image analysis with the mean-squared end-to-end model. The relationship between the persistence length and the thickness had exponents of 1.69-2.30, which is consistent with previous reports for other fibril types. In force spectroscopy experiments, the bundles stretched in a qualitatively different manner to fibrils, and some of the force curves were consistent with peeling fibrils away from bundles. The flexibility of pectin-linked nanofibril bundles is likely to be tunable by modulating the stiffness and length of fibrils and the ratio of pectin to fibrils, giving rise to a wide range of structures and functionalities.

Morphology of complexes formed between ß-lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins.

For the optimal use of ß-lactoglobulin nanofibrils as a raw material in biological composites an in-depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, ß-lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (∼48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between ß-lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the ß-lactoglobulin nanofibrils into ordered 'nanotapes'.

ß-Lactoglobulin nanofibrils can be assembled into nanotapes via site-specific interactions with pectin.

Controlling the self-assembly of individual supramolecular entities, such as amyloid fibrils, into hierarchical architectures enables the 'bottom-up' fabrication of useful bionanomaterials. Here, we present the hierarchical assembly of ß-lactoglobulin nanofibrils into the form of 'nanotapes' in the presence of a specific pectin with a high degree of methylesterification. The nanotapes produced were highly ordered, and had an average width of 180 nm at pH 3. Increasing the ionic strength or the pH of the medium led to the disassembly of nanotapes, indicating that electrostatic interactions stabilised the nanotape architecture. Small-angle X-ray scattering experiments conducted on the nanotapes showed that adequate space is available between adjacent nanofibrils to accommodate pectin molecules. To locate the interaction sites on the pectin molecule, it was subjected to endopolygalacturonase digestion, and the resulting products were analysed using capillary electrophoresis and size-exclusion chromatography for their charge and molecular weight, respectively. Results suggested that the functional pectin molecules carry short (<10 residues) enzyme-susceptible blocks of negatively charged, non-methylesterified galacturonic acid residues in the middle of their homogalacturonan backbones (and possibly near their ends), that specifically bind to sites on the nanofibrils. Blocking the interaction sites on the nanofibril surface using small oligomers of non-methylesterified galacturonic acid residues similar in size to the interaction sites of the pectin molecule decreased the nanotape formation, indicating that site-specific electrostatic interactions are vital for the cross-linking of nanofibrils. We propose a structural model for the pectin-cross-linked ß-lactoglobulin nanotapes, the elements of which will inform the future design of bionanomaterials.

Modulating ß-lactoglobulin nanofibril self-assembly at pH 2 using glycerol and sorbitol.

ß-Lactoglobulin (ß-lg) forms fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). When formed at protein concentrations <3%, these fibrils are made up of peptides produced from the acid hydrolysis of the ß-lg monomer. The present study investigated the effects of the polyhydroxy alcohols (polyols) glycerol and sorbitol (0-50% w/v) on ß-lg self-assembly at pH 2. Glycerol and sorbitol stabilize native protein structure and modulate protein functionality by preferential exclusion. In our study, both polyols decreased the rate of ß-lg self-assembly but had no effect on the morphology of fibrils. The mechanism of these effects was studied using circular dichroism spectroscopy and SDS-PAGE. Sorbitol inhibited self-assembly by stabilizing ß-lg against unfolding and hydrolysis, resulting in fewer fibrillogenic species, whereas glycerol inhibited nucleation without inhibiting hydrolysis. Both polyols increased the viscosity of the solutions, but viscosity appeared to have little effect on fibril assembly, and we believe that self-assembly was not diffusion-limited under these conditions. This is in agreement with previous reports for other proteins assembling under different conditions. The phenomenon of peptide self-assembly can be decoupled from protein hydrolysis using glycerol.

In vitro gastric digestion of heat-induced aggregates of ß-lactoglobulin.

In vitro gastric digestion of heat-induced aggregates of ß-lactoglobulin (ß-LG) in simulated gastric fluid was investigated using sodium dodecyl sulfate-PAGE (under nonreducing and reducing conditions), native PAGE, 2-dimensional electrophoresis, and size exclusion chromatography. Heating at 90°C significantly increased the digestibility of ß-LG, with a high initial digestion rate followed by a relatively constant rate of digestion at a high enzyme:substrate (E:S) ratio of 3:1. At a low E:S ratio (1:6), the rate of digestion of ß-LG was slower, and intermediate- and low-molecular-weight species could be seen. The high-molecular-weight nonnative aggregates (e.g., pentamers, tetramers, and trimers) were digested relatively rapidly, whereas some of the nonnative dimers were resistant to digestion and others were digested rapidly. The intermediate-molecular-weight species (21 to 23 kDa) were digested slowly. The digestibility of nonnative ß-LG aggregates varied significantly depending on the E:S ratio and the types of aggregate. Further investigation is necessary to identify and characterize slowly digested dimers and species of intermediate molecular weight.

Modelling Lactation Curves for Dairy Sheep in a New Zealand Flock.

Lactation curves were modelled for dairy sheep in a New Zealand flock, providing information on the lactation yields of milk, fat, protein, and lactose, corrected for 130 days of milking. From 169 ewes, a total of 622 test-day records were obtained during the milk production season of 2021-2022 (from October to January). The flock produced an average of 86.1 kg of milk, 5.1 kg of fat, 4.5 kg of protein, and 4.1 kg of lactose, and moderate to large coefficients of variation were observed (27-31%) for these traits. The lactation persistency of milk, fat, protein, and lactose yields ranged from 52.3 to 72.7%. Analyses of variance for total yield and persistency were performed with an animal model that included the fixed effects of age (parity number), litter size, coat colour, and milking frequency (days in twice-a-day milking) and random residuals. Age and milking frequency were the only factors that significantly affected the yields of milk, fat, protein, and lactose. Age significantly affected the lactation persistency of milk and lactose yields, whereas litter size affected the persistency of protein, and milking frequency affected the persistency of fat. This study on this single flock provides valuable experience for a larger-scale animal breeding programme in New Zealand.

The impact of heating and drying on protease activities of ruminant milk before and after in vitro infant digestion.

This study investigated the effect of heating (63°C/30 min or 75°C/15 s) and drying (spray-drying or freeze-drying) on plasmin, cathepsin D, and elastase activities in bovine, ovine, and caprine milk, compared to non-dried raw milk counterparts. Protease activities and protein hydrolysis were assessed before and after in vitro infant digestion with or without gastric and pancreatic enzymes. At 75°C/15 s, plasmin activity in caprine and ovine milk decreased (69-75%, p<0.05), while cathepsin D activity in spray-dried bovine milk heated increased (2.8-fold, p<0.05). Plasmin and cathepsin D activities increased (<1.2-fold, p<0.05) after in vitro digestion with pancreatin, regardless of milk species. Endogenous milk enzymes hydrolyzed more proteins than gastric enzymes during gastric digestion and contributed to small intestinal digestion. In summary, milk proteases remained active after processing with effects dependent on the species of milk, and they contributed to in vitro protein hydrolysis in the stomach and small intestine.

The use of confocal Raman microscopy and microfluidic channels to monitor the location and mobility of ß-carotene incorporated in droplet-stabilized oil-in-water emulsions.

This study sought to explore the combined use of confocal Raman microscopy and microfluidic channels to probe the location and mobility of hydrophobic antioxidant (ß-carotene) incorporated at the interface of food-grade droplet-stabilized emulsions (DSEs). Microfluidic channels were used to isolate emulsion droplets for efficient investigation of antioxidant mobility. This approach proved more conclusive than fixing the sample in agarose, because a single layer of droplets could be obtained. Results also indicated that the migration of ß-carotene incorporated in shell droplets of olive oil and trimyristin DSEs to core droplets was minimal and beta-carotene remained mostly localised at the interface even after 3 days of production. This work demonstrates that microfluidic isolation of emulsion droplets combined with confocal Raman microscopy can give new insights into the spatial variation of chemical composition within emulsions. This study revealed that the migration of ß-carotene between shell and core was minimal and hence it may be possible to concurrently deliver two incompatible compounds by spatially segregating them between shell and core compartments of DSEs.

Formation of ß-lactoglobulin nanofibrils by microwave heating gives a peptide composition different from conventional heating.

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of ß-lactoglobulin (ß-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of ß-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact ß-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of ß-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of ß-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.

Water dynamics in fresh and frozen yeasted dough.

Water is an integral part of wheat flour dough-the amount, physical state, and location of water are crucial to the formation of a dough that will hold gas and produce an open, aerated crumb structure in the final product. This has been understood for centuries by craft bakers, who were highly attuned to the "feel" of dough in their hands. In the 20th century, empirical instruments were invented that simulated part of the breadmaking process, and their limited predictive capacity made them valuable quality control tools. During the latter decades of the 20th century the cost and availability of advanced instrumental methods for characterizing foods improved dramatically, and facilitated a "fundamental science" approach to food research. The physicochemical mechanisms by which water exerts such a strong influence on the character of dough are now better understood. This review contrasts the empirical and fundamental view points, and summarizes recent knowledge about the roles of water in the manufacture of fresh and frozen yeasted dough.

Effect of calcium on the morphology and functionality of whey protein nanofibrils.

Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl(2), but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl(2) on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl(2) on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Small-amplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl(2) is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.

Soy Protein Pressed Gels: Gelation Mechanism Affects the In Vitro Proteolysis and Bioaccessibility of Added Phenolic Acids.

In this study, a model system of firm tofu (pressed gel) was prepared to study how the coagulation mechanism-acidification with glucono δ-lactone (GDL) or coagulation with magnesium sulphate (MgSO4)-affected the physical properties of the gels along with their in vitro proteolysis (or extent of proteolysis). The two types of gels were also fortified with 3.5 mM protocatechuic (PCA) and coumaric acid (CMA) to test whether they can be used as bioactive delivery systems. Texture analysis showed that all MgSO4-induced gels (fortified and control) had a higher hydration capacity and a weaker texture than the GDL-induced gels (p < 0.05). MgSO4 gels had almost double proteolysis percentages throughout the in vitro digestion and showed a significantly higher amino acid bioaccessibility than the GDL gels (essential amino acid bioaccessibility of 56% versus 31%; p < 0.05). Lastly, both gel matrices showed a similar phenolic acid release profile, on a percentage basis (~80% for PCA and ~100% for CMA). However, GDL gels delivered significantly higher masses of bioactives under simulated intestinal conditions because they could retain more of the bioactives in the gel after pressing. It was concluded that the coagulation mechanism affects both the macro- and microstructure of the soy protein pressed gels and as a result their protein digestibility. Both pressed gel matrices are promising delivery systems for bioactive phenolic acids.

Hemp globulin forms colloidal nanocomplexes with sodium caseinate during pH-cycling.

Seed from industrial hemp (Cannabis sativa L.) contains around 25% protein (mainly globulins) which is easily digested, but the low solubility of hemp globulins (HG) limits their application in many food systems. In this study, the solubility of HG was improved by blending HG with sodium caseinate (SC) and treating with a pH-cycling process. The pH-cycling involved adjusting the pH to 12 and reacting for 1 hr, followed by neutralisation to pH 7. Nanoparticles composed of HG and SC (Z-average diameter ≈ 130 nm) were formed after the pH-cycling, and the solubility of HG increased to > 80% when there was more than 1% of SC for 1% of HG. These HG|SC nanoparticles were monodisperse (PDI < 0.17) and ζ-potential was ≈ -17 mV. Hydrogen bonding is the main forces that assembles HG|SC nanoparticles because the nanoparticles dissociated by heat treatment (up to 60 °C) or urea, which is an effective hydrogen bond breaker. HG|SC nanoparticles will aggregate irreversibly above 60 °C, possibly due to thiol-disulphide exchange. The nanoparticles were heat-stable as the Z-average diameter was only 229 nm after heating (90 °C, 30 min). N-ethylmaleimide blocked free thiol groups on HG and resulted in less disulphide-linked HG aggregation after pH- cycling, which in turn lead to smaller HG|SC nanoparticles and a bimodal particle size distribution, indicating the importance of disulphide bond for the formation of monodisperse HG|SC nanoparticles. The soluble and heat-stable HG|SC nanoparticles could be used to increase the hemp protein content in beverages and emulsions.

In vitro dynamic gastric digestion of soya protein/milk protein blended beverages: influence of protein composition and co-processing.

The gastric digestion behaviours of blended protein beverages containing different ratios of casein, whey protein and soya protein that were heat-treated at 60 °C or 80 °C were investigated using an in vitro dynamic human gastric simulator. All beverages showed protein aggregation and curd formation under gastric conditions; the extent of protein aggregation/curd formation was dependent on the protein composition of the beverage. The beverages with high proportions of milk proteins showed a greater degree of curd formation during gastric digestion, due to the coagulation of casein micelles. These beverages also showed a lower rate of protein emptying from the stomach. Increasing the relative proportion of soya protein in the beverage increased the rate of protein emptying, because of changes in the curd structure caused by the interactions of soya protein with casein micelles. There was a relatively small effect of heating to 80 °C on the protein emptying rate and the protein content of the clot. The results suggest that protein beverages with different protein compositions could be developed to provide controlled delivery of proteins and amino acids.

Heat-Treatments Affect Protease Activities and Peptide Profiles of Ruminants' Milk.

Proteases present in milk are heat-sensitive, and their activities increase or decrease depending on the intensity of the thermal treatment applied. The thermal effects on the protease activity are well-known for bovine milk but poorly understood for ovine and caprine milk. This study aimed to determine the non-specific and specific protease activities in casein and whey fractions isolated from raw bovine, ovine, and caprine milk collected in early lactation, and to determine the effects of low-temperature, long-time (63°C for 30 min) and high-temperature, short-time (85°C for 5 min) treatments on protease activities within each milk fraction. The non-specific protease activities in raw and heat-treated milk samples were determined using the substrate azocasein. Plasmin (the main protease in milk) and plasminogen-derived activities were determined using the chromogenic substrate S-2251 (D-Val-Leu-Lys-pNA dihydrochloride). Peptides were characterized using high-resolution liquid chromatography coupled with tandem mass spectrometry. The activity of all native proteases, shown as non-specific proteases, was similar between raw bovine and caprine milk samples, but lower (P < 0.05) than raw ovine milk in the whey fraction. There was no difference (P > 0.05) between the non-specific protease activity of the casein fraction of raw bovine and caprine milk samples; both had higher activity than ovine milk. After 63°C/30 min, the non-specific protease activity decreased (44%; P > 0.05) for the bovine casein fraction only. In contrast, the protease activity of the milk heated at 85°C/5 min changed depending on the species and fraction. For instance, the activity decreased by 49% for ovine whey fraction, but it increased by 68% for ovine casein fraction. Plasmin and plasminogen were in general inactivated (P > 0.05) when all milk fractions were heated at 85°C/5 min. Most of the peptides present in heat-treated milk were derived from ß-casein and αS1-casein, and they matched the hydrolysis profile of cathepsin D and plasmin. Identified peptides in ruminant milk samples had purported immunomodulatory and inhibitory functions. These findings indicate that the non-specific protease activity in whey and casein fractions differed between ruminant milk species, and specific thermal treatments could be used to retain better protease activity for all ruminant milk species.

A protocol combining breath testing and ex vivo fermentations to study the human gut microbiome.

This protocol describes the application of breath testing and ex vivo fermentations to study the association between breath methane and the composition and functionality of the gut microbiome. The protocol provides a useful systems biology approach for studying the gut microbiome in humans, which combines standardized methods in human breath testing and fecal sampling. The model described is accessible and easy to repeat, but its relative simplicity means that it can deviate from human physiological conditions.