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.

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.

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.

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.

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.

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.

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.

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.

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.

Concentrated Pickering emulsions stabilised by hemp globulin-caseinate nanoparticles: tuning the rheological properties by adjusting the hemp globulin : caseinate ratio.

Industrial hemp (Cannabis sativa L.) is an underutilised novel protein source. However, the utilisation of hemp seed protein is limited by its low solubility in water. Soluble nanoparticles were made by complexing hemp globulin (HG) with sodium caseinate (SC) via a pH-cycling method. Oil-in-water Pickering emulsions were made with these co-assembled protein nanoparticles. The emulsions were composed of 70% oil phase and 30% water phase (v/v), and contained 2% protein (w/v, pure SC or HG-SC nanoparticles with an HG : SC ratio of 1 : 2 or 1 : 1). All emulsions were stable during 21 days of storage, as there was no phase separation, coalescence or flocculation. At day 0, all emulsions were solid-like (G' > G'') regardless of the protein composition. The rheological properties of the emulsions during storage could be tuned by controlling the HG : SC ratio in the HG-SC nanoparticles, i.e. the emulsions became more solid-like over time when there was more HG in the nanoparticles. In contrast, emulsions stabilised by pure SC became more liquid-like during storage. The internal structure and interactions within the emulsions were evaluated by fitting frequency sweep test data according to a co-operative theory of flow. The result suggested that the solid-like emulsion resulted from stronger short-range interactions between flocs of oil droplets, which developed during storage when there was more HG in the HG-SC nanoparticles, and not from the formation of a three-dimensional network. These HG-SC nanoparticles can be used to control the rheological properties of an emulsion during its shelf life.

Nanoemulsion structure and food matrix determine the gastrointestinal fate and in vivo bioavailability of coenzyme Q10.

In this work, we sought to incorporate coenzyme Q10-loaded nanoemulsions into a food system and to understand the impact of food digestion on the in vivo bioavailability of this bioactive compound. We selected octenyl succinic anhydride modified starch as emulsifier to prepare the nanoemulsions (with approximately 200 nm droplet diameter) after comparing with two other food-grade surfactants (whey protein isolate and lecithin) in terms of their colloidal stability during simulated gastrointestinal digestion. The change in ζ-potential revealed that the initial emulsifier might be partially replaced by bile salts under intestinal conditions, and the mixed micelles formed after digestion showed an apparent permeability coefficient of 4.79 × 10-6 cm/s in a rat intestinal epithelial cell line, without compromising the trans-epithelial electrical resistance. In a second step, a high protein beverage that incorporated the coenzyme Q10-loaded nanoemulsion was developed in a food pilot plant. The beverage had a particle size of D4,3 = 18 µm and D3,2 = 2.5 µm, corresponding to its different components. The changes in particle morphology and size distribution were analysed to understand the behaviour of this beverage during simulated gastrointestinal digestion. When coenzyme Q10 was encapsulated into the nanoemulsions and the beverage, its bioavailability in vivo increased 1.8- and 2.8-fold respectively, compared with coenzyme Q10 dissolved in oil. The higher coenzyme Q10 bioavailability in the beverage was probably because of a significantly higher level of lipolytic activity found for beverage than for nanoemulsions during gastrointestinal digestion. These results show the potential of using natural food materials to generate formulations to improve the bioavailability of bioactive compounds. More importantly, we highlight the influence of food digestion (i.e. lipolysis) on the absorption of hydrophobic bioactive components and suggest that food systems can be utilised as a dosage form to further enhance oral bioavailability.

Gastrointestinal digestion of Pickering emulsions stabilised by hydrophobically modified cellulose nanocrystals: Release of short-chain fatty acids.

This study aimed to deliver short-chain fatty acids (SCFAs, including propionic and butyric acids) using Pickering emulsions stabilised by hydrophobically modified cellulose nanocrystals (MCNCs). The emulsions (20 wt% oil, 1 wt% MCNCs) were subjected to two in vitro digestion pathways. In the first pathway, the emulsions were used for direct intestinal digestion by bypassing the gastric phase while in the second pathway, the emulsions were subjected to sequential gastrointestinal digestion. Flocculation of emulsion droplets occurred because of charge screening effects by the gastric electrolytes. Such gastric flocculation reduced the droplet surface area, overall lipolysis kinetics and consequently decreased the extent of SCFA release, latter was 40-45% in the gastric-bypassed emulsions and 30-35% in the sequentially-digested emulsions. High proportion of SCFAs remaining after the intestinal digestion (~65%) shows promise in the use of Pickering emulsions for the colon-targeted delivery of SCFAs.

Food matrix and co-presence of turmeric compounds influence bioavailability of curcumin in healthy humans.

The natural food-derived compound curcumin (from turmeric root) is known for its anti-inflammatory and anti-oxidant effects. However, due to its poor solubility when consumed in isolation, it is poorly bioavailable. In this crossover study we compared the bioavailability of curcumin from a meal containing either curcumin powder, turmeric powder or grated fresh turmeric root, all containing 400 mg of curcumin, along with mashed potatoes and cream. Healthy male participants consumed the meals following overnight fasting, and postprandial blood samples were taken to measure plasma curcuminoids (curcumin, dimethylcurcumin (DMC) and bisdimethylcurcumin (BDMC)). All plasma curcumin values refer to total curcumin (sum of free and conjugated curcumin). The meals were also analysed using confocal laser scanning microscopy to determine the location of curcuminoids. Both of the turmeric meals produced significantly higher amounts (p < 0.05) of plasma curcuminoids at 1-3 hours after the meal was consumed, as compared to the curcumin powder. Plasma curcumin Cmax was 4.9 ng ml-1 95% CI (confidence interval) [2.2, 7.5] for the fresh turmeric meal, 8.4 ng ml-1 95% CI [4.4, 12.48] for the turmeric powder meal and 0.19 ng ml-1 95% [-0.08, 0.47] for the curcumin powder meal. Plasma DMC and BDMC were significantly higher (p < 0.05) following the turmeric powder meal, compared with the fresh turmeric meal and the curcumin powder meal. Microscopy images showed that the curcuminoid particles were mostly confined within curcuminoid cells in the fresh turmeric meal. They were unconfined but in clusters in the turmeric powder meal, while the curcuminoid particles appeared smaller in the curcumin powder meal. Conclusion: curcumin bioavailability is enhanced when consumed as fresh or powdered turmeric, which could be due to the co-presence of other turmeric compounds and/or a turmeric matrix effect.

ε-Polylysine and ß-cyclodextrin assembling as delivery systems for gastric protection of proteins and possibility to enhance intestinal permeation.

An electrostatic nanocomplex between naturally occurring ε-poly-l-lysine (εPL) and ß-cyclodextrin sulphate (sCD) was designed, and its capacity to entrap four model proteins with high or low molecular weight and isoelectric point, i.e., lactoferrin, albumin, actinidin, and lysozyme, was investigated. The optimal formulations gave nanocomplexes with an average diameter around 276 ±â€¯16 nm, a ζ-potential of -39 ±â€¯1.5 mV, and a spherical shape with a core-shell structure. Different strategies were pursued to increase the entrapment efficiency for selected proteins, which led to 40-100% entrapment depending on the protein type. Under simulated gastric conditions with pepsin, the complexes protected lactoferrin and albumin against proteolysis, whereas actinidin and lysozyme were intrinsically stable. In Caco-2 cells, these complexes transiently decreased the trans-epithelial electrical resistance, indicating the potential to enhance the paracellular permeability of bioactive macromolecules. Thus, these εPL-sCD complexes would be a promising system for loading diverse proteins for gastric protection and enhancing intestinal absorption.

Food Proteins: Technological, Nutritional, and Sustainability Attributes of Traditional and Emerging Proteins.

Protein is an essential macronutrient and a key structural component of many foods. The nutritional and technological properties of food protein ingredients depend on their source, extraction and purification, modification during food manufacture, and interactions with other food components. In addition to covering these elements, this review seeks to highlight underappreciated aspects of protein environmental sustainability and explores the potential of cultured meat and insect-derived proteins.

Droplet-Stabilized Oil-in-Water Emulsions Protect Unsaturated Lipids from Oxidation.

Droplet-stabilized emulsions use fine protein-coated lipid droplets (the shell) to emulsify larger droplets of a second lipid (the core). This study investigated the oxidation resistance of polyunsaturated fatty acid (PUFA) oil within droplet-stabilized emulsions, using shell lipids with a range of melting points: olive oil (low melting), trimyristin (high-melting), and palmolein oil (intermediate melting point). Oxidation of PUFA oil was accelerated with a fluorescent lamp in the presence of ferrous iron (100 µM) for 9 days, and PUFA oxidation was monitored via conjugated dienes, lipid hydroperoxides, and hexanal levels. Oxidation was slower in droplet-stabilized emulsions than in conventional emulsions or control emulsions of the same composition as droplet-stabilized emulsions but different structure, and trimyristin gave the greatest oxidation resistance. Results suggest the structured interface of droplet-stabilized emulsions limits contact between pro-oxidants and oxidation-sensitive bioactives encapsulated within, and this antioxidative effect is greatly enhanced with solid surface lipids.

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.