Marsupials give birth to immunologically naïve young after a relatively short gestation period compared to eutherians. Consequently, the joey significantly relies on maternal protection, which is the focus of the present review. The milk and the pouch environment are essential contributors to maternal protection for the healthy development of joeys. In this review, we discuss bioactive components found in the marsupial pouch and milk that form cornerstones of maternal protection. These bioactive components include immune cells, immunoglobulins, the s100 family of calcium-binding proteins, lysozymes, whey proteins, antimicrobial peptides and other immune proteins. Furthermore, we investigated the possibility of the presence of plurifunctional components in milk and pouches that are potentially bioactive. These compounds include caseins, vitamins and minerals, oligosaccharides, lipids, and microRNAs. Where applicable, this review addresses variability in bioactive components during different phases of lactation, designed to fulfil the immunological needs of the growing pouch young. Yet there are numerous additional research opportunities to pursue, including uncovering novel bioactive components, investigating their mode of action, dynamics, stability, and ability to penetrate the gut epithelium ensuring systemic actions.

Methotrexate (MTX) is a cytotoxic immunosuppressant that is widely used in the treatment of tumours, rheumatoid arthritis and psoriasis. This study aims to evaluate the effects of whey proteins on MTX-induced liver and kidney damage by focusing on oxidant–antioxidant systems and eating habits. The study was conducted in four groups of thirty Sprague–Dawley rats (control, control + whey protein concentrate (WPC), MTX, MTX + WPC). A single dose of 20 mg/kg MTX was administered intraperitoneally to the MTX groups. Control and MTX groups were given 2 g/kg WPC by oral gavage every day for 10 d. At the end of day 10, blood samples were drawn and liver and kidney tissues were removed. MTX administration increased the lipid peroxidation level and decreased glutathione level, superoxide dismutase and glutathione-S-transferase activities in the liver and kidney. Administration of WPC significantly reduced the damage caused by MTX in the liver and kidney. While a decrease in serum urea level and an increase in serum creatinine level were detected in the MTX group, WPC administration reversed these results up to control group levels. Administration of WPC to the MTX group significantly reversed the histopathological damage scores of the liver and kidney. WPC administration ameliorated the MTX-induced oxidative damage in the liver and kidney tissues due to its antioxidant properties. Liver and kidney damage can be prevented by using whey proteins as a nutraceutical in MTX therapy. In conclusion, whey proteins demonstrated a protective effect against MTX-induced liver and kidney damage.

Recently there has been a considerable rise in the frequency of metabolic diseases, such as obesity, due to changes in lifestyle and resultant imbalances between energy intake and expenditure. Whey proteins are considered as potentially important components of a dietary solution to the obesity problem. However, the roles of individual whey proteins in energy balance remain poorly understood. This study investigated the effects of a high-fat diet (HFD) containing α-lactalbumin (LAB), a specific whey protein, or the non-whey protein casein (CAS), on energy balance, nutrient transporters expression and enteric microbial populations. C57BL/6J mice (n 8) were given an HFD containing either 20 % CAS or LAB as protein sources or a low-fat diet containing CAS for 10 weeks. HFD-LAB-fed mice showed a significant increase in cumulative energy intake (P=0·043), without differences in body weight, energy expenditure, locomotor activity, RER or subcutaneous and epididymal white adipose tissue weight. HFD-LAB intake led to a decrease in the expression of glut2 in the ileum (P=0·05) and in the fatty acid transporter cd36 (P<0·001) in both ileum and jejunum. This suggests a reduction in absorption efficiency within the small intestine in the HFD-LAB group. DNA from faecal samples was used for 16S rRNA-based assessment of intestinal microbiota populations; the genera Lactobacillus, Parabacteroides and Bifidobacterium were present in significantly higher proportions in the HFD-LAB group. These data indicate a possible functional relationship between gut microbiota, intestinal nutrient transporters and energy balance, with no impact on weight gain.

The experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving β-lactoglobulin (β-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for β-Lg isolation.

Non-fasting TAG – postprandial lipaemia (PPL) – are to a higher degree associated with cardiovascular risk compared with fasting TAG. Dietary protein, especially whey proteins (WP), may lower PPL. We hypothesised that a WP pre-meal (17·6 g protein) consumed 15 v. 30 min before a fat-rich meal reduces the PPL response in subjects with the metabolic syndrome (MetS) and that a WP pre-meal has more potent effects than casein and gluten pre-meals. A total of sixteen subjects with the MetS completed an acute, randomised, crossover trial. WP pre-meals were consumed 15 and 30 min, and casein and gluten 15 min before a fat-rich meal. Blood samples were drawn 360 min postprandially to determine metabolite and hormone responses, S-paracetamol (for assessment of gastric emptying) and amino acids. Insulin and glucagon responses were affected by both timing and protein type (for all P <0·01), with significantly higher concentrations for WP given at –15 min than WP at –30 min and higher responses compared with gluten for the first 30 min after pre-meal consumption (for all P <0·05). The PPL responses changed neither by timing nor by protein type. Glucose-dependent insulinotropic peptide but not glucagon-like peptide 1 responses differed between the three protein types. S-paracetamol concentration was higher for WP (–30 min) than for WP (–15 min) 15 min after the main meal (P = 0·028), and higher for casein and gluten than for WP at time point 30 min (for all P <0·05). In conclusion, the PPL response was not changed by ingestion of a 17·6 g protein pre-meal, whereas both timing and protein quality affected hormone secretion (insulin and glucagon).

This review summarises current knowledge on camel milk proteins, with focus on significant peculiarities in protein composition and molecular properties. Camel milk is traditionally consumed as a fresh or naturally fermented product. Within the last couple of years, an increasing quantity is being processed in dairy plants, and a number of consumer products have been marketed. A better understanding of the technological and functional properties, as required for product improvement, has been gained in the past years. Absence of the whey protein β-LG and a low proportion of к-casein cause differences in relation to dairy processing. In addition to the technological properties, there are also implications for human nutrition and camel milk proteins are of interest for applications in infant foods, for food preservation and in functional foods. Proposed health benefits include inhibition of the angiotensin converting enzyme, antimicrobial and antioxidant properties as well as an antidiabetogenic effect. Detailed investigations on foaming, gelation and solubility as well as technological consequences of processing should be investigated further for the improvement of camel milk utilisation in the near future.

Protein nanofibrils with 10–20 nm diameters were formed by heating whey protein solution at pH 2·0. Nanofibrils solution was deacidified slowly through dialysis followed by adding different amounts of CaCl2 (0–80 mM) into the dialysis water resulting in formation of a soft viscoelastic gel over time. The gel fabricated from the nanofibrils solution dialyzed against distilled water with 0 mM CaCl2 had zero ash content. Fourier transform infra-red spectroscopy revealed a change in the pattern of hydrogen bond formation in gel network by calcium chloride. The higher the ash content of gels, the lower was the storage modulus and fracture stress of samples. Gels with higher ash contents had a more porous microstructure which was attributed to the diminished hydrophobic interactions and hydrogen bonding among nanofibrils by the action of chloride. Higher ash contents also led to higher water holding capacity of gels which was attributed to the influence of the strongly hydrated calcium ions that interacted with the non-charged regions of proteins via site-specific interactions.

The present study examined the underlying mechanisms by which whey protein isolate (WPI) affects energy balance. C57BL/6J mice were fed a diet containing 10 % energy from fat, 70 % energy from carbohydrate (35 % energy from sucrose) and 20 % energy from casein or WPI for 15 weeks. Mice fed with WPI had reduced weight gain, cumulative energy intake and dark-phase VO2 compared with casein-fed mice (P< 0·05); however, WPI intake had no significant effects on body composition, meal size/number, water intake or RER. Plasma levels of insulin, TAG, leptin, glucose and glucagon-like peptide 1 remained unchanged. Notably, the intake of WPI reduced stomach weight and both length and weight of the small intestine (P< 0·05). WPI intake reduced the gastric expression of Wingless/int-1 5a (Wnt5a) (P< 0·01) and frizzled 4 (Fzd4) (P< 0·01), with no change in the expression of receptor tyrosine kinase-like orphan receptor 2 (Ror2) and LDL receptor-related protein 5 (Lrp5). In the ileum, WPI increased the mRNA expression of Wnt5a (P< 0·01) and caused a trend towards an increase in the expression of Fzd4 (P= 0·094), with no change in the expression of Ror2 and Lrp5. These genes were unresponsive in the duodenum. Among the nutrient-responsive genes, WPI specifically reduced ileal mRNA expression of peptide YY (P< 0·01) and fatty acid transporter protein 4 (P< 0·05), and decreased duodenal mRNA expression of the insulin receptor (P= 0·05), with a trend towards a decreased expression of Na–glucose co-transporter 1 (P= 0·07). The effects of WPI on gastrointestinal Wnt signalling may explain how this protein affects gastrointestinal structure and function and, in turn, energy intake and balance.

The prevalence of type 2 diabetes mellitus (T2DM) is increasing and it is estimated that by 2030 approximately 366 million people will be diagnosed with this condition. The use of dipeptidyl peptidase IV (DPP-IV) inhibitors is an emerging strategy for the treatment of T2DM. DPP-IV is a ubiquitous aminodipeptidase that cleaves incretins such as glucagon like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), resulting in a loss in their insulinotropic activity. Synthetic DPP-IV drug inhibitors are being used to increase the half-life of the active GLP-1 and GIP. Dietary intervention is accepted as a key component in the prevention and management of T2DM. Therefore, identification of natural food protein-derived DPP-IV inhibitors is desirable. Peptides with DPP-IV inhibitory activity have been identified in a variety of food proteins. This review aims to provide an overview of food protein hydrolysates as a source of the DPP-IV inhibitory peptides with particular focus on milk proteins. In addition, the proposed modes of inhibition and structure–activity relationship of peptide inhibitors are discussed. Milk proteins and associated peptides also display insulinotropic activity and help regulate blood glucose in healthy and diabetic subjects. Therefore, milk protein derived peptide inhibitors may be a unique multifunctional peptide approach for the management of T2DM.

The intake of whey protein isolate (WPI) is known to reduce high-fat diet (HFD)-induced body-weight gain and adiposity. However, the molecular mechanisms are not fully understood. To this end, we fed C57BL/6J mice for 8 weeks with diets containing 10 % energy as fat (low-fat diet, LFD) or 45 % energy as fat (HFD) enriched with either 20 % energy as casein (LFD and HFD) or WPI (high-fat WPI). Metabolic parameters and the hypothalamic and epididymal adipose tissue expression of energy balance-related genes were investigated. The HFD increased fat mass and plasma leptin levels and decreased the dark-phase energy intake, meal number, RER, and metabolic (VO2 and heat) and locomotor activities compared with the LFD. The HFD increased the hypothalamic tissue mRNA expression of the leptin receptor, insulin receptor (INSR) and carnitine palmitoyltransferase 1b (CPT1b). The HFD also reduced the adipose tissue mRNA expression of GLUT4 and INSR. In contrast, WPI reduced fat mass, normalised dark-phase energy intake and increased meal size in HFD-fed mice. The dietary protein did not have an impact on plasma leptin, insulin, glucose or glucagon-like peptide 1 levels, but increased plasma TAG levels in HFD-fed mice. At a cellular level, WPI significantly reduced the HFD-associated increase in the hypothalamic tissue mRNA expression of the leptin receptor, INSR and CPT1b. Also, WPI prevented the HFD-induced reduction in the adipose tissue mRNA expression of INSR and GLUT4. In comparison with casein, the effects of WPI on energy intake and hypothalamic and adipose tissue gene expression may thus represent a state of reduced susceptibility to weight gain on a HFD.

The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37°C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, β-lactoglobulin (β-LG) and α-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine β-LG was digested and the peptide profiles obtained were compared with those of the caprine β-LG in the digested whey. The bovine β-LG seemed to be more extensively cleaved than the caprine β-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred.

When κ-casein (κ-CN) was added to milk, and the milk was subsequently pH adjusted (pH 6·5−6·9) and heated (90°C/15 min), the serum contained considerably higher levels of denatured whey proteins than the milks without added κ-CN. When milk at pH 6·5−6·9 was heated at 90°C for different times, κ-CN was found in the serum in the early stages of heating and before significant levels of whey proteins were denatured. κ-CN reached its maximum level in the serum before the whey proteins were fully denatured. When milk at pH 6·5−6·9 was heated at 20−90°C for 15 min, κ-CN dissociated from the casein micelles at all temperatures, with the level in the serum increasing with the temperature and the pH at heating. κ-CN dissociated from the micelles at temperatures below those at which significant levels of the whey proteins were denatured. When taken together, the results from these experiments strongly indicate that the dissociation of κ-CN from the micelles can precede the interaction of the denatured whey proteins with κ-CN, and that there is a preferential interaction of the denatured whey proteins with serum-phase κ-CN.

The changes in milk fat globules and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure, were examined. The average fat globule size decreased with increasing homogenization pressure. The total surface protein (mg m−2) of concentrated milk increased after homogenization, the extent of the increase being dependent on the temperature and the pressure of homogenization, as well as on the preheat treatment. The concentrates obtained from high-preheated milks had higher surface protein concentration than the concentrates obtained from low-preheated milks after homogenization. Concentrated milks heat treated at 79°C either before or after homogenization had greater amounts of fat globule surface protein than concentrated milks heat treated at 50 or 65°C. This was attributed to the association of whey protein with the native MFGM (milk fat globule membrane) proteins and the adsorbed skim milk proteins. Also, at the same homogenization temperature and pressure, the amount of whey protein on the fat globule surface of the concentrated milk that was heated after homogenization was greater than that of the concentrated milk that was heated before homogenization. The amounts of the major native MFGM proteins did not change during homogenization, indicating that the skim milk proteins did not displace the native MFGM proteins but adsorbed on to the newly formed surface.

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55°C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20°C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16–18%) and PA (38–62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40°C and at the pressures 450 and 650 MPa at 55°C. Synergistic negative effects on the amount of native β-lactoglobulin were observed at 450 and 650 MPa and on the amount of native α-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native β-lactoglobulin and α-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 °C for 2 min and 95 °C for 8 min. The milk was acidified at 40 °C to a final pH of 4·4 using 20 g glucono-delta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100–600 MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by ~35%. Levels of non-micellar αs1- and β-caseins were increased by treatment [ges ]250 MPa, and were highest after treatment at 400–800 MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from ~82 for untreated milk to ~65 for milk treated at 800 MPa. Milk pH was increased by ~0·07 units after treatment at 100–800 MPa, with no significant difference between treatment pressures. Denaturation of α-lactalbumin occurred at pressures [ges ]400 MPa, and reached >90% after treatment at 800 MPa, whereas β-lactoglobulin (β-lg) was denatured >100 MPa, reaching ~100% after treatment at 400 MPa; after treatment [ges ]400 MPa, all β-lg was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250–800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 °C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.

Effects of atropine on blood plasma amino acid profile and on the yields and concentration of milk components were investigated in 12 Friesian cows in early lactation. Cows were housed indoors and fed with cut pasture ad libitum. Each cow received four treatments over 12 d during a replicated 4×4 Latin square experiment. Treatments were: control (saline); low dose (L; 30 μg atropine/kg body weight (BW)); medium dose (M; 40 μg atropine/kg BW); and 2×L dose, 2 h apart (2×L). On each of four treatment days, cows were milked at about 7.00, after which treatments were administered by subcutaneous injection. Cows were milked again at 2 h, 6 h and 10 h after injection. Milk samples were collected at each milking. Immediately after the 2 h milking, blood samples were drawn from each cow and the second injection was given for the 2×L treatment. Atropine reduced hourly milk yield, and concentrations and hourly yields of total protein, casein, whey protein, α-casein, β-casein, κ-casein, β-lactoglobulin and α-lactalbumin, but by differing amounts. Milk concentrations of bovine serum albumin and immunoglobulin G were increased by atropine, and overall yields of these proteins were mostly unchanged. Atropine lowered concentrations of most, but not all, amino acids in blood plasma, with essential amino acids reduced more than non-essential amino acids. Concentrations of α-amino N in whole blood, and glucose and insulin in blood plasma, fell after atropine injection. There was no difference between the L and M doses of atropine, but the 2×L dose had greater effects on milk composition than the single doses. For yields of milk and milk components, the effect of the 2×L dose was also more persistent. The results highlight the differential synthesis of individual milk proteins, and suggest that atropine might be useful for evaluating the mechanisms regulating milk protein composition.