Preparation of Nanoemulsions of Vitamin A and C by Microfluidization: Efficacy on the Expression Pattern of Milk-Specific Proteins in MAC-T Cells.

In this study, we prepared stabilized vitamin A and C nanoemulsions, and investigated their efficacy on milk-specific proteins in bovine mammary epithelial cells (MAC-T). Emulsions of vitamin A (vit-A) and C (vit-C) were prepared using Lipoid S 75 and microfluidization. The particle size and polydispersity index (PDI) of nanoemulsified vit-A and vit-C were studied. The cytotoxic effect of nanoemulsion-free and nanoemulsified vit-A and vit-C was determined by an MTT assay. In addition, the efficacy of nanoemulsified vit-A and vit-C on the in vitro expression pattern of milk-specific proteins in MAC-T cells was investigated by quantitative RT-PCR. The results showed that the efficacies of stabilized nanoemulsions of vit-A and vit-C were 100% and 92.7%, respectively. The particle sizes were around 475.7 and 225.4 nm, and the zeta potentials were around -33.5 and -21.3 mV, respectively. The expression changes of αs2-, ß- and κ-casein were higher in the presence of a stabilized nanoemulsion of vit-A, compared with nanoemulsion-free vit-A. Furthermore, the expression changes of αs2- and ß-casein were lower and that of κ-casein was higher in the presence of a stabilized nanoemulsion of vit-C, compared with nanoemulsion-free vit-C. Thus, our findings demonstrate the efficacy of nanoemulsified vit-A and vit-C in changing the expression of milk-specific proteins in MAC-T cells.

Lysine Stimulates Protein Synthesis by Promoting the Expression of ATB0,+ and Activating the mTOR Pathway in Bovine Mammary Epithelial Cells.

Background: l-lysine (Lys) is a critical dietary nutrient for mammary gland development and milk production. However, the specific pathways of Lys utilization and how milk protein synthesis is affected in bovine mammary epithelial cells (BMECs) are poorly understood. Objective: We aimed to investigate the effects of Lys on milk protein synthesis and the mechanism of Lys uptake and catabolism in BMECs. Methods: BMECs were cultured in 0, 0.5, 1.0, 1.5, 2.0, 5.0, and 10.0 mmol Lys/L to detect cell viability, or cultured in 0-2.0 mmol Lys/L with l-[ring-3H5] phenylalanine to study the effect of Lys on protein turnover, or cultured in Krebs buffer with [U-14C] l-Lys to quantify Lys metabolism. In some experiments, BMECs were cultured in a conditioned medium alone or including 1.0 mmol Lys/L and 2-amino-endo-bicyclo [2.2.1] heptane-2-carboxylic acid (BCH) for 24 h to analyze the expression of amino acid transporter B (0+) (ATB0,+), mammalian target of rapamycin (mTOR), and Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) pathways. Results: Including 1.0 mmol Lys/L in cultures increased cell viability by 17-47% and protein synthesis by 7-23%, whereas protein degradation was inhibited by 4-64% compared with BMECs cultured with 0, 0.5, or 2.0 mmol Lys/L (all P ≤ 0.05). Studies that used [U-14C] l-Lys showed that most Lys was incorporated into proteins (90%), whereas the remainder was either oxidized into CO2 (4%) or used as a substrate for aspartate (3%) and histidine synthesis (3%). Furthermore, Lys significantly increased expression of ATB0,+ (71% mRNA and 44% protein), STAT5 (27% mRNA and 21% phosphorylated proteins), and mTOR (51% mRNA and 22% phosphorylated proteins) compared with cells without Lys. Conclusions: Lys promoted protein synthesis, mostly through enhancing uptake by ATB0,+ and the mTOR and JAK2-STAT5 pathways. Understanding the utilization of Lys in BMECs provides insights into the role of amino acid nutrition in bovine milk production.

The effect of bleaching agents on the degradation of vitamins and carotenoids in spray-dried whey protein concentrate.

Previous research has shown that bleaching affects flavor and functionality of whey proteins. The role of different bleaching agents on vitamin and carotenoid degradation is unknown. The objective of this study was to determine the effects of bleaching whey with traditional annatto (norbixin) by hydrogen peroxide (HP), benzoyl peroxide (BP), or native lactoperoxidase (LP) on vitamin and carotenoid degradation in spray-dried whey protein concentrate 80% protein (WPC80). An alternative colorant was also evaluated. Cheddar whey colored with annatto (15 mL/454 L of milk) was manufactured, pasteurized, and fat separated and then assigned to bleaching treatments of 250 mg/kg HP, 50 mg/kg BP, or 20 mg/kg HP (LP system) at 50°C for 1 h. In addition to a control (whey with norbixin, whey from cheese milk with an alternative colorant (AltC) was evaluated. The control and AltC wheys were also heated to 50°C for 1 h. Wheys were concentrated to 80% protein by ultrafiltration and spray dried. The experiment was replicated in triplicate. Samples were taken after initial milk pasteurization, initial whey formation, after fat separation, after whey pasteurization, after bleaching, and after spray drying for vitamin and carotenoid analyses. Concentrations of retinol, a-tocopherol, water-soluble vitamins, norbixin, and other carotenoids were determined by HPLC, and volatile compounds were measured by gas chromatography-mass spectrometry. Sensory attributes of the rehydrated WPC80 were documented by a trained panel. After chemical or enzymatic bleaching, WPC80 displayed 7.0 to 33.3% reductions in retinol, ß-carotene, ascorbic acid, thiamin, α-carotene, and α-tocopherol. The WPC80 bleached with BP contained significantly less of these compounds than the HP- or LP-bleached WPC80. Riboflavin, pantothenic acid, pyridoxine, nicotinic acid, and cobalamin concentrations in fluid whey were not affected by bleaching. Fat-soluble vitamins were reduced in all wheys by more than 90% following curd formation and fat separation. With the exception of cobalamin and ascorbic acid, water-soluble vitamins were reduced by less than 20% throughout processing. Norbixin destruction, volatile compound, and sensory results were consistent with previous studies on bleached WPC80. The WPC80 colored with AltC had a similar sensory profile, volatile compound profile, and vitamin concentration as the control WPC80.

Arginine Supplementation Recovered the IFN-γ-Mediated Decrease in Milk Protein and Fat Synthesis by Inhibiting the GCN2/eIF2α Pathway, Which Induces Autophagy in Primary Bovine Mammary Epithelial Cells.

During the lactation cycle of the bovine mammary gland, autophagy is induced in bovine mammary epithelial cells (BMECs) as a cellular homeostasis and survival mechanism. Interferon gamma (IFN-γ) is an important antiproliferative and apoptogenic factor that has been shown to induce autophagy in multiple cell lines in vitro. However, it remains unclear whether IFN-γ can induce autophagy and whether autophagy affects milk synthesis in BMECs. To understand whether IFN-γ affects milk synthesis, we isolated and purified primary BMECs and investigated the effect of IFN-γ on milk synthesis in primary BMECs in vitro. The results showed that IFN-γ significantly inhibits milk synthesis and that autophagy was clearly induced in primary BMECs in vitro within 24 h. Interestingly, autophagy was observed following IFN-γ treatment, and the inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Furthermore, mechanistic analysis confirmed that IFN-γ mediated autophagy by depleting arginine and inhibiting the general control nonderepressible-2 kinase (GCN2)/eukaryotic initiation factor 2α (eIF2α) signaling pathway in BMECs. Then, it was found that arginine supplementation could attenuate IFN-γ-induced autophagy and recover milk synthesis to some extent. These findings may not only provide a novel measure for preventing the IFN-γ-induced decrease in milk quality but also a useful therapeutic approach for IFN-γ-associated breast diseases in other animals and humans.

Effects of rumen-protected choline supplementation on metabolic and performance responses of transition dairy cows.

The objective of this experiment was to compare metabolic and milk production parameters in dairy cows supplemented and nonsupplemented with rumen-protected choline (RPC) during the transition period. Twenty-three nonlactating, multiparous, pregnant Holstein cows were ranked by BW and BCS 21 d before expected date of calving and immediately were assigned to receive (n = 12) or not receive (control; n = 11) RPC until 45 d in milk (DIM). Cows supplemented with RPC received (as-fed basis) 50 and 100 g/d of RPC (18.8% choline) before and after calving, respectively. Before calving, cows were maintained in 2 drylot pens according to treatment with ad libitum access to corn silage, and individually they received (as-fed basis) 3 kg/cow daily of a concentrate. Upon calving, cows were moved to 2 adjacent drylot pens according to treatment, milked twice daily, offered (as-fed basis) 35 kg/cow daily of corn silage, and individually received a concentrate formulated to meet their nutritional requirements after milking. The RPC was individually offered to cows as a topdressing into the morning concentrate feeding. Before calving, cow BW and BCS were recorded weekly, and blood samples were collected every 5 d beginning on d -21 relative to expected calving date. Upon calving and until 45 DIM, BW and BCS were recorded weekly, individual milk production was recorded daily, and milk samples were collected once a week and analyzed for fat, protein, and total solids. Blood samples were collected every other day from 0 to 20 DIM and every 5 d from 20 to 45 DIM. Based on actual calving dates, cows receiving RPC or control began receiving treatments 16.8 ± 1.7 and 17.3 ± 2.0 d before calving, respectively. No treatment effects were detected (P ≥ 0.18) on postpartum concentrate intake, BW and BCS, or serum concentrations of cortisol, ß-hydroxybutyrate, NEFA, glucose, and IGF-I. Cows supplemented with RPC had greater (P ≤ 0.01) mean serum haptoglobin and insulin concentrations compared with control. Cows supplemented with RPC had greater (P < 0.01) milk protein, total solids (P < 0.01), and milk fat concentrations (P = 0.09) compared with control. No treatment effects were detected (P ≥ 0.43) for milk yield parameters, such as fat-corrected or solids-corrected milk yield. In conclusion, supplementing RPC to transition dairy cows increased haptoglobin and insulin concentrations and benefited milk composition.

Identification of MFG-E8 as a novel therapeutic target for diseases.

INTRODUCTION: Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a peripheral membrane glycoprotein has been widely studied in recent years due to its omnipresent locations, express and multiple functions. Traditionally, MFG-E8 was identified as an outstanding factor for phagocytosis of apoptotic cells and a significant factor in immune systems. Recent studies with some new findings have shed an interesting light on the old peripheral membrane glycoprotein in various diseases. AREAS COVERED: In inflammatory and the systemic lupus erythematosus-type autoimmune disease, age-related diseases and tumors, MFG-E8 plays a multifunctional role in attenuating inflammation and improving prognosis, healing wound and remodeling arterial and enhancing tumorigenicity and cancer metastasis. This review provides a comprehensive view on the latest advances in the field. The summarized knowledge will help to explore the potential therapeutic roles of MFG-E8 and to design MFG-E8-based strategies for the treatment of these diseases. EXPERT OPINION: Though the exact roles of MFG-E8 have not been fully elucidated in diseases. MFG-E8 may serve as a promising therapeutic strategy.

Regulation of milk protein solubility by a whey-derived proline-rich peptide product.

The effects of a bovine whey peptide product enriched in proline (wPRP) on the solubility of milk proteins were tested under ambient conditions or following heat treatment at 75 and 100 °C, for 1 and 15 min, followed by post-incubation storage at either ambient temperature or 4 °C for up to 7 d. wPRP promoted solubilisation of milk proteins in a concentration-dependent manner without heat treatment and also after heat treatment at 75 and 100 °C, and the effect was enhanced after storage under either ambient or refrigerated storage conditions. Interactions of wPRP and milk proteins were monitored by particle size analysis and tryptic digestion and specifically linked with solubilisation of αS1 casein (αS1-Cn), which supported observed changes in milk protein solubility. The results suggested that wPRP preferably prevented or reversed physical versus covalent protein aggregation, with the relaxation of hydrophobic interactions at 4 °C providing an additive effect. This application of wPRP represents a novel approach to stabilisation of dairy proteins following thermal processing with industrial usefulness yet to be explored.

Effect of bleaching permeate from microfiltered skim milk on 80% serum protein concentrate.

Whey proteins that have been removed before the cheese-making process are referred to as "native" whey proteins or milk serum proteins. Because serum proteins isolated directly from milk are not exposed to the cheese-making process, they are free from functional or sensory effects arising from this process. Whey proteins used in food and beverage applications are largely derived from annatto-colored Cheddar cheese. Some of the annatto is left in the whey and this color is converted to a colorless compound by bleaching. The effect of bleaching serum proteins on flavor and functionality of spray-dried protein provides a platform to investigate the effect of bleaching free from the confounding effects of cheese manufacture. The objective of this study was to characterize and compare the sensory and functional properties of 80% milk serum protein concentrate (SPC80) produced from bleached and unbleached microfiltration (MF) permeate made from skim milk with and without added annatto color. Colored and uncolored MF permeates were bleached with benzoyl peroxide (BP) or hydrogen peroxide (HP), ultrafiltered, diafiltered, and spray-dried. The SPC80 from unbleached colored and uncolored MF permeates were manufactured as controls. All treatments were manufactured in triplicate. All SPC80 were evaluated by sensory testing, instrumental analyses, functionality, color, and proximate analysis. The HP-bleached SPC80 was higher in lipid oxidation compounds than BP-bleached or unbleached SPC80, specifically hexanal, heptanal, nonanal, decanal, and 2,3-octadienone. The HP treatments were higher in aroma intensity and cardboard and fatty flavors compared with the unbleached and BP-bleached SPC80. The SPC80 bleached with BP had lower concentrations of norbixin compared with SPC80 bleached with HP. Functionality testing demonstrated that HP treatments had more soluble protein after 10min of heating at 90°C and pH 4.6 and pH 7 compared with the no bleach and BP treatments, regardless of additional color. Foams generated from bleached SPC80 were more stable than those from unbleached SPC80, and those bleached with HP were lower in yield stress than other SPC80. Overall, HP bleaching destroyed less norbixin and caused more lipid oxidation and subsequent off-flavors than did BP bleaching. However, the heat stability of SPC80 was enhanced by HP bleaching compared with control treatments or BP bleaching.

Starch addition in renneted milk gels: partitioning between curd and whey and effect on curd syneresis and gel microstructure.

Milk gels were made by renneting and acidifying skim milk containing 5 different starches, and then compressed by centrifugation to express whey and simulate curd syneresis during the manufacture of low-fat cheese. A series of 17 starches were examined, with 5 starches being selected for in-depth analysis: a modified waxy corn starch (WC), a waxy rice starch (WR), an instant tapioca starch (IT), a modified tapioca starch (MT), and dextrin (DX). Milks containing WC, WR, and DX were given a 72°C heat treatment, whereas those containing IT and MT had a 30-min treatment at 66°C that matched their optimum gelatinization treatments. Curd yields were calculated by weight, estimated starch content in whey was measured gravimetrically by alcohol precipitation, and starch retention in curd was calculated. Curd yields were 13.1% for the control milk (no added starch) and 18.4, 20.7, 21.5, 23.5, and 13.2% for the gels containing starches WC, WR, IT, MT, and DX, respectively. Estimated starch retentions in the curd were, respectively, 71, 90, 90, 21, and 1%. Laser scanning confocal microscopy was used to determine the location of the starches in the curd and their interaction with the protein matrix. Waxy corn, WR, and IT starches have potential to improve texture of low-fat cheese because they had high retention in the curd and they generated interruptions in the protein matrix network that may have helped limit extensive protein-protein interactions. Modified tapioca starch interfered with formation of the protein structure of the curd and produced a soft noncohesive gel, even though most (79%) of the MT starch was lost in the whey. Few distinct starch particles were present in the MT curd network. Dextrin was not retained in the curd and did not disrupt the protein network, making it unsuitable for use in low-fat cheese.

Exopolysaccharides modify functional properties of whey protein concentrate.

The objective of this research was to produce whey protein concentrate (WPC) with modified functionality using exopolysaccharide- (EPS) producing cultures. Two different EPS-producing cultures, Lactococcus lactis ssp. cremoris JFR and Streptococcus thermophilus, producing EPS1 and EPS2 respectively, were used in this study. One EPS-nonproducing commercial cheese culture (DVS 850; Chr. Hansen, Milwaukee, WI) was used as the control. Reconstituted sweet whey powder was used in this study to eliminate variations from fresh whey. Cultures grown overnight in reconstituted WPC (10% wt/vol) were added, directly or after overnight cooling (cooled EPS), at 2% (wt/vol) to 6% (wt/wt) solution of reconstituted whey. Whey was then high-temperature, short-time pasteurized at 75 °C for 35s and ultrafiltered to a volume reduction factor of 5. Ultrafiltered whey (retentate) was spray dried at inlet and outlet air temperatures of 200 and 90 °C, respectively, to obtain WPC. In general, the solubility of WPC was higher at pH 7 than at pH 3. Whey protein concentrate containing EPS2 exhibited higher protein solubility than did WPC containing no EPS. Also, the presence of EPS in WPC decreased protein denaturation. The emulsifying ability of WPC containing EPS was higher than that in control. Addition of EPS to WPC significantly enhanced its gelling ability. Foam overrun and hydrophobicity of WPC were not affected by addition of EPS. In conclusion, data obtained from this study show that EPS modify WPC functionality. The extent of modification depends on the type of EPS. Cooling of culture containing EPS before its addition to whey further reduced WPC protein denaturation and increased its solubility at pH 7 and gel hardness.

Effect of bleaching whey on sensory and functional properties of 80% whey protein concentrate.

Whey is a highly functional food that has found widespread use in a variety of food and beverage applications. A large amount of the whey proteins produced in the United States is derived from annatto-colored Cheddar cheese. Color from annatto is undesirable in whey and must be bleached. The objective of this study was to compare 2 commercially approved bleaching agents, benzoyl peroxide (BP) and hydrogen peroxide (HP), and their effects on the flavor and functionality of 80% whey protein concentrate (WPC80). Colored and uncolored liquid wheys were bleached with BP or HP, and then ultrafiltered, diafiltered, and spray-dried; WPC80 from unbleached colored and uncolored Cheddar whey were manufactured as controls. All treatments were manufactured in triplicate. The WPC80 were then assessed by sensory, instrumental, functionality, color, and proximate analysis techniques. The HP-bleached WPC80 were higher in lipid oxidation compounds (specifically hexanal, heptanal, octanal, nonanal, decanal, dimethyl disulfide, and 1-octen-3-one) and had higher fatty and cardboard flavors compared with the other unbleached and BP-bleached WPC80. The WPC80 bleached with BP had lower norbixin concentrations compared with WPC80 bleached with HP. The WPC powders differed in Hunter color values (L, a, b), with bleached powders being more white, less red, and less yellow than unbleached powders. Bleaching with BP under the conditions used in this study resulted in larger reductions in yellowness of the powders made from whey with annatto color than did bleaching with HP. Functionality testing demonstrated that whey bleached with HP treatments had more soluble protein after 10 min of heating at 90°C at pH 4.6 and pH 7 than the no-bleach and BP treatments, regardless of additional color. Overall, HP bleaching caused more lipid oxidation products and subsequent off-flavors compared with BP bleaching. However, heat stability of WPC80 was enhanced by HP bleaching compared with control or BP-bleached WPC80.

The use of lactoperoxidase for the bleaching of fluid whey.

Lactoperoxidase (LP) is the second most abundant enzyme in bovine milk and has been used in conjunction with hydrogen peroxide (H2O2) and thiocyanate (SCN⁻) to work as an antimicrobial in raw milk where pasteurization is not feasible. Thiocyanate is naturally present and the lactoperoxidase system purportedly can be used to bleach dairy products, such as whey, with the addition of very little H2O2 to the system. This study had 3 objectives: 1) to quantify the amount of H2O2 necessary for bleaching of fluid whey using the LP system, 2) to monitor LP activity from raw milk through manufacture of liquid whey, and 3) to compare the flavor of whey protein concentrate 80% (WPC80) bleached by the LP system to that bleached by traditional H2O2 bleaching. Cheddar cheese whey with annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a standard Cheddar cheesemaking procedure. Various levels of H2O2 (5-100 mg/kg) were added to fluid whey to determine the optimum concentration of H2O2 for LP activity, which was measured using an established colorimetric method. In subsequent experiments, fat-separated whey was bleached for 1h with 250 mg of H2O2/kg (traditional) or 20 mg of H2O2/kg (LP system). The WPC80 was manufactured from whey bleached with 250 mg of H2O2/kg or 20mg of H2O2/kg. All samples were subjected to color analysis (Hunter color values and norbixin extraction) and proximate analysis (fat, protein, and moisture). Sensory and instrumental volatile analyses were conducted on WPC80. Optimal LP bleaching in fluid whey occurred with the addition of 20mg of H2O2/kg. Bleaching of fluid whey at either 35 or 50°C for 1 h with LP resulted in > 99% norbixin destruction compared with 32 or 47% destruction from bleaching with 250 mg of H2O2/kg, at 35 or 50°C for 1 h, respectively. Higher aroma intensity and increased lipid oxidation compounds were documented in WPC80 from bleached whey compared with WPC80 from unbleached whey. Monitoring of LP activity throughout cheese and whey manufacture showed that LP activity sharply decreased after 30 min of bleaching (17.01 ± 1.4 to < 1 U/mL), suggesting that sufficient bleaching takes place in a very short amount of time. Lactoperoxidase averaged 13.01 ± 0.7 U/mL in unpasteurized, fat-separated liquid whey and 138.6 ± 11.9 U/mL in concentrated retentate (11% solids). Lactoperoxidase may be a viable alternative for chemical whey bleaching.

Effect of temperature and bleaching agent on bleaching of liquid Cheddar whey.

The use of whey protein as an ingredient in foods and beverages is increasing, and thus demand for colorless and mild-tasting whey protein is rising. Bleaching is commonly applied to fluid colored cheese whey to decrease color, and different temperatures and bleach concentrations are used. The objectives of this study were to compare the effects of hot and cold bleaching, the point of bleaching (before or after fat separation), and bleaching agent on bleaching efficacy and volatile components of liquid colored and uncolored Cheddar whey. First, Cheddar whey was manufactured, pasteurized, fat-separated, and subjected to one of a number of hot (68°C) or cold (4°C) bleaching applications [hydrogen peroxide (HP) 50 to 500 mg/kg; benzoyl peroxide (BP) 25 to 100 mg/kg] followed by measurement of residual norbixin and color by reflectance. Bleaching agent concentrations were then selected for the second trial. Liquid colored Cheddar whey was manufactured in triplicate and pasteurized. Part of the whey was collected (no separation, NSE) and the rest was subjected to fat separation (FSE). The NSE and FSE wheys were then subdivided and bleaching treatments (BP 50 or 100 mg/kg and HP 250 or 500 mg/kg) at 68°C for 30 min or 4°C for 16 h were applied. Control NSE and FSE with no added bleach were also subjected to each time-temperature combination. Volatile compounds from wheys were evaluated by gas chromatography-mass spectrometry, and norbixin (annatto) was extracted and quantified to compare bleaching efficacy. Proximate analysis, including total solids, protein, and fat contents, was also conducted. Liquid whey subjected to hot bleaching at both concentrations of HP or at 100mg/kg BP had greater lipid oxidation products (aldehydes) compared with unbleached wheys, 50mg/kg BP hot-bleached whey, or cold-bleached wheys. No effect was detected between NSE and FSE liquid Cheddar whey on the relative abundance of volatile lipid oxidation products. Wheys bleached with BP had lower norbixin content compared with wheys bleached with HP. Bleaching efficacy of HP was decreased at 4°C compared with 68°C, whereas that of BP was not affected by temperature. These results suggest that fat separation of liquid Cheddar whey has no effect on bleaching efficacy or lipid oxidation and that hot bleaching may result in increased lipid oxidation in fluid whey.

Potentiation of platelet-derived growth factor receptor-ß signaling mediated by integrin-associated MFG-E8.

OBJECTIVE: Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this α(v) integrin ligand enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice. Inhibition of MFG-E8 production or function attenuates platelet-derived growth factor-BB (PDGF-BB)-induced migration of pericyte/pericyte precursor-like 10T1/2 cells in vitro. Herein, we describe mechanisms by which MFG-E8 modulates PDGF-BB:PDGF receptor ß (PDGFRß) signaling in 10T1/2 cells. METHODS AND RESULTS: Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFRß and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFRß in PDGF-BB-treated 10T1/2 cells and reduced PDGFRß-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and α(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFRß was enhanced by PDGF-BB. Ubiquitination of PDGFRß was also increased in MFG-E8 small interfering RNA-transfected cells. CONCLUSION: Integrin α(v)-bound MFG-E8 associates with PDGFRß and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFRß, delays receptor degradation, potentiates downstream signaling, and enhances migration of 10T1/2 cells. MFG-E8 may promote angiogenesis, in part, via cell autonomous actions on pericytes or pericyte precursors that result in enhanced PDGF-BB:PDGFRß signaling mediated via integrin-growth factor receptor cross-talk.

Influence of bleaching on flavor of 34% whey protein concentrate and residual benzoic acid concentration in dried whey proteins.

Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations in dried whey products. No legal limit exists in the United States for BP use in whey, but international concerns exist. The objectives of this study were to determine the effect of hydrogen peroxide (HP) or BP bleaching on the flavor of 34% WPC (WPC34) and to evaluate residual BA in commercial and experimental WPC bleached with and without BP. Cheddar whey was manufactured in duplicate. Pasteurized fat-separated whey was subjected to hot bleaching with either HP at 500 mg/kg, BP at 50 or 100 mg/kg, or no bleach. Whey was ultrafiltered and spray dried into WPC34. Color [L*(lightness), a* (red-green), and b* (yellow-blue)] measurements and norbixin extractions were conducted to compare bleaching efficacy. Descriptive sensory and instrumental volatile analyses were used to evaluate bleaching effects on flavor. Benzoic acid was extracted from experimental and commercial WPC34 and 80% WPC (WPC80) and quantified by HPLC. The b* value and norbixin concentration of BP-bleached WPC34 were lower than HP-bleached and control WPC34. Hydrogen peroxide-bleached WPC34 displayed higher cardboard flavor and had higher volatile lipid oxidation products than BP-bleached or control WPC34. Benzoyl peroxide-bleached WPC34 had higher BA concentrations than unbleached and HP-bleached WPC34 and BA concentrations were also higher in BP-bleached WPC80 compared with unbleached and HP-bleached WPC80, with smaller differences than those observed in WPC34. Benzoic acid extraction from permeate showed that WPC80 permeate contained more BA than did WPC34 permeate. Benzoyl peroxide is more effective in color removal of whey and results in fewer flavor side effects compared with HP and residual BA is decreased by ultrafiltration and diafiltration.

Effect of liquid retentate storage on flavor of spray-dried whey protein concentrate and isolate.

The objective of this study was to determine the effects of holding time of liquid retentate on flavor of spray-dried whey proteins: Cheddar whey protein isolate (WPI) and Mozzarella 80% whey protein concentrate (WPC80). Liquid WPC80 and WPI retentate were manufactured and stored at 3°C. After 0, 6, 12, 24, and 48h, the product was spray-dried (2kg) and the remaining retentate held until the next time point. The design was replicated twice for each product. Powders were stored at 21°C and evaluated every 4 mo throughout 12 mo of storage. Flavor profiles of rehydrated proteins were documented by descriptive sensory analysis. Volatile components were analyzed with solid phase microextraction coupled with gas chromatography mass spectrometry. Cardboard flavors increased in both spray-dried products with increased retentate storage time and cabbage flavors increased in WPI. Concurrent with sensory results, lipid oxidation products (hexanal, heptanal, octanal) and sulfur degradation products (dimethyl disulfide, dimethyl trisulfide) increased in spray-dried products with increased liquid retentate storage time, whereas diacetyl decreased. Shelf stability was decreased in spray-dried products from longer retentate storage times. For maximum quality and shelf life, liquid retentate should be held for less than 12h before spray drying.

The effect of starter culture and annatto on the flavor and functionality of whey protein concentrate.

The flavor of whey protein can carry over into ingredient applications and negatively influence consumer acceptance. Understanding sources of flavors in whey protein is crucial to minimize flavor. The objective of this study was to evaluate the effect of annatto color and starter culture on the flavor and functionality of whey protein concentrate (WPC). Cheddar cheese whey with and without annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a mesophilic lactic starter culture or by addition of lactic acid and rennet (rennet set). Pasteurized fat-separated whey was then ultrafiltered and spray dried into WPC. The experiment was replicated 4 times. Flavor of liquid wheys and WPC were evaluated by sensory and instrumental volatile analyses. In addition to flavor evaluations on WPC, color analysis (Hunter Lab and norbixin extraction) and functionality tests (solubility and heat stability) also were performed. Both main effects (annatto, starter) and interactions were investigated. No differences in sensory properties or functionality were observed among WPC. Lipid oxidation compounds were higher in WPC manufactured from whey with starter culture compared with WPC from rennet-set whey. The WPC with annatto had higher concentrations of p-xylene, diacetyl, pentanal, and decanal compared with WPC without annatto. Interactions were observed between starter and annatto for hexanal, suggesting that annatto may have an antioxidant effect when present in whey made with starter culture. Results suggest that annatto has a no effect on whey protein flavor, but that the starter culture has a large influence on the oxidative stability of whey.

Deletion of arginine from an abomasal infusion of amino acids does not decrease milk protein yield in Holstein cows.

This study was undertaken to determine if a limited supply of Arg would alter milk and milk protein yields, as well as mammary uptake of AA and energetic substrates. Six lactating Holstein cows (199 ± 5 d in milk) were used in a replicated 3 × 3 Latin square balanced for residual effects with 14-d periods. The diet was formulated to supply 100% of the National Research Council net energy requirement and 72% of the metabolizable protein requirement. The treatments were randomly distributed as abomasal infusions of (1) water (CTL), (2) a mixture of essential AA (EAA) excluding Arg (ARG-), or (3) a mixture of EAA including Arg (ARG+). The profile of EAA in the infusates was the same as that found in casein with the exception that methionine was increased to maintain a 3:1 ratio of digestible lysine:methionine (total dietary+infusion). Milk protein yield was increased by the ARG+ compared with the CTL treatment and deletion of Arg in the infusate (ARG-) did not impair this response. Deletion of Arg from the EAA mixture decreased the mammary uptake of Arg relative to that of the CTL treatment, and although the uptake:output ratio decreased from 2.52 (ARG+) to 2.12 (ARG-), it was still largely in excess of Arg secretion in milk protein. Otherwise, Arg deletion did not affect any of the measured parameters (no significant difference between ARG- and ARG+) except Arg and urea arterial concentrations. In support of the increased yields of milk protein and lactose, mammary uptake of the group 2 AA (Ile, Leu, Lys, and Val) increased and the uptake:output ratio tended to increase from 1.04 to 1.23. The mammary uptake:milk protein output ratio was not different from 1 and not different among treatments for the group 1 AA (His, Met, Phe+Tyr, Trp). Mammary uptake of energetic substrates did not vary across treatments, although milk lactose yield increased with the ARG+ treatment relative to CTL. These results indicate that deletion of Arg has minimal effects on milk and milk component yields when the remaining EAA are supplied in sufficient amounts despite decreased mammary Arg uptake and that group 2 AA seem to be involved in the mammary gland to support the lactose yield response to EAA infusion.

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women.

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7-10 days.

Administration of bovine somatotropin in early lactation: a meta-analysis of production responses by multiparous Holstein cows.

A meta-analysis was conducted to assess production responses before 90 d in milk (DIM) when bovine somatotropin (bST) administration was initiated between 5 and 35 DIM. The database was developed from 13 studies of multiparous cows that were published between 1985 and 2006 and from an unpublished study that complied with the study selection criteria. The database included results from 842 cows and provided 50 treatment means for the effect of bST on 3.5% fat-corrected milk (FCM) in early lactation. Effects of bST were investigated using mixed model procedures that included fixed (intercept and slope) and random (intercept and slope) effects for independent variables. Yields of milk (38.6 +/- 1.3 kg/d) and FCM (37.6 +/- 1.6 kg/d) by control cows before 90 DIM were increased by 2.6 +/- 0.8 and 3.2 +/- 0.6 kg/d by bST administration. Fat content in milk from bST-treated cows was 0.31 +/- 0.10 percentage units greater than that from control cows (3.46 +/- 0.13%) but milk protein content (2.95 +/- 0.03%) was not altered by bST. Milk fat (1.39 +/- 0.10 kg/d) and protein (1.15 +/- 0.04 kg/d) yields by controls were increased 0.16 +/- 0.03 and 0.07 +/- 0.03 kg/d by bST, respectively. Dry matter intake and body weight loss were not altered by bST before 90 DIM, but duration of negative energy balance was prolonged and overall energy balance during this interval reduced when cows were treated with bST. Results are consistent with the premise that bST-treated cows partition nutrients and energy toward milk synthesis for a longer duration and thus likely need a longer interval to replenish their body reserves than cows not treated with bST. Production responses to bST were not altered when cows consumed typical early-lactation diets supplemented with fat except that supplemental fat tended to decrease the magnitude of the effect of bST on milk fat content and decreased the effect of bST on fat and protein yield. Yield of FCM increased curvilinearly with the amount of bST administered. Results indicate that initiation of bST administration to cows before 35 DIM increased FCM yield but the response was at the low end of that typically observed when bST administration is initiated in wk 9 of lactation.