X.Y. Mao

Effect of NaCl addition during diafiltration on the solubility, hydrophobicity, and disulfide bonds of 80% milk protein concentrate powder

We investigated the surface hydrophobicity index based on different fluorescence probes [1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-propionyl-2-(N,N-dimethylamino)-naphthalene (PRODAN)], free sulfhydryl and disulfide bond contents, and particle size of 80% milk protein concentrate (MPC80) powders prepared by adding various amounts of NaCl (0, 50, 100, and 150 mM) during the diafiltration process. The solubility of MPC80 powder was not strictly related to surface hydrophobicity. The MPC80 powder obtained by addition of 150 mM NaCl during diafiltration had the highest solubility but also the highest ANS-based surface hydrophobicity, the lowest PRODAN-based surface hydrophobicity, and the least aggregate formation. Intermolecular disulfide bonds caused by sulfhydryl-disulfide interchange reactions and hydrophobic interactions may be responsible for the lower solubility of the control MPC80 powder. The enhanced solubility of MPC80 powder with addition of NaCl during diafiltration may result from the modified surface hydrophobicity, the reduced intermolecular disulfide bonds, and the associated decrease in mean particle size. Addition of NaCl during the diafiltration process can modify the strength of hydrophobic interactions and sulfhydryl-disulfide interchange reactions and thereby affect protein aggregation and the solubility of MPC powders.

Zinc-binding capacity of yak casein hydrolysate and the zinc-releasing characteristics of casein hydrolysate-zinc complexes.

Many factors affect the bioavailability of dietary Zn, which leads to its low availability in some food systems and Zn nutrient deficiency. However, some proteins or peptides can form complexes with Zn and increase its absorption and bioavailability in intestinal conditions. The purpose of this work was to determine the Zn-binding activity of yak casein hydrolysate (YCH) and examine its stability, solubility, and dialyzability in a simulated intestinal environment. The Zn-binding activity of YCH, prepared using alcalase, pepsin, trypsin, Flavozyme (Novo Nordisk Biochem Inc., Franklinton, NC), or papain, was investigated. Evidence for the formation of complexes between Zn and YCH also were detected by UV-visible spectroscopy and Fourier transform infrared spectroscopy. Results were that YCH prepared with alcalase and trypsin possessed the highest Zn-binding capacity compared with YCH prepared with pepsin, Flavozyme, or papain. The 6-h YCH obtained with alcalase showed the highest Zn-binding capacity. Compared with native yak casein, the Zn-binding activity of YCH was significantly lower, but its solubility and dialyzability were markedly higher under intestinal basic pH ranges. This is important because high solubility and dialyzability is associated with better bioavailability. Both UV-visible spectroscopy and Fourier transform infrared spectroscopy spectra indicated that some sites of YCH can bind with Zn ions and form complexes that make Zn more soluble and dialyzable under simulated intestinal conditions. Therefore, YCH-Zn complexes may have potential to improve Zn bioavailability.

Preparation and characterization of β-lactoglobulin hydrolysate-iron complexes.

The purpose of this study was to determine the best preparation condition of β-lactoglobulin hydrolysate-iron complexes and characterize its structural transformation both before and after binding using the UV-visible absorption spectrum, Fluorescence spectrum, and Fourier transform infrared spectroscopy. Results showed that β-lactoglobulin hydrolysates obtained with alcalase after hydrolysis for 6h possessed the highest iron-binding capacity. The highest yield of complexes was obtained when the mass ratio between β-lactoglobulin hydrolysate and Fe(3+) reached 40:1, with the optimal pH value of 7.0. All of the spectra indicated that some sites such as amido bonds transformed during chelation, and nitrogen atoms could chelate with Fe(3+) to form coordinate bonds by offering electron pairs. Therefore, β-lactoglobulin hydrolysate-iron complexes may be good carriers for iron and possess great potential to be used as iron supplements.

Ameliorating effects of casein glycomacropeptide on obesity induced by high-fat diet in male Sprague-Dawley rats

The effect of casein glycomacropeptide (GMP) as a specific regulating mediator in obese rats induced by high-fat (HF) diet was investigated. Male obese Sprague-Dawley (SD) rats induced by high-fat diet for 8 weeks period were fed high-fat, high-fat with GMP of 100 mg/kg BW (HFLG), 200 mg/kg BW (HFMG) and 400mg/kg BW (HFHG) for 6 weeks. Compared with the high-fat control (HFC) group GMP supplementation significantly decreased adipose tissue weight, activity of fatty acid synthase (FAS) and glycerol-3-phosphate dehydrogenase (GPDH). Hepatic lipid droplet size, plasma and hepatic lipid levels markedly reduced. Moreover, GMP reduces plasma total cholesterol and low-density lipoprotein (LDL) cholesterol as well as hepatic-cholesterol and triglycerides. The liver steatosis observed in obese rats was also prevented by GMP supplement. In addition, GMP significantly diminished mitochondrial and liver malondialdehyde (MDA) production, and obviously elevated the activities of mitochondrial and hepatic superoxidase dismutase (SOD) and glutathione peroxidase (GSH-Px). Leptin production and proinflammatory cytokines such as TNF-α and IL-6 secretion decreased. Taken together, GMP can reduce lipid accumulation and enhance antioxidant capability of obese rats. It suggests that GMP can counteract high-fat diet-induced obesity, which might make it a potential ingredient with anti-obesity activity.

Endotoxin-Binding Peptides Derived from Casein Glycomacropeptide Inhibit Lipopolysaccharide-Stimulated Inflammatory Responses via Blockade of NF-κB activation in macrophages

Systemic low-grade inflammation and increased circulating lipopolysaccharide (LPS) contribute to metabolic dysfunction. The inhibitory effects and underlying molecular mechanisms of casein glycomacropeptide (GMP) hydrolysate on the inflammatory response of LPS-stimulated macrophages were investigated. Results showed that the inhibitory effect of GMP hydrolysates obtained with papain on nitric oxide (NO) production were obviously higher than that of GMP hydrolysates obtained with pepsin, alcalase and trypsin (p < 0.05), and the hydrolysate obtained with papain for 1 h hydrolysis (GHP) exhibited the highest inhibitory effect. Compared with native GMP, GHP markedly inhibited LPS-induced NO production in a dose-dependent manner with decreased mRNA level of inducible nitric oxide synthase (iNOS). GHP blocked toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway activation, accompanied by downregulation of LPS-triggered significant upregulation of tumor necrosis factor (TNF)-α and interleukin (IL)-1β gene expression. Furthermore, GHP could neutralize LPS not only by direct binding to LPS, but also by inhibiting the engagement of LPS with the TLR4/MD2 complex, making it a potential LPS inhibitor. In conclusion, these findings suggest that GHP negatively regulates TLR4-mediated inflammatory response in LPS-stimulated RAW264.7 cells, and therefore may hold potential to ameliorate inflammation-related issues.

Casein glycomacropeptide hydrolysate exerts cytoprotection against H2O2-induced oxidative stress in RAW 264.7 macrophages via ROS-dependent heme oxygenase-1 expression

The aim of this study was to investigate the antioxidant potential of bovine casein glycomacropeptide (GMP) and its hydrolysate, as well as to determine effects of GMP and its hydrolysate on hydrogen peroxide (H2O2)-induced oxidative stress in RAW 264.7 macrophages. In comparison with native GMP, GMP hydrolysate obtained with papain for 1 h hydrolysis (GHP) exerted higher free radical-scavenging capacity, ferrous ion (Fe2+)-chelating ability, and ferric reducing (FRAP) activity. GHP significantly blocked H2O2-induced intracellular reactive oxygen species (ROS) generation as well as cell death, and the cytoprotective effects of GHP were partially reversed by co-treatment with zinc(II)-protoporphyrin IX (ZnPPIX), a specific inhibitor of HO-1. GHP induced nuclear translocation of the nuclear factor-erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in RAW 264.7 macrophages. The chemical antioxidant, N-acetyl cysteine (NAC), significantly reduced GHP-induced HO-1 expression and Nrf2 activation by blocking intracellular ROS production. Additionally, pretreatment with GHP enhanced cellular antioxidant enzymes activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in H2O2-damaged cells. The antioxidant activity of GHP may be attributed to its amino acid profiles. Compared with native GMP, GHP had higher contents of alanine, glycine, glutamic acid, aspartic acid and branched chain amino acids (BCAAs, leucine, isoleucine and valine). BCAAs-enriched GHP may possess a potential to ameliorate oxidative stress-related diseases.

Casein glycomacropeptide-derived peptide IPPKKNQDKTE ameliorates high glucose-induced insulin resistance in HepG2 cells via activation of AMPK signaling

SCOPE Recently, casein glycomacropeptide (GMP)-derived peptide was found to possess potent antioxidant and anti-inflammatory activities. In this study, the improvement effects and underlying molecular mechanisms of GMP-derived peptide on hepatic insulin resistance were investigated. METHODS AND RESULTS The peptide IPPKKNQDKTE was identified from GMP papain hydrolysates by LC-ESI-MS/MS. Effects of IPPKKNQDKTE on glucose metabolism and expression levels of the hepatic insulin signaling proteins in high glucose-induced insulin-resistant HepG2 cells were evaluated. Results showed that IPPKKNQDKTE dose-dependently increased glucose uptake and intracellular glycogen in insulin-resistant HepG2 cells without affecting cell viability. IPPKKNQDKTE increased the phosphorylation of Akt and GSK3β and decreased the expression levels of p-GS, G6Pase and PEPCK. These IPPKKNQDKTE-mediated protection effects were reversed by PI3K/Akt inhibitor LY294002, showing the mediatory role of PI3K/Akt. Moreover, treatment with IPPKKNQDKTE reduced IRS-1 Ser307 phosphorylation and increased phosphorylation of AMPK. Knockdown AMPK using siRNA in HepG2 cells increased Ser307 phosphorylation of IRS-1 and reduced Akt phosphorylation in IPPKKNQDKTE-treated insulin-resistant cells. CONCLUSION IPPKKNQDKTE prevents high glucose-induced insulin resistance in HepG2 cells by modulating the IRS-1/PI3K/Akt signaling pathway through AMPK activation, indicating that IPPKKNQDKTE plays a potential role in the prevention and treatment of hepatic insulin resistance and type 2 diabetes.

In vitro iron absorption of α-lactalbumin hydrolysate-iron and β-lactoglobulin hydrolysate-iron complexes

To study the feasibility of promoting iron absorption by peptides derived from α-lactalbumin and β-lactoglobulin, the present work examined the transport of iron across Caco-2 monolayer cell as in vitro model. Caco-2 cells were seeded in bicameral chambers with α-lactalbumin hydrolysate-Fe (α-LAH-Fe) complex and β-lactoglobulin hydrolysate-Fe (β-LGH-Fe) complex, α-LAH and iron mixture, β-LGH and iron mixture, FeSO4 and ascorbic acid mixture, and FeSO4. In addition, the cytotoxicity of α-LAH-Fe and β-LGH-Fe complexes were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The iron absorption and ferritin content were assessed using the coupled in vitro digestion/Caco-2 cell model. Results support that peptide-iron complexes can promote ferritin formation and it is possible to apply β-LGH-Fe complexes as iron-fortified supplements with high iron absorbability.

Attenuating effect of casein glycomacropeptide on proliferation, differentiation, and lipid accumulation of in vitro Sprague-Dawley rat preadipocytes

Food components with the ability to suppress preadipocyte proliferation and intracellular lipid accumulation may be helpful in the prevention of obesity, which is a worldwide health concern. Casein glycomacropeptide (GMP), which has pronounced gastric inhibitory activity, could potentially possess fat synthesis inhibition properties and an obesity-alleviating capacity. The objective of the present study was to investigate the effect of GMP on the proliferation and differentiation of preadipocytes as well as triglyceride accumulation and glycerol-3-phosphate dehydrogenase activity in preadipocytes isolated from Sprague-Dawley rats. Different dosages (0, 0.31, 0.625, 1.25, 2.5, and 5.0 mg/mL) of GMP were co-incubated with preadipocytes. The proliferation activity of preadipocytes significantly decreased in the GMP-treated group compared with that of the control group without GMP supplementation. The GMP exhibited an inhibitory effect against preadipocyte proliferation in a dose-dependent manner; the maximal antiproliferative effect was obtained with 2.5 mg/mL. The GMP also attenuated differentiation, as revealed by decreased lipid content, and the effect was more pronounced when cells were treated with GMP before or at the beginning of differentiation induction than at later stages of cell differentiation. Cultured preadipocytes treated with GMP accumulated fewer triglycerides and had lower glycerol-3-phosphate dehydrogenase activity than did the control cells without GMP supplementation. In conclusion, GMP can inhibit the proliferation, differentiation, and lipid accumulation of preadipocytes in vitro.

Casein Glycomacropeptide Hydrolysates Exert Cytoprotective Effect against Cellular Oxidative Stress by Up-Regulating HO-1 Expression in HepG2 Cells

Oxidative stress is considered as an important mediator in the progression of metabolic disorders. The objective of this study was to investigate the potential hepatoprotective effects and mechanisms of bovine casein glycomacropeptide hydrolysates (GHP) on hydrogen peroxide (H2O2)-induced oxidative damage in HepG2 cells. Results showed that GHP significantly blocked H2O2-induced intracellular reactive oxygen species (ROS) generation and cell viability reduction in a dose-dependent manner. Further, GHP concentration-dependently induced heme oxygenase-1 (HO-1) expression and increased nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation. Moreover, pretreatment of GHP increased the activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2), which were shown to contribute to Nrf2-mediated HO-1 expression. Taken together, GHP protected HepG2 cells from oxidative stress by activation of Nrf2 and HO-1 via p38 MAPK and ERK1/2 signaling pathways. Our findings indicate that bovine casein glycomacropeptide hydrolysates might be a potential ingredient in the treatment of oxidative stress-related disorders and further studies are needed to investigate the protective effects in vivo.