A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells.

A phenylalanine (Phe)-restricted diet is indispensable for individuals suffering from phenylketonuria (PKU). Our previous study reported a low-Phe-containing whey protein hydrolysate (LPH) prepared from a selected whey protein hydrolysate (TA2H). This study aimed to investigate the osteogenic activity of LPH and TA2H in MC3T3-E1 preosteoblast cells and explore the underlying mechanism. Results showed that the treatment of TA2H and LPH (at the final concentrations of 100-1000 µg/mL) had a stimulatory effect on the proliferation, differentiation, and mineralization of MC3T3-E1 cells. The LPH of 1000 µg/mL significantly increased cell proliferation (2.15- ± 0.11-fold) and alkaline phosphatase activity (1.22- ± 0.07-fold), promoted the protein and mRNA levels of runt-related transcription factor 2 (Runx2, 2.50- ± 0.14-fold and 2.97- ± 0.23-fold, respectively), enhanced the expression of differentiation biomarkers (type-I collagen, osteocalcin, and osteopontin), increased calcium deposition (1.56- ± 0.08-fold), and upregulated the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand. The exploration of signaling pathways indicated that the activated p38-dependent Runx2 signaling contributed to the LPH-induced osteogenesis. These results provided evidence, for the first time, that a prepared low-Phe whey protein hydrolysate positively modulated the activity of osteoblasts through the p38/Runx2 pathway, thereby providing a new osteoinductive protein substitute to make functional PKU food.

Evaluation of the anti-osteoporotic effect of a low-phenylalanine whey protein hydrolysate in an ovariectomized mice model.

A phenylalanine (Phe)-restricted diet is indispensable to control the blood Phe for individuals with phenylketonuria (PKU), who are also confronted with progressive bone impairment. Thus, the development of a low-Phe protein substitute that could positively regulate bone metabolism is desired for their bone health. Our previous study reported the preparation of a low-Phe containing whey hydrolysate (LPH) from a selected whey protein hydrolysate (TAH). However, the effect of LPH on the bone status is unknown. In this study, we used an ovariectomized (OVX) mice model to evaluate the anti-osteoporotic potential of oral administration of whey protein concentrate (WPC, protein control), TAH, and LPH on bone physiology and bone metabolism. The results showed that after 12 weeks of treatment, the decreased bone mineral density, the deteriorated trabecular microarchitecture, and the reduced ultimate load due to ovariectomy were significantly attenuated by two whey protein hydrolysates (TAH and LPH); meanwhile, the body weight, uterine weight, bone composition, and the femoral elastic load of OVX mice had not been significantly affected by whey samples. In addition, LPH and TAH dual-regulated bone remodeling in OVX mice through triggering osteogenesis (promoted the expression of runt-related protein 2 (Runx2) and osteoformation markers) and inhibiting osteoresorption as well as inflammation. The modulated mitogen-activated protein kinase signaling and the inhibited nuclear factor κB signaling by LPH and TAH might relate to the dual-regulatory activities on bone. Overall, in the OVX mice model, LPH exerted higher osteoprotective potential than TAH of the same dose by activating the bone formation markers and inhibiting the inflammatory status. The current study demonstrated for the first time the potential use of a low-Phe whey hydrolysate, a protein substitute for PKU individuals, in the prevention of osteoporosis.

Purification and characterization of hypoglycemic peptides from traditional Chinese soy-fermented douchi.

Douchi is a popular soy-fermented food that originated in China with documented hypoglycemic effects. However, the responsible molecules and the mechanism underlying their beneficial effects remain unclear. Therefore, in this study, we aimed to identify the responsible peptide(s) in douchi. A peptide extract of douchi was isolated step-wise by the C18 Sep-Pak technique, size exclusion chromatography, and semi-preparative liquid chromatography, and then the peptides were sequenced by UPLC-MS/MS. A total of 21 peptides were identified, of which three peptides, P3 (HPFR), P5 (VY), and P7 (SFLLR), were shown to improve glucose uptake in L6 cells. Both P5 and P7 increased glucose transporter 4 (GLUT4) translocation via the activation of AMPK and MAPK signaling pathways, but not the insulin-signaling pathway; adding an AMPK or an MAPK inhibitor prevented P5 or P7-induced glucose uptake as well as AMPK and MAPK activation. Our study showed that P5 and P7 could promote glucose uptake via AMPK and MAPK signaling pathways. In this study, two hypoglycemic peptides from douchi have been characterized for the first time.

Peptide Analogues of VPP and IPP with Improved Glucose Uptake Activity in L6 Myotubes can be Released from Cereal Proteins.

VPP (Val-Pro-Pro) and IPP (Ile-Pro-Pro) are two famous antihypertensive peptides with possible benefits for type 2 diabetes mellitus (T2DM). The study was aimed to investigate the effect of peptide analogues of VPP and IPP on glucose uptake activity in L6 myotubes. The analogues were designed by replacing the N-terminal, middle, or C-terminal amino acid residues of VPP and IPP with one amino acid at a time from five amino acid groups (polar, nonpolar, basic, acidic, and aromatic amino acids). Among 26 tripeptides tested, IQP, IPQ, VPE, and VEP showed significantly higher glucose uptake activity than their parent peptides, and all were successfully released from rice proteins at the contents of 5415.82 ± 63.34, 1586.77 ± 14.94, 354.07 ± 6.56, and 596.10 ± 2.32 ng/mg dry basis, respectively, and quantified by liquid chromatography-mass spectrometry (MS)/MS using multiple reaction monitoring. All four peptides were shown to promote glucose uptake via the adenosine monophosphate-activated protein kinase pathway accompanied by glucose transporter type 4 (Glut4) translocation rather than the insulin signaling pathway.

Peptides in Brewed Wines: Formation, Structure, and Function.

The traditional low-alcoholic beverages, such as grape wine, sake, and rice wine, have been consumed all over the world for thousands of years, each with their unique methods of production that have been practiced for centuries. Moderate consumption of wine is generally touted as beneficial for health, although there is ongoing debate for the responsible components in wine. In this review, the structural and functional characteristics, the formation mechanisms, and their health-promoting activities of peptides in three brewed wines, grape wine, Chinese rice wine (also called Chinese Huangjiu or Chinese yellow wine), and Japanese sake, are discussed. The formation of peptides in wine imparts sensorial, technological, and biological attributes. Prospects on future research, with an emphasis on the peptide characterization, formation mechanism, physiological activity, and molecular mechanisms of action, are presented.

Milk proteins and their derived peptides on bone health: Biological functions, mechanisms, and prospects.

Bone is a dynamic organ under constant metabolism (or remodeling), where a delicate balance between bone resorption and bone formation is maintained. Disruption of this coordinated bone remodeling results in bone diseases, such as osteoporosis, the most common bone disorder characterized by decreased bone mineral density and microarchitectural deterioration. Epidemiological and clinical evidence support that consumption of dairy products is beneficial for bone health; this benefit is often attributed to the presence of calcium, the physiological contributions of milk proteins on bone metabolism, however, are underestimated. Emerging evidence highlighted that not only milk proteins (including individual milk proteins) but also their derived peptides positively regulate bone remodeling and attenuate bone loss, via the regulation of cellular markers and signaling of osteoblasts and osteoclasts. This article aims to review current knowledge about the roles of milk proteins, with an emphasis on individual milk proteins, bioactive peptides derived from milk proteins, and effect of milk processing in particular fermentation, on bone metabolism, to highlight the potential uses of milk proteins in the prevention and treatment of osteoporosis, and, to discuss the knowledge gap and to recommend future research directions.

Comparison of Predictors of COVID-19 Vaccination Intention Between Healthcare Workers and Non-Healthcare Workers in China.

BACKGROUND: Vaccination is an effective strategy to mitigate the spread of COVID-19. This study aimed to compare predictors of vaccination intention between healthcare workers (HCWs) and non-healthcare workers (non-HCWs) in China. METHODS: A web-based cross-sectional survey was conducted among HCWs and non-HCWs. Several bivariate analysis techniques, eg, crosstab with Chi-square, independent t-test and single factor ANOVA, were performed to analyze the correlation. After that, a series of multivariate binary regressions were employed to determine predictors of vaccination intention. RESULTS: Intention was closely and significantly related with gender, perceived vaccination knowledge, perceived importance and effectiveness of vaccine to prevent COVID-19. HCWs and non-HCWs were heterogeneous, since vaccination intention, perceived knowledge, and attitudes (eg, importance, severity, risk) toward COVID-19 or vaccine had statistically significant difference between the two groups. With comparison of predictors of vaccination intention, for HCWs, demographic factors were the major predictors of COVID-19 vaccination intention. Female HCWs and HCWs with a Master's or higher degree were more hesitant about vaccination (P = 0.01 and P < 0.001, respectively), while HCWs had greater vaccination intention as their age increased (P = 0.02). For non-HCWs, perceived vaccination knowledge was the major predictor of COVID-19 vaccination intention (P < 0.001). Additionally, perceived importance and effectiveness of vaccine were predictors for both HCWs and non-HCWs. CONCLUSION: Vaccination intention of HCWs was greater than that of non-HCWs in China. Measures should be taken to improve the vaccination rate based on the predictors of vaccination intention identified in this study. For HCWs, especially those with a high level of education or who were females, the safety and effectiveness of vaccines in use may reinforce their vaccination intention. For non-HCWs, popularization of general medical knowledge, including of vaccine-preventable diseases, may increase their vaccination intention.

Naturally occurring low molecular peptides identified in egg white show antioxidant activity.

Egg white is a rich source of bioactive proteins that are essential to provide protection to the embryo in yolk. There is an ongoing interest in identifying novel egg white proteins; it is not known however if peptides are also present in egg white. The objectives of the study were to identify low molecular peptides occurring naturally in egg white using a peptidomics approach and to determine their potential antioxidant activity. A total of 45 peptides were identified but surprisingly all are originated from egg white minor proteins (except ovotransferrin); three most abundant peptides, STDVPRDPWVWGSAHPQAQHTR, GDPSAWSWGAEAHS, and ALGEDIVDLDSFSEQH are derived from ovocleidin, zona pellucida glycoprotein C (ZPC), and sulfhydryl oxidase 1, respectively. Neuropeptide Y was identified for the first time in egg whites. The concentrations of eight most abundant peptides in egg white ranged from 0.004 to 0.292 mg/g, determined by triple quadrupole mass spectrometer in multiple-reaction monitoring (MRM) mode. Six peptides were found to have antioxidant activities based on reduced formation of superoxide and increased levels of superoxide dismutase (SOD) and catalase (CAT) in cells. Our study provided evidence on the presence of naturally occurring antioxidant peptides in egg white.

Preparation, Bioavailability, and Mechanism of Emerging Activities of Ile-Pro-Pro and Val-Pro-Pro.

Ile-Pro-Pro and Val-Pro-Pro are two most well-known food-derived bioactive peptides, initially identified as inhibitors of angiotensin I-converting enzyme (ACE) from a sample of sour milk. These two peptides were identified in fermented and enzymatic hydrolyzed cow and non-cow (that is, goat, sheep, buffalo, yak, camel, mare, and donkey) milk, as well as sourdough prepared from wheat, rye, and malt. Similar to other bioactive peptides, bioavailability of these peptides is low (about 0.1%), reaching picomolar concentration in human plasma; they showed blood pressure lowering activity in animals and in human, via improved endothelial function, activation of ACE2, and anti-inflammatory property. Emerging bioactivities of these two peptides toward against metabolic syndrome and bone-protection received limited attention, but may open up new applications of these peptides as functional food ingredients. Further studies are warranted to determine the best source as well as to identify novel enzymes (particularly from traditional fermented milk products) to improve the efficiency of production, to characterize possible peptide receptors using a combination of omics technology with molecular methods to understand if these two peptides act as signal-like molecules, to improve their bioavailability, and to explore new applications based on emerging bioactivities.

Evaluation of astaxanthin incorporated collagen film developed from the outer skin waste of squid Doryteuthis singhalensis for wound healing and tissue regenerative applications.

The present investigation was aimed to evaluate in vivo wound healing activity of astaxanthin incorporated collagen hydrogel film biomaterials extracted from the outer skin waste of squid Doryteuthis singhalensis, to releases antibiotic, delivering potentialities of excisional and incisional wound model in Wistar rats. These results suggested that the astaxanthin incorporated collagen film (ACF) and gentamicin incorporated collagen film (GCF) exhibited excellent wound healing activity (71%) in both full thickness excision and linear incision in rats. The in-vitro antioxidant abilities of extracted astaxanthin exhibited strongly significant 1,1­diphenyl­2­picrylhydrazyl (DPPH) radical scavenging activity. In addition, tensile strength, epithelialization, hydroxyproline content and protein content in ACF and GCF treated groups were significantly increased. Histopathological assessment revealed an increase in collagen content, fibroblasts, granulation, thickness of scar formation, effective neovascularization and faster epithelialization within the short duration after the treatment of ACF and GCF compared to the control groups. The structure of prepared ACF and GCF biomaterials were characterized by SEM, EDS, and XRD. The in vivo biological study of the collagen-based film releases the antibiotic substance. The composite of collagen based biomaterials displays a promising biocompatibility through the dermal wound healing process as well as an evidence of biodegradability. Thus, the marine-derived biomaterials gave a substantial pledge for the development of biodegradable materials in drug delivery and soft tissue regeneration process.