Bromelain-generated biopeptides from stone fish protein exhibit strong inhibitory effect against ACE and can potentially serve as designer food (DF) with blood pressure lowering effect. Contextually, the DF refer to the biopeptides specifically produced to act as ACE-inhibitors other than their primary role in nutrition and can be used in the management of hypertension. However, the biopeptides are unstable under gastrointestinal tract (GIT) digestion and need to be stabilized for effective oral administration. In the present study, the stone fish biopeptides (SBs) were stabilized by their encapsulation in sodium tripolyphosphate (TPP) cross-linked chitosan nanoparticles produced by ionotropic gelation method. The nanoparticles formulation was then optimized via Box-Behnken experimental design to achieve smaller particle size (162.70 nm) and high encapsulation efficiency (75.36%) under the optimum condition of SBs:Chitosan mass ratio (0.35), homogenization speed (8000 rpm) and homogenization time (30 min). The SBs-loaded nanoparticles were characterized for morphology by transmission electron microscopy (TEM), physicochemical stability and efficacy. The nanoparticles were then lyophilized and analyzed using Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD). The results obtained indicated a sustained in vitro release and enhanced physicochemical stability of the SBs-loaded nanoparticles with smaller particle size and high encapsulation efficiency following long period of storage. Moreover, the efficacy study revealed improved inhibitory effect of the encapsulated SBs against ACE following simulated GIT digestion.
Recent biotechnological advances in the food industry have led to the enzymatic production of angiotensin I-converting enzyme (ACE)-inhibitory biopeptides with a strong blood pressure lowering effect from different food proteins. However, the safe oral administration of biopeptides is impeded by their enzymatic degradation due to gastrointestinal digestion. Consequently, nanoparticle (NP)-based delivery systems are used to overcome these gastrointestinal barriers to maintain the improved bioavailability and efficacy of the encapsulated biopeptides. In the present study, the ACE-inhibitory biopeptides were generated from stone fish (Actinopyga lecanora) protein using bromelain and stabilized by their encapsulation in chitosan (chit) nanoparticles (NPs). The nanoparticles were characterized for in vitro physicochemical properties and their antihypertensive effect was then evaluated on spontaneously hypertensive rats (SHRs). The results of a physicochemical characterization showed a small particle size of 162.70 nm, a polydispersity index (pdi) value of 0.28, a zeta potential of 48.78 mV, a high encapsulation efficiency of 75.36%, a high melting temperature of 146.78 °C and an in vitro sustained release of the biopeptides. The results of the in vivo efficacy indicated a dose-dependent blood pressure lowering effect of the biopeptide-loaded nanoparticles that was significantly higher (p < 0.05) compared with the un-encapsulated biopeptides. Moreover, the results of a morphological examination using transmission electron microscopy (TEM) demonstrated the nanoparticles as homogenous and spherical. Thus, the ACE-inhibitory biopeptides stabilized by chitosan nanoparticles can effectively reduce blood pressure for an extended period of time in hypertensive individuals.
This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young's modulus, Poisson's ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m-1, 123.4700 GPa, 0.3000 and 0.0693 V·m·N-1, respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young's modulus of the cells are determined to be 10.8867 ± 0.0094 N·m-1 and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young's modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment.
Protein hydrolysates produced from different food sources exhibit therapeutic potential and can be used in the management of chronic diseases. This study was targeted to optimise the conditions for the hydrolysis of stone fish protein to produce antioxidant hydrolysates using central composite design (CCD) by response surface methodology (RSM). The stone fish protein was hydrolysed under the optimum predicted conditions defined by pH (6.5), temperature (54°C), E/S ratio (1.5%), and hydrolysis time (360 min). The hydrolysates were then evaluated for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) scavenging activity and ferrous ion- (Fe2+-) chelating activity. Results validation showed no significant difference between the experimental values of DPPH• scavenging activity (48.94%) and Fe2+-chelating activity (25.12%) obtained at 54.62% degree of hydrolysis (DH) compared to their corresponding predicted values of 49.79% and 24.08% at 53.08% DH, respectively. The hydrolysates demonstrated non-Newtonian behavior (n < 1) with stronger shear-thinning effect and higher viscosities at increasing concentration. Thus, RSM can be considered as a promising strategy to optimise the production of stone fish protein hydrolysates containing antioxidant peptides. It is hoped that this finding will enhance the potential of stone fish protein hydrolysates (SHs) as therapeutic bioactive ingredient in functional foods development.
Three novel peptide sequences identified from palm kernel cake (PKC) generated protein hydrolysate including YLLLK, WAFS and GVQEGAGHYALL were used for stability study against angiotensin-converting enzyme (ACE), ACE-inhibition kinetics and molecular docking studies. Results showed that the peptides were degraded at different cleavage degrees of 94%, 67% and 97% for YLLLK, WAFS and GVQEGAGHYALL, respectively, after 3 h of incubation with ACE. YLLLK was found to be the least stable (decreased ACE-inhibitory activity) compared to WAFS and GVQEGAGHYALL (increased ACE-inhibitory activity). YLLLK showed the lowest Ki (1.51 mM) in inhibition kinetics study when compared to WAFS and GVQEGAGHYALL with Ki of 2 mM and 3.18 mM, respectively. In addition, ACE revealed the lowest Kmapp and Vmaxapp and higher catalytic efficiency (CE) in the presence of YLLLK at different concentrations, implying that the enzyme catalysis decreased and hence the inhibition mode increased. Furthermore, YLLLK showed the lowest docking score of -8.224 and seven interactions with tACE, while peptide GVQEGAGHYALL showed the higher docking score of -7.006 and five interactions with tACE.
Stone fish is an under-utilized sea cucumber with many health benefits. Hydrolysates with strong ACE-inhibitory effects were generated from stone fish protein under the optimum conditions of hydrolysis using bromelain and fractionated based on hydrophobicity and isoelectric properties of the constituent peptides. Five novel peptide sequences with molecular weight (mw) < 1000 daltons (Da) were identified using LC-MS/MS. The peptides including Ala-Leu-Gly-Pro-Gln-Phe-Tyr (794.44 Da), Lys-Val-Pro-Pro-Lys-Ala (638.88 Da), Leu-Ala-Pro-Pro-Thr-Met (628.85 Da), Glu-Val-Leu-Ile-Gln (600.77 Da) and Glu-His-Pro-Val-Leu (593.74 Da) were evaluated for ACE-inhibitory activity and showed IC50 values of 0.012 mM, 0.980 mM, 1.310 mM, 1.440 mM and 1.680 mM, respectively. The ACE-inhibitory effects of the peptides were further verified using molecular docking study. The docking results demonstrated that the peptides exhibit their effect mainly via hydrogen and electrostatic bond interactions with ACE. These findings provide evidence about stone fish as a valuable source of raw materials for the manufacture of antihypertensive peptides that can be incorporated to enhance therapeutic relevance and commercial significance of formulated functional foods.
We evaluated the acute (single-dose) and subacute (repeated-dose) oral toxicity of alcalase-hydrolyzed whey protein concentrate. Our acute study revealed no death or treatment-related complications, and the median lethal dose of whey protein concentrate hydrolysate was >2,500 mg/kg. In the subacute study, when the hydrolysate was fed at 3 different concentrations (200, 400, and 800 mg/kg), no groups showed toxicity changes compared with controls. Then, whey protein concentrate hydrolysate was orally administered to spontaneously hypertensive rats. Results revealed significant reductions in blood pressure in a dose-dependent manner, and dosing at 400 mg/kg led to significant blood pressure reduction (-47.8 mm Hg) compared with controls (blood pressure maintained) and the findings of previous work (-21 mm Hg). Eight peptides-RHPEYAVSVLLR, GGAPPAGRL, GPPLPRL, ELKPTPEGDL, VLSELPEP, DAQSAPLRVY, RDMPIQAF, and LEQVLPRD-were sequentially identified and characterized. Of the peptides, VLSELPEP and LEQVLPRD showed the most prominent in vitro angiotensin-I converting enzyme inhibition with half-maximal inhibitory concentrations of 0.049 and 0.043 mM, respectively. These findings establish strong evidence for the in vitro and in vivo potential of whey protein concentrate hydrolysate to act as a safe, natural functional food ingredient that exerts antihypertensive activity.