Hypertension, afflicting more than 20% of adults worldwide, is a major risk factor for cardiovascular diseases. Spent hens, a major egg industry byproduct, can be used as the starting material for preparing antihypertensive peptides. Therefore, the objectives of this study were to identify both ACE inhibitory (ACEi) peptides and ACE2 upregulating (ACE2u) peptides in spent hens (SPH-T) and to determine gastrointestinal stability of these peptides. In addition,
structure-activity relationships and molecular interactions between ACE and ACEi peptides were also explored.
Spent hen carcasses were purchased from a local supermarket. Spent hen muscle proteins were extracted according to a pH-shift method with slight modifications. The protein extract (~93% protein) was dissolved in ddH2O (5%, w/w), pre-heated at 90 °C for 10 min for protein denaturation, prior to a 3 h of hydrolysis by thermoase PC10F (4% enzyme/substrate, E/S, w/w protein in a jacket beaker, connected with a Titrando (Metrohm, Herisan, Switzerland) and a circulating water bath (Brinkman, Mississauga, ON, Canada) for pH and temperature control, respectively. After the hydrolysis, the slurry was heated at 95 °C for 10 min to inactive the enzyme. After centrifuged at 10,000 g for 15 min (4 °C), the supernatant was collected, freeze-dried, and kept at −20 °C for further analysis.
Our recent study indicated that SPH-T had both high ACEi and ACE2u activities. Five potent ACEi peptides and four ACE2u peptides were identified from SPH-T using the conventional activity- guided fractionation method. The structural requirements of the identified ACEi peptides corresponded well with the structure-activity relationship reported in literature; the molecular docking study confirmed that these peptides formed H-bonds with ACE. A lack of sufficient number of ACE2u peptides makes it impossible to draw any structure-activity relationship, which relies heavily on a continuous discovery of new peptides.
Our study supports the potential use of spent hens as antihypertensive functional food ingredients and nutraceuticals. However, the in vivo efficacy of the identified ACEi and ACE2u peptides are yet to be determined in order to confirm their antihypertensive effect. Biotransformation of spent hens into high-valued health-beneficial compounds will contribute to a more sustainably growing egg industry, which also shed light on valorization of other under-utilized protein-rich agricultural byproducts.
The study explored the use of spent hen, a major egg industry byproduct, as the starting material for preparing antihypertensive peptides. While previous studies were focused mainly on ACE inhibitory (ACEi) peptides, this work also studied peptides with ACE2 upregulating (ACE2u) activity, an emerging target for treating hypertension. Spent hen muscle protein hydrolysate prepared by thermoase (SPH-T) exhibited both ACEi and ACE2u activities. After ultrafiltration and chromatographic fractionation, five potent ACEi peptides, VRP, LKY, VRY, KYKA, and LKYKA, with IC50 values of 0.034–5.77 μg/mL, respectively, and four ACE2u peptides, VKW, VHPKESF, VVHPKESF and VAQWRTKYETDAIQRTEELEEAKKK, which increased ACE2 expression by 0.52–0.84 folds, respectively, were identified; VKW also showed ACEi activity. All peptides, except for VRP, are susceptible to degradation during the simulated gastrointestinal digestion. Our study supports the potential use of spent hens as antihypertensive functional food ingredients and nutraceuticals.
