Identification and Effects of Skim Milk-Derived Bioactive Antihypertensive Peptides

Bioactive peptides are generated during milk fermentation or enzymatic hydrolysis. Lactobacillus (L) helveticus is commonly used to produce some types of fermented milk products. Fermented milk derived bioactive peptides are known to be beneficial in human health. Anti-hypertensive peptides play a dual role in the regulation of hypertension through the production of the vasoconstrictor angiotensin II and its inactivation of the vasodilator bradykinin. MALDI MS/MS, nano-LC/MS/MS and RP-HPLC were used to isolate peptides showing angiotensin converting enzyme inhibition (ACE-I) from 12% fermented skim milk using a combination of L. helveticus and Flavourzyme®. The fermentation procedure facilitated the identification of 133 anti-hypertensive peptides and 75% short chain amino acids, and the three with the highest ACE-I activity reduced blood pressure in a rat model of hypertension. The freeze- dried extract was supplemented in rodent chow. In this study 14-week-old male spontaneously hypertensive rats were fed for 10 weeks with the identified peptides added to chow and compared to controls supplemented with skim milk powder. Blood pressure (BP) decreased significantly (p < 0.05) from 6 to 10 weeks of FS groups (120/65 mmHg) compared with the NFS control groups, where the BP increased significantly (220/150 mmHg) (p < 0.05). The F6 fraction provided bioactive peptides with stronger antihypertensive properties than other fractions. Skim milk fermented by L. helveticus and Flavourzyme® generates several bioactive peptides which have a blood pressure lowering effect in hypertensive disease.

Critical timing of ACEi initiation prevents compensatory glomerular hypertrophy in the remaining single kidney

Increasing evidence suggests that single in kidney states (e.g., kidney transplantation and living donation) progressive glomerulosclerosis limits kidney lifespan. Modeling shows that post-nephrectomy compensatory glomerular volume (GV) increase drives podocyte depletion and hypertrophic stress resulting in proteinuria and glomerulosclerosis, implying that GV increase could serve as a therapeutic target to prevent progression. In this report we examine how Angiotensin Converting Enzyme inhibition (ACEi), started before uninephrectomy can reduce compensatory GV increase in wild-type Fischer344 rats. An unbiased computer-assisted method was used for morphometric analysis. Urine Insulin-like growth factor-1 (IGF-1), the major diver of body and kidney growth, was used as a readout. In long-term (40-week) studies of uni-nephrectomized versus sham-nephrectomized rats a 2.2-fold increase in GV was associated with reduced podocyte density, increased proteinuria and glomerulosclerosis. Compensatory GV increase was largely prevented by ACEi started a week before but not after uni-nephrectomy with no measurable impact on long-term eGFR. Similarly, in short-term (14-day) studies, ACEi started a week before uni-nephrectomy reduced both GV increase and urine IGF-1 excretion. Thus, timing of ACEi in relation to uni-nephrectomy had significant impact on post-nephrectomy “compensatory” glomerular growth and outcomes that could potentially be used to improve kidney transplantation and live kidney donation outcomes.

TORTILLAS MADE WITH EXTRUDED FLOURS OF BLUE MAIZE AND CHÍA SEEDS AS AN NUTRITIOUS AND NUTRACEUTICAL FOOD OPTION

Tortillas with adequate nutrients and phytochemicals as a recommended option may improve health in older adults. In this research tortillas were prepared with native blue maize (Zea mays L.) and chía (Salvia hispanica) seeds processed by alkaline extrusion. The hypothesis was that alkaline extrusion would generate flours of both seeds with improved nutraceutical and nutritional characteristics compared to traditional products. The aim was then to evaluate extruded defatted chía flour addition on nutritional and nutraceutical properties of tortillas made with extruded native blue maize flour. Tortillas were prepared with a mixture 75% extruded blue maize flour +25% extruded defatted chía flour. Other tortillas from 100% extruded blue maize flour and commercial lime-cooked maize flour (Blue MASECA®) were used as control and reference. Nutritional, antioxidant, antihypertensive and hypoglycaemic properties of tortillas were evaluated in vitro. A completely randomized experimental design with one factor (type of tortilla) was used for data analysis. Tortillas added with extruded chía flour had higher protein, dietary fibre, essential amino acid (EAA) profile, in vitro protein digestibility (IVPD), and calculated protein efficiency ratio (C-PER) than control and reference tortillas. Tortillas with addition of extruded chía flour also had better antioxidant activity [oxygen radical absorbance capacity, (ORAC) 18006 vs 15531 Trolox equivalent, (TE) μmol per 100g of dry weight, (DW) sample], and better antihypertensive [(ACE) angiotensin-converting enzyme inhibition, IC50=0.47 vs 0.65 extract mg mL-1] and hypoglycaemic (IC50=20.07 vs 25.81 extract mg mL-1 to α-amylase inhibition, and IC50=16.25 vs 19.69 extract mg mL-1 to α-glucosidase inhibition) potential than tortillas from 100% extruded blue maize flour. The use of extruded chía flour and blue maize flour allowed obtaining tortillas with enhanced nutritional, antioxidant, antihypertensive, and hypoglycaemic properties. The extrusion process consumes few water volumes without generating polluting effluents. An appropriate public policy in Mexico may promote these tortillas to be used to fight against malnutrition, and to reduce incidences of chronic degenerative diseases such as hypertension or diabetes.

Abstract P504: Transient Angiotensin Converting Enzyme Inhibition Protects Against Future Fibrogenic Responses Via Sex-specific Mechanisms

Following washout of angiotensin converting enzyme inhibitor (ACEi) treatment in hypertensive rats (SHR), there is an increase in the proportion of homeostatic cardiac fibroblasts (CFs) characterized by a less fibrogenic gene profile. The present study investigated the impact of this shift in CFs on subsequent Ang II-induced oxidative stress and fibrogenic responses in the left ventricle (LV). Male and female SHRs (11 wk old) underwent a 6-week treatment scheme: 2-week ACEi (enalapril, 30mg/kg/day) or vehicle treatment, followed by a 2-week washout period, and then a 2-week Ang II (400ng/kg/min, s.c.) or vehicle infusion (n=5-11/group/sex). RT-qPCR for collagens and immunoblotting for LOX, Postn, OPN, NOX2, SOD2, and catalase was performed. In males, Ang II-induced increases in ColI, III, and IV expression were significantly attenuated by prior ACEi. While in females, Ang II significantly increased the expression of ColI and III similarly, regardless of prior ACEi treatment; highlighting a sex-specific impact of ACEi. After Ang II infusion, both Postn and LOX are reduced in LV of male and female SHR previously treated with an ACEi - suggesting reduced collagen cross-linking. Oxidative stress induced by NOX2 has been linked to OPN in fibrotic tissue. Ang II-induced increases in NOX2 are attenuated in LV of both sexes, while OPN is only reduced in females previously treated with an ACEi. NOX2 is significantly positively correlated with OPN in males only suggesting a sex-specific link between OPN and oxidative stress. Antioxidant (SOD2, catalase) levels were positively correlated to NOX2 in females, but not males. This suggests that females more efficiently neutralize oxidative stress than males - regardless of prior ACEi. Taken together, the persistent genomic and phenotypic changes in CFs following transient ACEi render the heart resistant to future fibrotic and oxidative effects of Ang II, but the mechanisms of protection may differ by sex. Collectively, these data reveal that there are sex-specific processes that govern collagen production vs. cross-linking and oxidative stress. Future studies will elucidate the mechanistic underpinnings of sex-specific cellular memory following transient ACEi with a goal of identifying novel therapeutic targets.

In Vitro Antioxidant and Antihypertensive Activity of Edible Insects Flours (Mealworm and Grasshopper) Fermented with Lactococcus lactis Strains

The objective of the present study was to evaluate the potential antioxidant and angiotensin converting enzyme inhibition (ACEI) activity of edible insect flours fermented with Lactococcus lactis strains. For the fermentation, mealworm and grasshoppers flours were dissolved (0.5% w/v) in buffer solution (pH 7.0) and individually inoculated (3%) with Lactococcus lactis strains (NRRL B-50571, NRRL B-50572). The samples were incubated for 72 h at 30 °C, and the pH was recorded. The degree of hydrolysis (DH) and protein content were determined. The total polyphenol compounds, antioxidant activity (ABTS, DPPH, ORAC, and FRAP), and ACEI of the <3 kDa fractions were analyzed. The pH of the fermented samples decreased to 3.5–3.9 (p < 0.05). The fermented grasshopper flour showed an increased DH (0.42%) and overall higher total polyphenol content (8.23 mg Gallic Acid Equivalent/mL). In general, the highest antioxidant activity was for the grasshopper fractions fermented for 24 h by Lactococcus lactis NRRL B-50572, which also showed 23.47% ACEI inhibition with an IC50 of 0.97 mg/mL. The peptide profile obtained increased after fermentation, being higher for the mealworm flour fermented sample. This study presents, for the first time, the use of specific strains of Lactococus lactis for fermenting edible insect-derived products in the production of bioactive compounds with potential antioxidant and antihypertensive activity.

Roles of Dietary Bioactive Peptides in Redox Balance and Metabolic Disorders

Bioactive peptides (BPs) are fragments of 2–15 amino acid residues with biological properties. Dietary BPs derived from milk, egg, fish, soybean, corn, rice, quinoa, wheat, oat, potato, common bean, spirulina, and mussel are reported to possess beneficial effects on redox balance and metabolic disorders (obesity, diabetes, hypertension, and inflammatory bowel diseases (IBD)). Peptide length, sequence, and composition significantly affected the bioactive properties of dietary BPs. Numerous studies have demonstrated that various dietary protein-derived BPs exhibited biological activities through the modulation of various molecular mechanisms and signaling pathways, including Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2/antioxidant response element in oxidative stress; peroxisome proliferator-activated-γ, CCAAT/enhancer-binding protein-α, and sterol regulatory element binding protein 1 in obesity; insulin receptor substrate-1/phosphatidylinositol 3-kinase/protein kinase B and AMP-activated protein kinase in diabetes; angiotensin-converting enzyme inhibition in hypertension; and mitogen-activated protein kinase and nuclear factor-kappa B in IBD. This review focuses on the action of molecular mechanisms of dietary BPs and provides novel insights in the maintenance of redox balance and metabolic diseases of human.

Physical Activity and Inhibition of ACE Additively Modulate ACE/ACE-2 Balance in Heart Failure in Mice

Angiotensin-converting enzyme inhibition (ACE-I) and physical activity favorably modulate the ACE/ACE-2 balance. However, it is not clear whether physical activity and ACE-I could synergistically modulate ACE/ACE-2 balance in the course of heart failure (HF). Here, we studied the effects of combined spontaneous physical activity and ACE-I–based treatment on angiotensin (Ang) pattern and cardiac function in a mouse model of HF (Tgαq*44). Tgαq*44 mice with advanced HF (at the age of 12 months) were running spontaneously in a running wheel (exercise training group, ExT) and/or were treated with ACE inhibitor (ACE-I, perindopril, 10 mg/kg) for 2 months. Angiotensin profile was characterized by an LC-MS/MS-based method. The cardiac performance was assessed in vivo by MRI. Ang-(1–7)/Ang II ratio in both plasma and the aorta was significantly higher in the combined treatment group than the ACE-I group or ExT alone, suggesting the additive favorable effects on ACE-2/Ang-(1–7) and ACE/Ang II axes’ balance induced by a combination of ACE-I with ExT. The basal cardiac performance did not differ among the experimental groups of Tgαq*44 mice. We demonstrated additive changes in ACE/ACE-2 balance in both plasma and the aorta by spontaneous physical activity and ACE-I treatment in Tgαq*44 mice. However, these changes did not result in an improvement of failing heart function most likely because the disease was at the end-stage. Ang-(1–7)/Ang II balance represents a valuable biochemical end point for monitoring therapeutic intervention outcome in heart failure.