β-lactoglobulin peptides obtained by chymotrypsin hydrolysis

Objective: Whey proteins, as β-lactoglobulin, have biological activity. Controlled hydrolysis of this protein could generate peptides with some biological function. The aim of this work was to analyze the peptides resulting from the in vitro hydrolysis with chymotrypsin in order to evaluate the presence of bioactive peptides. Design/methodology/approach: Chymotrypsin was used in the hydrolysis of β-lactoglobulin, and its peptides were evaluated by ultrafiltration, electrophoresis, and mass spectrometry. Findings/conclusion: Results showed that 2 h of chymotrypsin hydrolysis (T1) released peptides with molecular weight values of 8 and 9 KDa, while 4 h of hydrolysis (T2) produced peptides with molecular mass weight values of 7 and 5 KDa. The mass spectrometry (MALDI-TOF) showed six peaks and five of them were comparable with those obtained by in silico hydrolysis results (done previously by Fonseca Ayala, 2018). The identified peptides (DTDYK, DAQSAPL and LKPTPEGDL) in the fraction <1 kDa showed inhibitory activity of angiotensin converting enzyme and inhibitory activity of enzyme dipeptidyl peptidase IV according BIOPEP database. These results showed that β-lactoglobulin peptides obtained by chymotrypsin hydrolysis could have biological activity that can be used in different types of industries as pharmaceutical and food. Limitations on study/implications: The in vitro evaluation of the biological activity of the characterized peptides is necessary.

An LC-MS/MS Method for the Pharmacokinetic and in Vitro Metabolism Studies of Praeruptorin A in Rat

Objective: The study aims to investigate the pharmacokinetic profile of Praeruptorin A and khellactone and in vitro hydrolysis of praeruptorin A to khellactone in different biological samples. Methods: A LC-MS/MS method was established. Analytes and internal standard (IS) were isolated using the protein precipitation method and then separated on a Thermo BDS Hypersil C18 (2.1 mm×50 mm, 2.4μm) column using a mobile phase consisting of 0.05% formic acid solution and acetonitrile. Samples were analyzed in positive electrospray-ionization (ESI) mode using multiple reaction monitoring (MRM). Results: The calibration plots gave desirable linearity (r2>0.99) in the concentration range from 0.99-990.0 and 2.0-2000.0 ng/mL for Praeruptorin A and khellactone, respectively. In addition, the LOQs of these analytes were sufficient for vivo pharmacokinetic study and vitro hydrolysis study of Praeruptorin A. The intra-batch and inter-batch precision were all within 14.05%, and the accuracy was between 89.39% and 109.50%. The extraction efficiency of PA and khellactone ranged from 76.35 ~ 89.58%. The matrix effects of analytes and the IS were between 89.67% ~ 105.26%. Conclusion: The liver CYPs mediated by the metabolism of PA may contribute to the systemic exposure of its active metabolite, khellactone, in rats.

The Influence of Hypericum perforatum L. Addition to Wheat Cookies on Their Antioxidant, Anti-Metabolic Syndrome, and Antimicrobial Properties

The aim of this study was to characterize wheat cookies enriched with 0.5% and 1.0% of Hypericum perforatum L. (St. John’s wort, SJW) and determine their pro-health properties in vitro after hydrolysis in simulated gastrointestinal conditions. The results indicated that 1.0 SJW was characterized by the highest content of polyphenols, flavonoids, and phenolic acids (2.32 mg mL−1, 4.93 µg mL−1, and 12.35 µg mL−1, respectively). The enriching cookies had no effect on water absorption capacity (WAC) and oil absorption capacity (OAC). After in vitro hydrolysis, the highest peptide content was noted in 1.0 SJW (0.52 mg mL−1), and the bioactive compounds were characterized by high potential bioaccessibility (PAC), but poor bioavailability (PAV). The addition of SJW increased the ACE, α-amylase, and LOX inhibitory effect, but reduced the inhibition of pancreatic lipase. The highest antioxidant activity was noted for 1.0 SJW. The results showed that only 0.5 SJW and 1.0 SJW had slight antimicrobial activity against E. coli ATCC 25922 and B. cereus ATCC 14579 with MIC = 12.5 mg mL−1. Fractions with molecular mass <3.0 kDa were characterized with the highest p-coumaric acid content. The results show that SJW cookies had a higher content of bioactive compounds and more potent anti-metabolic syndrome effects.

Bioactivity of Hydrolysates Obtained from Chicken Egg Ovalbumin Using Artichoke (Cynara scolymus L.) Proteases

The aim of this work was to obtain chicken egg ovalbumin hydrolysates using aspartic proteinases present in extracts from the artichoke flower (Cynara scolymus L.) and evaluate their antioxidant, antimicrobial, and angiotensin I-converting enzyme (ACE) inhibitory activity in vitro. Hydrolysis time and molecular weight (<3 kDa) had a significant influence on the hypertensive and antioxidant activity of the hydrolysates. The <3 kDa fraction of the 16 h hydrolysate had an ACE inhibitory activity with an IC50 of 64.06 µg peptides/mL. The fraction <3 kDa of ovalbumin hydrolysate at 2 h of hydrolysis showed a DPPH radical scavenging activity of 30.27 µM of Trolox equivalents/mg peptides. The fraction <3 kDa of the hydrolysate of 16 h had an ABTS+ caption activity of 4.30 mM of Trolox equivalents/mg peptides. The fraction <3 kDa of the hydrolysate of 2 h had an iron (II) chelating activity of 32.18 µg peptides/mL. From the peptide sequences identified in the hydrolysates, we detected four peptides (from the BIOPEP database) that were already in their bioactive form (IAAEVYEHTEGSTTSY, HLFGPPGKKDPV, PIAAEVYEHTEGSTTSY, and YAEERYPIL), and are reported to display antioxidant and ACE inhibitory activity.

134 Effects of Aspergillus oryzae prebiotic on in vitro digestibility and fermentability of fibrous ingredients and diets

It has been an increasing interest to develop strategies to improve utilization of fibrous ingredients in swine. The objective of this study was to determine the effects of an Aspergillus oryzae prebiotic (AOP, Amaferm®) on in vitro digestibility and fermentability of energy and dry matter using a 3-step in vitro enzymatic digestion and fermentation. Three diets were formulated based on corn-DDGS (DDGS), rice bran (RB), and wheat middlings (WM). During in vitro hydrolysis and fermentation, 0.05% AOP was added to the diets (DDGS+AOP, RB+AOP, and WM+AOP) and sole ingredients. Each 2-g sample was hydrolyzed for 2 h with pepsin and for a subsequent 4 h with pancreatin. Hydrolyzed residues were filtered, washed, dried, weighed, pooled within the same sample, and used for subsequent fermentation using swine fecal inoculum. Volume of gas produced was recorded at 11 time points during 72 h of incubation. Parameters of gas production kinetics were calculated using a nonlinear monophasic model, and differences among treatment groups were compared using a mixed model. Supplementation of AOP increased (P &lt; 0.05) in vitro digestibility of gross energy (IVDGE) and dry matter (IVDDM) in the diets and AOP tended to increase (P = 0.06) dry matter fermentability of the diets. Supplementation of AOP during fermentation did not (P &gt; 0.1) affect maximum gas production in ingredients or diets. AOP decreased the half-time to asymptote (T/2) in the ingredient of DDGS+AOP compared with that of DDGS and increased the fraction rate of degradation at T/2 in the diet of RB+AOP compared with that of RB. In conclusion, supplementation of AOP increased energy and dry matter digestibility of the diets during in vitro hydrolysis and modified gas production kinetics during in vitro fermentation and the effects were dietary- and ingredient-type dependent.