The instantaneous controlled pressure drop process (or D.I.C process: “Detente Instantanee Controlee”) was used as pre-treatment prior to pectin acid extraction from orange peel. This process involves subjecting the orange peel for a short time to steam pressure varying from 100 to 700 kPa, followed by an instantaneous decompression to vacuum at 5 kPa. Effects of processing pressure, moisture content of peels before the thermomechanical treatment and processing time were examined with response surface methodology. The optimal conditions were determined and the responses surfaces were plotted from the mathematical models. The Fisher test and p-value indicated that both processing pressure and the moisture content of peels before the pre-treatment had highly significant effect on the pectin yield. The quadratic effect of processing pressure as well as the interaction effects of the initial moisture content and processing time had also a significant effect on the response. Moreover, the kinetics of pectin extraction showed that after few minutes of hydrolysis, the yields of pectin were systematically higher than that of control sample and this is important from industrial point of view because the hydrolysis of pectin is generally performed in 10-15 minutes.
Abstract Second order derivative spectroscopy in the ultraviolet (UV) region has the potential to detect subtile differences in the environments of the aromatic residues of proteins. We show here that second order derivative UV spectrum of protein can be a tool to determine quantitatively the aromatic amino acid apparent composition “AAAAC” at protein surface. This non destructive method allows us to obtain conformational information for protein structure-function research and for protein identification in chromatographic process.
Goat whey was hydrolysed by pepsin in an ultrafiltration membrane enzymic reactor coupled with a 30 kDa mineral membrane. Peptides collected in the permeate were resolved using reversed-phase HPLC. Their sequences were determined by amino acid analysis, second order derivative spectra analysis and mass spectrometry. Owing to the resistance of β-lactoglobulin (β-lg) towards pepsin, the majority of peptides identified were derived from α-lactalbumin (α-la). Pepsin showed a broad specificity of hydrolysis sites and generated a wide range of products from dipeptides to very large peptides containing disulphide bridges. The molecular masses of peptides resulting from α-la degradation were between 150 and 6900 Da: 36% were < 600 Da, 24% were 600–2000 Da and 40% were > 2000 Da.
Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.
Bovine globin has been incubated with mice peritoneal macrophages in order to study its hydrolysis by lysosomal enzymes, among which chiefly cathepsin D. Analysis of resulting peptides, by reversed‐phase high‐performance liquid chromatography (RP‐HPLC), shown the release of a bioactive peptide, VV‐hemorphin‐7. When a carboxyl proteinase inhibitor such as pepstatin A was added, no hemorphin was generated. Our results clearly demonstrated that VV‐hemorphin‐7 generation was principally due to cathepsin D. This study allowed us to hypothetize a possible pathway for in vivo hemorphins appearance from globin catabolism by macrophages.
Angiotensin-I-Converting Enzyme (ACE) inhibitors peptides were produced from unsupplemented acid goat whey fermented aerobically for 168 h by Kluyveromyces marxianus and Lactobacillus rhamnosus . This yeast-lactobacillus association is GRAS. Two novel lactokinins were identified: NYW and W with IC 50 of 20 and 0·86 μ m respectively. They both were resistant toward simulated gastrointestinal digestion. In addition, WLAHK was found in the hydrolysate. These three sequences belong to f (99–110) of α-la which seems to be a lactokinin cryptic zone. W was the major molecule released by the fermentation process. Considering that W is the precursor of serotonin, the hydrolysate produced could be of interest for the generation of functional health ingredient involved in regulation of affective disorders and hypertension.
To determine the angiotensin-I converting enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates, (N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-hippuryl-His-Leu hydrate salt (HHL)), and a natural one, angiotensin-I. The IC50 value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nanomolar range (1.79-15.1 nM) when synthetic substrates were used, whereas it exhibited IC50 of micromolar range (16.71 μM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC50 of micromolar range (17.13-146 μM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK), and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC50 displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK, and VIY show IC50 values over the IC50 value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW, and LKP exhibited IC50 value lower than the IC50 value of losartan for all substrates tested and were thus considered as good candidates for effectively decreasing hypertension. It appears that the comparison of IC50 is not consistent when IC50 values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references such as captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.
