This study describes the characterisation of whey protein hydrolysates obtained from tryptic hydrolysis to assess their application as ingredients with angiotensin-converting-enzyme (ACE) inhibitory action. The levels of a-lactalbumin (alpha-la) and P-lactoglobulin (beta-lg) remaining after hydrolysis were quantified. Peptides were separated by RP-HPLC, and Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), the most potent beta-lg-derived ACE-inhibitory peptide was monitored. A correlation curve was established for the production of this peptide as a function of hydrolysis time. Heat-induced gelation of hydrolysates was studied by small-deformation rheology. The gelation times and the strength of the final gels were highly dependent on the degree of hydrolysis. Smaller peptides liberated by hydrolysis contributed to the inability of whey protein hydrolysates to gel. (c) 2006 Elsevier Ltd. All rights reserved.

The simultaneous separation and quantification of two casein peptides (IPP, VPP) presenting potent inhibitory activity of angiotensin-converting-enzyme (ACE) and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 mL/min, using a mixture of two solvents. Solvent A was 0.1% TFA in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by UV detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 mg/mL for VPP, 0.005-1.0 mg/mL for IPP, and 0.05-3.0 mg/mL for casein. R 2 invariably exceeded 0.999. The detection limits were 0.004 for VPP, 0.002 mg/mL for IPP, and 0.02 mg/mL for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing VPP, IPP, and casein. The RSD values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of VPP, IPP, and casein in commercial fermented milks labeled as presenting anti hypertensive properties, but also, in milk with different degrees of fermentation by L Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse phase-HPLC analysis. The reduction of kappa-, alpha-, and beta-caseins (CN), alpha-lactalbumin (alpha-LA), and beta-lactoglobulin (beta-LG) contents during 48 and 90 h of incubation of pasteurized milk (100 mL) and respective cheese whey with kefir grains (6 and 12 g) at 20 degrees C was monitored. Significant proteolysis of alpha-LA and kappa-, alpha-, and beta-caseins was observed. The effect of kefir amount (6 and 12 g/100 mL) was significant for alpha-LA and alpha- and beta-CN. alpha-Lactalbumin and beta-CN were more easily hydrolyzed than alpha-CN. No significant reduction was observed with respect to beta-LG concentration for 6 and 12 g of kefir in 100 mL of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of alpha-LA and beta-LG: alpha-LA was hydrolyzed between 60 and 90% after 12 h (for 6 and 12 g of kefir) and no significant beta-LG proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of alpha-LA and kappa-, alpha-, and beta-caseins. Hydrolysis is dependent on the kefir: milk ratio and incubation time. beta-Lactoglobulin is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase beta-LG digestibility in whey-based or whey-containing foods.

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S](+) cluster. Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in Fe-57-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis, The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.

Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alpha H195Q, alpha H195N, and alpha Q191 K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N-2 with the alpha H195Q and alpha H195N variants, but not with the alpha Q191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alpha H195Q or alpha H195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alpha Q191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze Fe-57 Mossbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alpha H195Q and alpha H195N MoFe proteins can bind N-2, but alpha Q195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N-2 reduction. (C) 2007 Elsevier Inc. All rights reserved.