Mercedes Ramos

Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis.

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 microg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 microg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 < 40 microg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 microM, respectively.

Stability to gastrointestinal enzymes and structure–activity relationship of β-casein-peptides with antihypertensive properties

Physiological digestion plays a key role in the formation and degradation of angiotensin-converting enzyme (ACE)-inhibitory peptides. In this study, we evaluated the impact of a simulated gastrointestinal digestion on the stability of eight peptides previously identified in fermented milk with antihypertensive activity. Two of these identified peptides with sequences LHLPLP and LVYPFPGPIPNSLPQNIPP, possess ACE-inhibitory activity in vitro and antihypertensive activity in vivo. The results showed that LHLPLP was resistant to digestive enzymes. In contrast, LVYPFPGPIPNSLPQNIPP was totally hydrolyzed and its activity decreased after incubation with pepsin and a pancreatic extract. The peptide LHLPLP was incubated with ACE and was found to be a true inhibitor of the enzyme and to exhibit a competitive inhibitor pattern. A structure-activity relationship study of this peptide was carried out by synthesizing several modified peptides related to the sequence LHLPLP. The substitution of amino acid Leu in the penultimate position by Gly improved the ACE-inhibitory activity twofold and the substitution of Pro at C-terminal position by Arg increased the activity twofold, with an IC50 of LHLPLR as low as 1.8 microM.

Short-term effect of egg-white hydrolysate products on the arterial blood pressure of hypertensive rats.

In the present study we evaluate the blood pressure-lowering effect of the following products: the hydrolysate obtained from egg white (EW) by enzymatic treatment with pepsin (HEW), the peptide fraction of HEW with molecular mass lower than 3000 Da (HEW<3000 Da), and three peptide sequences isolated from HEW<3000 Da (Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu: YAEERYPIL); (Arg-Ala-Asp-His-Pro-Phe-Leu: RADHPFL); and (Ile-Val-Phe (IVF)). These peptides, and also HEW and HEW<3000 Da, had been characterized previously in vitro as potent inhibitors of angiotensin-converting enzyme (ACE). EW and the products mentioned earlier were orally administered by gastric intubation, to 17-20-week-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. We measured the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of the rats by the tail cuff method before administration and also 2, 4, 6, 8 and 24 h post-administration. Distilled water served as negative control, and we used captopril (50 mg/kg) as positive control to carry out similar experiments with a known ACE inhibitor. HEW, HEW<3000 Da and the three peptide sequences decreased SBP and DBP in SHR but they did not modify these variables in WKY rats. The peptide sequences YAEERYPIL, RADHPFL and IVF showed a potency to decrease blood pressure greater than HEW or HEW<3000 Da. The results obtained suggest that the studied products could be used as a functional food with potential therapeutic benefit in the prevention and treatment of hypertension.

Influence of skimmed milk concentrate replacement by dry dairy products in a low fat set-type yoghurt model system. I: Use of whey protein concentrates, milk protein concentrates and skimmed milk powder

Ten commercial samples of dry dairy products used for protein fortification in a low fat yoghurt model system at industrial scale were studied. The products employed were whey protein concentratres, milk protein concentrates, skimmed milk concentrates and skimmed milk powder which originated from different countries. The gross chemical composition of these dried products were determined, including polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing of the proteins, and minerals such as Na, Ca, K and Mg. Yoghurts were formulated using a skim milk concentrated as a milk base enriched with different dry dairy products up to a 43 g kg−1 protein content. Replacement percentage of skim milk concentrated by dry dairy products in the mix was between 1.49 and 3.77%. Yoghurts enriched with milk protein concentrates did not show significantly different viscosity (35.12 Pa s) and syneresis index (591.4 g kg−1) than the two control yoghurts obtained only from skimmed milk concentrates (35.6 Pa s and 565.7 g kg−1) and skimmed milk powder (32.77 Pa s and 551.5 g kg−1), respectively. Yoghurt fortified with the whey protein concentrates, however, was less firm (22.59 Pa s) and had less syneresis index (216 g kg−1) than control yoghurts. Therefore, whey protein concentrates may be useful for drinking yoghurt production. © 1999 Society of Chemical Industry

Genetic polymorphism of ovine milk proteins: its influence on technological properties of milk - a review.

Current knowledge of the genetic polymorphism in ovine milk proteins including heterogeneity detected and their relationships with the technological properties of milk is reviewed. In the casein fraction a great heterogeneity has been determined either by the presence of genetic variants or other factors such as a discrete phosphorylation level and the coexistence of protein forms of different chain length. In the whey fractions, three genetic variants (A, B, and C) are described for β-lactoglobulin (β-LG), and two for α-lactalbumin (α-LA). The topics discussed include the rennetability of ovine milk, cheese yield and susceptibility of β-LG genetic variants to heat denaturation as well as the findings concerning the relationships between ovine milk protein polymorphism and milk production and composition. However, further investigation is needed in order to better outline the relevant features of polymorphism and technological properties of ovine milk.

Application of NMR spectroscopy to milk and dairy products

NMR spectroscopy is a technique in increasing use for dairy research. It provides us with unique information that can be applied to research or to quality control of dairy samples. In addition, it is a non-destructive and very versatile technique, providing data on the same sample under different parameters. This review intends to give an overview of the type of information that can be obtained, based on some results obtained in dairy research. It includes the application of NMR for qualitative and quantitative analysis, monitoring reactions in vivo, isotopic analysis, study of the physical state of milk fat and water, structural characterization and studies on the conformational and aggregation state of proteins.

Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening

Abstract Changes in the microflora, physicochemical characteristics and the levels of proteolysis and lipolysis in Gamonedo cheese were examined over a 180 day ripening period. The total microbial count was high throughout manufacture and the ripening period. Growth of lactic acid bacteria was greater in the interior of the cheese than on the surface and among the lactic acid bacteria, lactobacilli predominated. Levels of enterococci and micrococci reached high numbers during the latter stages of maturation which suggests that these organisms may play a significant role in ripening. Of the lactic acid bacteria. Lactobacillus plantarum was present at the highest levels, followed by L. casei spp. casei . Low levels of lactic streptococci and leuconostocs were detected and the species identified included Lactococcus lactis spp. lactis, Leuconostoc mesenteroides spp. mesenteroides and L. paramesenteroides . Levels of proteolysis and lipolysis were high: water soluble nitrogen, NPN and NNH 2 levels in the mature cheese were 57·3%, 51·7% and 31·2% of total N, respectively. The degradation of α s - and β-caseins was almost complete at the end of ripening. The level of total FFA was 75 684 mg/kg.

Application of capillary electrophoresis to the study of proteolysis of caseins

Capillary electrophoresis using hydrophillically coated capillary and a low pH buffer containing urea has been used to follow the proteolytic action of plasmin and chymosin on isolated casein fractions, whole casein and individual milk samples selected on the basis of their genetic variants. Several of the main casein breakdown products were identified. These included, among others, γ 1 -casein (CN) A 1 , γ 1 -CN A 2 , γ 1 -CN B, γ 1 -CN C, γ 2 -CN A, γ 1 -CN A 3 , γ 2 -CN B, γ 3 -CN A and γ 3 -CN B, as well as proteose peptones, arising from the action of plasmin on the different genetic variants of β-CN. α s1 -I-CN and α s1 -CN f(1-23) from α s1 -CN , and para-κ-CN and caseinomacropeptide from κ-CN produced by chymosin action were also separated. The knowledge of their migration times provided information on the extent and origin of casein hydrolysis in both milk and cheese, as found in samples of proteolysed milk or fresh cheese

Capillary electrophoretic analysis of genetic variants of milk proteins from different species

Polymorphism of bovine, ovine and caprine milk proteins was studied by CE. Identification of some rare bovine variants was carried out by isoelectric focussing (IEF) using PhastSystem. Genetic variants A and D of bovine alpha s2-casein, beta-casein variants A1, A2, A3, B and C and alpha s1-casein variants B and C were determined by CE. In addition, the different casein fractions including some genetic variants of ovine and caprine milk were identified by CE. In order to carry out this identification, collected fractions from a cation-exchange FPLC separation were injected by CE.

Free amino acids by high performance liquid chromatography and peptides by gel electrophoresis in Mahon cheese during ripening

Abstract The free amino acids and peptides contents of Mahon cheese during four months of ripening were studied using high performance liquid chromatography and gel electrophoresis, respectively. The total content of free amino acids generally increased throughout the ripening period, except in one batch. Phenylalanine, valine, proline, glutamic acid and isoleucine were the most abundant free amino acids in all the tests throughout the four month period. They accounted for between 67 and 80% of the free amino acids. No difference was found between the pattern of traditionally made and industrially made Mahon cheeses. Electrophoresis of soluble nitrogen showed the main whey proteins as well as other bands corresponding to peptides.