Lourdes Amigo

β-Lactoglobulin as source of bioactive peptides

Summary.β-Lactoglobulin (β-Lg) is currently an important source of biologically active peptides. These peptides are inactive within the sequence of the precursor protein, but they can be released by in vivo or in vitro enzymatic proteolysis. Once released, these peptides play important roles in the human health, including antihypertensive, antioxidant and antimicrobial activities as well as opioid-like features and ability to decrease the body-cholesterol levels. Bioactive peptides derived from β-Lg are currently a point of intensive research. Their structure, biological significance and mechanism of action are briefly presented and discussed in this review.

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.

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.

Identification of Antibacterial Peptides from Bovine κ-Casein

The objective of the present study was to identify antimicrobial peptides present in several digests of commercial caseins with gastric enzymes. The most active hydrolysate against Escherichia coli ATCC 25922 and Listeria innocua CECT 910T corresponded to a pepsin digest of bovine kappa-casein. The protein digest was first separated by semipreparative high-performance liquid chromatography (HPLC), and the most active fractions were again subjected to a second chromatographic step. Finally, identification of the active peptides was carried out by online and offline HPLC-electrospray ionization-tandem mass spectrometry. By means of this technique, 21 peptides were identified in the active HPLC fractions. Although most were derived from bovine kappa-casein, some of the identified fragments corresponded to beta-casein and alpha(s)-casein fragments, a result of the presence of small amounts of these proteins in the preparation of kappa-casein. Some of the peptides identified were chemically synthesized and showed antibacterial effects against several gram-positive and gram-negative bacteria. Among the synthesized peptides, kappa-casein f(18-24), f(30-32), and f(139-146) were most effective against all bacteria tested. The antibacterial effect of these peptides is discussed in relation to their amino acid sequences.

Assessment of the quality of dairy products by capillary electrophoresis of milk proteins.

This paper presents an overview of existing capillary electrophoretic methods for the study of milk proteins. The main methods of analysis of caseins, whey proteins and peptides are examined with particular attention to their application to the evaluation of the quality of dairy products. Aspects such as the study of protein polymorphism, evaluation of heat treatments, detection of adulteration and assessment of proteolysis are considered in detail.

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

F. AMARITA, T. REQUENA, G. TABORDA, L. AMIGO AND C. PELAEZ. 2001.

Effects of concentrates with different contents of protected fat rich in PUFAs on the performance lactating Granadina goats: Part II. Milk production and composition

Abstract With the aim of obtaining higher quality goats milk, three groups of Granadina goats in the middle of their second lactation were fed diets consisting of forage and concentrate fractions. To the concentration fractions was added 0, 9 or 12% of a fat protected against rumen metabolism that was particularly rich in polyunsaturated fatty acids (PUFAs). The composition of the protected fat was determined as was the milk yield and the chemical composition of the milk. The PUFAs content of the protected fat was 14.3%, with the degree of saponification equal to 84.9%. Daily milk production and milk dry matter, protein, fat and lactose concentration did not vary significantly in relation to diet. In all cases, all values depended on the corresponding energy intake. However, milk fat from goats fed protected fat contained higher concentrations of PUFAs. Also, fat addition caused some differences in the fractional composition of milk protein. It is concluded that milk fat from goats can be modified by nutritional means to obtain a healthier product.

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.