Isolation and characterisation of antibacterial peptides derived from the f(164–207) region of bovine αS2-casein

A chymosin digest of sodium caseinate, which showed antibacterial activity against Listeria innocua, was fractionated using reverse phase high performance liquid chromatography and the purified antibacterial peptides were characterised by mass spectrometry, N-terminal amino acid sequencing and comparison to peptide masses of theoretical enzymic digests of milk proteins. Five antibacterial peptides, Cr1, Cr3, Cr4, Cr5 and Cr7 corresponding to amino acid residues 181–207, 180–207, 175–207, 164–207 and 172–207 of bovine α_S2-casein, respectively, were isolated. The minimal inhibitory concentration of peptides Cr1, Cr4 and Cr5 was determined against a range of Gram-positive and Gram-negative bacteria and showed similar activities to those of the bacteriocin peptide, nisin and the antibacterial peptide, lactoferricin B against certain Gram-positive bacteria. A partially purified chymosin digest of sodium caseinate (Cr_MIX) was prepared and observed to be heat stable for up to 15 min on exposure to 121 °C. Although Cr_MIX showed bactericidal activity against Salmonella Typhimurium in 0.1% (w/v) peptone medium, no antibacterial activity was observed when tested in skim milk at the same concentration.     

Plasmin digestion of photooxidized milk proteins

Plasmin-mediated hydrolysis of 6 different milk protein preparations [α_S-casein (α_S1 + α_S2), β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, and lactoferrin] was found to be very dependent on photooxidation of the said proteins. Changes in plasmin proteolysis were investigated in a peptide-mapping study applying liquid chromatography-mass spectrometry. The changes were seen in the formation of peptides formed by plasmin-mediated hydrolysis after photooxidation, which was initiated with the naturally occurring photosensitizer riboflavin in all the milk protein preparations studied. The changes in the plasmin-mediated hydrolysis of photooxidized proteins are discussed in relation to changes introduced in the protein structure upon photooxidation. Plasmin-mediated hydrolysis of α_S-casein, consisting of a mixture of α_S1- and α_S2-casein and a preparation of β-casein, was most highly affected by photooxidation, which is in agreement with the fact that those 2 proteins have been found to be most labile toward photooxidation. Changes in the formation of potential angiotensin-I-converting enzyme-inhibitory peptides as well as peptides proposed to have antibactericidal activities by plasmin were observed by oxidation of milk proteins before plasmin-mediated hydrolysis.     

Proteolysis of milk proteins lactosylated in model systems

Five different milk proteins (alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin, and lactoferrin) and a peptide substrate were applied as substrates for the investigation of how lactosylation affected proteolysis by different proteases. After a lactosylation period of 4 days in aqueous solution, at 65 degrees C and pH 6.8 in a protein: lactose ratio of 1000 the proteins were enzymatically hydrolyzed by the three milk relevant proteases plasmin, cathepsin D, and chymosin. Lactosylation of all substrates affected hydrolysis by plasmin negatively, with the largest effect on the globular proteins. This could be explained by modification of lysine residues, being the preferred cleavage site for plasmin, but also the residue generally preferred for lactosylation. Lactosylation of the caseins and of beta-lactoglobulin did not affect subsequent cleavage by cathepsin D and chymosin significantly, but for beta-lactoglobulin, both the secondary as well as the tertiary structure were affected by lactosylation. In contrast, decreased hydrolysis by cathepsin D and chymosin was observed for lactoferrin after lactosylation. Decreased hydrolysis may be caused by a more compact tertiary structure induced by lactosylation of lactoferrin, as indicated by fluorescence spectroscopy measurements.     

Ripening of extra-hard cheese made with mesophilic DL-starter

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on α_s1- and β-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from α_s1-CN f1–23, and the peptide β-CN 29–93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used.     

Antioxidant activity of yoghurt peptides: Part 2 – Characterisation of peptide fractions

The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultra filtration using membranes with cut off sizes of 30, 10 and 3 kDa. The peptides in these fractions were identified by LC–MS/MS. The identified peptides comprised a few N-terminal fragments of αs₁-, αs₂-, and κ-casein, and several fragments from β-casein. Almost all the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained a considerable amount of free amino acids such as His, Tyr, Thr and Lys, which have been reported to have antioxidant properties. Thus, our findings confirm that the antioxidant effects of the peptide fractions from yoghurt are due to the presence of certain peptides and free amino acids with recognised antioxidant activity in these fractions.     

Impact of milk processing on the generation of peptides during digestion

Milk processing may induce changes in dairy product composition and influence digestibility and nutrient bioavailability. Differences in protein degradation and peptide generation were studied for β-lactoglobulin and α_S1-casein from commercially available dairy products before, during, and after in vitro digestion. All major milk proteins, except β-lactoglobulin, were degraded to smaller peptides during the gastric phase in all investigated products. After the gastric phase, a shortened fragment of β-lactoglobulin was identified in the non-fermented dairy products, underlining differences in protein conformation due to the fermentation process. During the gastric phase, greater numbers of small peptides were generated from α_S1-casein than from β-lactoglobulin. The monitoring of generation of specific β-lactoglobulin and α_S1-casein peptide profiles by liquid chromatography–mass spectrometry allowed the identification of potential bioactive peptides. Peptides with satiety-influencing DPP-4 inhibiting properties were monitored and quantities were compared between products to identify promising targets for the development of new health promoting products.     

Primary proteolysis studied in a cast cheese made from microfiltered milk

Primary proteolysis was studied in a starter-free cheese model made from microfiltered (MF) milk (19.0% casein,<0.2% whey proteins). Specificity of plasmin and chymosin activity was investigated in the pH range 5.0–6.0, by analysis of peptide composition using high-performance liquid chromatography and liquid chromatography–mass spectrometry. Hydrolysis experiments with purified caseins were performed to aid identification of peptides released by specific activities. Plasmin had no activity in cheese below pH 5.4, while its activity increased from pH 5.4 to 6.0. Chymosin activity on the Phe₂₃–Phe₂₄ bond of α_S1-casein had an optimum pH around 5.3, while release of the bitter peptide β-casein (f193–209), effected by chymosin, was highest at pH 6.0. At pH <5.3, the specificity of chymosin on α_S1-casein changed, and the peptide bond Leu₂₀–Leu₂₁ was cleaved at an increasing rate with decreasing pH. Demineralisation of the MF retentate generally increased proteolytic activity.     

Effects of natural trypsin inhibitor from soybean on texture deterioration of the bay scallop (Argopecten irradians) during cold storage and its mechanism

In this study, the inhibitory effects of natural trypsin inhibitor from soybean (SBTI) on texture deterioration of adductor muscle of scallop (AMS) during cold storage and its mechanism were revealed by determining the changes of structural proteins of AMS in situ and in vitro, respectively. Results indicated that the endogenous proteolysis resulted in the disintegration of connective tissue proteins, the degradation of myosin and actin, as well as the reduction of α-helix, the increase of β-sheet and amino acid side chains, and the exposure of tryptophan residues of isolated myofibrillar proteins (MPs). These changes in structural proteins contribute to the texture deterioration of AMS as reflected in a significant decrease in hardness, chewiness, adhesiveness and springiness, but a significant increase in cohesiveness. SBTI significantly inhibited the conformational changes of structural proteins caused by endogenous proteolysis, improved the MP gel-forming ability and consequently retarded the texture deterioration of AMS.     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

Proteomic characterisation of hydrothermal redox damage

BACKGROUND: Peptide and protein damage contributes to the loss of quality and value in protein‐based food and textile products as well as to the degeneration of biological tissues such as hair and skin. The effects of elevated temperature on such substrates at the molecular level are, however, relatively unknown. This paper examines the response of peptides and proteins to hydrothermal damage using mass spectrometry and reports the location of molecular markers of hydrothermal damage within wool proteins. RESULTS: The hydrothermal exposure of model peptides containing the oxidatively sensitive residues tryptophan and tyrosine revealed the formation of a number of products such as hydroxytryptophan and dihydrophenylalanine. A variety of degradation products were also observed in intermediate filament proteins, including products arising from deamidation and from oxidation of histidine, tyrosine and tryptophan residues. CONCLUSION: The products observed to form during hydrothermal exposure indicated the involvement of reactive oxygen species. Molecular markers were identified within a proteinaceous system to allow the evaluation of damage type or severity. These findings have important implications for the thermal processing of foods and textiles. Copyright © 2011 Society of Chemical Industry     

发酵对酸肉蛋白质结构的影响

以猪背最长肌为原料制备酸肉,研究蛋白质结构在发酵中的变化。结果表明,随发酵时间延长,蛋白质静电作用力逐渐减弱,疏水相互作用和二硫键作用显著增强后呈小幅度波动变化。蛋白紫外吸收结果显示,肌原纤维蛋白芳香族氨基酸残基偏向更加疏水的环境移动,肌浆蛋白酪氨酸、色氨酸残基微环境极性增加。内源荧光图谱显示,荧光强度总体呈下降趋势且相对发酵0 d时发生红移,表明色氨酸发生了氧化降解,其残基主要暴露到极性环境中。拉曼光谱分析表明,随发酵进行,蛋白质α-螺旋减少,β-折叠增多,发酵110 d酸肉蛋白二级结构可能发生了重排现象。结果表明长时间发酵可使酸肉蛋白质二级结构和三级结构发生变化,这些变化可从蛋白质结构水平上解释发酵对酸肉品质的影响。     

Aggregation and conformational changes of bovine β-lactoglobulin subjected to dynamic high-pressure microfluidization in relation to antigenicity

Our previous research indicated that dynamic high-pressure microfluidization (DHPM) had a significant effect on the antigenicity of β-lactoglobulin (β-LG). In this study, aggregation and conformational changes subjected to DHPM (0.1-160 MPa) were investigated in relation to antigenicity. When DHPM pressure increased from 0.1 to 80 MPa, disaggregation of β-LG samples and partial unfolding of the molecule were accompanied by an increase in β-LG antigenicity, which was reflected in the decrease of particle size, increase of free sulfhydryl (SH) contents and β-strands contents, and slight exposure of aromatic amino acid residues. At pressures above 80 MPa, the reaggregation of β-LG may contribute to the decrease in antigenicity, which was reflected by an increase in particle size, the formation of aggregates, a decrease of in SH and β-strands contents, and slight changes in aromatic amino acid residues. Aggregation and conformational changes of β-LG under DHPM was related to its antigenicity.     

Changes in myofibrillar structure of silver carp (Hypophthalmichthys molitrix) as affected by endogenous proteolysis under acidic condition

Effect of endogenous proteolysis on myofibrillar conformational changes under acidic condition was investigated by spectroscopic techniques. The result of intrinsic tryptophan fluorescence showed that tryptophan residues were exposed to polar environment by acid denaturation of protein and participation of endogenous proteolysis. Extrinsic fluorescence indicated that average surface hydrophobicity (S_hANS) decreased by reduction of pH but increased by endogenous proteolysis. Second‐derivative UV‐spectroscopy implied that the decrease of S_hANS under acidic condition may be due to burying of some aromatic amino acid residues into hydrophobic cores induced by acid‐induced aggregation, such as tyrosine, while S_hANS increased by exposure of some hydrophobic amino acids due to the effect of endogenous proteases. Raman spectra verified the changes in microenvironment of tryptophan and tyrosine residues, and showed that aliphatic amino acids were mainly exposed to polar solvent by both acid denaturation and endogenous proteolysis. In addition, new disulphide bonds were less likely to form by acid‐induced aggregation and proteolytic cleavage, while random coils increased and smaller water domains were found.     

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from α_S1-CN, 813 from α_S2-CN, and 234 from κ-CN. The majority of β-CN- and α_S1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from α_S2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, α_S2-CN, as well as α_S1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and α_S1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of α_S2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.     

地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响

利用地衣芽孢杆菌凝乳酶制作切达干酪和切达干酪类似物,分析干酪成熟过程中各蛋白水解指标的变化规律,以揭示地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响。结果表明,CDF组（添加地衣芽孢杆菌D3.11凝乳酶所制切达干酪）、CD3组（添加地衣芽孢杆菌D3.11凝乳酶但未添加发酵剂制成的干酪类似物）和CCF组（添加商品凝乳酶所制切达干酪）干酪蛋白含量、pH 4.6-可溶性氮、12%三氯乙酸-可溶性氮、5%磷钨酸-可溶性氮、总游离氨基酸含量均随着成熟时间延长呈显著增加趋势,并且成熟期间CDF组干酪均显著高于CCF组干酪（P＜0.05）;十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明,CDF组干酪α-酪蛋白水解程度较大;pH 4.6-可溶性肽段分析表明,随着干酪的成熟,总肽含量呈先增加后下降趋势,但疏水性肽与亲水性肽的比值呈持续下降趋势,在成熟第6个月时,CDF组、CD3组和CCF组干酪疏水性肽与亲水性肽比值分别为2.668、2.822、3.788。主成分分析表明,3 组干酪的蛋白水解程度与成熟度呈正相关,与疏水性肽和亲水性肽的比值呈负相关。以上结果表明,利用地衣芽孢杆菌凝乳酶制作的干酪蛋白水解度更高,但其疏水性肽比例较小,研究结果可为地衣芽孢杆菌凝乳酶在干酪生产中的应用提供理论依据。     

Photo-oxidation of whey proteins: Molecular markers of modification

Oxidative damage significantly affects the food industry, with progressive modification of protein foods and ingredients altering structure, shelf-life, digestibility, nutritional value and function. A redox proteomic damage scoring system was applied to profile and track protein primary level photo-oxidative modification in UVB-irradiated bovine milk whey proteins, lactoferrin and β-lactoglobulin. Lactoferrin oxidation increased significantly after ultraviolet B (UVB) exposure, with the redox score increasing from 0.38 in the control to 1.00 in the irradiated sample. β-lactoglobulin oxidation also increased significantly, from a relatively high baseline redox score of 1.07 in the control to 1.67 in the irradiated sample. A potential marker peptides set for tracking photo-oxidation in milk whey proteins was characterised, with six lactoferrin and four β-lactoglobulin peptides identified. This is anticipated to be of significant utility in monitoring and tracking relative levels of modification, and therefore exposure to oxidative insult, in dairy products through processing, storage, and retail and consumer handling.     

Charakterisierung pankreatischer Casein-Plasteine

Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of β-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from α_s-casein, were determined.     

Release of pyrraline in absorbable peptides during simulated digestion of casein glycated by 3-deoxyglucosone

The glycation product 6-(2-formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine (pyrraline), which is formed in the final stage of the Maillard reaction, is taken up in significant amounts with the daily diet. The high bioavailability of pyrraline known from human balance studies can be partly explained by the fact that pyrraline-containing dipeptides are substrates of the human peptide transporter 1 (PEPT1). In the present study we assessed whether pyrraline-containing peptides are formed during luminal digestion of glycated proteins. For this, casein and β-casein, respectively, were enriched with peptide-bound pyrraline by incubation with 3-deoxyglucosone (3-DG), and the modified casein samples were subjected to simulated gastrointestinal digestion. The digestibility of modified casein decreased with increasing pyrraline concentration as measured by analytical size-exclusion chromatography of the digested peptide mixtures. After digestion, 50–60 % of pyrraline was bound in peptides smaller than 1,000 Da isolated by ultrafiltration. Only <4 % of pyrraline was released as the free amino acid. High-pressure liquid chromatography in combination with mass spectrometry of small peptides from digestion mixtures, however, revealed the formation of a large number of different pyrraline dipeptides, which can theoretically be absorbed from the gut by the intestinal peptide transporter PEPT1. Moreover, the N-terminal arginine residue of β-casein modified with 3-DG to a pyrraline analogue at its amino group was also released. The high occurrence of potentially absorbable pyrraline peptides and the minor occurrence of non-absorbable free pyrraline after luminal digestion imply that the decrease in digestibility can yet enhance the bioavailability of pyrraline.     

Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis

The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of β-casein had a positive effect on the cheese yield expressed as grams of cheese per 100 g of milk, whereas several other minor fragments of β-, α_s1-, and α_s2-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.     

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC₅₀ = 287 μg/mL) and (ii) from β-lactoglobulin (IC₅₀ = 128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC₅₀).