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Effect of enzymatic hydrolysis on surface activity and surface rheology of type I collagen

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Abstract

We describe the adsorption behaviour and rheological properties of a calf skin type I collagen, and of its hydrolysates obtained using a Clostridium histolyticum collagenase (CHC) under moderate conditions (pH 7, 37°C). The effect of CHC concentration (2×10(-9)-2×10(-6)M) and incubation time (35-85min) was studied and optimised to achieve the highest decrease of surface tension and the highest dilational surface viscoelasticity of the adsorbed layers. SDS-PAGE electrophoresis and reverse-phase high performance liquid chromatography (RP-HPLC) were used to characterise the hydrolysis products. The results show that even simple modifications (heat treatment, pH change, partial hydrolysis) of collagen enhances its surface properties, especially in terms of surface dilational elasticity modulus. The use of low enzyme concentration (CHC-to-collagen molar ratio of 4×10(-3)) and short incubation time (<45min) results in moderately hydrolysed products with the highest ability to lower surface tension (γ=53.9mNm(-1)) forming highly elastic adsorbed layers (surface dilational elasticity, E'=74.5mNm(-1)). Copyright © 2015 Elsevier B.V. All rights reserved.

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... To the best of our knowledge, studies of surface activity of collagen proteins have been limited to the fibrillar type I collagen. For this purpose, the protein has been modified using enzymatic hydrolysis with either collagen-specific enzymecollagenase [16] or non-specific one -alcalase [32]. Other modifications include: pH variation (in the range 1.8-9.0) ...
... Other modifications include: pH variation (in the range 1.8-9.0) [16], temperature treatment (in the range 21-90 • C) [20] and chemical acetylation using lauryl chloride or succinic anhydride [33,34]. ...
... On the other hand, with increasing CTAB concentration the mechanical properties of the mixed adsorbed layers clearly deteriorate (Fig. 3). Similarly to other low-molecular weight synthetic surfactants, CTAB itself forms adsorbed layers with poor mechanical properties [16,20]. Thus, worsening of the mechanical properties of the mixed layers suggests that the protein and surfactant molecules are co-adsorbing at the interface, but the collagen's ability to form elastic networks is reduced by binding with CTAB. ...
Article
We describe the effect of three synthetic surfactants (anionic – sodium dodecyl sulfate (SDS), cationic – cetyltrimethylammonium bromide (CTAB) and nonionic – Triton X-100 (TX-100)) on surface properties of the type I calf skin collagen at the air/water interface in acidic solutions (pH 1.8). The protein concentration was fixed at 5 × 10⁻⁶ mol L⁻¹ and the surfactant concentration was varied in the range 5 × 10⁻⁶ mol L⁻¹–1 × 10⁻⁴ mol L⁻¹, producing the protein/surfactant mixtures with molar ratios of 1:1, 1:2, 1:5, 1:10 and 1:20. An Axisymmetric Drop Shape Analysis (ADSA) method was used to determine the dynamic surface tension and surface dilatational moduli of the mixed adsorption layers. Two spectroscopic techniques: UV–vis spectroscopy and fluorimetry allowed us to determine the effect of the surfactants on the protein structure. The thermodynamic characteristic of the mixtures was studied using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC).
... The downstream processing necessary to recover and purify the microbial surfactants is time-consuming and generates high costs [4]. Even though purification is still an issue for animal-derived biosurfactants, the yield is no longer a problem, since the waste products from meat and leather industries can be used [5]. ...
... In contrast to the majority of other collagens, which are homotrimeric, the type I collagen is a heterotrimer, consisting of two identical ␣1 chains and one ␣2: [␣1(I)] 2␣ 2(I). Some aspects of surface activity of the modified type I collagen have already been investigated in the past [5,[20][21][22]. The beneficial effect of temperature denaturation on surface activity of collagen type I at different pH has been described in our previous report [5]. ...
... Some aspects of surface activity of the modified type I collagen have already been investigated in the past [5,[20][21][22]. The beneficial effect of temperature denaturation on surface activity of collagen type I at different pH has been described in our previous report [5]. We also recently studied the effect of low-molecular-weight surfactants on the native type I collagen at low pH (1.8) [23]. ...
Article
The effect of three synthetic surfactants on surface activity and surface dilatational rheology of temperature-denatured type I collagen at water/air interface is described. An anionic (sodium dodecyl sulfate, SDS), a cationic (cetyltrimethylammonium bromide, CTAB) and a nonionic (Triton X-100, TX-100) surfactants were employed at variable concentrations (5 × 10⁻⁶ mol L⁻¹–1 × 10⁻⁴ mol L⁻¹). With the protein concentration fixed at 5 × 10⁻⁶ mol L⁻¹, the protein/surfactant mixtures with molar ratios of 1:1, 1:2, 1:3, 1:5, 1:10 and 1:20 were obtained. An Axisymmetric Drop Shape Analysis (ADSA) method was used to determine the dynamic and equilibrium surface tension, as well as the surface dilatational moduli of the mixed adsorption layers formed at the water/air interface at pH 4.5. For the collagen-SDS mixtures, analogous studies were also performed at the water/tetradecane and water/olive oil interfaces. These results were complemented with the foam and emulsion formation ability tests, and the oil-in-water emulsions were characterized using Dynamic Light Scattering (DLS) and visual assessment. The most beneficial effect on surface activity, mechanical properties of adsorbed layer and foaming ability was observed for the mixtures of temperature-denatured collagen with SDS. The rheological parameters of the adsorbed layers formed by the same mixtures at the water/olive oil and water/tetradecane interfaces worsened significantly (values of rheological parameters E’ and E” are lower than values of E’ and E” of the adsorbed layers formed at the water/air interface). The corresponding emulsions, despite very low interfacial tension values, phase-separated within hours. We hypothesize that the poor kinetic stability was caused by flocculation due to collagen bridging between the oil droplets, resulting in emulsion creaming.
... Sufficient concentration and curing time will be able to break hydrogen and covalent bonds of a protein that is difficult to dissolve. As stated by Kezwoń et al. (2016), hydrolysis depends not only the concentration of the enzyme, but also the length of incubation of the protein with the enzyme. The longer the incubation time, the greater the opportunity for enzymes to cut protein molecules into peptides. ...
Article
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Ensuring food safety in livestock requires specific feeding technology in agriculture by using feed additives in the form of antibiotics, prebiotics, probiotics, acidifiers, hormones and enzymes. Bioactive peptides improve the health status of humans and animals. Protein hydrolysis produce peptides that are safe, quickly metabolizable, less risky for livestock products to have contaminant residue. Bioactive peptides are still bound to the original protein, so they need to be released through an enzymatic process. This research explored the potential of chicken slaughterhouse waste to produce biopeptides by hydrolyzing proteins using various proteolytic enzymes. The slaughterhouse waste included chicken feet, intestines, filleting waste and blood plasma. The proteolytic enzymes used were papain, bromelain, protease by Rhizopus oligosphorus, probiotic protease. The observed variables were dissolved protein content with and without precipitation, protein hydrolyzate content, and the degree of enzyme hydrolysis. The research was conducted using exploratory methods. The results showed that the dissolved protein content in the chicken slaughterhouse waste protein concentrate was 1,585 mg/ml (feet), 2,361 mg/ml (intestines), 1,787 (filleting waste) and 2,372 mg/ml (blood plasma). Blood plasma protein concentrate showed the highest yield among other chicken slaughterhouse waste protein concentrates, namely 0.14 mg/ml (hydrolysis of papain), 0.18 mg/ml (hydrolysis of bromelain), 0.56 mg/ml (hydrolysis of R.oligosphorus protease) 0.68 mg/ml (hydrolysis of probiotic proteases). The highest degree of hydrolysis was shown in blood plasma hydrolyzates using probiotic protease enzymes, namely 28.72%. The highest antioxidant activity was 92.92% as observed in chicken feet protein hydrolyzate which was hydrolyzed using papain. Therefore, chicken feet, intestines and fillet waste can produce protein concentrates through precipitation using ammonium sulfate, and plasma using acetone. The highest protein concentration was in blood plasma protein which also produced the highest hydrolysis from hydrolyzing blood plasma proteins with hydrolyzed probiotic protease. The highest antioxidant activity was observed in chicken feet protein hydrolyzate which was hydrolyzed using papain enzyme and incubated for 6h.
... Collagen is the second-largest renewable biomass resource in nature and exists widely in vertebrates, as skin, bone, cartilage, and tendon [1,2]. Skin collagen exists in the form of collagen fiber, and its basic unit is the collagen molecule with a molecular weight of approximately 300 kDa [3]. When treated by physical, chemical, or biological methods, insoluble collagen fiber can be hydrolyzed into the compounds with relatively low molecular weight and good hydrophilicity, known as collagen hydrolysates (CHs) [4,5]. ...
Article
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A series of collagen hydrolysates (CHs) were prepared from pigskin shavings by using pepsin (PCH), trypsin (TCH), Alcalase (ACH), HCl (HCH), and NaOH (NCH). Their physicochemical properties, including degree of collagen hydrolysis, molecular weight distribution, electric charge, and microstructure, were investigated, and their flocculation performance was evaluated in a kaolin suspension, at varied pHs and concentrations. PCH exhibited high flocculation capability under acidic and neutral conditions, and its efficiency for removing suspended particles was approximately 80% at a concentration of 0.05 g/L. TCH, ACH, HCH, and NCH showed almost no flocculation capability. The flocculation capability of PCH could be mainly due to a combination of optimal molecular weight distribution and electric charge. This study could provide an environment-friendly natural flocculant and also proposes a promising approach for the reuse of collagen wastes. Graphical Abstractᅟ
... Innocente et al. (1998) reported proteose peptone 3 produced from the milk fat globule membrane protein could reduce air/water surface tension to as low as 32.11 dynes/cm. Kezwoń et al. (2015) found that moderately hydrolyzed collagen could lower air/water surface tension to 53.9 dynes/cm. In this study, the air/water surface tension reduction capability of shrimp waste-based product was studied. ...
Article
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In this study, shrimp waste was enzymatically hydrolyzed to generate a green dispersant and the product was tested for crude oil dispersion in seawater. The hydrolysis process was first optimized based on the dispersant effectiveness (DE) of the product. The functional properties of the product were identified including stability, critical micelle concentration, and emulsification activity. Water was confirmed as a good solvent for dispersant generation when compared with three chemical solvents. The effects of salinity, mixing energy, and temperature on the dispersion of the Alaska North Slope (ANS) crude oil were examined. Microtox acute toxicity test was also conducted to evaluate the toxicity of the produced dispersant. In addition, DE of the product on three different types of crude oil, including ANS crude oil, Prudhoe Bay crude oil (PBC), and Arabian Light crude oil (ALC) was compared with that of the Corexit 9500, respectively. The research output could lead to a promising green solution to the oil spill problem and might result in many other environmental applications.
... Lafarga, et al. (2016) menyatakan bahwa penggunaan enzim papain dengan aktifitas > 3 U/mg dan konsentrasi serta lama pemeraman yang cukup akan mampu memutus ikatan hidrogen maupun kovalen suatu protein yang sulit terlarut. Ditambahkan oleh Kezwoń et al. (2016) Tabel 2 menunjukkan bahwa nilai konsentrasi protein terlarut yang diperoleh dari sampel larutan bagian atas (A) lebih tinggi dari pada sampel larutan bagian bawah (B) dengan kisaran masing-masing 1,31 -1,62 mg/ml dibanding 0,74 -0,76 mg/ml. Nilai tersebut sesuai dengan prinsip umum sentrifugasi bahwa protein yang mempunyau ukuran dan berat partikel lebih rendah dan seragam akan berada di atas begitu juga sebaliknya. ...
Article
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The objective of this study was to optimization antioxidant activity of active peptides of chicken feet with papain hydrolysis. The research method was experimental design conducted using Factorial Completely Randomized Design. Treatments included papain concentration (control (0%) , 1%, 2% and 4%) and Incubation time (24 hours, 36 hours and 48 hours) were repeated four times. The variables observed were proximate analysis,microstructure, dissolved protein concentration and antioxidant activity. The results indicate that the treatment of papain concentration and incubation time variations in chicken feet protein extraction gave a differenceinfluence (P
... Collagen is an amphoteric macromolecule that possesses a pI value between 7 and 8. On the hydrolysis process, the pI value is shifted to lower values between 3.68 and 5.7. This change will depend on the amino acid sequences and distribution of amino acid residues according to the type or time of hydrolysis [65,[70][71][72]. The composition and degree of hydrolysis of collagen are factors that increase functional properties such as antioxidant capacity, antimicrobial activity, and higher bioavailability. ...
Article
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Hydrolyzed collagen (HC) is a group of peptides with low molecular weight (3–6 KDa) that can be obtained by enzymatic action in acid or alkaline media at a specific incubation temperature. HC can be extracted from different sources such as bovine or porcine. These sources have presented health limitations in the last years. Recently research has shown good properties of the HC found in skin, scale, and bones from marine sources. Type and source of extraction are the main factors that affect HC properties, such as molecular weight of the peptide chain, solubility, and functional activity. HC is widely used in several industries including food, pharmaceutical, cosmetic, biomedical, and leather industries. The present review presents the different types of HC, sources of extraction, and their applications as a biomaterial.
... Furthermore, each source of collagen has numerous subtypes of collage that exist in different tissues and aid the specific tilized functions within the body [5,13]. Different sources, types, and extraction methods possess various characteristics [13,136,137], such as viscosity [138,139], isoelectric point (pI) [140], and molecular weight [141,142], which ultimately allows for the ability to control the scaffold properties and EC processing. ...
Article
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As the most prevalent structural protein in the extracellular matrix, collagen has been extensively investigated for biofabrication-based applications. However, its utilisation has been impeded due to a lack of sufficient mechanical toughness and the inability of the scaffold to mimic complex natural tissues. The anisotropic alignment of collagen fibres has been proven to be an effective method to enhance its overall mechanical properties and produce biomimetic scaffolds. This review introduces the complicated scenario of collagen structure, fibril arrangement, type, function, and in addition, distribution within the body for the enhancement of collagen-based scaffolds. We describe and compare existing approaches for the alignment of collagen with a sharper focus on electro-compaction. Additionally, various effective processes to further enhance electro-compacted collagen, such as crosslinking, the addition of filler materials, and post-alignment fabrication techniques, are discussed. Finally, current challenges and future directions for the electro-compaction of collagen are presented, providing guidance for the further development of collagenous scaffolds for bioengineering and nanotechnology.
... Newly formed fragments of protein macromolecules form new bonds. As a result, macromolecules appear that differ from the original architecture [19][20][21][22]. This is the scaffold that represents hydrogel with certain characteristics of elasticity, transparency and density [23]. ...
Article
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: Enzymatic hydrolysis of native collagen and fibrinogen was carried out under comparable conditions at room temperature. The molecular weight parameters of proteins before and after hydrolysis by thrombin were monitored by gel-penetrating chromatography (GPC). An analysis of the experiment results shows that the molecular weight parameters of the initial fibrinogen (Fn) and cod collagen (CC) are very similar. High molecular CC decays within the first minute, forming two low molecular fractions. The main part (~80%) falls on the fraction with a value of Mw less than 10 kDa. The initial high molecular fraction of Fn with Mw ~320–340 kDa is not completely hydrolyzed even after three days of control. The presence of low molecular fractions with Mw ~17 and Mw ~10 kDa in the solution slightly increases within an hour and noticeably increases for three days. The destruction of macromolecules of high molecular collagen to hydrolysis products appears almost completely within the first minute mainly to the polymer with Mw ~10 kDa, and enzymatic hydrolysis of fibrinogen proceeds slower than that of collagen, but also mainly to the polymer with Mw ~10 kDa. Comparative photos of the surfaces of native collagen, fibrinogen and the scaffold based on them were obtained.
... Proteins that help with structural abilities, as well as mechanical properties of the tissue, should match the original tissue to a reasonable extent so that the process would have more chance to be successful. It is assumed Some decellularization agents and protocols destroy basic ECM elements; for example, detergents may disrupt collagen structure; therefore mechanical strength of ECM undergoes changes or most detergents eliminate the amount of GAG, thus decrease the viscoelasticity feature of ECM (Kezwoń et al. 2016;Conconi et al. 2005). ...
Chapter
The extracellular matrix (ECM) of mammalian organs and tissues has been applied as a substitute scaffold to simplify the restoration and reconstruction of several tissues. Such scaffolds are prepared in various arrangements including sheets, powders, and hydrogels. One of the more applicable processes is using natural scaffolds, for this purpose discarded tissues or organs are naturally derived by processes that comprised decellularization of following tissues or organs. Protection of the complex structure and 3D (three dimensional) ultrastructure of the ECM is extremely necessary but it is predictable that all protocols of decellularization end in disruption of the architecture and potential loss of surface organization and configuration. Tissue decellularization with conservation of ECM bioactivity and integrity can be improved by providing well-designed protocols regarding the agents and decellularization techniques operated during processing. An overview of the characterization of decellularized scaffolds and the role of reagnets can validate the applied methods' efficacy.
... Similarly, extensive digestion of the soy hydrolysate and soy-fermented foods, including dephosphorylation, deglycosylation and proteolysis, resulted in several peptides with variable surface activity (31.6 < γ < 71.3 mN/m) [72]. These observations agree with conclusions from other studies on protein hydrolysates, that maximum surface activity is usually observed for intermediate degree of hydrolysis [73][74][75][76]. Luckily, if the peptides issued from extensive hydrolysis of soy proteins become too hydrophilic to provide sufficient amphiphilicity, the lipophilic-hydrophilic balance can be restored, e.g. by their acylation. ...
Article
The present review, dedicated to Prof. Zbigniew Adamczyk on the occasion of his 70th anniversary, covers the literature data on surface tension and surface compression (dilational) rheology of the adsorbed layers of 21 plant seed proteins (10 leguminous and 11 non-leguminous plants). They are typically analyzed as protein concentrates or isolates, the latter usually obtained by isoelectric precipitation or diafiltration. Despite generally lower solubility, as compared to their animal counterparts (lactoglobulins, caseins, albumins, etc.), the plant seed proteins are also capable of lowering surface tension and forming viscoelastic adsorbed layers. Many seed proteins serve mostly as amino acids reservoirs for the future seedling (storage proteins), hence their instantaneous amphiphilicity is not always sufficient to induce strong adsorption at the aqueous-air interface. They can be, however, conveniently unfolded, hydrolyzed and/or chemically/enzymatically modified to expose more hydrophilic or hydrophobic patches. As shown in numerous contributions reviewed below, the resulting shift of the hydrophilic-lipophilic balance can boost their surface activity to the level comparable to that of many animal proteins or low molecular weight surfactants. An important advantage of the plant seed proteins over the animal ones is their much lower environmental cost and abundance in many plants (e.g. ~40% in sunflower or soybean seeds).
... Чаще других в случае биополимеров используют белки, такие как коллаген, фибрин, фиброин шелка и др. При получении скаффолдов применяют протеолитические ферменты [8][9][10][11], гидролизующие пептидную связь белков с целью образования новых связей уже в скаффолде, при этом отмечается влияние молекулярно-массовых характеристик (ММХ) используемого полимера на свойства получаемых матриц [12][13][14]. Изменения молекулярных и надмолекулярных параметров биополимеров клеточных матриц при формировании скаффолдов, влияние этих параметров на биомиметические свойства при построении тканеинженерных конструкций зависят не только от природы используемого белкового материала, но и от природы фермента [15,16]. ...
Article
High-molecular collagen was isolated from cod skin and subjected to enzymatic hydrolysis with pancreatin and thrombin. The resulting products were characterized by molecular weight parameters. It is shown that formation of two oligomeric fractions with molecular masses of 9 kDa (>80%) and 17–18 kDa (2–4%) is characteristic of both the pancreatin and thrombin hydrolysis of the peptide bonds in collagen.
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Collagen solutions in the presence of 0.1–2.0M acetic acid (AA) were investigated to understand the interaction between collagen molecules and an acidic solvent. Fluorescence measurements of pyrene showed that the critical aggregation concentration (CAC) of collagen increased from 0.518 to 1.581 mg/mL for AA concentrations ranging from 0.1 to 2.0M, indicating that the aggregated state of collagen molecules was associated with AA concentration. The size of the collagen aggregates, determined by dynamic light scattering, demonstrated that their disaggregation was enhanced with increasing electrostatic repulsion between the collagen chains. The variations in the intrinsic viscosity and Huggins coefficient depended on the molecular interaction among the collagen molecules. Furthermore, the increased AA concentration has a different influence on the rigidity of collagen molecules above and below the CAC. This observation was manifested by the changes in the morphology of collagen molecules observed by atomic force microscopy. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45255.
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A combination of proteolysis and dilational rheology has been used to study the behavior of films of beta-casein (beta-CN) and of peptides spread at the oil-water interface. Identification of the peptides produced by trypsin hydrolysis of beta-CN in emulsion at 37 degrees C provided information on the structure of beta-CN adsorbed at the oil-water interface. Good interface properties were observed for beta-CN or its peptides, probably because of the amphipathic nature of beta-CN or a synergistic effect between hydrophilic and hydrophobic peptides. Remarkable surface activity was found for the amphipathic peptide beta-CN (f114-169). Rheological studies had shown that interface films made with peptide fractions or with beta-CN were elastic rather than viscous. Film made with the purified peptide beta-CN (f114-169) was merely elastic at the triolein-water interface. A decrease of the viscoelastic modulus was observed for aging beta-CN film but not for aging peptide films; The beta-CN decrease was related to the flexibility of its structure. When the interface is increased by the dilation of an aqueous droplet plunged into oil, beta-CN may expose new polypeptide trains to cover the increased interface, unlike peptides with simpler structures.
Article
Spread monolayers of the fibril protein collagen were studied at the air-water interface in the presence of denaturants, urea and thiourea. The most prominent feature of spread collagen monolayers at the air-water interface is the ability to form supramolecular structures (fibrils), which themselves can form monolayers with collapse points of their own. The surface pressure isotherms of collagen monolayers have two “quasi-linear” centers, which are separated by a plateau and correspond to liquid-expanded and liquid-condensed states; this unique capability makes collagen different from other proteins. When in monolayer, collagen acquires the same level of structural organization as in the bulk. In the presence of denaturants, subphase characteristics of collagen monolayers change rapidly and irreversibly. Thiourea exerts more pronounced denaturing action on collagen monolayers than urea; this effect increases with exposure time and denaturant concentration. A hypothetical mechanism of thiourea-induced denaturation of fibril proteins is proposed according to which interactions between hydrophobic C=S groups of thiourea and nonpolar surface groups of the protein lead to reorientation of carbonyl groups to formation of intrinsic hydrogen bonds with NH2-groups of thiourea eventually resulting in the rupture of intrinsic hydrogen bonds and denaturation of the protein.
Article
Enzymic hydrolysis was applied for the efficient recovery of the protein sources from the fish processing by-product, cod frame. The enzyme used for the hydrolysis was crude proteinase extracted from tuna pyloric caeca. The resultant hydrolysate, cod frame protein hydrolysate (CFPH), was separated based on the molecular weight of the peptides in the hydrolysate and several functional properties were examined, including physicochemical properties (emulsifying and foaming property) and bioactivities (antioxidative and angiotensin I converting enzyme (ACE) inhibitory activity) to determine its potential functions. CFPH was processed through a series of ultrafiltration (UF) membranes with molecular weight cut-off(MWCO) of 30, 10, 5 and 3 kDA, and four types of permeates including 30-K (permeate from 30 kDA), 10-K (permeate from 10 kDA), 5-K (permeate from 5 kDA) and 3-K hydrolysate (permeate from 3 kDA) were obtained. 10- and 30-K hydrolysates showed excellent emulsion properties and whippability. The 10-K hydrolysate showed high antioxidative activity, while the 3-K hydrolysate had excellent ACE inhibitory activity. In terms of all functional properties tested, the fractionated hydrolysates were superior to the original non-separated hydrolysate. These results suggested that separating hydrolysate enhanced several functional properties.
Article
Collagen, gelatin and collagen hydrolysate were prepared from bovine limed split wastes by different preparative processes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight distribution of collagen was very narrow (about 200 and 100kDa for β and α chains respectively) compared with those of gelatin (less than 300kDa and wide distribution) and collagen hydrolysate (less than 50kDa and wide distribution). The isoelectric points of collagen, gelatin and collagen hydrolysate were 8.26, 4.88 and 4.54 respectively determined by Zeta potential titration. Circular dichroism (CD) spectra revealed that there were two peaks, a positive peak around 221nm and a negative peak around 192nm for collagen, which are the characteristics of collagen triple helix. However, gelatin and collagen hydrolysate lacked any positive peaks around 220nm, suggesting random coils. The denaturation temperature of collagen was about 37.5°C determined by the viscosity method, the helix-coil transitions for gelatin and collagen hydrolysate were not present in the heating process. Collagen reaggregated to fibrils at 35°C monitored at 313nm. In contrast, gelatin and collagen hydrolysate lost the ability of fibril formation. Collagen was more resistant to trypsin hydrolysis compared with gelatin and collagen hydrolysate. In addition, the collagen membrane exhibited superior features such as higher enthalpy, greater network structure and better physical/mechanical properties compared with those of the gelatin membrane. Therefore, collagen isolated from limed split wastes can be a high value product due to its special characteristics and has many potential future applications in biomaterials, functional additives, cosmetics and pharmaceutical industries.
Article
The dynamic surface tension and surface dilational rheology of native collagen (type I, from bovine calf skin) were analyzed in the temperature range from 21 to 34.5 °C. In addition, the effect of heating the collagen solutions up to 90 °C followed by cooling down to room temperature, on surface tension and rheological parameters of the adsorbed layers was studied. As a water-soluble fibrous protein with little exposed hydrophobicity, native collagen is weakly surface active. However, its layers formed by spontaneous adsorption on the water/air surface display exceptional dilational rheology parameters, comparable with those of globular proteins: at high frequency limit (0.1 Hz) the layers are predominantly elastic, with the storage modulus, E′ = 58 mN/m for collagen concentration of 1.3 × 10−5 M (4 g/L). Increasing temperature during adsorption helps to reduce the surface tension, but the resulting layers show diminished surface elastic response. On the other hand, the samples heated at elevated temperatures and measured after cooling down to the room temperature maintain their high surface elasticity. The results suggest that collagen, even in its native form (not processed to gelatin) has a potential to stabilize biocompatible foams/emulsions by forming mechanically strong adsorbed layers.
Article
Effect of a model bovine milk protein, β-casein, on surface activity of Quillaja bark saponin (QBS) from Sigma was studied at three fluid/fluid interfaces: air/water, tetradecane/water and olive oil/water. In all cases, the protein concentration was fixed at 10−6 mol L−1, and QBS concentration was varied between 5·10−7 and 1·10−3 mol L−1. Dynamic interfacial tension on the timescale 5 s–3600 s was measured using a drop shape analysis technique. For the air/water system, they were complemented with short-term (50 ms–5 s) measurements using a maximum bubble pressure technique. The dynamic results together with the extrapolated equilibrium surface pressures are discussed from the point of view of a complexation between β-casein and QBS, with the surface activity of the complex changing with its stoichiometry. At low biosurfactant/protein ratios, the interfacial tension at all three interfaces passes through a maximum, corresponding to a transient decrease of both foam and emulsion formation ability. In addition, the effect of QBS on deterioration of β-casein's surface activity upon ageing at room temperature is discussed.
Article
A series of collagen hydrolysate-based surfactants (CHBS) were prepared by grafting different amount of oleoyl group onto collagen hydrolysate (CH). Their physicochemical properties and surface activities were investigated, and their emulsifying ability on rapeseed oil–water emulsion was evaluated. The results showed that, as increasing grafting degree of oleoyl group, the free amino content and isoelectric point of CHBSs and the size of CHBS particles formed in aqueous solution declined, while their molecular weight and surface hydrophobicity increased. The CHBSs with high grafting degree, such as CHBS-30, CHBS-40, and CHBS-50 (postfix datum represents mmol amount of oleic acid chloride to react with 10 g collagen hydrolysate), exhibited satisfactory wetting capacity and foaming capacity, and the CHBSs with low grafting degree, such as CHBS-10 and CHBS-20, presented good emulsifying capacity. The surface activity of CHBSs was closely correlated with the hydrophobicity of their molecules, as well as their capacity to reduce the surface tension of air–water interface. In addition, CHBS-20 showed good emulsifying ability on rapeseed oil–water emulsion in neutral and alkaline pH, even in the condition of low salt concentration.
Article
Protein was extracted from soy residue (okara) at alkaline pH, and was modified by mild acid treatments. The degree of deamidation and peptide bond hydrolysis ranged from 10 to 70% and 6 to 15%, respectively. Size exclusion gel filtration chromatography revealed that there was a progressive degradation of the okara protein. Solubility was increased markedly by the modification, while other functional properties such as emulsifying and foaming properties were also improved. The okara protein products have good essential amino acid profiles, and acid modification also increased the in vitro digestability and available lysine content of okara protein. The results indicate that changes in functional properties of okara protein by acid modification were attributed to changes in physicochemical properties such as decreases in molecular size, higher net titratable charge and surface hydrophobicity. The low solubility of okara protein makes it difficult to be incorporated into many food systems. Improved solubility and other functional properties by acid modification will enhance the utilization okara protein as food ingredient.
Article
The surface activity of pepsin-solubilized type I collagen acylated by lauroyl chloride and succinic anhydride was investigated in this article. Compared with native collagen, acylated collagen exhibited better surface activity such as oil absorption capacity, emulsion activity and stability, foam expansion, and foam stability but presented lower water absorption capacity. Acylated collagen also had higher emulsion activity and stability than Tween 80, whereas Tween 80 displayed higher foam expansion and stability than acylated collagen. After acylation, the surface tension of collagen decreased with the storage time increased, the hydrophobicity and the wetting power increased with the increase of the concentration. Meanwhile, the emulsion activity and stability of acylated collagen decreased with the increase of NaCl concentration ranging from 20 to 100 mmol/L. Under neutral pH value, acylated collagen reached higher emulsion activity and stability at acylated collagen concentration of 0.4 mg/mL and lower temperature (<35°C). © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40174.
Article
Mixed protein–surfactant adsorption layers at liquid interfaces are described including the thermodynamic basis, the adsorption kinetics and the shear and dilational interfacial rheology. It is shown that due to the protrusion of hydrophobic protein parts into the oil phase the adsorption layers at the water–hexane interface are stronger anchored as compared to the water-air surface. Based on the different adsorption protocols, a sequential and a simultaneous scheme, the peculiarities of complexes between proteins and added surfactants are shown when formed in the solution bulk or at a liquid interface. The picture drawn from adsorption studies is supported by the findings of interfacial rheology.
Article
Hydroxyapatite/collagen composites (HAp/Col) were synthesized, in vitro, using the self-organization mechanism, by the co-precipitation of collagen, extracted from pork skin, and aqueous H3PO4 and Ca(OH)2. The effects of pH and temperature, on the microstructure and morphology of HAp/Col composites, were extensively studied.It was shown that the yield of the composite is closely related to the pH value, during preparation. At high pH, the white precipitate of the composites was formed in a large quantity. In contrast, the yield was quite low, at low pH, owing to the dissolution of the composite precipitates. The electrophoretogram of the as-prepared composite shows that the α1(I) chain was dominant and that there was no protein residue in the supernatant of the reaction solution. Accordingly, it is reasonable to assume that the collagen molecules had completely reacted with hydroxyapatite.A closer look at the structural evolution of HAp/Col composites revealed that, at pH = 5, there was no sign of interfacial interaction between hydroxyapatite and collagen, during the first 36 h. In contrast, under alkaline conditions, the amount of α(I) chains was indeterminate, at the start of precipitation, while the concentration of β-chains decreased gradually. The chemical structure of HAp/Col, as determined by FTIR spectra, revealed that the bending vibration of phosphate contours can be attributed to the dissolution of HAp, at low pH. The crystalline of HAp was readily discerned, for all samples, as revealed by the XRD patterns. The morphology of the HAp/Col, prepared under neutral to alkaline conditions (7 ≤ pH ≤ 9), exhibited a compact, coral reef-like structure. In summary, HAp/Col composites can be synthesized, in vitro, under conditions native to animal physiology. The desirable conditions for synthesis are a higher pH (8 to 9) and a temperature of 40 °C.
Article
The effects of concentration, pH value and ionic strength on the kinetic self-assembly of acid-soluble collagen from walleye pollock (Theragra chalcogramma) skin were investigated. A two-phase kinetic process was provided which included the formation of nucleus center and nucleus growth, the first phase being the controlled step for collagen self-assembly. Collagen showed marked assembly behavior when concentration reaching and above 0.6 mg/mL, and higher concentration could accelerate collagen self-assembly. Rate constants of the first and second assembly phase both increased with pH to a maximum around pH 7.2 and then decreased, indicating that pH 7.2 was the optimum pH value for collagen self-assembly. The kinetics of collagen self-assembly could be modulated by NaCl concentration. The concentration of NaCl from 30 to 60 mM was more suitable to self-assemble for pollock skin collagen.
Article
Drop and bubble shape tensiometry is a modern and very effective tool for measuring dynamic and static interfacial tensions. An automatic instrument with an accurate computer controlled dosing system is discussed in detail. Due to an active control loop experiments under various conditions can be performed: constant drop/bubble volume, surface area, or height, trapezoidal, ramp type, step type and sinusoidal area changes. The theoretical basis of the method, the fitting procedure to the Gauss-Laplace equation and the key procedures for calibration of the instrument are analysed and described.The interfacial tension response to transient and harmonic area perturbations yields the dilational rheological parameters of the interfacial layer: dilational elasticity and exchange of matter function. The data interpretation with the diffusion-controlled adsorption mechanism based on various adsorption isotherms is demonstrated by a number of experiments, obtained for model surfactants and proteins and also technical surfactants. The application of the Fourier transformation is demonstrated for the analysis of harmonic area changes. The experiments shown are performed at the water/air and water/oil interface and underline the large capacity of the tensiometer.
Article
The stabilization of foam by proteins has been mostly studied in relation to the food industry. The main aim of the research is to understand the relation between proteins used and the product properties. The molecular properties of proteins and their foam forming and stabilizing properties are typically linked to the adsorption kinetics and the interfacial properties. Additionally, the properties of thin liquid films formed between neighboring air bubbles are considered. While there are several rules of thumb describing the relations between the different parameters and processes it seems that there is not yet a ‘unifying’ theory on protein stabilized foams. If the different processes could be described by quantitative parameters the applications of traditional proteins and the use of proteins from novel sources could be optimized. However, even for simple protein systems there is a lack of such quantitative rules, and as a result the advancement in the understanding of protein foam seems to progress slowly. This is attributed to the complexity of the system by some authors, but by viewing the literature it also becomes apparent that certain ideas seem to resist change. There are some interesting articles that offer a different point of view. In this article we aim to provide an insight in the different ways in which proteins and their role in foamed systems are described. Based on recent results, it seems that protein adsorption and subsequent changes in interfacial properties could be described in colloidal terms such as the net charge, exposed hydrophobicity and size of the proteins. Such a description can help to understand the behavior of single-component systems, but can also add to the understanding of the more complex systems that seem to attract more and more interest in recent years. An example of the value of using new viewpoints is the exchange of information between fields of food and non-food foams. Examples in this field are the use of particles to stabilize foams, or the production of very stable microbubbles.
Article
A quantitative analysis of the long time period approximation of the diffusion-controlled model is presented and the interval of its validity is defined to interpret adsorption kinetics data. An estimate is also given of the error which results from linear extrapolation of equilibrium surface tension values.
Article
HPLC resolution of fibrillar collagen chains, and particularly the α2 chain from β and γ components, has continued to provide a challenge. A single column method for the clear resolution of the denatured chains of hoki (Macruronus novaezelandiae) skin type I collagen is presented. The effect of changing chromatographic parameters (flow rate, loading, temperature, gradient and solvent, pH) was examined. The α1 and α3 chains were readily resolved under all the conditions studied. The α2 chain was the most difficult component to resolve but also gave the largest response to changes in solvent gradient and pH.
Article
A new kind of acylated collagen with water solubility and better surface activity was prepared via reaction of pepsin-solubilized calf skin collagen with lauroyl chloride and succinic anhydride in this paper. The equilibrium surface tension and the isoelectric point were 55.92 mN/m and 4.93 respectively, suggesting that acylated collagen had surface activity as well as water solubility. Meanwhile, the results of Fourier transform infrared spectroscopy analyses and electrophoresis patterns demonstrated that the triple helix conformation of collagen was not destroyed, but the subunits of acylated collagen shifted to higher molecular weight than those of native collagen. Scanning electron microscope and differential scanning calorimeter measurements revealed that lyophilized acylated collagen exhibited relatively well-distributed pore structure and its denaturation temperature was ahout 9.0°C higher than that of native collagen. Additionally, the increase of the diameter of the fibrils was observed by atomic force microscopy. Acylated collagen with water solubility and better surface activity might broaden the application of collagen-based materials to cosmetics, drug delivery and pharmacotherapy.
Article
Using optical rotation to study the triple helix reversion of gelatin in aqueous solutions demonstrates that the reversion is a combination of first-order and second-order kinetic processes. On the basis of this observation, we propose a new two-step mechanism of triple helix formation in polypeptides, that leads to an expression distinct from the one obtained by Flory and Weaver. The rate-limiting step is formation of a two-stranded nucleus, which can be intramolecular (first order) or intermolecular (second order). The triple helix is formed by subsequent wrapping of a third strand onto this nucleus. We estimate the minimum stable helix length and the size of the loop at the end of an intramolecular helix from our kinetics analysis. The new two-step mechanism of triple helix formation is consistent with all existing literature data and allows prediction of concentration and temperature dependencies of helix formation rate.
Article
Collagen self-assembly of silver-line grunt type I collagen solution was done at a concentration of 1 mg/mL and at pH 5, 6, 7 and 8 with incubation temperatures of 25, 30 and 35C. The self-assembly of collagen showed typical sigmoid curves and had a short lag phase at pH 5, 6, 7 and 8. The lowest absorbance was found at pH 5.0, whereas pH 6.0 gave the highest absorbance and the fastest growth phase. The absorbance gradually increased at lower temperature, but rapidly increased at higher temperature. The rate of reconstruction also increased when the temperature increased from 20 to 30C, but the rate decreased when the temperature rose to 35C. The rate increased rapidly with increasing pH from 5.0 to 6.0, but decreased with raising pH from 6.0 to 8.0. The scanning electron micrographs of the collagen fibrils reconstructed at pH 6.0 were thicker, more uniform and more interwoven than fibrils formed at pH 7.0.
Article
Endo-protease treatments achieving low degrees of hydrolysis (DH 2% and 4%) were used to improve functional properties of hexane-extracted soy flour (HESF), extruded-expelled partially defatted soy flour (EESF), ethanol-washed soy protein concentrate (SPC), and soy protein isolate (SPI). These substrates had protein dispersibility indices ranging from 11% to 89%. Functional properties, including solubility profile (pH 3 to 7), emul-sification capacity and stability, foaming capacity and stability, and apparent viscosity were determined and related to surface hydrophobicity and peptide profiles of the hydrolysates. Protein solubilities of all substrates increased as DH increased. Emulsification capacity and hydrophobicity values of the enzyme-modified HESF and EESF decreased after hydrolysis, whereas these values increased for SPC and SPI. Emulsion stability was improved for all 4% DH hydrolysates. Hydrolyzed SPC had lower foaming capacity and stability. For substrates other than SPC, foaming properties were different depending on DH. Hydrolysis significantly decreased the apparent viscosities regardless of substrate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated differences in the molecular weight profiles of the hydrolysates. HESF and EESF, which had high proportions of native-state proteins, showed minor changes in the peptide profile due to hydrolysis compared with SPC and SPI.
Article
Casein was hydrolyzed by trypsin to 5% degree of hydrolysis, and the hydrolysate was fractionated by ultrafiltration. While screening for the best emulsifying properties, the retentate fraction was systematically fractionated by ammonium sulfate precipitation, ion exchange, and reverse-phase high-performance liquid chromatography (HPLC). Residue from 30% ammonium sulfate saturation yielded 3 fractions on anion exchange chromatography. Preparative reverse-phase HPLC was used to isolate 4 peptides with good emulsifying properties for characterization. Computational methods were used to complement amino acid sequencing, and the peptides were identified as originating from αs-1 casein (f167-208), β-casein (f48-63), and (f129-184). A 4th peptide could not be conclusively identified. Properties of the emulsifying peptides are discussed.
Article
The adsorption dynamics and equilibrium properties of hen egg-white lysozyme at the air/water interface is studied using bubble and drop profile analysis technique, ellipsometry and infrared reflection absorption spectroscopy (IRRAS). The set of equilibrium data can be described by a recently developed thermodynamic model. Using the equilibrium adsorption parameters the adsorption dynamics can be well described by a diffusion controlled mechanism at the lower concentrations and a mixed model at higher lysozyme concentrations where conformational changes in the surface layer become time determining.
Article
Spread monolayers of the fibril protein collagen were studied at the air-water interface in the presence of denaturants, urea and thiourea. The most prominent feature of spread collagen monolayers at the air-water interface is the ability to form supramolecular structures (fibrils), which themselves can form monolayers with collapse points of their own. The surface pressure isotherms of collagen monolayers have two “quasi-linear” centers, which are separated by a plateau and correspond to liquid-expanded and liquid-condensed states; this unique capability makes collagen different from other proteins. When in monolayer, collagen acquires the same level of structural organization as in the bulk. In the presence of denaturants, subphase characteristics of collagen monolayers change rapidly and irreversibly. Thiourea exerts more pronounced denaturing action on collagen monolayers than urea; this effect increases with exposure time and denaturant concentration. A hypothetical mechanism of thiourea-induced denaturation of fibril proteins is proposed according to which interactions between hydrophobic C=S groups of thiourea and nonpolar surface groups of the protein lead to reorientation of carbonyl groups to formation of intrinsic hydrogen bonds with NH2-groups of thiourea eventually resulting in the rupture of intrinsic hydrogen bonds and denaturation of the protein.
Article
Composition, functional properties and antioxidative activity of a protein hydrolysate prepared from defatted round scad (Decapterus maruadsi) mince, using Flavourzyme, with a degree of hydrolysis (DH) of 60%, were determined. The protein hydrolysate had a high protein content (48.0%) and a high ash content (24.56%). It was brownish yellow in colour (L∗ = 58.00, a∗ = 8.38, b∗ = 28.32). The protein hydrolysate contained a high amount of essential amino acids (48.04%) and had arginine and lysine as the dominant amino acids. Na+ was the predominant mineral in the hydrolysate. The protein hydrolysate had an excellent solubility (99%) and possessed interfacial properties, which were governed by their concentrations. The emulsifying activity index of the protein hydrolysate decreased with increasing concentration (p < 0.05). Conversely, the foaming abilities increased as the hydrolysate concentrations increased (p < 0.05). During storage at 25 °C and 4 °C for 6 weeks, the antioxidative activities and the solubility of round scad protein hydrolysate slightly decreased (p < 0.05). Yellowness (b∗-value) of the protein hydrolysate became more intense as the storage time increased but the rate of increase was more pronounced at 25 °C than at 4 °C.
Article
We analysed existing and newly derived asymptotic solutions of the adsorption kinetic equations for the liquid phase interface in the regions of infinitely small and infinitely great surface lifetimes (t) for the cases of one and of a few surfactants, on non-deforming and deforming surfaces, under non-stationary, stationary and quasi-stationary conditions, assuming either the diffusion adsorption mechanism or mixed adsorption mechanism. It was proved that in the region t → ∞, the adsorption barrier does not influence the dynamic surface tension σ, but the role of surface-active contaminants is significant. In contrast, in the region t → 0, the role of contaminants is small, but the adsorption barrier influences the dynamic surface tension substantially. The dynamic surface tension of sodium alkyl sulphate solutions was measured by the maximum bubble pressure method, in the t range 0.001–10 s. In the region t → ∞ we obtained good agreement of experimental results with asymptotic formulae. The diffusion adsorption mechanism of the surfactant solutions studied was confirmed and we also estimated the concentration values of the surfactant admixtures. Small additions of the more active surfactant sodium tetradecyl sulphate to sodium dodecyl sulphate substantially influences the shape of the σ—t curve in the region t → ∞, increasing (in full accordance with theoretical considerations) the tangent value of the curve inclination of the dependence of σ on t−1/2. In the regions t → 0, long-chained high molecular weight sodium alkyl sulphates adsorb according to the diffusion mechanism, whereas for sodium decyl and dodecyl sulphates the existence of the adsorption barrier was confirmed. We corroborated experimentally the absence of any influence of surfactant admixtures on the values of dynamic surface tension at t → 0.
Article
Acid-soluble collagen (ASC) was extracted from the skin of walleye pollock (Theragra chalcogramma) and partially characterized. It exhibited a maximum absorbance at 220 nm, but little absorbance near to 280 nm. Amino acid composition and SDS-PAGE suggested that the collagen might be classified as type I collagen. Moreover, FTIR investigations showed the existence of helical arrangements of collagen. The denaturation temperature (Td) and shrinkage temperature (Ts) were 24.6 °C and 47 °C, respectively, both lower than those of mammalian collagens. However, Td of walleye pollock skin collagen was higher than that of cod skin collagen reported previously. These results indicate that walleye pollock skin is a potential source of collagen and provide the theoretical basis for further research.
Article
Enzymic hydrolysis was applied for the efficient recovery of the protein sources from the fish processing by-product, cod frame. The enzyme used for the hydrolysis was crude proteinase extracted from tuna pyloric caeca. The resultant hydrolysate, cod frame protein hydrolysate (CFPH), was separated based on the molecular weight of the peptides in the hydrolysate and several functional properties were examined, including physicochemical properties (emulsifying and foaming property) and bioactivities (antioxidative and angiotensin I converting enzyme (ACE) inhibitory activity) to determine its potential functions. CFPH was processed through a series of ultrafiltration (UF) membranes with molecular weight cut-off (MWCO) of 30, 10, 5 and 3 kDA, and four types of permeates including 30-K (permeate from 30 kDA), 10-K (permeate from 10 kDA), 5-K (permeate from 5 kDA) and 3-K hydrolysate (permeate from 3 kDA) were obtained. 10- and 30-K hydrolysates showed excellent emulsion properties and whippability. The 10-K hydrolysate showed high antioxidative activity, while the 3-K hydrolysate had excellent ACE inhibitory activity. In terms of all functional properties tested, the fractionated hydrolysates were superior to the original non-separated hydrolysate. These results suggested that separating hydrolysate enhanced several functional properties.
Article
Adsorption of purified diphosphorylated Al-ovalbumin at the air–water interface was studied by ellipsometry, surface tension, and shear elastic constant measurements. The value of pH did not significantly affect the final value of surface concentration. It affected slightly the kinetics of surface pressure increase and the final value of surface pressure. The interfacial rheology was affected strongly by pH. The interface exhibited a maximum of the shear elastic constant at a pH close to the isoelectric pH of ovalbumin. The bulk protein concentration also had a more pronounced effect on the surface rheology when the protein net charge was low. At a pH where the protein net charge is negative, an increase of the ionic strength increased the final value of the shear elastic constant. The results suggest that interactions between adsorbed ovalbumin molecules, which form slowly in the adsorbed layer upon conformational rearrangements, impart rigidity to the interface, and that these intermolecular associations are hindered at high protein net charge.
Article
Antioxidative activity and functional properties of protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) meat, hydrolyzed by Alcalase 2.4L (HA) and Flavourzyme 500L (HF) with different degrees of hydrolysis (DH) were investigated. As the DH increased, DPPH radical-scavenging activity and reducing power of HA decreased (p < 0.05) but no differences were observed for HF (p > 0.05). Metal chelating activity of both HA and HF increased with increasing DH (p < 0.05). HF generally had a higher (p < 0.05) chelating activity than had HA at the same DH tested. At low DH (5%), HA exhibited a better DPPH radical-scavenging activity while, at high DH (25%), HF had a higher (p < 0.05) reducing power. For the functional properties, hydrolysis by both enzymes increased protein solubility to above 85% over a wide pH range (2–12). When the DH increased, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity, foam stability) of hydrolysates decreased (p < 0.05), possibly caused by the shorter peptide chain length. At the same DH, the functionalities of protein hydrolysate depended on the enzyme used. The results reveal that antioxidative activity and functionalities of protein hydrolysates from yellow stripe trevally meat were determined by the DH and by the enzyme type employed.
Article
In this paper, we did a study of the physicochemical, dielectric and piezoelectric properties of collagen films, considering the development of new biomaterials which have potential applications in coating of cardiovascular prostheses, support for cellular growth and in systems for controlled drug delivery. The piezoelectric strain tensor element d14, the elastic constant S55, and the dielectric permittivity ε11 were measured for collagen films. It was observed that the collagen samples submitted to different pH treatment lead to different electrical behavior. For sample S2 (pH=5.2) one has a higher denaturation temperature (Td=80.55°C), lowest density (ρ=909.6 kg m−3), highest dielectric function, lowest frequency constant product (f×L=320.33 kHz.m) and higher piezoelectricity (d14=0.122 pC/N) which is our major interest in this study. The loss factor Q−1 measured for all the samples shows that for all the electrodes dimensions sample S2 present the highest loss factor which is between 0.68 and 0.76. We believe that the different pH treatment is critical to the final level of organization of the microscopic structure of the sample, which could result in an increase of the piezoelectricity.
Article
The structural properties of collagen have been the subject of numerous studies over past decades, but with the arrival of new technologies, such as the atomic force microscope and related techniques, a new era of research has emerged. Using microthermal analysis, it is now possible to image samples as well as performing localized thermal measurements without damaging or destroying the sample itself. This technique was successfully applied to characterize the thermal response between native collagen fibrils and their denatured form, gelatin. Thermal transitions identified at (150 ± 10)°C and (220 ± 10)°C can be related to the process of gelatinization of the collagen fibrils, whereas at higher temperatures, both the gelatin and collagen samples underwent two-stage transitions with a common initial degradation temperature at (300 ± 10)°C and a secondary degradation temperature of (340 ± 10)°C for the collagen and of (420 ± 10)°C for the gelatin, respectively. The broadening and shift in the secondary degradation temperature was linked to the spread of thermal degradation within the gelatin and collagen fibrils matrix further away from the point of contact between probe and sample. Finally, similar measurements were performed inside a bone resorption lacuna, suggesting that microthermal analysis is a viable technique for investigating the thermomechanical response of collagen for in situ samples that would be, otherwise, too challenging or not possible using bulk techniques.
Article
Collagens presently used in tissue engineering are primarily of bovine or porcine origin. However, a risk of a spongiform encephalopathy epidemic has limited the use of collagen from these sources. Keeping the aforementioned perspective in mind, we explored the possibility of using domestic goat available in the subcontinent as a potential source of collagen for tissue-engineering application. This article delineates the isolation, physico-chemical characterization, biocompatibility study and wound healing application of acid soluble caprine (goat) tendon collagen (GTC). Physico-chemical characterization of 1% acetic acid extracted GTC was done by SDS-PAGE, amino-acid composition analysis, FT-IR and CD spectroscopy. Results revealed that GTC was comprised of type-I collagen. Biocompatibility study showed that GTC augmented cell adhesion, cell cycle progression and proliferation. Immuno-cytochemical analysis in conjugation with traction force microscopy further confirmed a superior focal adhesion complex mediated cell-substrate interaction in GTC. Finally, in vivo study in mice model revealed that GTC has low immunogenicity and it augments healing process significantly. Throughout the study, calf skin collagen (CSC) was used as standard for comparative evaluation. In conclusion, it can be said that GTC may find its application as biomaterial in skin tissue engineering.
Article
Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.
Article
Peptides offer interesting alternatives to conventional surfactants in applications where renewability, biocompatibility, or added functionality may be desired. This review offers a brief overview of different classes of surface-active peptides and lipopeptides, covering molecules obtained from natural sources as well as those obtained by design. Bacterial lipopeptides are cyclic molecules containing a single fatty acyl moiety, which can exhibit ultralow interfacial tension as well as antimicrobial activities. Bacterial lipopeptides have been proposed for industrial applications such as bioremediation and oil recovery, but they suffer the dual disadvantages of being difficult to bioproduce at low cost and not being easily genetically engineered. A class of synthetic molecules related to bacterial lipopeptides are the peptide amphiphiles, in which a peptide headgroup is combined with a peptide or nonpeptide hydrophobic tail. Self-assembly of peptide amphiphiles has largely been studied in bulk solution rather than at interfaces, meaning that very little information is available on the interfacial properties of these designer molecules. A larger body of information is available for protein hydrolysates, products of the partial breakdown of low-cost proteins (usually food proteins) into a complex mixture of small peptides. Partial hydrolysis can improve the functional properties of many proteins, but the outcomes are difficult to predict or control, and useful functional properties may be associated with only a few minor components in the digest mix. Finally, designed peptide surfactants (Pepfactants), recently reported by the authors, are facially amphipathic molecules that self-assemble at fluid interfaces to give cohesive films stabilizing foams and emulsions. A change in the bulk solution conditions can switch off the interfacial film, leading to rapid foam or emulsion collapse. Pepfactants can be genetically engineered and bioproduced using standard methods, which represents an advantage over bacterial lipopeptides. While peptides have not been widely used in surfactant applications so far, recent developments may facilitate the incorporation of these interesting molecules into industrial and consumer products in the near future.
Article
Bovine β-casein (βCN) was hydrolyzed by plasmin. The hydrolysate was fractionated by ultrafiltration and selective precipitation, which resulted in several peptide fractions of which the peptide composition was monitored by reversed-phase high-performance liquid chromatography. Poorly soluble, hydrophobic peptide fractions, containing peptides from the C-terminal half of the βCN sequence, possessed improved foam-forming and -stabilizing properties compared to those of intact βCN, especially at pH 4.0. Soluble peptide fractions, containing a variety of peptides from the “middle” part of the βCN sequence in different proportions, possessed improved emulsion-forming capacity at pH 6.7, compared to that of intact βCN, and showed large variations in emulsion stability. The fraction containing the hydrophilic N-terminal part of βCN showed inferior foam, emulsion, and surface-active properties, especially at pH 6.7. The differences in functionality found between the various peptide fractions may be attributed either to synergistic effects between peptides or to a specific functionality of some individual peptides. Keywords: β-Casein; peptides; plasmin; foam; emulsions
Article
The dynamic surface dilational elasticity, surface pressure, and adsorbed amount of the mixed solutions of beta-lactoglobulin and guanidine hydrochloride were measured as a function of surface age and denaturant concentration. It was shown that the conformational transition from compact globules to disordered protein molecules in the surface layer leads to strong changes in the surface elasticity kinetic dependencies and thereby can be easily detected by measuring the surface dilational rheological properties. The corresponding changes of the kinetic dependencies of the surface pressure and adsorbed amount are not so pronounced but correlate with the results on surface dilational elasticity.
Article
Studies on the structure and stability of peptides and proteins during l-->d configurational change are certainly important for the designing of peptides with new biological activity and protein engineering. The l-->d amino acid (d AA) changes have been observed in aged proteins such as collagen. Hence, in this study, an attempt has been made to explore the effect of the replacement of l amino acid (l AA) in the model collagen-like peptides with d AA and the origin of structural stability (destability) has been traced using the molecular dynamics (MD) method employing the AMBER force field. Our results reveal that the substitution of d AA produces a large local disruption to the triple-helical structure. Formation of a kink (bulge) at the site of substitution is observed from the detailed analysis of MD trajectory. However, this local perturbation of kinked helix changes the direction of the helices and affects the relative orientation of the respective AA residues for helix-helix interaction, enough to affect the overall stability of the model collagen-like peptide. The destabilization energy per d Ala substitution is 7.87 kcal/mol, which is similar to the value for the Gly-->Ala mutation in collagen. Since the Gly-->Ala mutation is involved in genetic disorders such as osteogenesis imperfecta (OI), the l-->d configurational change may produce a similar effect on collagen.
Article
A true collagenase was isolated from the culture fluid of a marine bacterium which has been designated Vibrio B-30 (ATCC 21250). Collagenase production was obtained only in media containing collagen or certain degradation products of collagen. Partial purification on DEAE-cellulose and Sephadex G-200 columns produced active enzyme which was free of nonspecific proteases but which contained two collagenases. The two collagenases have the same apparent molecular size, and evidence is presented to support the theory that one collagenase is derived from the other. Vibrio B-30 collagenase appears to be a tetramer with a molecular weight of about 105 000 composed of two different subunits (mol wt 24 000 and 28 000). Some of the properties of the Vibrio collagenase are compared with those of Clostridium histolyticum collagenase. Molecular weights, subunit structures, specificity and mode of collagen hydrolysis, insensitivity to diisopropyl fluorophosphate and calf serum, and sensitivity to certain metal ion complexing agents and isopropyl alcohol are similar for the collagenases from both organisms. However, Vibrio B-30 collagenase and Clostridium collagenase differ immunologically and electrophoretically.
Article
Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.
Article
Seven collagenases denoted by the letters alpha, beta, gamma, delta, epsilon, zeta and eta have been purified to homogeneity from the culture filtrate of Clostridium histolyticum. All seven enzymes are zinc proteinases that require calcium ions for activity and have essential carboxyl, tyrosyl and lysyl residues. These enzymes can be divided into two classes on the basis of the sequence homologies in their polypeptide chains, as revealed from a comparison of their tryptic digests. This division into classes is also supported by a comparison of their specificities toward peptide substrates, their interaction with substrate-analog inhibitors, and their mode of attack of triple helical collagens. The sequence specificities of these enzymes have been studied in detail. The specificities of the two classes are similar, but complementary. Both classes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities, where the latter is thought to facilitate removal of Gly-X-Y triplets from the C-terminus of collagen fragments. The mode of attack of these collagenases on triple helical type I, II and III collagens is very similar for the enzymes within each class, but different for the two classes. The class I enzymes first hydrolyze loci near the ends of the triple helical domains of these collagen molecules, while the class II enzymes make their initial cleavages in the interior. The sites of these initial cleavages are being sequenced and preliminary results indicate that they do not resemble the tissue collagenase cleavage site with respect to either their imino acid content or distribution. The kinetic parameters for the hydrolysis of type I, II and III collagens have been measured and are similar in magnitude to those for the tissue collagenases. Synthetic peptide substrate-analog inhibitors have been prepared for both classes of collagenases and shown to be transition-state-analog inhibitors.
Article
Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified. Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar. Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3. This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes. Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity. C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site. The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related. Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3. C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining.
Article
A comprehensive approach for the structural microanalysis of collagen based of collagen based on high-performance liquid chromatography (h.p.l.c.) has been developed using calf skin type I collagen as a model system. The alpha, beta and gamma components were separated, after heat denaturation, on a TSK 4000 SW gel permeation column, using a nonvolatile buffer. Monitoring at 210 nm permits the detection of 1 microgram of a single chain. The alpha 1(I) and alpha 2(I) chains were completely resolved using a large-pore reversed-phase column (Vydac 201 TP 4.6) eluted by an aqueous acetonitrile gradient (24-48%) containing 0.01 M heptafluorabutyric acid as an ion-pairing agent. The purified alpha 1(I) chain was digested with CNBr and the resulting fragments separated in the same chromatography system with a gradient containing a 12.8-44.8% acetonitrile gradient. The purified alpha 1(I)CB 3 peptide was further cleaved with trypsin and the resulting peptides separated first by a similar chromatography with a 4-32% acetonitrile gradient. Resolution of some poorly separated peptides was obtained by a rechromatography using trifluoroacetic acid as counterion. The isolated peptides were hydrolyzed and identified by their amino-acid composition. Sequencing of h.p.l.c.-purified alpha 1(I)CB 3 was also performed to demonstrate the suitability of the technique for the preparation of peptides for amino-acid sequencing. This study demonstrates that detailed structural analysis can be performed on 3 mg of a purified collagen.
Article
The objective of this study was to characterize the changes in peptide solubility resulting from changing some physicochemical conditions in a tryptic hydrolysate of beta-lactoglobulin (beta-LG). The turbidity (500 nm) of a 1% solution of tryptic peptides was measured at pH 3-10, at 5, 25, and 50 degrees C, in the presence of different salt concentrations (0, 0.5, and 1 M NaCl), in the presence of denaturing and reducing agents (6 M urea, 5% SDS, or 5% beta-mercaptoethanol), and under an electric field (isoelectric focusing). The results reveal an increase in turbidity of the peptide solution at pH 4, but a slight increase in turbidity was also observed at pH 8, which is attributable to peptides linked by disulfide bridges. The effect of temperature and ionic strength on the turbidity occurring at pH 4 indicates that mainly hydrophobic interactions are involved in the aggregation process. The material in the precipitate at pH 4 was identified as the peptides beta-LG 1-8, 15-20, and 41-60 and non-hydrolyzed alpha-lactalbumin. These results suggest that a limited number of peptides are involved in the aggregation process observed at pH 4, some of which having bioactive (beta-LG 15-20, ACE inhibitor, and opioid) or emulsifying properties (beta-LG 41-60). Aggregation of these peptides at acidic pH indicates that a simple acidification step could represent an easy process for isolating peptidic fractions enriched in bioactive or functional peptides.