Radical scavenging properties of protein hydrolysates from Jumbo flying squid (Dosidicus eschrichitii Steenstrup) skin gelatin

Gelatin extracted from Jumbo flying squid skin was hydrolyzed with serials of protease. The scavenging effects of gelatin hydrolysates on superoxide and hydroxyl radical were assessed by chemiluminescence analysis. Properase E and pepsin have shown efficient hydrolysis of squid skin gelatin to obtain high radical scavenging activity peptides. The conditions chosen for enzymic hydrolysis of squid skin gelatin with properase E were pH 9.0, 45 °C, 3 h reaction time, and enzyme‐to‐substrate ratio of 1:50. Hydrolysates combining properase E and pepsin showed the best radical scavenging activity. For fragmented hydrolysates by ultrafiltration, fractions UF‐2 (M_w < 2000 Da) had high yield and radical scavenging activity. Copyright © 2006 Society of Chemical Industry     

Partial characterization of collagen from purple sea urchin (Anthocidaris crassispina) test

Summary As part of a study to establish an effective use of underutilized resources, purple sea urchin test collagen was prepared by limited pepsin digestion and characterized. This collagen consisted of two α chains and has a chain composition of (α1)₂α2 heterotrimer. The primary structure of the purple sea urchin collagen is different from that of a mammalian such as porcine. Sea urchin collagen has a denaturation temperature of 28 °C which is about 9 °C lower than that of the porcine skin collagen. The yield of collagen was about 35% on the basis of lyophilized dry weight. As the purple sea urchin has the potential as an alternative source of collagen for use in various fields such as foods, medicine, and cosmetics, it is necessary to improve the denaturation temperature of the sea urchin.     

Bovine collagen. II. Electrophoresis of collagen fractions

Summary. Vertical starch gel electrophoresis was used to separate components of neutral salt soluble (NSS) and acid soluble (AS) collagens, and of eucollagens, from muscle, skin and tendon of bovines of different age. The relative proportions of the separated components were assessed spectrophotometrically.
The electrophoretograms indicated that the greater difficulties of extraction which distinguish AS from NSS collagen reflect a greater measure of cross-bonding between the α chains of the former.
They also indicated that there was a marked increase in the proportion of components having intra molecular cross bonds, in both NSS and AS collagens and in the three tissues studied, between the 5th and 9th month of gestation. In older animals there were also indications of an enhanced degree of inter molecular cross bonding.
Such changes help explain the increased toughness of the meat from older bovines despite a lower percentage of collagen.     

Effect of dry heat on the in vitro digestibility and trypsin inhibitor activity of chickpea flour

The effect of a dry heat treatment on trypsin inhibitors, protein quality and molecular weight of products obtained from enzymatic hydrolysis of chickpea flours was analysed in order to use chickpeas as food protein hydrolysates. Chickpea flour obtained from dehusked seeds was processed under different conditions, either by heating or enzymatic treatment. Heat treatment at 140 °C for varying times (1–24 h) inactivated trypsin inhibitors and facilitated enzymatic treatment but showed an unacceptable loss in the nutritional quality of the protein for heating times longer than 6 h. Enzymatic treatment with a commercial protease, Alcalase 0.6L, at pH 8 and 50 °C, increased the protein nutritional value of the chickpea by breaking the protein chains into shorter peptide chains more suitable to human nutrition.     

不同来源胶原蛋白抗冻活性的研究

采用酶法制备不同来源的胶原蛋白,并对其纯度及氨基酸组成进行鉴定。在此基础上,利用差示扫描量热仪及低温显微镜对其热滞活性（thermal hysteresis activity,THA）和重结晶抑制效应（ice recrystallizationinhibition,IRI）进行了研究。结果表明：所制备的胶原蛋白均为典型的Ⅰ型胶原,纯度较高,其分子质量约为330 kD;相比于牛血清白蛋白,不同来源的胶原蛋白中,猪皮胶原具有较高的热滞活性,即当保留温度为－0.2 ℃,体系冰晶含量φ≤5% 时,THA为0.52 ℃;在猪皮胶原蛋白体系中形成的冰晶为不规则的圆球形,不易对细胞及组织造成伤害,且经过一次循环后,冰晶无显著增大。     

Bioactive peptides from collagen hydrolysates from squid (Dosidicus gigas) by-products fractionated by ultrafiltration

The bioactive properties of peptide fractions obtained from the hydrolysis of squid (Dosidicus gigas) by-products collagen, using Protease type XIV and ultrafiltration (UFI), were studied. The basic objective was to improve the bioactivity of squid hydrolysates via the application of UFI. Peptide fractions obtained after UFI had higher antioxidant and antimutagenic activities, but the antiproliferative activity did not improve after UFI. Peptides <5 kDa (Fraction F3) showed higher antioxidant and antimutagenic activities, as well as lower antioxidant and antiproliferative activities than both, peptides >10 kDa (F1) and those within the range of >5 to <10 kDa (F2). Band at lower field observed in FT-IR spectra and proton-peaks observed at higher ¹H-NMR fields, both associated to aromatic amino acids, as well as to other antioxidant amino acids such as hydroxyproline, glycine, arginine and lysine, may explain F3 bioactivity. Ultrafiltration can, therefore, be used to improve some bioactivities of squid collagen hydrolysates.     

Temperature effects on type I pepsin‐solubilised collagen extraction from silver‐line grunt skin and its in vitro fibril self‐assembly

BACKGROUND: Fish skin is a potential source of collagen. Increasing the extraction temperature increases the yield of collagen. However, it may also result in degradation of the peptide chains, thus damaging the 3D structure of collagen that is vital for its application as a biomaterial. This work investigated the effects of extraction temperature on the yield and characteristics, including fibril self‐assembly, of type I pepsin‐solubilised fish skin collagen. RESULTS: Pepsin‐solubilised collagens were extracted from fresh skin of silver‐line grunt at 4, 10, 20 and 28 °C for 6 h. Extraction at 10 °C gave the highest yield of collagens (439.32 ± 96.43 mg g^?1 fresh skin, dry basis), which were identified as type I and comprised β, α1 and α2 subunits. Extraction at higher temperatures (20 and 28 °C) resulted in the formation of low‐molecular‐weight peptide fragments, thus reducing the yield of the resultant type I collagen. The denaturation temperatures of collagens extracted at 4 and 10 °C, as determined by thermal analysis using differential scanning calorimetry, were 39.5 and 37.5 °C respectively. In vitro fibril self‐assembly of 1 mg mL^?1 collagen solution (pH 6) incubated at 25 °C was only observed with collagens extracted at 4 and 10 °C. The 10 °C collagen not only showed a higher rate of self‐assembly, but its matrix also had a larger fibril diameter of 0.50 ± 0.07 µm (compared with 0.41 ± 0.07 µm for the 4 °C collagen) after 4 h of incubation. CONCLUSION: The results indicated strong effects of extraction temperature on the yield and characteristics of the collagen obtained. Extraction of pepsin‐solubilised collagen from silver‐line grunt skin at 4–10 °C gave a high yield of type I collagen with molecular integrity suitable for tissue‐engineering applications. Copyright © 2010 Society of Chemical Industry     

Isolation and characterisation of collagen from the outer skin waste material of cuttlefish (Sepia lycidas)

In an investigation into making more effective use of underutilized resources, collagen was prepared from the outer skin of cuttlefish (Sepia lycidas). Initial extraction of the cuttlefish outer skin in acetic acid yielded only 2% of collagen (dry weight basis). On subsequent digestion of the residue with 10% pepsin (w/v), a solubilized collagen (PSC) was obtained in a yield of 35% (dry weight basis). With respect to PSC, it had a chain composition of (α 1)₂α2 heterotrimer similar to Japanese common squid. Moreover, the denaturation temperature of this collagen was 27°C which is about 10°C lower than that of porcine collagen. This report indicates that cuttlefish waste materials have potential in supplementing the skin of land vertebrates as a source of collagen.     

不同蛋白酶对复合骨素酶解液呈味物质的影响

为研究不同蛋白酶对复合骨素（牛骨素和鸡骨素）酶解液呈味物质的影响,选取复合蛋白酶（P）、菠萝蛋白酶（B）和复合风味蛋白酶（F）,制备单一和组合酶解液,对酶解液肽分子质量分布、核苷酸含量、游离氨基酸含量等呈味物质指标进行综合评价。结果表明：P＋F组酶解液的水解度最大,酶解液中小分子质量肽（＜200 Da）分布比例最大;P＋F组酶解液的游离氨基酸含量最高,鲜味和甜味氨基酸是复合骨素酶解液的主要呈味成分;P＋F组的5’-核苷酸（5’-肌苷酸、5’-鸟苷酸、5’-腺苷酸）总含量较高。因此,复合蛋白酶和复合风味蛋白酶处理的复合骨素酶解液整体风味最佳。     

Influence of enzymatic hydrolysis,pH and storage temperature on the emulsifying properties of canola protein isolate and hydrolysates

The aim of this work was to enhance emulsification properties of canola proteins through enzymatic proteolysis and pH variaton. Canola protein isolate (CPI) and hydrolysates (CPHs) were used to form emulsions at pH 4.0, 7.0 and 9.0 followed by storage at 4 or 25 °C for 7 days. Controlled enzymatic hydrolysis led to increased peptide bond cleavage with time (0.23 g/100 g in CPI to 7.18 g/100 g after 24‐h Alcalase hydrolysis). Generally, oil droplet sizes were smaller for emulsions made at pH 9.0, which suggest better quality than those made at pH 4.0 and 7.0. Trypsin hydrolysate emulsions were the most physically stable at pH 7.0 and 9.0; in contrast, the pepsin hydrolysate emulsions were unstable at all conditions. The results suggest that selective enzymatic hydrolysis could play an important role in enhancing successful incorporation of canola proteins and peptides into food systems as protein emulsifiers.     

Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella)

Pepsin-soluble collagen was extracted from the skin of grass carp (Ctenopharyngodon idella) with a yield of 46.6%, on a dry weight basis. Electrophoretic patterns showed that the collagen contained α1 and α2 chains, similar to those of calf skin collagen. The imino acid content of the collagen from grass carp skin was much lower than those of mammalian’s collagens, as also were the transition temperature and denaturation temperature which were only 24.6 °C and 28.4 °C respectively. Peptide maps of the collagen digested by trypsin and V8 protease showed different peptide fragments from those of calf skin collagen and other fish skin collagens, suggesting differences in amino acid sequences and collagen conformation. In addition, the lyophilized collagen sponge from grass carp skin had a uniform and regular network structure, just like calf skin collagen sponge. These results suggest that grass carp skin has potential for use as a supplementary source of collagen.     

Characterization of Acid-soluble Collagen from Pacific Whiting Surimi Processing Byproducts

ABSTRACT: Three different solid byproducts (skin, frame, and refiner discharge) from Pacific whiting surimi manufacturing are a good resource for collagen extraction according to their total protein concentrations and other biochemical properties. Denaturation temperature of acid-soluble collagens was 23.3°C for refiner discharge, 21.7 °C for skin, and 20.6°C for frame. Based on the functional properties, acid-soluble collagen from refiner discharge was the best and showed potential as an ingredient in processed food manufacturing.     

High pressure can reduce the antigenicity of bovine whey protein hydrolysates

The combined effect of high pressure (HP) and enzymatic treatments on the proteolysis and antigenicity of hydrolysates from bovine whey proteins (WP) was studied. Four food grade protease preparations (Alcalase, Neutrase, Corolase 7089 and Corolase PN-L) were used. Hydrolysis was performed at 40 °C for Corolase PN-L and 50 °C for the other enzymes, for 15 min, after or during HP treatment. The degree of hydrolysis and RP-HPLC peptide profile were evaluated. An indirect ELISA test using polyclonal rat anti β-lactoglobulin antibodies was used to determine the residual antigenicity. The results showed that HP treatment enhanced the hydrolysis of bovine WP. For most enzymes, the best results were obtained at a pressure of 300 MPa. For two enzymes, Corolase PN-L and Neutrase, differences in peptide profiles were obtained due to the pressure applied during the enzymatic hydrolysis. Based on these differences, the residual antigenicity of these preparations were determined. An important reduction was found in the antigenicity of the hydrolysates obtained with Corolase PN-L and Neutrase in combination with HP treatment (300 MPa), prior to or during enzymatic hydrolysis, respectively. These results suggest that HP can enhance the WP hydrolysis, and, depending on the choice of enzymes, reduce the residual antigenicity of the hydrolysates. The reduced antigenicity of hydrolysates obtained by the combined treatments could have a relevant application in the development of hypoallergenic infant formulae.     

Studies on genetic variants of α-lactalbumin and β-lactoglobulin from milk of native Portuguese ovine and caprine breeds

Summary α-Lactalbumin (α-La) and β-lactoglobulin (β-Lg) fractions were obtained from Portuguese native breeds of ewes and goats by preparative gel filtration and further purified by ion exchange; their genetic variants were characterized by isolectric focusing, and β-Lg isolated was further characterized by differential scanning calorimetry. Separation of β-Lg and α-La by molecular exclusion from native whey was relatively easy, whereas β-Lg from both breeds accounted for a single peak via ion exchange under various gradients of NaCl. Isoelectric focusing has indicated that α-La from ovine and caprine wheys appears as a single variant in each case, as well as β-Lg from caprine whey; however, β-Lg from ovine whey appears as two peaks, tentatively denoted as β-Lg A and B. Further comparison with bovine whey made it possible to rank whey proteins by increasing value of pI as follows: bovine β-Lg A, bovine α-La, bovine β-Lg B, ovine and caprine α-La, ovine β-Lg A, and finally ovine β-Lg B and caprine β-Lg. β-Lg from goat's whey showed the highest onset temperature of denaturation in the presence (78–97 °C) or absence (90–100 °C) of NaCl for every pH tested; when NaCl was present, a good correlation between pI and onset temperature of denaturation was obtained for pH values in the range 3.5–7.0.     

The role of collagen in the quality and processing of fish

Collagen in the muscles of fish constitutes the main component of the connective tissue membranes joining individual myotomes and is responsible for the integrity of the fillets. The content of collagen in fish muscles is from about 0.2 to 1.4% and in squid mantel about 2.6%. Fish and invertebrata collagens contain slightly more essential amino acids than intramuscular bovine connective tissue collagen. The invertebrata collagens are exceptionally rich in sugars linked mainly O-glycosidically to hydroxylysine residues. During maturation of fish the proportion of collagen to total protein in the muscles increases while the extent of crosslinking does not change significantly. The thermal properties of fish collagens depend significantly on the content of hydroxyproline and proline residues which in turn is correlated to the temperature of the habitat. Generally the shrinkage temperature of fish skin collagens is about 20 degrees C lower than that of mammalian hide collagens. In several species of fish the weakening of the connective tissues post mortem may lead to serious quality deterioration that manifests itself by disintegration of the fillets, especially under the strain of rough handling and of rigor mortis at ambient temperature. Thermal changes in collagen are the necessary result of the cooking of fish, squid, and minced fish products and contribute to the desirable texture of the meat. However, they may lead to serious losses during hot smoking due to a reduction in the breaking strength of the tissues when heating is conducted at high relative humidity. Because of the high viscosity of gelatinized collagen, it is not possible to concentrate the fish stickwaters, a proteinaceous byproduct of the fish meal industry, to more than 50% dry matter. Better knowledge of the contents and properties of fish collagens could be helpful in rationalizing many aspects of fish processing.     

Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus)

Extraction and some properties of pepsin-solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus) were investigated. Addition of bigeye snapper pepsin (BSP) at a level of 20 kUnits/g of defatted skin resulted in an increased content of collagen extracted from bigeye snapper skin. The yields of collagen from bigeye snapper skin extracted for 48 h with acid and with BSP were 5.31% and 18.74% (dry basis), respectively. With pre-swelling in acid for 24 h, collagen extracted with BSP at a level of 20 kUnits/g of defatted skin for 48 h had a yield of 19.79%, which was greater than that of collagen extracted using porcine pepsin at the same level (13.03%). The skin collagen was characterised to be type I with no disulfide bond. Electrophoretic study revealed slight differences in molecular weight between acid-solubilised collagen and all pepsin-solubilised collagens. The molecular weights of α1 and α2 chains in acid-solubilised collagen were estimated to be 120 and 112 kDa, respectively, whereas α1 and α2 chains of pepsin-solubilised collagens had molecular weights of 118 and 111 kDa, respectively. The result suggested that these pepsin-solubilised collagens might undergo partial cleavage in the telopeptide region by pepsin treatment. The maximum transition temperature (T_max) of acid-solubilised collagen was observed at 32.5 °C, which was slightly higher than that of pepsin-solubilised collagens (by about 1 °C). Generally, all collagens were highly solubilised in the pH range of 2–5 and sharply decreased at the neutral pH. No changes in solubility were observed in the presence of NaCl up to 3% (w/v) and the decrease was more pronounced with increasing NaCl concentration.     

Collagen of the skin of ocellate puffer fish (Takifugu rubripes)

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from ocellate puffer fish skin and partially characterized. With respect to the pepsin-solubilized collagen, it was a heterotrimer with a chain composition of (α1)₂α2. The patterns of peptide fragments were different from skin collagens of other species. The denaturation temperature was 28 °C, about 9 °C lower than that of porcine skin collagen. On the other hand, the yields of acid-solubilized and pepsin-solubilized collagens were very high, 10.7% and 44.7%, respectively, on a dry weight basis. These results suggest that ocellate puffer fish skin has potential as an alternative source of collagen for use in various fields.     

In vitro amylolytic degradation of natural and graft copolymerised cassava and potato starches

 Various enzymatic hydrolyses to maximise degradation of the starch moiety of grafted starch (starch–g-polyacrylonitrile) were tried. The percentage of α- and β-amylolyses of grafted starch were 55 and 50 compared to 80 and 70 respectively for natural starches. Sequential degradation with α-amylase and glucoamylase of grafted starch showed 70% hydrolysis. The maltooligosaccharide profile by HPLC of the hydrolysates of grafted starch showed oligomers up to a degree of polymerisation (DP) 3, whereas the natural starch hydrolysates showed up to DP 7. The percentage hydrolysis, as well as the enzyme degradation profile, remained similar for both potato and cassava starches. Further treatment of the grafted starch hydrolysates with Bacillus cereus cells showed the presence of very low molecular weight polyacrylonitrile chains grafted onto maltooligosaccharides. The size exclusion chromatography analysis of grafted starch indicated the amylose component of starch that undergoes graft copolymerisation. Received: 30 December 1999     

Studies on collagen from the skin of channel catfish (Ictalurus punctaus)

Acid-soluble and pepsin-soluble collagens (ASC and PSC) were extracted from the skin of channel catfish (Ictalurus punctaus) and partially characterized . The collagen obtained in the experiment contained more than 23% glycine as the most abundant amino acid. The denaturation temperature of acid-soluble collagen was 32.5 °C, about 5 °C lower than that of the porcine skin collagen. SDS–PAGE showed that the collagens were composed of two distinct alpha chains, which is similar to the porcine type I collagen. The contents of the skin ASC and PSC, on a dry weight basis, were 25.8% and 38.4%, respectively. These results suggest that channel catfish skin has potential as a supplement to the sources of vertebrate collagens.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE. 9. Effect of Temperature and pH on Tropomyosin-Troponin and Purified α-Actinin from Rabbit Muscle

SUMMARY– A study was done on the effects of in vitro storage of purified α-actinin, troponin, tropomyosin, and the tropomyosin-troponin complex on the activity of these protein fractions in the ATPase and superprecipitation assays. Storage was done at various combinations of temperatures between 0 and 40°C and pH values between 5.7 and 7.0. Even after 40 hr of storage, activities of purified tropomyosin and the tropomyosin-troponin complex were not affected by any combination of temperature and pH included in this study, but activities of purified α-actinin and troponin were almost completely lost after 16 hr at 40°C and pH 5.7. Storage for 40 hr at low pH (5.7) and low temperatures (0°C) did not affect the activity of either α-actinin or troponin, but 40 hr of storage at high temperatures (40°C) and neutral pH caused some loss in activity for both these proteins. This loss of activity caused by 40°C, pH 7.0 storage was much more noticeable in the case of troponin than in the case of α-actinin. Storage periods of 40 hr or longer were required before any loss of α-actinin activity could be detected at pH 7.0 and 40°C. Since most meat animal carcasses are chilled soon after exsanguination and attain muscle temperatures of 25°C or lower before the pH falls below 6.2, it is probable that α-actinin and tropomyosin-troponin activity remain almost unchanged in meat handled through normal market channels. However, myofibrillar tissue in those porcine animals whose musculature undergoes a very rapid post-mortem decline in pH so that values of 5.7 or less are reached while muscle temperatures are still 37°C or higher may lose much of its α-actinin and tropomyosin-troponin activity during the first 24 hr post-mortem.