Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin

Acid‐solubilized collagen (ASC) and pepsin‐solubilized collagen (PSC) were isolated from the skin of bigeye snapper (Priacanthus macracanthus) with yields of 64 and 11 g kg^?1 wet weight, respectively. Both ASC and PSC were characterized as type I collagens with no disulfide bonds. Peptide maps of ASC and PSC digested by V8 protease and lysyl endopeptidase showed some differences in peptide patterns and were totally different from those of calf skin collagen. The maximum solubility was observed at pH 4 and 5 for ASC and PSC, respectively. A sharp decrease in solubility of both collagens in acetic acid was found with NaCl concentration above 30 g l^?1. Thermal transitions of ASC and PSC in deionized water were observed with T_max of 30.37 and 30.87 °C, respectively, and were lowered in the presence of acetic acid (0.05 mol kg^?1 solution). Therefore, ASC was a major fraction in bigeye snapper skin and it exhibited some different characteristics to PSC. Copyright © 2005 Society of Chemical Industry     

Isolation and Characterization of Collagen from the Body Wall of Sea Cucumber Stichopus monotuberculatus

To exploit a new collagen resource from the body wall of tropical sea cucumber, pepsin‐solubilized collagen of Stichopus monotuberculatus (PSC‐Sm) was isolated and characterized with UV‐vis spectra, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), amino acid composition, enzyme‐digested peptide maps, Fourier transform infrared spectroscopy (FTIR), maximum transition temperature (T_m), and solubilities. The maximum absorbance of PSC‐Sm was exhibited at 218 nm in UV‐vis spectra. The triple helical structure and activity of PSC‐Sm could be indicated by FTIR. SDS‐PAGE showed that the triple helix of PSC‐Sm was formed as (α₁)₃ by 3 α₁ chain homologous with molecular weight of 137 kDa. The T_m of PSC‐Sm and calf skin collagen (CSC) were 30.2 and 35.0 ºC, respectively, which consistent with the result of FTIR that CSC contained more stable triple‐helix than PSC‐Sm. Peptide maps were different between PSC‐Sm and CSC, indicating the differences in their amino acid compositions and sequences. The maximum and minimum solubilities of PSC‐Sm were observed at pH 2.0 and 4.0, respectively. A sharp decrease in solubility appeared when NaCl concentration was between 3% and 5%. These results showed that collagen from S. monotuberculatus had the type I collagen characteristics and good thermal stability, and therefore, it could be used as an alternative resource of collagen.     

Characterization and subunit composition of collagen from the body wall of sea cucumber Stichopus japonicus

Pepsin-solubilized collagen (PSC) without telopeptides was prepared from the body wall of the sea cucumber Stichopus japonicus and isolated by selective precipitation with NaCl. The PSC exhibited a maximum absorbance at 220 nm. The subunit of PSC was isolated by Sephacryl S-300 HR. The results of SDS-PAGE suggested that purified collagen from S. japonicus was a 1α trimer (about 135 kDa) while 1α chain resembling α₁ chain of type I collagen of vertebrate. The thermal stability temperature (T_s) was 57.0 °C as measured by DSC, about 5.0 °C lower than that of type I collagen of calf. Peptide mapping and amino acid analysis of PSC also revealed the difference between invertebrate and vertebrate. However, the presence of (α₁)₃ trimers was evident.     

Characteristics of collagen from the skin of clown featherback (Chitala ornata)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of clown featherback (Chitala ornata) were isolated and characterised. Yields of ASC and PSC were 27.64 and 44.63% (dry weight basis) with total collagen recovery of 82.08%. Both collagens contained glycine as the major amino acid with relatively high content of proline, hydroxyproline and glutamic acid/glutamine. Nevertheless, they had the low content of cysteine, histidine and tryrosine. The collagen was characterised as type I, comprising (α1)₂α2‐heterotrimer. Pepsin‐aided process did not affect triple‐helical structure of PSC as determined by FTIR spectra. Thermal transition temperature of ASC (36.28 °C) was slightly higher than that of PSC (35.23 °C). However, no differences in isoelectric point (5.54–5.68) between ASC and PSC were observed. Therefore, collagen from the skin of clown featherback could be successfully extracted for further applications.     

Collagens from the skin of arabesque greenling (Pleurogrammus azonus) solubilized with the aid of acetic acid and pepsin from albacore tuna (Thunnus alalunga) stomach

BACKGROUND: Due to the low extraction efficiency of collagen from fish skin by the typical acid solubilization process, pepsin has been widely used to aid further extraction of collagen from the residue. The aim of this study was to characterize collagen from the skin of arabesque greenling extracted with the aid of albacore tuna pepsin, in comparison with collagen obtained from the acid solubilization process. RESULTS: Acid‐solubilized collagen (ASC) from the skin of arabesque greenling was extracted with acetic acid. Pepsin‐solubilized collagen (PSC) was further extracted from the skin residue with the aid of pepsin from albacore tuna. The yields of ASC and PSC were 303 and 140 g kg^?1 (dry weight), respectively. Both collagens contained α‐ and β‐chains as their major components and were characterized as type I collagen. Both collagens contained glycine as a major amino acid and had imino acid content of 157–159 residues per 1000 residues. The degradation induced by lysyl endopeptidase and V8‐protease was more pronounced in PSC compared with ASC. Maximal transition temperatures of both collagens were in the range of 15.4–15.7 °C. Fourier transform infrared spectra revealed some differences in molecular order between ASC and PSC. Nevertheless, the triple‐helical structure of PSC was still predominant. Based on ζ‐potential, pI of ASC and PSC was estimated to be 6.31 and 6.38, respectively. CONCLUSION: Isolation of collagens from the skin of arabesque greenling could be achieved by acid or albacore tuna pepsin solubilization. However, there was a slight difference in properties between ASC and PSC. Copyright © 2010 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.     

Study on the self‐assembly property of type I collagen prepared from tilapia (Oreochromis niloticus) skin by different extraction methods

Type I collagen was prepared from tilapia (Oreochromis niloticus) skin by acetic acid and pepsin process at 4 °C, respectively (ASC and PSC), and hot‐water method separately at 25, 35 and 45 °C (C‐25, C‐35 and C‐45). Their structure and self‐assembly property were discussed. SDS‐PAGE patterns suggested that pepsin hydrolysis and the 35 and 45 °C extraction produced collagen with much reduced proportions of α‐ and β‐chains. Fourier transform infrared spectroscopy spectra revealed that pepsin hydrolysis did not change the conformation of collagen, but higher extraction temperature did. Self‐assembly curves and atomic force microscopy (AFM) observations showed that only ASC, PSC and C‐25 could self‐assemble into fibrils with D‐periodicity, but the reconstruction rate of C‐25 was lower. Besides, PSC had relatively higher resolution ratio compared with others. Overall, pepsin‐extracted collagen displayed higher solubility and better fibril‐forming capacity, having the potential of applying in biomaterials and food‐packaging materials.     

Combining culture‐dependent and culture‐independent molecular methods for the isolation and purification of a potentially novel anaerobic species from pit mud in a Chinese liquor distillery

Strain S12–27–1‐3‐5 (a potentially novel anaerobic species) with a 16S rRNA sequence homology of <97% was isolated and purified from pit mud by combining culture‐dependent and culture‐independent molecular methods, such as cloning of 16S rRNA, amplified rRNA restriction analysis, and denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the 16S rRNA gene indicated that strain S12–27–1‐3‐5 was related to Aminobacterium mobile strain ILE‐3 DSM 12262^T and Aminobacterium colombiense strain DSM 12261^T (95 and 96% similarity value, respectively). The results verified that cloning of the 16S rRNA was efficient to identify whether a potentially new bacterial taxon existed in impure isolates and that the DGGE method was a powerful tool for screening the target bacteria and for identifying duplicate strains. Therefore, the application of the culture‐independent molecular methods for the isolation and purification of a potentially novel species was effective. Strain S12–27–1‐3‐5 (= DSM 27871 = JCM 19605 = CICC 10731^T) was an anaerobic amino acid‐degrading bacterium. The results of fermentation experiments demonstrated that strain S12–27–1‐3‐5 produced volatile fatty acids (VFAs) and the presence of Methanosarcina barkeri enhanced the generation of VFAs, which contribute to the aroma composition of Chinese liquor. This work could enrich the species resources and promote the development and utilization of an uncultured species. Copyright © 2016 The Institute of Brewing & Distilling     

Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by‐products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G‐75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G‐75 gel filtration, resulting in a 5.42‐fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N‐α‐benzoyl‐L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N‐α‐benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °C respectively using BAEE as a substrate. The trypsin kinetic constants K_m and k_cat on BAPNA were 1.67 mmol L^?1 and 3.87 s^?1 respectively, while the catalytic efficiency k_cat/K_m was 2.31 s^?1 L mmol^?1. CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish‐processing and food industries. Copyright © 2008 Society of Chemical Industry     

Purification of Polyphenoloxidase from the Purple‐fleshed Potato (Solanum tuberosum Jasim) and its Secondary Structure

ABSTRACT: Polyphenoloxidase (PPO) was purified from purple‐fleshed potatoes (Solanum tuberosum Jasim) using membrane concentration, ammonium sulfate fractionation, Resource Q ion exchange chromatography, and Sephacryl S‐200 HR gel permeation chromatography. PPO was purified 78‐fold from a crude extract. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results showed that the purified enzyme has a major subunit molecular weight of 40 kDa. To elucidate the secondary structure of the purified PPO, circular dichroism (CD) was performed. The CD spectrum of the purified enzyme showed that PPO contains 35% α‐helix, 30% β‐turn, and 35% random coil structure.     

Detection of five novel GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of blacktip shark (Carcharhinus limbatus) were isolated and characterized. The yield of ASC (20.01%) was much higher than that of PSC isolated from the residue of ASC extraction (0.86%). Both collagens had protein as their major constituent with the trace amounts of ash and fat. Based on protein patterns and TOYOPEARL^® CM-650M column chromatography, both collagens contained α- and β-chains as their main components and were characterized as type I collagen with the cross-link of α2-chain. Similar peptide maps of both collagens, digested by either V8 protease or lysyl endopeptidase, were observed but they were totally different from those of type I collagen from calf skin hydrolyzed by the same enzyme. Thermal transition temperature (T _max) of ASC and PSC were 34.23 and 34.37 °C, respectively. Fourier-transform infrared spectra suggested that both collagens were in triple-helical structure. From zeta potential analysis, isoelectric points (pI) of ASC and PSC were estimated to be 6.78 and 7.02, respectively. Thus, blacktip shark skin may serve as an alternative source of collagen and acid solubilization process could be implemented with ease and high yield.     

Isolation and Characterisation of collagen from the skin of brownbanded bamboo shark (Chiloscyllium punctatum)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of brownbanded bamboo shark (Chiloscyllium punctatum) were isolated and characterised. The yield of ASC and PSC were 9.38% and 8.86% (wet weight basis), respectively. Based on protein patterns and TOYOPEARL^® CM-650M column chromatography, both collagens contained α- and β-chains as their major components. These were characterised as type I collagen with the cross-link of α2-chain. As digested by V8-protease and lysyl endopeptidase, peptide maps of both ASC and PSC were similar, but differed from that of type I collagen from calf skin. Fourier transform infrared (FTIR) spectra of both collagens were similar and pepsin hydrolysis had no effect on triple-helical structure of collagen. Transition temperature (T_max) of ASC and PSC were 34.45 and 34.52 °C, respectively, as determined by differential scanning colorimetry (DSC). From zeta potential study, the isoelectric points of ASC and PSC were estimated to be 6.21 and 6.56, respectively. Therefore, the skin of brownbanded bamboo shark could serve as an alternative source of collagen for different applications.     

Purification and characterisation of a novel α‐L‐rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae

A novel α‐L‐rhamnosidase was isolated and purified from Aspergillus oryzae NL‐1. The enzyme was purified 13.2‐fold by ultrafiltration, ion exchange and gel filtration chromatography with an overall recovery of 6.4% and specific activity of 224.4 U/mg, and the molecular mass of its subunit was approximately 75 kDa. Its optimal temperature and pH were 65 °C and 4.5, respectively. The enzyme was stable in the pH range 3.5–7.0, and it showed good thermostability at higher temperatures. The K_M, kcat and kcat/K_M values were 5.2 mm , 1624 s^?1 and 312 s^?1 mm ^?1 using pNPR as substrates, respectively. Moreover, the enzyme exhibited transglycosylating activity, which could synthesise rhamnosyl mannitol through the reactions of transglycosylation with inexpensive rhamnose as the glycosyl donor. Our findings indicate that the enzyme has potential value for glycoside synthesis in the food industry.     

Isolation and characterisation of a novel angiotensin I‐converting enzyme‐inhibitory peptide derived from douchi,a traditional Chinese fermented soybean food

BACKGROUND: Douchi, a traditional fermented soybean food, has recently attracted a great deal of attention owing to its superior physiological activity. In the present study the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of typical douchi procured from various regions of China was analysed. An ACE‐inhibitory peptide derived from the most potent douchi was also isolated and characterised. The pattern of ACE inhibition and resistance to hydrolysis by gastrointestinal proteases of this peptide are described. RESULTS: ACE‐inhibitory activities were detected in all douchi samples, with IC₅₀ values ranging from 0.204 to 2.011 mg mL^?1. Among the douchi samples, a Mucor‐type douchi exhibited the most potent ACE‐inhibitory activity (IC₅₀ = 0.204 mg mL^?1). A novel ACE‐inhibitory peptide was then isolated from this Mucor‐type douchi using ultrafiltration followed by Sephadex G‐25 column chromatography and reverse phase high‐performance liquid chromatography. The amino acid sequence of the purified peptide was identified by Edman degradation as His‐Leu‐Pro (IC₅₀ = 2.37 µmol L^?1). The peptide is a competitive inhibitor and maintained its inhibitory activity even after incubation with some gastrointestinal proteases. CONCLUSION: The present study shows that peptides derived from soybean fermentation during douchi processing could be the main contributor to the ACE‐inhibitory activity observed. Copyright © 2009 Society of Chemical Industry     

Acid‐soluble and pepsin‐soluble collagens from grass carp (Ctenopharyngodon idella) skin: a comparative study on physicochemical properties

In this study, acid‐soluble (ASC) and pepsin‐soluble (PSC) collagens with triple helical structures were successfully extracted from the skin of grass carp (Ctenopharyngodon idella) by two different extraction approaches. SDS‐PAGE pattern revealed that ASC and PSC are type I collagens with typical α1, α2 and β‐chains. In addition, the intensity of χ‐chain (trimer) in ASC was higher than that of PSC, representing the presence of the high proportion of intra‐ and intermolecular cross‐links of extracted collagens with large molecular weight using the acid method. Differential scanning calorimetry (DSC) results demonstrate that T_d (69.04 °C) of ASC was higher than T_d (62.20 °C) of PSC. Both ASC and PSC had the highest solubility at acidic pHs or at a low concentration of NaCl (<2%, w/v). The results of FTIR suggested the ASC and PSC maintained in the helical secondary structure at high degree.     

Microwave‐Assisted Extraction,Chemical Structures,and Chain Conformation of Polysaccharides from a Novel Cordyceps Sinensis Fungus UM01

Cordyceps sinensis is a well‐known tonic food with broad medicinal properties. The aim of the present study was to investigate the optimization of microwave‐assisted extraction (MAE) and characterize chemical structures and chain conformation of polysaccharides from a novel C. sinensis fungus UM01. Ion‐exchange and gel filtration chromatography were used to purify the polysaccharides. The chemical structure of purified polysaccharide was determined through gas chromatography‐mass spectrometry. Moreover, high performance size exclusion chromatography combined with refractive index detector and multiangle laser light scattering were conducted to analyze the molecular weight (M_w) and chain conformation of purified polysaccharide. Based on the orthogonal design L₉, optimal MAE conditions could be obtained through 1300 W of microwave power, with a 5‐min irradiation time at a solid to water ratio of 1:60, generating the highest extraction yield of 6.20%. Subsequently, the polysaccharide UM01‐S1 was purified. The UM01‐S1 is a glucan‐type polysaccharide with a (1→4)‐β‐d ‐glucosyl backbone and branching points located at O‐3 of Glcp with a terminal‐d ‐Glcp. The M_w, radius of gyration (R_g) and hydrodynamic radius (R_h) of UM01‐S1 were determined as 5.442 × 10⁶ Da, 21.8 and 20.2 nm, respectively. Using the polymer solution theory, the exponent (ν) value of the power law function was calculated as 0.38, and the shape factor (ρ = R_g/R_h) was 1.079, indicating that UM01‐S1 has a sphere‐like conformation with a branched structure in an aqueous solution. These results provide fundamental information for the future application of polysaccharides from cultured C. sinensis in health and functional food area.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Thermodynamic characteristics of copigmentation reaction of acylated anthocyanin isolated from blue flowers of Scutellaria baicalensis Georgi with copigments

Anthocyanin extracts are increasingly used as food colorants. So far, anthocyanins have not been broadly used in foods and beverages, since they are not as stable as synthetic dyes. Copigmentation between anthocyanins and copigments is the main colour‐stabilizing mechanism. The process of copigmentation between isolated acylated anthocyanin and rutin, QSA or baicalin has been observed using UV–vis spectrophotometry. The thermodynamic parameters were correlated to the structure and position of the substituents in the interacting molecules. The acylated anthocyanin was isolated from cultivars of Scutellaria baicalensis Georgi flowers and purified by column chromatography by our own method and has been identified by ¹H‐/¹³C‐NMR spectroscopy and electrospray mass spectrometry as delphinidin‐3‐O‐(6‐O‐malonyl)‐β‐D ‐glucopyranosyl‐5‐O‐β‐D ‐glucopyranoside. Copyright © 2004 Society of Chemical Industry     

Chemical Properties of a Polysaccharide Purified From Solid‐State Fermentation of Auricularia Auricular and its Biological Activity as a Hypolipidemic Agent

A water‐soluble crude polysaccharide was extracted by hot water from Auricularia auricular mycelium grown under solid‐state fermentation (SSF). The crude polysaccharide was purified by DEAE Sephadex A‐50 and Sephadex G‐200 chromatography. Fourier transform infrared spectroscopy and nuclear magnetic resonance (¹H NMR) spectroscopy were used to investigate the structure of the purified A. auricular polysaccharide (AAP‐I) and revealed that it is α‐glycosidically linked. After 14 and 28 days of AAP‐I orally administered, the AAP‐I significantly decreased the levels of total cholesterol, triglyceride, and low‐density lipoprotein cholesterol in mice in which hyperlipidemia had been induced by a high fat diet (P < 0.05). The results revealed that AAP‐I from SSF of A. auricular mycelium possesses potent hypolipidemic properties. The polysaccharide may be useful as a functional food additive and a hypolipidemic agent.