Hideki Kishimura

Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were extracted from scale of spotted golden goatfish (Parupeneus heptacanthus) with the yields of 0.46% and 1.20% (based on dry weight basis), respectively. Both ASC and PSC were characterised as type I collagen, containing α1 and α2 chains. β and γ components were also found in both collagens. Based on FTIR spectra, the limited digestion by pepsin did not disrupt the triple helical structure of collagen. ASC and PSC contained glycine (336-340 residues/1000 residues) as the major amino acid and had imino acids of 186-189 residues/1000 residues. Maximal transition temperatures (Tmax) were 41.58 and 41.01°C for ASC and PSC, respectively. From zeta potential analysis, net charge of zero was found at pH 4.96 and 5.39 for ASC and PSC, respectively. Both collagens exhibited high solubility in acidic pH (2-4) and were soluble in the presence of NaCl at concentration up to 20 and 30g/l for ASC and PSC, respectively.

Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera

Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of N(alpha)-p-Tosyl-L-arginine methyl ester hydrochloride were approximately pH 8.0 and 55 degrees C, respectively. A. pectinifera trypsin was unstable at above 50 degrees C and below pH 5.0, and was not activated by adding Ca(2+). The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found.

Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus)

BACKGROUND Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by-product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin-solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of beta-chain but a lower content of alpha-chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189-193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6-31.3 degrees C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple-helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15-7.46 and 5.97-6.44 respectively. CONCLUSION Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found.

Characteristics and gel properties of gelatin from skin of seabass (Lates calcarifer) as influenced by extraction conditions

Characteristics and gel properties of gelatin from seabass skin, as influenced by extraction conditions, were studied. Yields of gelatin extracted at 45 and 55 °C for various times were 51.6-57.3% and 62.0-66.4% (dry weight basis), respectively. All gelatins contained β-chain and α-chains as the predominant components and showed a high imino acid content (198-202 residues/1000 residues). Generally, the gel strength of gelatins decreased as the extraction temperature and time increased. Gelatin extracted at 45 °C for 3h exhibited the highest gel strength (369 g). Gelling and melting temperatures for seabass skin gelatin were 19.5-20.0 and 26.3-27.0 °C, respectively. All gelatins could be set at 25 °C within 30 min, however gelatins extracted at 45 °C had a shorter setting time than those extracted at 55 °C (P<0.05). Gelatin from seabass skin showed a higher gel strength than bovine gelatin and could be used as a potential replacement for land animal gelatins.

Comparative study on molecular characteristics of acid soluble collagens from skin and swim bladder of seabass (Lates calcarifer)

Acid soluble collagens (ASCs) from skin and swim bladder of seabass (Lates calcarifer) were isolated and comparatively characterised. Higher yield (28.5%) was obtained for ASC from swim bladder, compared with that from skin (15.8%). ASCs from both skin and swim bladder had the similar protein patterns and were identified to be type I. Both α- and β-chains constituted as major components. Fourier transform infrared (FTIR) spectra revealed that both ASCs were triple helix in structure. ASC from both sources contained glycine as the major amino acid with imino acids (proline and hydroxyproline) of 194-195 residues/1000 residues). Peptide maps of both ASCs digested by chymotrypsin and trypsin showed slight differences, suggesting some differences in their primary structure. The thermal transition temperature of swim bladder ASC (35.02°C) was slightly higher than its skin counterpart (33.33°C). Based on zeta potential analysis, ASCs from skin and swim bladder had a net charge of zero at pH 6.46 and 6.64, respectively. Therefore, both the skin and swim bladder of seabass could be used potentially for collagen extraction.

Comparative study on chemical compositions and properties of protein isolates from mung bean, black bean and bambara groundnut

BACKGROUND Different legume seeds may have different protein compositions and properties, thereby affecting applications in food systems. This study aimed to extract and characterize protein isolates from legumes grown in Thailand, including mung bean (MBPI), black bean (BBPI) and bambara groundnut (BGPI). RESULTS All protein isolates had a protein content in the range of 85.2-88.2%. The highest trypsin inhibitory activity was found in BGPI. All protein isolates exhibited satisfactory balanced amino acids with respect to the FAO/WHO pattern. MBPI and BBPI had three predominant proteins with a molecular weight (MW) range of 42-54 kDa, whereas BGPI had two dominant proteins with MW of 52 and 62 kDa. Based on differential scanning calorimetric analysis, MBPI and BGPI had two endothermic peaks, whereas three peaks were found for BBPI. All protein isolates exhibited similar FTIR spectra, indicating similarity in functional group and structure. All protein isolates showed a minimum protein solubility at around pH 4-5. CONCLUSION All protein isolates were important sources of proteins with high lysine content. Isolates from different legumes showed slight differences in physiochemical and thermal properties. Those isolates can be used as proteinaceous ingredients in a variety of food products such as salad dressing, meat products and desserts.

Characteristics of collagens from the swim bladders of yellowfin tuna (Thunnus albacares)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were extracted from the swim bladders of yellowfin tuna (Thunnus albacares) with yields of 1.07% and 12.10%, respectively. Based on electrophoretic patterns, both ASC and PSC consisted of two α-chains (α1 and α2) and were characterised to be type I collagen. ASC had higher β-chains than PSC. Imino acid contents of ASC and PSC were 128 and 169 residues/1,000 residues, respectively. Fourier transform infrared (FTIR) spectra of both ASC and PSC were similar and revealed the presence of a triple helix. Both ASC and PSC had the highest solubility at acidic pHs. From zeta potential analysis, a net charge of zero was found at pH 6.05 and 5.93 for ASC and PSC, respectively. Tmax of ASC and PSC were 32.97 and 33.92°C, respectively. The swim bladders of yellowfin tuna could therefore serve as an alternative source of collagen for future 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 alpha- and beta-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 degrees 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 zeta-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.

Chemical compositions and nutritional value of Asian hard clam (Meretrix lusoria) from the coast of Andaman Sea.

Chemical compositions and nutritive value of the edible portions including foot, mantle and viscera of Asian hard clam (Meretrix lusoria) harvested from the coast of Andaman Sea were determined. Proximate compositions varied with portions tested. Edible portions had moisture (76.23-84.22%) and protein (9.09-12.75%) as the major components. Carbohydrate (0.32-7.89%), fat (1.58-6.58%) and ash (1.23-2.58%) were also found at various levels, dependent upon portions. Myofibrillar proteins were observed as the major fraction in foot (40.54%) and mantle (31.65%), whilst non-protein nitrogen constituents were dominant in the viscera (36.85%). All portions contained a large amount of essential amino acids (167.66-187.63 mg/g sample), in which leucine (30.91-36.96 mg/g sample) and lysine (35.24-36.03 mg/g sample) were predominant. They were rich in polyunsaturated fatty acids (46.84-49.18% of total fatty acid) with high level of DHA (13.33-16.47 % of total fatty acids) and EPA (4.75-7.11% of total fatty acids). Cholesterol of 0.07-0.21% wet weight was detected. All portions were also rich in macro- (Na, K, Ca and Mg) and micro- (Fe, Zn, Cu and Cr) minerals. Therefore, Asian hard clam is an excellent source of several nutrients, which could be beneficial for the health of the consumers.

Bacterial expression and characterization of starfish phospholipase A2

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.