Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus)

Trypsin-like enzyme was partially purified from the intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus) by a simple three steps procedure: heat treatment, ammonium sulphate precipitation and Sephadex G-75 filtration. The enzymes from the intestine and pyloric caeca were 96- and 57.7-fold purified with yield values of 68.1% and 26.1%, respectively. The pyloric caeca enzyme collected from the Sephadex G-75 filtration showed a single band in SDS–PAGE (24.5 kDa). Both enzymes presented identical optima pH (9.0) and temperature (55 °C). After incubation at 45 °C for 30 min, enzymes obtained from intestine remained fully activity while a loss of activity (10%) of enzyme extracted from pyloric caeca was registered. Michaelis constant was not significantly different for trypsin-like enzyme from pyloric caeca (1.82 ± 0.19 mM) and that from the intestine (1.94 ± 0.45 mM) acting on benzoyl-dl-arginine-p-nitroanilide (BAPNA). Finally, their activities were inhibited by the following ions in decreasing order: Al³⁺ > Zn²⁺ > Hg²⁺ = Cu²⁺ > Cd²⁺. The effects of Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, K¹⁺, Li¹⁺ and Co²⁺ showed to be less intensive. The similarities between them provide basis for the proposition of obtaining an attractive protease preparation from the tons of intestine and pyloric caeca, that are usually discarded, from this fish which is an important species exported by North-eastern Brazilian fishery industry.     

Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius)

The objective of this study was to extract and characterize acid soluble collagens (ASCs) and pepsin soluble collagens (PSCs) from the skin and bone of Spanish mackerel (Scomberomorous niphonius) and to provide a simultaneous comparison of the four collagens. The yields of ASC-S (ASC from skin), PSC-S (PSC from skin), ASC-B (ASC from bone) and PSC-B (PSC from bone) were 13.68 ± 0.35, 3.49 ± 0.24, 12.54 ± 0.83 and 14.27 ± 0.66% (on wet weight basis), respectively. The four collagens contained glycine (341.6–352.6 residues/1000 residues) as the major amino acid and the contents of imino acids were between 177.1 and 184.3 residues/1000 residues. Amino acid composition, SDS-PAGE and FTIR confirmed that ASC-S, ASC-B and PSC-B were mainly composed of type I collagen with slight molecular structure differences, and PSCs had lower content of high-molecular weight cross-links than that of ASCs. The denaturation temperatures of ASC-S, PSC-S, ASC-B and PSC-B were 15.12, 14.66, 18.02 and 16.85 °C, respectively, which were much lower than those of collagens from the mammalian and tropical fish species due to low imino acid contents. All the collagens were soluble at acidic pH (1–4) and lost their solubility when the NaCl concentrations were above 2% (w/v). The four lyophilized collagens displayed a uniform and regular network ultrastructure based on the ultrastructural analysis. The isolated collagens from Spanish mackerel could serve as an alternative source of collagens for further application in food and neutraceutical industries.     

Isolation and characterisation of collagens from the skin,scale and bone of deep-sea redfish (Sebastes mentella)

To make more effective use of underutilised resources, collagens from skin, scale and bone (SKC, SCC and BOC) of deep-sea redfish were isolated with acetic acid and characterised for their potential in commercial applications. The abundant ash and fat in the materials could be removed effectively by EDTA and hexane treatment in 24 h, with high recoveries of protein. The yield of SKC (47.5%) was significantly higher than that of SCC and BOC (6.8% and 10.3%, respectively). The denaturation temperatures of SKC, SCC and BOC were 16.1 °C, 17.7 °C and 17.5 °C, respectively, which were lower than those of most other fish species. The amino acid profiles of these collagens were similar with a low imino acid content, which might be the reason for the low denaturation temperature. All the collagens were type I mainly and maintained their triple helical structures well with slight molecular structure differences. SKC possessed a higher degree of intermolecular cross-linking and molecular order, but the extent of peptide chain unwinding was also higher, due to the existence of fewer hydrogen bonds, compared to SCC and BOC.     

IgE reactivity to type I collagen and its subunits from tilapia (Tilapia zillii)

Acid-soluble collagen (ASC) was isolated from the skin of tilapia (Tilapia zillii) via acetic acid (HAc) extraction and NaCl precipitation. ASC from tilapia consists of α chains (α1 and α2), β chains and γ chains and is classified as type I collagen. A comparison of the properties of tilapia collagen and silver carp parvalbumin showed that tilapia collagen was less stable under heat treatment and more resistant to pepsin digestion. Both tilapia collagen and silver carp parvalbumin were degraded at pH 2.0 but stable at pH 3.0–11.0. Subunits α1 and α2 were further purified from tilapia collagen by carboxymethyl (CM) cellulose column chromatography with linear gradient elution and stepwise elution, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoblotting results demonstrated the specific IgE activity of different fish-allergenic patients’ sera towards the α1 and α2 chains of tilapia collagen. It can be inferred that tilapia collagen and its subunits are all potential allergens.     

Chromatographic separation and physicochemical properties of collagen species in the skin of deep-sea redfish (Sebastes mentella)

Type I collagen (PSC-I) and type V collagen (PSC-V) were directly separated by DEAE-cellulose chromatography without salt precipitation from the skin of deep-sea redfish after limited pepsin digestion. The yields of PSC-I and PSC-V were 79.4% and 8.4%, respectively. The denaturation temperature of PSC-V (17.8 °C) was significantly higher than that of PSC-I (15.9 °C), as a result of its higher total content of imino acid (17.1%) and hydroxylation degree of proline and lysine (44.7%) than those of PSC-I (14.7% and 35.8%). However, the intrinsic viscosity and viscosity transition temperature span of PSC-I (14.4 dL/g and 8 °C) were considerably higher than those of PSC-I (13.5 dL/g and 5 °C), which could be attributed to its greater extent of triple helix and more intermolecular crosslinks as shown by SDS-PAGE pattern and infrared spectra.     

Isolation and characterisation of acid-solubilised collagen from the skin of Nile tilapia (Oreochromis niloticus)

Acid-solubilised collagen (ASC) was extracted from the skin of Nile tilapia (Oreochromis niloticus) and characterisation was studied. The results indicated that the yield of ASC was 39.4% on the basis of dry weight. This ASC was rich in glycine (35.6%). The amount of imino acids, proline and hydroxyproline, in ASC was 210 residues per 1000 residues. The ultraviolet (UV) absorption spectrum of ASC showed that the distinct absorption was at 220 nm. ASC showed transition curve at maximum temperature (T_max) of 32.0 °C in 0.05 M acetic acid, about 12 °C lower than that of calf skin collagen. Maximum solubility (0.75 mg/ml) in 0.5 M acetic acid was observed at pH 3. Solubility reached the minimum at pH 7. A sharp decrease in solubility was observed in 2% (w/v) NaCl or above. Biochemical studies indicated that ASC was composed of the α1α2α3 heterotrimers.     

Chilled storage of golden gray mullet (Liza aurata)

This study determined quality changes of whole ungutted golden gray mullet (Liza aurata) while stored in ice or in a refrigerator (without ice). Changes in microbiological quality (total viable and psychrophilic counts, lactic acid bacteria and Enterobacteriaceae), chemical quality (pH, total volatile basic nitrogen, trimethylamine, peroxide value, thiobarbituric acid, and free fatty acids), and raw fish sensory attributes were evaluated during 16 days of storage. The sensory attributes of golden gray mullet correlated well with the microbiological analyses (r = 0.92). Based on the overall raw acceptability sensory scores and the microbiological tests, the shelf life of the raw golden gray mullet was 10 days in ice and about 14 days in a refrigerator.     

Partial characterisation of gelatinolytic activities in herring (Clupea harengus) and sardine (Sardina pilchardus) possibly involved in post-mortem autolysis of ventral muscle

The present work aims at identifying enzymatic activities that may contribute to the post-mortem autolysis of the ventral muscle, known as belly bursting, in herring (Clupea harengus). Gelatinolytic proteases extracted from several herring tissues and also from a sardine (Sardina pilchardus) tissue were partially characterised using gelatin zymography, inhibitor analysis, immunodetection with anti-trypsin antibody and MALDI-TOF/TOF peptide sequencing. The results indicate the presence of gelatinolytic trypsin-like serine proteases and metalloproteases in several samples including the ventral muscle of herring. MS/MS analysis gave partial sequences of peptides from some of the proteases, and these were identified as elastase, trypsin and aspartyl aminopeptidase. It is likely that belly bursting in herring is caused by leakage of enzyme-rich fluids from the intestine and/or pyloric caeca which may also contain exogenous proteases from the digestive systems of the prey.     

Collagen from common minke whale (Balaenoptera acutorostrata) unesu

Collagen was prepared from common minke whale unesu and characterised. The yield of collagen was high, about 28.4% on a wet weight basis. By SDS–PAGE and CM-Toyopearl 650 M column chromatography, the collagen was classified as type I collagen. The denaturation temperature of the collagen was 31.5 °C, about 6–7 °C lower than that of porcine collagen. Attenuated total reflectance-FTIR analysis indicated that acid-soluble collagen from common minke whale unesu held its triple helical structure well, but the structures of porcine skin collagen and pepsin-solubilized collagen from common minke whale unesu were changed slightly, due to the loss of N- and C-terminus domains.     

Antioxidant activity of hydrolysates obtained from scallop (Patinopecten yessoensis) and abalone (Haliotis discus hannai Ino) muscle

Scallop (Patinopecten yessoensis) and abalone (Haliotis discus hannai Ino) muscle were hydrolysed with commercially available food-grade proteases. The resulting hydrolysates showed DPPH and hydroxyl radicals scavenging abilities, reducing power, and ferrous ion chelating capacity. The antioxidant activities of hydrolysate of abalone foot muscle (HAFM) increased with increasing incubation time during the whole hydrolysis process in 180 min. Whereas, the antioxidant activities of hydrolysate of scallop adductor muscle (HSAM) increased at initial stage and peaked after 25-30 min of hydrolysis, and then gradually decreased thereafter. Compared with HAFM, HSAM with comparable hydrolysis time contained more free amino acids (FAA) and small-sized peptides (below 500 Da), which may account for the differences in antioxidant activities versus hydrolysis time curves of the two hydrolysates. The above results indicate that limited hydrolysis of proteins can increase their antioxidant activity, whereas extensive hydrolysis can decrease it.     

Potent antibacterial property of APC protein from curry leaves (Murraya koenigii L.)

A monomeric protein with molecular mass of ∼35 kDa, isolated from Murraya Koenigii L. (curry leaves) shows potent antibacterial activity. The protein designated as APC (antioxidant protein from curry leaves) demonstrated potent antibacterial activity against all the human pathogenic strains tested. APC effectively inhibited Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Salmonella typhi and Bacillus subtilis. The inhibition is comparable to that of commercial antibiotics chloramphenicol, streptomycin and gentamycin. APC inhibited bacterial growth, with MIC values ranging from 13 to 24 μg/ml, which are comparable to MIC values of standard antibiotics. APC is devoid of ribonuclease/deoxyribonuclease and protease activity. APC is non-toxic at tested doses. These results encourage further studies of APC as a potent therapeutic agent.     

Chemical composition and antioxidant activity of Gaylussacia brasiliensis (camarinha) grown in Brazil

This paper presents the centesimal and mineral composition, fatty acid profile of the lipidic fraction, phenolic and anthocyanin contents, and antioxidant activity of Gaylussacia brasiliensis fruit. The results indicated the following composition: moisture (81.30%), lipids (0.62%), proteins (0.56%), carbohydrates (10.74%), dietary fiber (6.53%), and ash (0.25%). The main elements comprising the mineral composition were K, Mg, Ca, and Fe. The fatty acid composition was characterized by a high content of polyunsaturated fatty acids (62.2%) and a high PUFA/SFA ratio (2.92). The G. brasiliensis fruit contained considerable amounts of phenolics (492.87 mgAG/100 g) and anthocyanins (240.43 mg/100 g), which contribute to its high antioxidant activity. This study highlights the potential of this fruit as an important source of both nutritional and bioactive compounds available in the native Brazilian flora.     

Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes

The sour orange (Citrus aurantium) juice is commonly used as flavoring and acidifying agent for vegetable salads and appetizers in Turkey. It was aimed to determine the survival and growth pattern of Salmonella Typhimurium and Listeria monocytogenes in sour orange juice. Different concentrations of neutralized and un-neutralized juice samples were inoculated with each of the test microorganisms (∼6 log CFU/mL) separately and then incubated at 4 °C and 37 °C for seven days. It was detected both of the test microorganisms could survive and even grow in neutralized juice samples at 37 °C for two days. However, none of them could survive at the end of seventh day of incubation at 37 °C. Low incubation temperature (+4 °C) increased the survival of the tested microorganisms. Also, it was detected that L. monocytogenes were less resistant to the variable conditions than S. Typhimurium. It was concluded that the antimicrobial effect of sour orange juice mainly depends on the low pH value of the product. However, incubation time and temperature are also effective on the survival of the tested pathogens.     

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.     

Extraction of pepsin-soluble collagen from grass carp (Ctenopharyngodon idella) skin using an artificial neural network

A multilayer feed-forward neural network trained with an error back-propagation algorithm was used to evaluate the effects of pepsin amount, reaction time and pH on the yield of pepsin-soluble collagen. A positive correlation was observed between the yield and the amount of pepsin and also the reaction time. The yield increased with an increase of pH to nearly 3, thereafter yield decreased. The trained network gave a regression coefficient (r²) of 0.97 and a mean squared error (MSE) of 0.21, which implied a good generalisation of the network. Based on the genetic algorithm, the optimal extraction conditions to obtain the highest yield were determined to be pH 3.4, 53.3 unit/mg of pepsin and 35.2 h. The predicted yield value was 30.3%. As the estimated optimal extraction conditions were used in the actual preparation of the pepsin-soluble collagen, the yield was measured experimentally to be 29.3 ± 0.8%, which was not significantly different (p > 0.05) from the predicted value. The response surface plots showed the yield of pepsin-soluble collagen as a function of two factors under various extraction conditions.     

Sensory,microbiological and chemical assessment of the freshness of red mullet (Mullus barbatus) and goldband goatfish (Upeneus moluccensis) during storage in ice

The shelf life of red mullet and goldband goatfish during ice storage were studied in terms of sensory, microbiological and chemical changes. The sensory acceptability limit was 8 days for goldband goatfish (Upeneus moluccensis) and 11 days for red mullet (Mullus barbatus) stored in ice. The TVC level was correlated with sensory assessment. The TVC exceeded 7 log cfu g⁻¹ after 8 days for goldband goatfish, and 11 days for red mullet. At the end of storage period, pH, TVB-N, TBA, FFA and PV for red mullet were 7.84, 47.19 mg/100 g, 0.69 mg MA kg⁻¹, 1.17% oleic acid and 1.58 meq O₂/kg and for goldband goatfish they were 7.53, 43.97 mg/100 g, 0.74 mg MA kg⁻¹, 1.62% oleic acid and 1.68 meq O₂/kg, respectively. In red mullet, agmatine, serotonin, histamine and dopamine became the dominant amines, reaching 7.30, 5.97, 2.52 and 2.31 mg/100 g, respectively. Also the dominant amines for goldband goatfish were 4.37, 3.88, 3.38 and 2.00 mg/100 g for histamine, agmatine, dopamine and putrescine, respectively.     

Type I collagen from the skin of ornate threadfin bream (Nemipterus hexodon): Characteristics and effect of pepsin hydrolysis

Type I collagen from the skin of ornate threadfin bream (Nemipterus hexodon) was purified and characterised. Purified type I collagen contained [α1(I)]₂α2(I) as the dominant component with the co-presence of α1(I)α2(I)α3(I). It was rich in glycine and alanine with high content of imino acids (188 residues/1000 residues). The maximum transition temperature (T_m) and the total denaturation enthalpy (ΔH) of purified type I collagen was 33.35 °C and 0.819 J/g, respectively. The isoelectric point (pI) of purified type I collagen was estimated to be 6.40. After hydrolysis of purified type I collagen using pepsin, the band intensity ratios of α1/α2-chains were increased (P < 0.05). The cross-linked components were effectively hydrolysed by pepsin 1 and 2 from skipjack tuna stomach and porcine pepsin at 4 °C without the cleavage of β- and α-chains. At 50 °C, they were more susceptible to porcine pepsin hydrolysis, followed by pepsin 2 and 1, respectively.     

Pepsinogen and pepsin from the stomach of smooth hound (Mustelus mustelus): Purification,characterization and amino acid terminal sequences

Pepsinogen from the stomach of smooth hound (Mustelus mustelus) was purified to homogeneity by 20–70% ammonium sulphate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose anion exchange chromatography with a 9.4-fold increase in specific activity and 38.36% recovery. Upon activation at pH 2.0, M. mustelus pepsinogen was converted to active form in one-step pathway. Molecular weights of the purified pepsinogen and the active pepsin were estimated to be 40,000 and 35,000 Da using SDS-PAGE and gel filtration, respectively. The optimum pH and temperature for the pepsin activity were pH 2.0 and 40 °C, respectively, using haemoglobin as a substrate. Activity was completely inhibited by Pepstatin A but not by phenylmethylsulphonyl fluoride, a serine-protease inhibitor and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The N-terminal amino acid sequences of the first 15 amino acids of the activation segment of the pepsinogen and the first 20 amino acids of the active pepsin were LLRVPLRKGKSTLDV and ATEPLSNYLDSSYFGDISIG, respectively. M. mustelus pepsinogen, which showed high homology to rat C pepsinogen, had Thr-Leu-Asp sequence at amino acid positions 12–14 not found in all pepsinogen sequences. A remarkable substitution was found in the activation segment of M. mustelus pepsinogen: the Arg-13 conserved in all gastric proteinases, whose sequences are known, is replaced by Leu-13.     

Characteristics of lipid components,fatty acid distributions and triacylglycerol molecular species of adzuki beans (Vigna angularis)

Fatty acid (FA) distributions and molecular species of triacylglycerols (TAG) isolated from total lipids extracted from adzuki beans (Vigna angularis) were analysed by a combination of AgNO₃-TLC and GC, and were investigated in relation to the content of endogenous antioxidants determined by HPLC. δ-Tocopherol was present in the highest concentration (30.5 mg/kg beans), and γ-tocopherol in small amounts (12.8 mg/kg beans). The major lipid components were phospholipids (74.3%), TAG (13.5%), hydrocarbons (4.6%) and steryl esters (4.0%), whilst other components were also present in minor proportions (0.5–1.3%). Seventeen different molecular species were detected. The major TAG components were SMD (5.0%), S₂T (19.2%), SD₂ (13.7%), SMT (9.3%), MD₂ (4.5%), SDT (7.0%), D₃ (8.8%) and ST₂ (15.8%) (where S, M, D, and T denote a saturated FA, a monoene, a diene, and triene, respectively). These results would be useful to both consumers and producers for manufacture of traditional adzuki foods in Japan.     

Purification and characterization of soluble invertases from suspension-cultured bamboo (Bambusa edulis) cells

An alkaline invertase (IT I) and an acid invertase (IT II) were purified from the soluble fraction of suspension cultured bamboo cells. Both purified invertases were homogeneous as examined by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and were identified as β-fructofuranosidases able to attack the β-fructofuranoside from the fructose end. With respect to sucrose hydrolysis, the optimal pHs were 7.0 and 4.5 for IT I and IT II, respectively. The Km’s were 10.9 and 3.7 mM. The IT I and IT II molecular masses were 240 and 68 kDa, respectively, as estimated by gel filtration. The isoelectric points were 4.8 and 7.4. IT I was a homotetrameric enzyme activated by bovine serum albumin (BSA). IT II was a monomeric enzyme activated by BSA, concanavalin A (ConA) and urease. Both isoforms were significantly inhibited by heavy metal ions Ag⁺ (5 mM) and Hg²⁺ (1 mM), and mercaptide forming agent ρ-chloromercuribenzoic acid (PCMB; 0.5 mM).