[Show abstract][Hide abstract] ABSTRACT: Cysteine proteinase inhibitors (CPI) were purified to 59 and 54 fold from black gram (Vignaraungo (L.) Hepper) and rice bean (Vignaumbellata Thunb.), respectively, by using heal treatment, followed by chromatography on a carboxymethyl (CM)-papain-Sepharose affinity column. The purified inhibitors were highly inhibitory to papain and Pacific whiting cathepsin L in a concentration dependent manner. They were detected as a dark band on tricine-SDS-PAGE gel stained for inhibitory activity. The apparent molecular weights of purified CPI from black gram and rice bean seeds were estimated to be 12, 000 daltons. The purified inhibitors were thermostable up to 90C and active in the neutral and alkaline pH ranges.
[Show abstract][Hide abstract] ABSTRACT: Pacific whiting surimi wash water (SWW) proteinase was recovered by ohmic heating, ultrafiltration, and freeze-drying. By these processes, 5.9-fold purification was achieved. The most efficient step was ohmic heating, which concentrated the proteinase by 4.8 fold. Specific activity of the recovered SWW proteinase on casein and Z-Phe-Arg-NMec was 28.2 and 0.17 U/mg protein, respectively. The SWW proteinase showed good hydrolytic activity towards casein, acid-denatured hemoglobin and myofibrils. Acidification increased specific activity on all substrates tested but reduced thermal stability. β-Mercaptoethanol, dithiothreitol and urea enhanced activity against Z-Phe-Arg-NMec. Proteinase activity on Z-Phe-Arg-NMec showed an optimum pH of 4.0. The recovered proteinase showed 18.5% residual activity after 7 week storage at 4C.
[Show abstract][Hide abstract] ABSTRACT: Isoelectric focusing (IEF) polyacrylamide gel containing an 80% pH 4–6.5 and 20% pH 3–10 ampholyte mixture greatly improved protein banding pattern for species identification of water extracts of raw pink, white and rock shrimp compared with the system using only the pH 3–10 range ampholyte. Identification of a specific species in mixture samples was achieved by the detection of water-extractable shrimp specific protein bands present in the gel. Sodium dodecyl sulfate (SDS) was a better protein extractant than water for cooked shrimp. Both water and SDS extracts of cooked shrimp showed specific protein banding patterns and improved resolution for species identification.
[Show abstract][Hide abstract] ABSTRACT: Commonly used protease assays and substrates were compared for sensitivity and simplicity in analyzing proteolytic activity in Pacific whiting causing gel weakening of surimi during heat-setting. Assay based on detection of trichloroacetic acid (TCA)-soluble products, using azocasein as substrate, showed highest sensitivity. By that assay, optimal pH of the protease was 5.5, and optimal temperature, 55°. The validity of the assay for measuring activity was confirmed by pH profiles of residual proteolytic and autolytic activities of uncooked surimi. These analyses showed pH profiles similar to those of fish juice with a pH optimum of 5.5.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACTA proteinase in Pacific whiting surimi wash water (SWW) was characterized to be cathepsin L based on molecular mass (Mr), substrate specificity, and SDS-substrate gel electrophoresis. The proteinase was highly active on Z-Phe-Arg-NMec, and the native Mr was 27,400 based on size exclusion (SEC)-HPLC. Acidification of the SEC-HPLC fractions showed a two-fold increase in activity on Z-Phe-Arg-NMec. SWW proteolytic activity was found at Mr 54,200 on SDS-substrate gel. However, acidification shifted the activity zone to Mr 39,500 corresponding to cathepsin L. No evidence of activity by calpain or cathepsin B or H was found in Pacific whiting SWW.
[Show abstract][Hide abstract] ABSTRACT: Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.
[Show abstract][Hide abstract] ABSTRACT: Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and urea gel isoelectric focusing (IEF) were used to identify species-specific protein bands of raw and cooked fish and surimi samples from Alaska pollock (Theragra chalcogramma) and red hake (Urophycis chuss). In raw samples, species-specific bands were found in the water extracts, while in cooked samples 1% SDS and 8M urea extracts were more effective for species identification in both fish and surimi.
[Show abstract][Hide abstract] ABSTRACT: Proteins were extracted from raw or cooked pink (Penaeus duorarum), white (Penaeus setiferus) or rock shrimp (Sicyonia brevirostris) with five different solutions: water, water homogenate adjusted to pH 8.0, 0.1M NaCl, 1% SDS, or 8M urea. Each extract from each species was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Water extraction showed highly species-specific banding patterns for raw shrimp, while patterns for SDS extracts were not as species-specific. However, SDS extracts provided the greatest information on species variation of cooked shrimp. SDS-PAGE was useful in distinguishing shrimp of different genus. This technique was tested and proven in a blind study to be useful for species identification and detection (within 10% of actual amount) of fabricated products.
[Show abstract][Hide abstract] ABSTRACT: Isoelectric focusing (IEF) using 9.2M urea and 6.2% (v/v) ampholytes in a polyacrylamide gel was used to separate protein banding patterns for species identification of pink, white and rock shrimp. A 17-hr focusing time was used. IEF of water extracts of raw shrimp showed excellent banding patterns useful for distinguishing each shrimp species. For cooked shrimp, water and sodium dodecyl sulfate (SDS) were better protein extractants. Detection of shrimp species in a mixture was difficult due to similar banding patterns between closely related species.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stability of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and volatile and oxidized volatile compounds in 2 types of DHA-enriched fish oil, triacylglycerol (TG) and ethyl ester (EE), were studied during storage at 80 °C with aeration. The rate of DHA autoxidation was higher than that of EPA. DHA in EE form was more susceptible to autoxidation than in TG form. Thirty-one volatile compounds were identified in EE and 23 volatile compounds in TG. (E)-2-pentenal, 2-(1-pentenyl) furan, and (E,E)-2,4-heptadienal were commonly detected as oxidized volatile compounds from TG and EE fish oil. These volatile oxidized compounds might be formed mainly from the oxidation of DHA and EPA, the main fatty acids of the oil.
[Show abstract][Hide abstract] ABSTRACT: Testing for Morganella morganii, a prolific histamine former, was carried out in fish and processing plant to determine its origin and contamination source using PCR assay coupled with Southern hybridization. M. morganii was mostly found in mackerel and sardines but rarely in albacore. It was most frequently present in the gill followed by the skin but rarely in the intestine and cavity. No M. morganii was found in the processing plant but was detected on the surfaces of conveyer belts and plastic totes during processing. The compiled results indicated that histamine-forming bacteria are endogenous to fish and pointed out the importance of sanitation in the processing plant to prevent cross-contamination.
[Show abstract][Hide abstract] ABSTRACT: Morganella morganii was studied for its growth and histamine formation in mackerel, albacore, mahi-mahi, and salmon stored at various temperatures from -30 °C to 37 °C. The optimal temperature for histamine formation was 25 °C. Mackerel, albacore, and mahi-mahi were shown as good substrates for histidine decarboxylation by M. morganii at elevated temperatures (> 15 °C). M. morganii inoculated in all fish species including salmon formed histamine above the FDA guideline. Their growth was controlled by cold storage of the fish at 4 °C or below, but histamine formation was controlled only by frozen storage. Although histamine was not detected in any frozen samples, it accumulated rapidly in the previously frozen fish stored at 25 °C.
[Show abstract][Hide abstract] ABSTRACT: A unique biomarker, h-caldesmon, was identified and purified from bovine intestine smooth muscle. It was used to develop monoclonal antibodies (MAbs) for use in immunochemical assays to detect prohibited meat and bone meal (MBM) in animal feed. This biomarker with a molecular weight of 150 kDa was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be present in MBM samples that were obtained from different manufacturers. The presence of this biomarker in MBM and smooth muscle was also demonstrated by immunostaining with MAb 8B4 in Western blot assay. h-Caldesmon in intestinal smooth muscle was demonstrated to be stable after autoclaving at 130 °C for up to 1 h. Because MAb 8B4 was sensitive in detecting MBM in animal feed at >0.05%, it can be used in immunoassays for MBM detection.
[Show abstract][Hide abstract] ABSTRACT: Multidrug-resistant enteric bacteria were isolated from turkey, cattle, and chicken farms and retail meat products in Oklahoma. Among the isolated species, multidrug-resistant Klebsiella pneumoniae was prevalently isolated from most of the collected samples. Therefore, a total of 132 isolates of K. pneumoniae were characterized to understand their potential roles in the dissemination of antibiotic-resistance genes in the food chains. Multidrug-resistant K. pneumoniae was most frequently recovered from a turkey farm and ground turkey products among the tested samples. All isolates were resistant to ampicillin, tetracycline, streptomycin, gentamycin, and kanamycin. Class 1 integrons located in plasmids were identified as a common carrier of the aadA1 gene, encoding resistance to streptomycin and spectinomycin. Production of beta-lactamase in the K. pneumoniae isolates played a major role in the resistance to beta-lactam agents. Most isolates (96%) possessed bla(SHV1). Five strains were able to express both SHV-11 (pI 6.2) and TEM-1 (pI 5.2) beta-lactamase. Transfer of these antibiotic-resistance genes to Escherichia coli was demonstrated by transconjugation. The bacterial genomic DNA restriction patterns by pulsed-field gel electrophoresis showed that the same clones of multidrug-resistant K. pneumoniae remained in feathers, feed, feces, and drinking water in turkey environments, indicating the possible dissemination of antibiotic-resistance genes in the ecosystem and cross-contamination of antibiotic-resistant bacteria during processing and distribution of products.
Journal of food protection 11/2005; 68(10):2022-9. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130 degrees C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90 degrees C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130 degrees C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.
Journal of food protection 10/2005; 68(9):1860-5. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sixty-four multidrug-resistant isolates of Proteus mirabilis were collected from retail meat products in Oklahoma. The isolates showed four different patterns of antibiotic resistance based on their resistant phenotype and genotypes. Most of these isolates were resistant to ampicillin, tetracycline, gentamycin, and kanamycin. Class 1 integrons were detected as a common carrier of the antibiotic-resistant genes, such as aadA1, aadB, and aadA2. A few isolates (9%) contained class 2 integrons with three gene cassettes included: dhfr1, sat1, and aadA1. These isolates were even resistant to nalidixic acid due to mutations in gyrA and parC. All ampicillin-resistant isolates contained blaTEM-1. Plasmids that contained class 1 or 2 integrons and blaTEM-1 were able to be transferred from P. mirabilis isolates into Escherichia coli by conjugation, indicating that conjugal transfer could contribute to the dissemination of antibiotic resistance genes between the Enterobacteriaceae species.
Journal of food protection 08/2005; 68(7):1408-13. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An antimicrobial peptide was purified from fermented skate skin extract using the solid-phase extraction and separation on HPLC reversed-phase chromatography. Amino acid sequence of the purified peptide (Peak A) having an antimicrobial activity revealed the presence of many cationic residues of the total 28 amino acids. Its molecular mass was found to be 3059 Da. This result was in excellent agreement with the theoretical molecular mass calculated from the amino acid sequence. The synthetic kenojeinin I had inhibitory effects on B. subtilis (MIC, 12 microg/ml), E. coli (28 microg/ml), and S. cerevisiae (12 microg/ml). These results indicate that fermented skate skin is potentially antimicrobial.
[Show abstract][Hide abstract] ABSTRACT: Histamine and biogenic amine contents in retail canned anchovies were determined. Bacterial strains were isolated, and their histamine-producing capability was determined. The majority of canned anchovy products (80%) had histamine levels below the FDA guideline of 50 ppm. The sensory quality of products was relatively good. A few samples contained high levels of histamine (>1000 ppm). Overall, histamine contents in the products showed great lot-to-lot variations. Spermine and tyramine were commonly detected in all samples analyzed, regardless of their histamine contents. Bacterial counts in the products were mostly below the detection limit (102 CFU/g), and bacteria were frequently recovered with the enrichment of test samples in tryptic soy broth supplemented with 0.5% or 5% NaCl. Only Bacillus spp., the nonhistamine formers, were isolated from these test products. Prolific histamine-forming bacteria were not detected in these canned anchovies.
[Show abstract][Hide abstract] ABSTRACT: For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.
Journal of Agricultural and Food Chemistry 01/2005; 52(25):7580-5. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Histamine and other biogenic amines were evaluated in canned anchovies recalled by the U.S. Food and Drug Administration. In addition, bacteria were isolated from the products and identified to species. The recalled products were divided into 2 groups of high and low histamine, depending on the histamine contents as determined by the AOAC method. The high histamine group had the histamine contents >200 ppm, and 24 of the 30 cans analyzed belonged to this group. The most prevalent biogenic amine in this group was histamine followed by cadaverine. On the other hand, the low histamine group of 6 cans contained approximately 50 ppm histamine. The most prevalent biogenic amine found in this group of samples was cadaverine at levels >200 ppm. Other biogenic amines, such as putrescine, serotonin, and spermidine, were also detected in all the products, although at varied levels. Aerobic and anaerobic bacterial counts, if present in all of the recalled products, were below the detection limit of 102 colony-forming units (CFU)/g. Bacteria were recovered only after enrichment of the test samples. They were mostly halophilic bacteria. Bacillus spp. were most frequently identified, followed by Staphylococcus spp. However, these isolates produced negligible amounts of histamine in culture broth, indicating that they are not the contributors to histamine accumulation in the canned anchovies.