Mohamed M. Abouzied

Fermentation of starch to ethanol by a complementary mixture of an amylolytic yeast andSaccharomycescerevisiae

SummarySynergistic coculture of an amylolytic yeast (Saccharomycopsisfibuligera) andS.cerevisiae, a non-amylolytic yeast, fermented unhydrolyzed starch to ethanol with conversion efficiencies over 90% of the theoretical maximum. Fermentation was optimal between pH 5.0 to 6.0. Using a starch concentration of 10% (w/v) and a 5% (v/v) inoculum ofS.fibuligera, increasingS.cerevisiae inoculum from 4% to 12% (w/v) resulted in 35–40% (w/v) increase in ethanol yields. Anaerobic or “limited aerobic” incubation almost doubled ethanol yields.

Polyspecific and autoreactive IgA secreted by hybridomas derived from Peyer's patches of vomitoxin-fed mice: characterization and possible pathogenic role in IgA nephropathy.

A total of 122 immunoglobulin (Ig)A-producing hybridoma clones were isolated from the Peyers patches of vomitoxin-fed BALB/c mice and the resultant antibodies were characterized for their antigenic specificity and pathogenic potential. When reactivity was tested against a panel consisting of DNA, sphingomyelin, thyroglobulin, collagen, casein, cardiolipin and bovine serum albumin conjugates of phosphorylcholine, inulin and trinitrophenol that were representative of self and non-self antigens, approximately 95% of the monoclonal IgAs bound to at least one of the panel antigens and 80% bound to more than one antigen. The polyspecificity of some of the monoclonal IgAs was further suggested by demonstrating the capacity of one antigen to inhibit binding of monoclonal IgA to another antigen. Protein staining and Western blotting of gradient native polyacrylamide gels, indicated that trimeric IgA predominated in the isolated monoclonal IgAs. Repeated injections of mice with representative monoclonal IgAs induced microhaematuria in three of four of the clones tested but not IgA deposition in the kidney glomerulus. In addition, three of the four monoclonal IgAs caused IgG and C3 deposition in the kidney mesangium. These and previous results suggest that dietary vomitoxin promotes the polyclonal activation and expansion of IgA-secreting B cells at the Peyers patch level and that resultant polyspecific, autoreactive IgA may contribute to kidney pathogenesis.

Lactate dehydrogenase as safe endpoint cooking indicator in poultry breast rolls : development of monoclonal antibodies and application to sandwich enzyme-linked immunosorbent assay (ELISA)

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect lactate dehydrogenase (LDH) as a marker protein for verifying endpoint cooking of uncured poultry products. Monoclonal antibodies were prepared against chicken muscle LDH and used with rabbit polyclonal antibodies developed against turkey or chicken muscle LDH for capture and detection in the assay, respectively. Minimum assay detection limits for turkey and chicken muscle LDH were 1 ng/ml. Turkey and chicken muscle LDH, but not LDH from other species cross reacted in the ELISA. The ELISA was further verified using extracts of turkey breast rolls processed to internal temperatures between 68.3 and 72.1°C. The LDH content of extracts diluted 3- to 6-fold was below 15 ng/ml for turkey rolls processed to 70.9 and 72.1°C. At a 6-fold dilution, LDH content of extracts from rolls processed to 69.7°C was approximately 10 times greater than those processed to 70.9°C. A survey of market precooked poultry products indicated assay validity with precooked turkey roast, but not turkey hams with maximum internal temperature requirements of 68.3°C. Results suggested the sandwich ELISA should be applicable for determining whether turkey breast rolls are processed to the required U.S. Department of Agriculture endpoint temperature of 71.1°C.

Production of a hybrodoma cell line secreting retinoic acid-specific monoclonal antibody

A stabilized hybridoma cell line secreting anti-retinoic acid monoclonal antibodies of subclass IgG1 with κ chains was produced by fusing NS-1 myeloma cells with the spleen cells from BALB/c female mice immunized with all-trans-4-oxoretinoic acid-oxime-chicken IgG conjugate. The antibody titer of mice ascitic fluid ranged from 112,800to125,600, as determined by competitive indirect enzyme-linked immunosorbent assay (ELISA). 50% inhibition dosage of all-trans-retinoic acid at a 120,000 dilution of mice ascitic fluid was 6.6 ng/ml, as determined by ELISA. The anti-retinoic acid monoclonal antibody was generated in mice ascitic fluid and purified by protein G affinity chromatography. Cross-reactivity of the monoclonal antibody was determined at 0.1 μg/ml concentration of retinoids and indicated high specificity to both all-trans-retinoic acid (86% inhibition) and 13-cis-retinoic acid (87% inhibition), and strong cross-reactivity with 4-oxoretinoic acid (77%) and 4-oxoretinoic acid oxime (109%). Specificity was confirmed by the horseradish peroxidase-linked immunostaining method and immunoradioassay. The affinity constant of the monoclonal antibody, K, was determined to be 3.6 × 109 1/mol. A calibration curve for retinoic acid using the monoclonal antibody to retinoic acid was developed; the detection limit for all-trans-retinoic acid is 1 ng/ml in the competitive indirect ELISA. The antibody counteracts the effect of retinoic acid on growth inhibition and differentaition in HL-60 cells.

Glucose feedback inhibition of amylase activity in Aspergillus sp. and release of this inhibition when cocultured with Saccharomyces cerevisiae

Abstract The effect of starch and glucose concentration on amylase activity produced by Aspergillus niger, A. foetidus and A. awamori was determined. Up to 15 U ml −1 of amylase activity was observed in extracellular fluid of cultures grown with 1–2% starch for 7 days; however, no amylase activity was detectable in the extracellular fluid of otherwise identical cultures grown with 3–5% starch, even though these cultures contained substantial concentrations of glucose (16–20 mg ml −1 ) which could only have been derived from starch hydrolysis. When extracellular fluid from the 5% starch cultures was dialysed to remove soluble sugar, high level of amylase activity was observed. Addition of increasing amounts of glucose to the dialysed extracellular fluid resulted in increasing levels of inhibition of amylase activity. Coculture of the Aspergillus species with Saccharomyces cerevisiae , an efficient sugar fermenting yeast, in 5% starch medium resulted in low sugar concentration and high amylase activity (>18 U ml −1 ) in the extracellular fluid. Amylase activity in cultures grown with glucose was comparable to that observed in cultures grown with glucose plus starch or starch only. These results lead us to conclude that amylase activity in the Aspergillus species studied is subject to feedback inhibition by glucose but is not subject to catabolite repression by glucose or starch as previously believed. Furthermore, the amylase activity in these organisms appears to be inducible by starch.

Detection of fumonisins in Fusarium cultures, corn, and corn products by polyclonal antibody-based ELISA : Relation to Fumonisin B1 detection by liquid chromatography

A specific sheep polyclonal antiserum was applied to the competitive direct enzyme-linked immunosorbent assay (CD-ELISA) of fumonisins in Fusarium cultures, fresh corn, animal feed, and human foods, and the results were related to fumonisin B1 (FB1) levels determined by liquid chromatography (LC). The limit of detection of FB1 for the CD-ELISA was 0.1 ng/ml. Cross reactivity (defined as [ng/ml FB1 required for 50% inhibition]/[ng/ml of analogue required for 50% inhibition] × 100) for the antiserum in the ELISA was 100, 24, and 30%, for FB1 FB2, and FB3, respectively. FB1 was detected in F. moniliforme cultures grown on corn (≤760 μg/g) but not in those for F. graminearum . Fumonisin estimates were 2.8-fold higher by CD-ELISA than FB1 estimates by LC. Upon using the antiserum in a commercial CD-ELISA format (Veratox), mean recovery of FB1 from spiked corn (0.1 to 3 μg/g)was 85.2 ± 25.3%, whereas the mean recovery by LC was 74.1 ± 6.2%. Analyses of 43 fresh corn, 28 animal feed, and 14 human food samples revealed that estimates of total fumonisins by the commercial ELISA were 2.9-, 2.2-, and 1.3-fold higher than FB1 values determined by LC. For each sample type, the ELISA significantly correlated with LC (r > 0.9; P < 0.05). Taken together, the results suggest that the polyclonal antibody-based CD-ELISA could be a reliable first-tier screening procedure for fumonisins in corn, animal feed, and human food when used in conjunction with LC confirmation. The differences between the two methods suggests the further presence of FB2 and FB3 or structurally related compounds.

Lactate dehydrogenase polyclonal antibody sandwich ELISA for determination of endpoint heating temperatures of ground beef

An enzyme-linked immunosorbent assay (ELISA) for the protein marker lactate dehydrogenase (LDH) was developed to determine endpoint heating temperature (EPT) of ground beef. Ground beef was placed in 10 by 75 mm tubes and heated to temperatures between 62 and 74°C at 2°C intervals. Electrophoresis and immunoblotting of meat extracts indicated a decrease in the intensity of the LDH band as temperature was increased. Polyclonal antibodies (PAb) against bovine muscle LDH were produced and a sandwich ELISA devised using biotinylated PAb as the detecting antibody. The LDH content decreased (P < 0.05) in ground beef heated between 66 and 74°C and was less than 4 μg/g of meat at the recommended minimum endpoint of 69°C. The ELISA was used to estimate the EPT of commercially cooked beef patties.

Detection of Zearalenone By Tandem Immunoaffinity-Enzyme-Linked Immunosorbent Assay and Its Application to Milk

A hybridoma-based method utilizing tandem affinity chromatography and enzyme-linked immunosorbent assay (ELISA) was devised to detect zearalenone. A zearalenone specific monoclonal antibody was attached to Sepharose for initial sample clean-up. Zearalenone was eluted with methanol and then quantified by competitive direct ELISA. Average ELISA recoveries from the column for water spiked with zearalenone at levels of 1, 5, 10, 25, and 50 ng/ml were 107, 86, 95, 95, and 92%, respectively, with a mean recovery of 95%. Mean interwell and interassay coefficients of variation were 9.7 and 8.9%, respectively. Average recovery by the method from milk spiked with zearalenone at levels of 1, 5, 10, 25, and 50 ng/ml was 187, 113, 107, 110, and 112%, respectively, with a mean recovery of 126%. Mean interwell and interassay coefficients of variation were 14.5 and 9.1%, respectively. Zearalenone was not detectable in 12 commercial milk samples assayed by the tandem method.

Fusarenon X and nivalenol production by Gibberella zeae in liquid and rice cultures

Toxigenesis of Gibberella zeae strains NHL-F-1372 and NHL-F-1373 was compared in liquid and rice culture. Growth of both strains in glucose-yeast extract-peptone medium (GYEP) for 25 days resulted in peak levels of fusarenon X (FX) ranging from 40–200 mg/L with lower levels of nivalenol (NIV) (10 mg/L). Growth of these strains in modified Fries medium amended with 4% corn steep liquor (CSL) resulted in a much lower total 8-ketotrichothecene yield than in GYEP, with NIV being the primary compound produced. Although FX appeared initially in this latter medium, the toxin disappeared concurrently as the pH exceeded 8.0. Growth rates and total mycelial accumulation were lower in GYEP cultures than in the modified Fries with CSL cultures. Appearance of FX and NIV in modified Fries medium with CSL paralleled the order of appearance of these compounds in rice, but the total trichothecene yield in rice was much higher. In general, growth and toxigenesis by the nivalenol-producing fusaria in liquid and rice cultures was qualitatively similar to that previously found for deoxynivalenol-producing isolates.