Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Expression and purification of the recombinant mustard trypsin inhibitor 2 (MTI2) in Escherichia coli

The mustard trypsin inhibitor 2, MTI2, was expressed in Escherichia coli. A specific procedure for its production and purification is described. The recombinant protein was recovered by protein extraction from the insoluble fraction, then renatured and purified by ion exchange and gel filtration chromatography. Finally, the inhibitory activity against trypsin was also determined.     

Studying the growth boundary and subsequent time to growth of pathogenic Escherichia coli serotypes by turbidity measurements

The presence of Escherichia coli in contaminated food products is commonly attributed to faecal contamination when they are improperly handled and/or when inactivation treatments fail. Adaptation of E. coli at low pH and a_w levels can vary at different temperatures depending on the serotype, thus more detailed studies are needed. In this work, a screening to assess the growth of four pathogenic serotypes of E. coli (O55:H6; O59:H21; O158:H23 and O157:H7) was performed. Subsequently, boundary models were elaborated with the fastest serotype selected at different temperatures (8, 12 and 16 °C), and inoculum levels (2, 3 and 4 log cfu/mL) as function of pH (7.00–5.00) and a_w (0.999–0.960). Finally, the growth kinetics of E. coli was described in the conditions that allowed growth. Results obtained showed that the serotypes O157:H7 and O59:H21 did not grow at more stringent conditions (8 °C; pH 5.50), while the E. coli O158:H23 was the best adapted, resulting in faster growth. The logistic regression models presented a good adjustment to data observed since more than 96.7% of cases were correctly classified. The growth interface was shifted to more limited conditions as the inoculum size was higher. Detection times (t_d, h) and their variability were higher at low levels of the environmental factors studied. This work provides insight on the growth kinetics of E. coli at various environmental conditions.     

The combination of stable isotope abundance ratios of H,C, N and S with Sr/Sr for geographical origin assignment of orange juices

Within the EU-project “Pure Juice” established stable isotope methods (δ²H, δ¹³C, δ¹⁵N) have been applied and improved in order to determine and verify the geographical origin of orange juices. In addition, new approaches employing analyses of δ³⁴S and ⁸⁷Sr/⁸⁶Sr have been developed and tested. Approximately 150 authentic orange juice samples from several regions in North- and South-America, Africa and Europe have been analysed. A discrimination of orange producing regions, based on the results which ultimately depend on geographical, climatic and lithological differences was successfully performed. Furthermore, we demonstrate that blending of single strength juice by adding concentrate can be revealed by comparing ⁸⁷Sr/⁸⁶Sr of soluble and insoluble components of the juices. We conclude that regional assignment of orange juice samples is most successful when single parameters are combined in a “multi-element approach”.     

Effect of Lactobacillus buchneri,Lactobacillus fermentum,Leuconostoc mesenteroides inoculants,or a chemical additive on the fermentation,aerobic stability,and nutritive value of crimped wheat grains

The preservation of crimped wheat grains by three bacterial inoculants or a chemical additive was compared. Crimped wheat grain [56.8 g dry matter (DM)/kg] was conserved in 1.75-kg plastic bag, mini-silos without treatment, with 4L/tonne of Crimpstore (CS; an additive containing a mixture of ammonium formate, propionate, ethyl benzoate, and benzoate, SAS Kelvin Cave, Ltd., UK) or 1 x 10(5) cfu/g of each of three inoculant additives containing Lactobacillus fermentum (A), Leuconostoc mesenteroides (B), and Lactobacillus buchneri (C). Six replicates were conserved per treatment. Ensiling DM losses, chemical composition, fermentation characteristics, and aerobic stability were measured in the silages after 68 d of ensiling. All the silages were well fermented and remained stable for 84 h after aeration. Subsequently, the rate of deterioration was slowest in crimped grains treated with CS treatment, followed by those treated with inoculant C, while those treated with inoculant A deteriorated most rapidly. Residual water-soluble carbohydrate concentration was higher in crimped grains treated with CS than those treated with the inoculants. Ammonia nitrogen concentrations were lowest in CS-treated crimped grains, followed by inoculants C and A. DM losses were greater in CS-treated crimped grains than in crimped grains treated with inoculants A and C. In vivo digestibility was also measured in Texel-cross lambs fed a grass silage basal diet supplemented with the additive-treated crimped grains or a conventional, lamb finisher concentrate. Dry matter intake and digestibility were unaffected by treatment. In conclusion, bacterial inoculants containing L. buchneri are promising preservatives for crimped wheat grains.