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Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

BackgroundGamma-aminobutyric acid (GABA) plays a significant role in the food and drug industries. Our previous study established an efficient fed-batch fermentation process for Lactobacillus brevis NCL912 production of GABA from monosodium l-glutamate; however, monosodium l-glutamate may not be an ideal substrate, as it can result in the rapid increase of pH due to decarboxylation. Thus, in this study, l-glutamic acid was proposed as a substrate. To evaluate its potential, key components of the fermentation medium affecting GABA synthesis were re-screened and re-optimized to enhance GABA production from L. brevis NCL912.ResultsThe initial fermentation medium (pH 3.3) used for optimization was: 50 g/L glucose, 25 g/L yeast extract, 10 mg/L manganese sulfate (MnSO4·H2O), 2 g/L Tween-80, and 220 g/L l-glutamic acid. Glucose, a nitrogen source, magnesium, and Tween-80 had notable effects on GABA production from the l-glutamic acid-based process; other factors showed no or marginal effects. The optimized levels of the four key components in the fermentation medium were 25 g/L glucose, 25 g/L yeast extract FM408, 25 mg/L MnSO4·H2O, and 2 g/L Tween-80. A simple and efficient fermentation process for the bioconversion of GABA by L. brevis NCL912 was subsequently developed in a 10 L fermenter as follows: fermentation medium, 5 L; glutamic acid, 295 g/L; inoculum, 10% (v/v); incubation temperature, 32 °C; and agitation, 100 rpm. After 48 h of fermentation, the final GABA concentration increased up to 205.8 ± 8.0 g/L.Conclusionsl-Glutamic acid was superior to monosodium l-glutamate as a substrate in the bioproduction of GABA. Thus, a high efficacy bioprocess with 205 g/L GABA for L. brevis NCL912 was established. This strategy may provide an alternative for increasing the bioconversion of GABA.

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Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity-related metabolic complications, which caused by excess energy intake and physical inactivity apart from genetic defects. The mechanisms that promote disease progression from NAFLD to further liver injury are still unclear. We hypothesize that the progression involved "2nd hit" is strongly influenced by the type of fatty acids in diets. Flow cytometric analysis showed that medium-chain fatty acid (MCFA) markedly decreased the percentage of late apoptotic and necrotic cells compared with long-chain fatty acid (LCFA), and MCFA inhibited the activities of caspase-3 and -9 in human liver cells with steatosis. Western blot analysis found that the levels of inflammatory markers (interleukin [IL]-6, IL-1-β, and tumor necrosis factor-α) were substantially reduced by MCFA compared with LCFA. Proteomic analysis further showed that LCFA inhibited the expression of antioxidant enzymes, and increased the expression of proteins associated with oxidative stress. It was found that LCFA (palmitate), not MCFA induced apoptosis, oxidative stress and chronic inflammatory responses in the hepatic cells with steatosis. In conclusion, reasonable selection of dietary fats has potential to translate therapeutically by ameliorating disease progression in patients with NAFLD.

Accumulation of lipids in the liver can lead to cell dysfunction and steatosis, an important factor in pathogenesis causing non-alcoholic fatty liver disease. The mechanisms related to lipid deposition in the liver, however, remain poorly understood. This study was aimed to investigate the effects of medium-chain fatty acid (MCFA) on the lipolysis and expression of lipid-sensing genes in human liver cells with steatosis. A cellular steatosis model, which is suitable to experimentally investigate the impact of fat accumulation in the liver, was established in human normal liver cells (LO2 cells) with a mixture of free fatty acids (oleate/palmitate, 2:1) at 200 μm for 24 h incubation. MCFA was found to down-regulate expression of liver X receptor-α, sterol regulatory element binding protein-1, acetyl-CoA carboxylase, fatty acid synthase, CD 36 and lipoprotein lipase in this cellular model, and have positive effects on adipose triglyceride lipase and hormone-sensitive lipase. These results suggest that MCFA may reduce lipid accumulation by regulating key lipid-sensing genes in human liver cells with steatosis.