Metabolome sampling is one of the most important factors that determine the quality of metabolomics data. The main steps in metabolite sample preparation include quenching and metabolite extraction. Quenching with 60% (v/v) cold methanol at -40 °C has been most commonly used for Saccharomyces cerevisiae, and this method was recently modified as "leakage-free cold methanol quenching" using pure methanol at -40 °C. Boiling ethanol (75%, v/v) and cold pure methanol are the most widely used extraction solvents for S. cerevisiae. In the present study, metabolome sampling protocols, including the above methods, were evaluated by analyzing 110 identified intracellular metabolites of S. cerevisiae using gas chromatography/time-of-flight mass spectrometry. According to our results, fast filtration followed by washing with an appropriate volume of water can minimize the metabolite loss due to cell leakage as well as the contamination by extracellular metabolites. For metabolite extraction, acetonitrile/water mixture (1:1, v/v) at -20 °C was the most effective. These results imply that the systematic evaluation of existing methods and the development of customized methods for each microorganism are critical for metabolome sample preparation to facilitate the reliable and accurate analysis of metabolome.

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Microwave sterilization was performed to inactivate the spores of biofilms of involved in foodborne illness. The sterilization conditions, such as the amount of water and the operating temperature and treatment time, were optimized using statistical analysis based on 15 runs of experimental results designed by the Box-Behnken method. Statistical analysis showed that the optimal conditions for the inactivation of biofilms were 14 ml of water, 108°C of temperature, and 15 min of treatment time. Interestingly, response surface plots showed that the amount of water is the most important factor for microwave sterilization under the present conditions. Complete inactivation by microwaves was achieved in 5 min, and the inactivation efficiency by microwave was obviously higher than that by conventional steam autoclave. Finally, confocal laser scanning microscopy images showed that the principal effect of microwave treatment was cell membrane disruption. Thus, this study can contribute to the development of a process to control food-associated pathogens.

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Lignocellulose contains a large amount of cellulose but is recalcitrant to enzymatic hydrolysis, which yields sugars for fuels or chemicals. Various pretreatment methods are used to improve the enzymatic digestibility of cellulose in lignocellulose. Depending on the lignocellulose types and pretreatment methods, biomass compositions and physical properties significantly vary. Therefore, customized enzyme mixtures have to be employed for the efficient hydrolysis of pretreated lignocellulose. Here, using three recombinant model enzymes consisting of endoglucanase, cellobiohydrolase, and xylanase with a fixed amount of β-glucosidase, the optimal formulation of enzyme mixtures was designed for two differently pretreated rice straws (acid-pretreated or alkali-pretreated rice straw) by the mixture design methodology. As a result, different optimal compositions for the enzyme mixtures were employed depending on the type of pretreatment of rice straw. These results suggest that customized enzyme mixtures for pretreated lignocellulosic biomass are necessary to obtain increased sugar yields and should be considered in the industrial utilization of lignocellulose.

We have investigated the effects of supercritical carbon dioxide (SC-CO2) modified with water as a cosolvent on the inactivation of Penicillium oxalicum spores inoculated on barley grains and also on the germination yield of the SC-CO2treated barley grains. After SC-CO2 treatment at different conditions of temperature, cosolvent (water) content and treatment times, the number of colonyforming units (cfus) of fungal spores and the germination of SC-CO2-treated barley grains were analyzed. Among the SC-CO2 treatment parameters, the cosolvent content was the most significant factor affecting the inactivation yields of P. oxalicum spores. When the pressure of SC-CO2 was fixed at 10 MPa, the optimal conditions obtained by ridge analysis of response surface methodology were cosolvent content of 231 mL, temperature of 44C and treatment duration of 12 min, which resulted in a 6.8 log10 reduction of cfu. However, the germination yield of barley grains decreased significantly by treating with SC-CO2 modified with water. For example, the addition of only 300 mL water in the SC-CO2 treatment reduced the germination yield from 65.4% to 1.4% at the same SC-CO2 treatment conditions (40C, 10 MPa, and 20 min).

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