Simultaneous improvement of thermostability and maltotriose-forming ability of a fungal α-amylase for bread making by directed evolution.

For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.

A novel high maltose-forming α-amylase from Rhizomucor miehei and its application in the food industry.

A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.

Current status and demand of health services in primary and middle school clinic in 16 provinces of China / 中国学校卫生

Objective@#To analyze the current situation and needs of primary and secondary school health clinic services, and to provide a reference for strengthening school health service in China.@*Methods@#Investigation provinces were selected according to China s three major economic zones, 1 or 2 survey centers were selected from each province, 16 provinces and 26 survey centers were selected. In each survey centers, six schools from primary and secondary schools, junior high schools and high schools in urban and rural areas were randomly selected as research sites. Each research site randomly selected 10 students in each grade(grade three, four, five from primary schools) to fill in the questionnaire anonymously. A total of 4 661 valid questionnaires were collected.@*Results@#Among the services provided by the school clinics to students, "trauma treatment" "consulting health problems" "physical examination" accounted for 49.4%, 40.5% and 39.0% respectively. Within the past one year, 45.1% students went to the school clinic for consultation or consultation 1-3 times, 6.4% for 4-5 times, and 3.7% for 6 times or more. When students were sick in school, 65.8%(primary school), 64.0%(junior high school), and 54.4%(high school) of them selected to go to the school clinic. When suddenly suffered trauma in school, 78.2%(primary school), 73.2%(junior high school) and 69.6%(high school) of the students went to the school clinic.@*Conclusion@#Primary and middle school students have a great demand for school health services, and it is necessary to continue to strengthen the construction of primary and secondary school health clinics.

High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production.

A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.