In order to improve the production of the milkclotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, optimal conditions were obtained using the Box-Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milkclotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.Keywords Optimization Á Culture Conditions Á Milkclotting enzyme Á Wheat bran Many bacteria especially several species belonging to Bacillus are known to produce variety of extracellular enzymes and they have a wide range of industrial applications [1]. Most commercial amylases are produced from a small subgroup of Bacillus species such as B. amyloliquefaciens, B. subtilis, and B. cereus [1][2][3][4].Various bacterial protease have been widely used such as B. subtilis, B. licheniformis, and B. mojavensis [5][6][7]. Xylanase from bacteria, such as B. pumilus, are being applied in textile processing [8]. Numerous bacteria belonging to Bacillus such as B. licheniformis, B. subtilis and B. subtilis natto have been suggested as promising microbial rennet producers [9][10][11][12].Wheat bran, an agro-industrial residue, a cheap source of energy has high potential in the area of fermentation for the production of enzymes. Wheat bran contains cellulose material, starch, crude protein, trace elements and other certain ingredients, which can be used as carbon and nitrogen sources to promote the growth of microorganisms and enzyme production. There are several reports describing wheat bran as potent substrate for enzyme production [12,13]. We recently reported several bacteria producing milk-clotting enzyme isolated from the yak grazing soil in the north-eastern Tibetan Plateau. Among these bacteria, B. amyloliquefaciens D4 possessed high rennet-producing capacity in wheat bran juice [14,15]. The objective of this work was to attempt to optimize the culture conditions to increase milk-clotting enzyme production from wheat bran by B. amyloliquefaciens D4.The producer strain was B. amyloliquefaciens D4(CGMCC 3290), which was deposited in the China General Microbiological Culture Collection Center (Beijing, China). The strain was propagated at 37°C on lysogeny broth (LB) agar slants (1.0 % (w/v) peptone, 1.0 % (w/v) beef extract, 0.5 % (w/v) NaCl, and 2.0 % (...

Background The Tibetan Plateau has an abundance of yak milk resources. The complex microbiota found in traditional fermented yak milk produced and sold by local Tibetans endows the yak milk with unique quality characteristics such as tissue morphology, flavor, and function. However, the diversity of bacterial flora in traditional fermented yak milk have not been elucidated. Methods In this study, 15 samples of fermented yak milk were collected for 16S rRNA high-throughput sequencing to analyze the bacterial community composition and function. Results After filtering for quality, 792,642 high-quality sequences were obtained, and 13 kinds of different phyla and 82 kinds of different genera were identified, of which the phylum Firmicutes (98.94%) was the dominant phylum, Lactobacillus (64.73%) and Streptococcus (28.48%) were identified as the dominant genus, in addition, the bacterial community richness and diversity were higher in Manang Village, followed by Bola Village. Bacterial community richness and diversity in Huage Village were relatively low. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification, the microorganisms in traditional fermented yak milk have rich metabolic functions (77.60%). These findings suggest that a large number of bacteria in traditional fermented yak milk contain abundant metabolic genes and can carry out a variety of growth and metabolic activities. This study established a theoretical foundation for further exploring the microbial flora of traditional fermented yak milk in Gannan.