Microbial proteases are more economical than plant- and animal-derived proteases due to their ease of production and high activity. This study aimed to optimize the production of proteases from fermentative food-derived microorganisms. Five strains with proteolytic activity among 50 Bacillus sp. were first screened. Two strains with high protease activity were identified: Bacillus amyloliquefaciens SRCM 102139 and Bacillus subtilis SRCM 104999. SRCM 102139 strain and SRCM 104999 strain had the highest protease activity in 0.8% glucose and 0.3% yeast extract, and in 0.8% starch and 0.1% soy peptone, respectively. The production of protease for two strains was optimized by the Central Composite Design (CCD) under response surface methodology. The optimal conditions for protease production in SRCM 102139 were 0.5% and 0.347%, pH 6.0, for carbon (glucose) and nitrogen (yeast extract springer 0202) sources, respectively, with a predicted value of 0.929 U/mL. Additionally, the optimal conditions for protease production in SRCM 104999 were 0.5% and 0.5%, pH 6.7, for carbon (starch) and nitrogen (soy peptone HSP-349) sources, respectively, with a predicted value of 0.431 U/mL. The actual protease activities of SRCM 102139 and SRCM 104999 under the established conditions were 0.926 U/mL and 0.428 U/mL, respectively, closely matching the predicted values.

Single seeds of common buckwheat cultivar Suwon No. 1 when subjected to SDS-PAGE revealed very high polymorphism. High variation existed for protein or protein subunits with molecular weight 54-47kDa, 45-25kDa and 16-11kDa. The electrophoregram showed variation for globulin as well as other protein fractions. About 300 proteins were separated by two-dimensional electrophoresis in common buckwheat (Fagopyrum esculentum Moench.) seed. Seed maturation is a dynamic and temporally regulated phase of seed development that determines the composition of storage proteins reserves in mature seeds. Buckwheat seeds from 5, 10, 15, 20, and 25 days after pollination and matured stage were used for the analysis. This led to the establishment of high-resolution proteome reference maps, expression profiles of 48 spots. It was identified 48 proteins from MALDI-TOF/MS analysis of wild buckwheat seed storage proteins. The 48 proteins were found identical or similar to those of proteins reported in buckwheat and other plants; it is belonging to 9 major functional categories including seed storage proteins, stress/defense response, protein synthesis, photosynthesis, allergy proteins, amino acid, enzyme, metabolism, and miscellaneous. It appears that the major allergenic storage protein separated played the important role in buckwheat breeding and biochemical characterization.

To facilitate the introgression of F. esculentum into the traits of F. homotropicum, several accessions of the hybrids between these two species were pollinated with F. esculentum as the recurrent parent. The embryo in vitro rescue was performed to increase the recovery of backcross progenies. The F2 generation was more amenable than F1 hybrids to produce backcross progenies. The F1 hybrids were backcrossed twice with common buckwheat (pin-type F. esculentum) (recurrent backcrossing). Also, alternate backcrosses with common buckwheat and F. homotropicum (congruity backcrossing) were carried out. Pollen tube growth of BCF1 × F. esculentum (thrum) and F. homotropicum × BCF1 was the disturbed penetration exceeded for all initial interspecific hybrids, and its requirement was proportionally lower when the common buckwheat was used as the recurrent parent and as the last parent of congruity hybrids. Effects of both common buckwheat and F. homotropicum on seed success rate for hybridization were observed. Growth of hybrid embryos before rescue, regeneration of mature hybrids all increased recurrent and congruity backcrosses and inter-crosses between F1 plants and selected fertile plants of the second congruity backcrosses.