β-Glucosidase Production By Submerged Fermentation And Its Application

In natural aromatic plants there are many glycosidically bound flavour precursor compounds,and the potential aromatic aglycones can be released by beta-glucosidase which is responsiblefor hydrolyzing flavourless glycosides to flavour compounds. In this way the amount of flavorextracts from natural aromatic plants can be enhanced, which is beneficial to the natural perfumeindustry. In this paper, the production of beta-glucosidase by submerged fermentation and theapplication of this enzyme in flavouring extract were preliminarily investigated.Used Aspergillus ficuum as the starting strain for stepwise mutation by UV irradiation,N-methyl-N'-nitro-soguanidine (NTG) treatment, 60Co irradiation, and a beta-glucosidaseproducing mutant N-9 was obtained, and the activity of beta-glucosidase production wasincreased from 4.04IU/mL to 6.86 IU/mL.The effects of various factors on beta-glucosidase production were also studied, such ascarbon sources, nitrogen sources together with initial pH, culture temperature, aeration andculture time. The optimum 50mL culture media in a 250mL flask consisted of 36g/L bran, 24g/Lbean dregs, 4g/L NH4Cl, 0.4g/L MgSO4·7H2O, 0.12g/L SDS, 0.06 g/L L-phenylalanine withinitial pH 5.5. The strain was cultured in a 180r/min rotating shaker at 30℃ for 6d, with theenzyme activity yielded at 8.50IU/mL.An Amberlite XAD-2 resin column was used to isolate and separate free and glycosidicallybound aroma compounds (FAC and GBAC) from rose. GBAC were then hydrolyzed bybeta-glucosidase, and FAC and the hydrolyzed fractions of GBAC were subjected to GC-MS.Many flavor compounds were found in the latter, such as Linalool oxide, L-terpineol, myrtenol,3-O-alfa-ionol, hexa-hydro-farnesol, 3-keto-beta-ionone, isovanillic acid, geranyl isovalerate,geranyl vinyl ether, etc. Glucose, galactose and fructose were found in the hydrolyzed fractionsof GBAC by HPLC, which showed that there were many alcohols, terpenes and other aromaticcompounds existing in the form of glucoside, galactoside or both of glucoside and galactoside.The conditions of hydrolytic pretreatment of rose by beta-glucosidase were investigated. Theamount of beta-glucosidase and water added were respectively 30IU/g dried flower and 6mL/gdried flower, and the materials were hydrolyzed at 40℃ for 24h with the agitation of 150r/min,then followed by steam distillation for 120min. The results of GC showed that the amount ofaroma extracts after hydrolysis was increased by 280% than that of the control, and somearomatic compounds were found such as linalool oxide, benzyl alcohol, tetrahydro-ionol,herboxide etc., which were not found in the control. Through the organoleptic evaluation, aromaextracts with enzymatic pretreatment smelled more elegant and of fresh rose floral aroma. Therewas significant difference between these two rose aroma extracts at 1% significant level.