Transformation Of Acetoin In Vinegar And The Acetoin Reductase From Acetobacter Pasteurianus

Vinegar is a worldwide popular condiment,with huge differences of place-to-place specific raw materials and manufacturing processes.Acetoin is considered as one of the most aroma-active compounds in vinegar,which affects the flavor of vinegar.However,there was few studies on the origin and transformation of acetoin in vinegar,and the molecular mechanism of acetoin was unknown.Acetobacter pasteurianus is one of the most important acid producing bacteria in vinegar,but the conversion of acetoin in Acetobacter pasteurianus has not been studied yet.In addition,2,3,5,6-Tetramethylpyrazine?TMP?is an important health functional composition in vinegars,but there are controversial viewpoints about its formation mechanism and scarce relevant records on TMP content enhancement in vinegar products.Moreover,no gas chromatography method was present for simultaneous determination of acetoin and TMP in vinegar.The main content of this article includes the following four aspects.Firstly,a simple and accurate gas chromatography method was developed for the simultaneous determination of TMP and acetoin in vinegars.The limits of detection?LOD?of TMP and acetoin were 0.025 mg/kg and 1.36 mg/kg,respectively;the limits of quantitation?LOQ?of them were 0.083 mg/kg and 4.53 mg/kg,respectively.The linearity ranges for TMP and acetoin were from their LOQ to 236 mg/kg and 10,000 mg/kg,respectively.The precision for acetoin?2.16%RSD?was better than that of TMP?6.28%RSD?.The accuracy of the method was expressed by the recovery rate,the average recoveries were 101.94±1.96%and96.67±1.22%for TMP and acetoin,respectively.Compared with other analytical methods,this method was superior due to the advantages of fast,economical,accurate,etc.Secondly,second order polynomial?Poly2D?regression models was builted with the concentration of TMP,acetoin and ammonium for the first time.The 137 vinegars were divided into group??51 Shanxi solid fermentation vinegars?,group??45 no-Shanxi solid fermentation vinegars?,and group??41 liquid fermentation vinegars?.The Poly2D models for Group I had higher R² values and lower percentages of root mean square error of calibration?RMSEC?and prediction?RMSEP?than those from Group II,so the models for Group?had better performances than Group II.Thirdly,it was confirmed that acetoin and ammonium were the precursors of TMP in vinegars by isotopic tracing for the first time.Preparation of ¹³C labelled acetoin by Bacillus sp.H-18W fermentation using D-glucose-1-¹³C,and ¹³C labelled acetoin together with ¹⁵N labelled NH₄⁺were set as tracer.GC-MS detection proved that the acetoin and ammonium were the precursor of TMP.It was of great significance to guide the production of vinegar and improve the quality of vinegar.Finally,a novel acetoin reductase from Acetobacter pasteurianus was studied.The protein was encoded by ap3 comprises 253 amino acids and belongs to short chain dehydrogenase family?SDR?.However,this enzyme could catalyze?2R,3R?-2,3-BD as substrate,and this characteristic was similar to the medium chain dehydrogenase family.The reduction reaction could take acetoin and diacetyl as substrates with NADH,and diacetyl was optimal substrate with optimal pH and temperature being pH 5 and 40?,respectively.The oxidation reaction could take?2R,3R?-2,3-BD and meso-2,3-BD as substrates with NAD⁺,and?2R,3R?-2,3-BD as optimal substrate,with optimal pH and temperature being pH 9 and 50?.Compared with other acetoin reductase,this enzyme had low K_m value.It could catalyze the conversion of diaceyl and?2R,3R?-2,3-BD to?3R?-acetoin,and the interchange between meso-2,3-BD and?3S?-acetoin.