Aspergillus oryzae is an excellent strain for soy sauce fermentation because of its complicated enzyme system, especially protease. The aim of this study was to investigate the key enzymes and flavors during soy sauce fermentation, and a comparative assessment of extracellular enzymes during various fermentation stages at the proteomic level via iTRAQ analysis is presented. Many important enzymes related to the amino acid and glucose metabolisms participated in the material decomposition under high-salt stress. Dipeptidase, dipeptidyl aminopeptidase, leucine aminopeptidase, aspartic protease pep1, and extracellular metalloproteinase played positive roles during the early stage of soybean mash fermentation, whilst leucine aminopeptidase A and extracellular metalloproteinase NpI were the dominant proteolytic enzymes during the later period of fermentation. At the same time, β-glucosidase and β-xylanase exerted great effects upon glucose metabolism throughout the fermentation process. The results show that protease and amylolytic enzymes are complementary in the formation of flavors such as alcohols, acids, esters, aldehydes, furans, and pyrazines during soy sauce fermentation.

Aspergillus oryzae is a common starter in the soy sauce industry and struggles to grow under complex fermentation conditions. However, little is known about the flavor formation mechanism under osmotic conditions (low-temperature and high-salt) in A. oryzae. This work investigated the flavors and the relative protein expression patterns by gas chromatography-mass spectrometry (GC-MS) and proteomic analysis. Low-temperature and a high-salt content are unfavorable to the secretion of hydrolases and the formation of fragrant aldehydes. The aldehyde contents under osmotic conditions were reduced to 1.4–3.7 times lower than that of the control. Besides, copper amine oxidases which decreased under low-temperature stress and salt stress were shown to be important in catalyzing the oxidative deamination of several amine substrates to fragrant aldehydes. Furthermore, alcohol dehydrogenase and polyketide synthase are beneficial to the formation of alcohols and aromatic flavors under low-temperature stress and salt stress. Particularly, the ethanol content under 16 °C stress was 3.5 times higher than that under 28 °C.