The Evolution And Formation Mechanism Of Key Aroma Compounds During The Process Of High-salt Liquid Fermentation Soy Sauce

Aroma Characteristic, which is based on the mixture and interactions of different volatile compounds, is one of the most important parameters for the quality of high-salt liquid fermentation soy sauce(HLFSS). Clarifying the volatile compounds and their evolutions during fermentation of HLFSS is a prerequisite to regulate and control the soy sauce aroma. It is of great important for the improvement of quality and international competitiveness. However, previously researches on the aroma of HLFSS were not systematic and in-depth.The thesis was concerned with the volatile and aroma-active compounds of HLFSS. Optimized flavor research approaches were employed to detect the volatile and aroma profiles of HLFSS qualitatively and quantitatively. The effects of four key processes on generation of aroma compounds in HLFSS are investigated, and the major formation stages of the volatiles were confirmed. Combined with the evolution of non-volatiles of koji and moromi fermentation processes, the formation mechanism and regulation approach were explored. The main research contents and results are as follows:(1) A method based on simultaneous distillation extraction(SDE) was developed for the analysis of soy sauce volatiles. The optimum conditions of SDE were: 2h extraction of 100 m L soy sauce with 100 m L saturated brine, using 50 m L dichloromethane as extraction solvent. Optimization of headspace solid-phase micro-extraction(SPME) focusing on type of fibre, exposure time, extraction temperature and ionic strength was supported by gas chromatography-mass spectrometry(GC-MS) and D-GC-O. The optimized conditions were: CAR/PDMS fibre, Na Cl concentration of 270 g/L, extraction temperature of 45 °C and 30 min exposure time. Finally, the usefulness of three extraction methods: SPME, SDE, and liquid-liquid extraction(LLE) for isolation of flavor compounds from HLFSS were compared. Results showed that SPME technique was good for the extraction of high volatile compounds(i.e. sulfur-containing compounds), while LLE was good for the extraction of furan(one)s and acids. Aldehydes were the major compounds of SDE.(2) SPME coupled with GC-MS were used to obtain wider information about the chemical composition of the volatiles in 14 different brands and grades of HLFSS. A total of 114 compounds were identified, of which 38 volatiles were in common and 12 volatiles were first identified in soy sauce. Alcohols(38%)、acids(16%) and esters(13%) were the main peaks present in the chromatography of soy sauces. Then, considering the solvent-free nature of SPME, three aroma dilution methods were compared to develop a reliable SPME-AEDA approach. A strategy by combination of different extract methods and GC-O/MS was used for the identification of aroma-active compounds in HLFSS. A total of 61 aroma-active areas had been detected, and 4 of them were first smelled in HLFSS. Results showed that the aroma of HLFSS was constituted by 15 categories aromas, of which caramel-like, roast, fruity and malty aroma volatiles were abundant. The most intense aroma-active components in HLFSS were: 2-phenylethanol, 3-methylbutanal, 2-methylbutanal, 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl 2-methylpropanoate, 3-(methylthio)propanal, phenylacetaldhyde, maltol, HEMF(2 isomers), sotolone, HDMF and 2-methoxyphenol.(3) Considering the complex aroma composition and matrix of soy sauce, two techniques(SPME and LLE) were used to quantify the key aroma-active compounds in 27 commercial soy sauces produced through three different fermentation processes(HLFSS; low-salt solid-state fermentation soy sauce, LSFSS; Koikuchi soy sauce, KSS). OAVs, aroma reconstitution and omission tests were examined to identify contribution of each aroma compound on the flavor of HLFSS. In addition, principle component analysis was conducted to analysis the difference among the aroma of soy sauces with different processes. Results showed that KSS was smelled with intense alcoholic and ester aroma, LSFSS was mainly consisted of acids, malty and smoky aroma. And HLFSS, with stable aroma characteristic in commercial products, contained the advantages of these two kinds soy sauce. The types of odorants occurring in the three soy sauce groups were similar, although their intensities significantly differed. Furthermore, the concentration range of soy sauce aroma was from 0.01 μg/kg to 5.2×103 mg/kg. OAVs results showed that 23 aroma compounds had average OAVs > 1, among which 3-methylbutanal, ethyl acetate, 2-methylbutanal, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-(methylthio)propanal, phenylacetaldhyde, and dimethyl trisulfide exhibited OAVs >1 in all the samples.(4) The effects of 4 different fermentation processes on the flavor components were investigated by SPME-GC-MS. The generations of key aroma compounds through the soy sauce koji and moromi fermentation stages were investigated detailed. Results showed that the aroma of soy sauce significantly affected by four steps of soy sauce production: heat treatment of raw materials, koji culturing, moromi fermentation including aging, and pasteurization. The koji stage produced most of the aldehydes, while esters, acids, and furan(one)s were mainly generated during moromi process. In addition, the reduction of esters and alcohols and the increment of phenols were caused by pasteurization. Generally, the aroma compounds, related to the amino acid and carbohydrate metabolism, would be produced during the whole fermentation process, while the volatiles related to fat metabolism would increase in the koji stage and decreased in the moromi stage.(5) Evolution of non-volatile compounds, especially the protein and lipid, during koji culturing and moromi fermentation processes were investigated. To get an insight into the formation mechanism of these aroma compounds, preliminary reaction models and metabolism regulations were tested. Results showed that triacylglycerol(TAG) and phospholipid(PL) would degradation as the fermentation proceeded. Among the TAG, the degradation of LLn Ln and LLP were most obviously. Preferential degradation of PLs to liberate free fatty acids(FFAs) was also observed, and the control of PL utilization during fermentation was a potential method to improve soy sauce’s characteristic flavor. Additionally, linoleic acid was utilized fastest according to the fatty acid composition of total lipids, and there was a correlation between the metabolic rate of linoleic acid and the production rate of C8 volatile compounds during koji culturing. The concentrations of soluble nitrogen(SN) and amino acid nitrogen(AN) increased significantly in the koji stage, providing the foundations of materials and enzymes for the rapid growth of total nitrogen(TN) and AN at the initial moromi stage. Moreover, hydrophobic amino acids(i.e. Leu, Lys, Ile, Val, and Phe) had been released more quickly in the moromi fermentation process, providing important precursors for the formation of Strecker aldehydes. In conclusion, harvested koji provides various types of enzyme, part of aroma compounds, and volatile precursors for subsequent fermentation. The quality of koji would greatly influence the formation of soy sauce aroma.