Mechanism Of Aroma Production For The Improvement Of Soy Sauce Flavor By Candida Etchellsii

Halophilic aromatic yeast bacteria were usually added to enhance the flavor and qualityin the production process of high-salt liquid soy sauce fermentation. Although it is wellknown that such microorganisms play an important role to the flavor of the soy sauce duringthe fermentation process, it is still not well understood as the formation of the flavor is arather slow metabolic process. Much of the research work was centered on how to screen andselect the superior strain of high-salt-tolerant halophilic aromatic yeast in order to improve theflavor and quality of the soy sauce. However, little is known about the physiologicalcharacteristics of the yeast. Understanding of the types of soy sauce flavor and the mechanismfor the production of specific quality as well as consistency of the flavor generated by thehigh-salt-tolerant halophilic aromatic yeast in the production of soy sauce are absolutelyessential.In this paper, halophilic aromatic yeast was isolated on the basis of the growing andmetabolic functions from soy sauce. Outlines of this thesis are shown as follows:(1) Our studies on the soy sauce established the model for its flavor quality evaluation.Initially,39types of flavor compounds in six different types of soy sauce were identified anddetermined by headspace solid phase micro extraction gas chromatography mass spectrometry.Organic acids and free amino acids were analyzed by HPLC. The model was establishedbased on principal component analysis method for assessing and ranking of flavor quality ofsoy sauce. It was proven very useful for the evaluation of flavor quality model and for sensoryevaluation of six types of soy sauce. Using principal component analysis which simplifiescomplex information, our correlative evaluation model was established, tested by comparingtraditional sensory evaluation method, providing a new methodology for objective evaluationof the flavor quality of soy sauce.(2) Salt-tolerant yeasts are very important for the flavor formation in soy saucefermentation production. A halophilic aromatic yeast was isolated on the basis of themolecular biological and metabolic functions from soy sauce. The ITS nucleotide sequencealignment method was used to identify the strain as Candida etchellsii by subjecting thesequence to NCBI-BLAST in comparison with that of the C. etchellsii strain Miso0208(atypical high-salt-tolerant halophilic aromatic yeast strain). Organic acids, amino acids andvolatile flavor compounds were produced by the yeast strain which were analyzed by HPLCand SPME-GC/MS methods. Tartaric acid (0.979±0.040) g/L, formic acid (0.636±0.030) g/L,lactic acid (2.80±0.10) g/L, α-alkone glutaric acid (0.132±0.015) g/L, citric acid (2.59±0.10)g/L and succinic acid (3.03±0.20) g/L were detected at72h of fermentation, respectively. Freeand acid hydrolyzed amino acids at levels of (3.7355±0.0027) g/L and (11.5604±0.0037) g/L,respectively,4-ethyl guaiacols as well as other volatile flavor compounds were also detected.(3) It describes the establishment of flow cytometric methods for C. etchellsii yeast andtheir application in4-hydroxy-2(or5)-ethyl-5(or2)-methyl-3(2H)-furanone (HEMF, animportant aromatic agent for the food industry) fermentation. It focused on the developmentand the application of a rapid and reliable staining method for the characterization of C. etchellsii cells. Experiments were carried out during the shake flask batch cultivation of yeastin the medium under aerobic conditions at30oC. Stained samples were analyzed by flowcytometer. Propidium iodide was used to determine cell viability in native sample. To furtherunderstand the roles for HEMF in this process, Pearson correlation analysis was used toevaluate cellular data from the fermentation process at various sodium chloride levels.Comparison of HEMF concentration to that of biomass, cell viability and sodium chloridelevel demonstrated high correlation with HEMF. Lastly, studies on the effects of variouslevels of NaCl plus addition of amino acids on HEMF production by C. etchellsii. Using the2-step process with initial200g/L NaCl and subsequently220g/L NaCl after40h offermentation with the addition of alanine, arginine and glycine (both1.67g/L) in the medium.The shake flask fermentation results indicated that110.74mg/L of HEMF was produced bythe C. etchellsii yeast. Results from the7L fermentor experiments shows that HEMFproduction reached121.51mg/L. The2-step,2-level NaCl with various amino acid additivesprocess in the fermentation medium significantly improves the production of HEMF by the C.etchellsii yeast.(4) In order to enhance the adaptability of an aromatic yeast in soy sauce mash, the yeastwas acclimatized by gradually increasing sodium chloride concentration in medium. Resultsshowed that the tolerant acclimation of aromatic yeast to sodium chloride increased from180g/L to240g/L after5batches acclimation. Biological characteristics of three high sodiumchloride tolerant strains were studied after acclimatized. The results showed that total free andacid hydrolysis amino acid reached to2.01g/L and7.00g/L respectively by C. etchellsii with200g/L sodium chloride in medium. Comparison of the strain in other different concentrationof sodium chloride in medium, the volatile ester content by C. etchellsii was reached to thehighest of0.80g/L. The salt tolerance enhancement made the aromatic yeast better adapted tothe high salinity environment in soy sauce fermentation. Four high-salt tolerant strains of C.etchellsii isolated from soy sauce mashes were inoculated into new soy sauce fermentationproduction media with180g/L,200g/L,220g/L and240g/L sodium chloride with biomassof about10g/L. Soy sauce inoculated with200g/L sodium chloride showed high contents offree and acid hydrolyzed amino acids. In200g/L sodium chloride free and acid hydrolyzedamino acid content of6-month-old soy sauce fermentation samples inoculated with C.etchellsii was5.14%and9.71%higher than that of the control. Major volatile organiccompounds in soy sauce were4-hydroxyl-2,5-dimethyl-3(2H)-furanone (HDMF),2-ethyl-4-hydroxyl-5-methyl-3(2H)-furanone (HEMF) and4-ethyl guaiacol. C.etchellsii-inoculated soy sauce samples demonstrated the ability to increase HEMF whichcompound contributed to its caramel odor. The addition of200g/L sodium chloride led to theC. etchellsii yeast in the soy sauce fermentation media to increase the formation of desirableamino acids and volatile flavor compounds and thus significantly improving the quality of theresulting soy sauce.(5) A novel two-stage inoculation process using the C. etchellsii yeast was developed andoptimized. It was proven to be a feasible reproducible process for industrial application forthe improvement of the flavor and quality of soy sauce production. The first-stage consists ofa5%culture inoculum of log phase cells at30-day. Subsequently, a20%culture inoculum of stationary phase cells was added at60-day. The resulting amino nitrogen and soluble salt-freesolid yield reached (9.15±0.12) g/L and (269.60±3.15) g/L in30oC incubator experiments,increased by23.1%and17.6%, respectively as compared to the control without cultureinoculation. Maximal free amino acid yield of (58.21±1.77) g/L was achieved and39types ofvolatile flavor compounds content was (17.81±0.45) g/L, which were1.76%and178.7%higher than the control.