ORIGINAL PAPER

Identification and analysis of the metabolic functionsof a high-salt-tolerant halophilic aromatic yeastCandida etchellsii for soy sauce production

Jie Feng • Xiao-Bei Zhan • Dong Wang •

Li-Min Zhang • Chi-Chung Lin

Received: 13 July 2011 / Accepted: 3 November 2011 / Published online: 13 November 2011

� Springer Science+Business Media B.V. 2011

Abstract Salt-tolerant yeasts are very important for the fla-

vor formation in soy sauce fermentation production. A halo-

philic aromatic yeast was isolated on the basis of the molecular

biological and metabolic functions from soy sauce. The ITS

nucleotide sequence alignment method was used to identify the

strain as Candida etchellsii by subjecting the sequence to

NCBI-BLAST in comparison with that of the C. etchellsii strain

Miso 0208 (a typical high-salt-tolerant halophilic aromatic

yeast strain). Organic acids, amino acids and volatile flavor

compounds were produced by the yeast strain which were

analyzed by HPLC and 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), a-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 at 72 h of fermentation,

respectively. Free and acid hydrolyzed amino acids at levels of

3.7355 ± 0.0027 and 11.5604 ± 0.0037 g/l, respectively,

4-ethyl guaiacols as well as other volatile flavor compounds

were also detected.

Keywords Soy sauce � Halophilic aromatic yeast �Identification � Metabolic characteristics � Solid phase

micro-extraction gas chromatography mass spectrum

(SPME-GC/MS)

Introduction

Chinese soy sauce is a very important traditional condi-

ment. It has more than 3,000 years of documented history

in China started as early as the Zhou Dynasty (Zhang and

Tao 2009). The process of soy sauce fermentation pro-

duction has been based on the enzymatic activities of rel-

evant microorganisms. Various agricultural product-based

substrates were hydrolyzed and fermented by the enzy-

matic catalysis with appropriate microorganisms during the

process of soy sauce production. It involves complex multi-

step enzymatic transformations with various microbial

species to produce traditional soy sauce. Many different

metabolites are released after microbial autolysis which

constitutes a rich sauce paste.

Historically, soy sauce production facilities used natural

and traditional fermentation process lacking proper scien-

tific control leading to variability of soy sauce quality

under relatively inferior hygienic condition. The traditional

natural sauce fermentation production process did not use

pure microbial cultures. Consequently, the traditional pro-

cess lacks the understanding of the microbial physiological

functions as well as without proper scientific production

process control and specifications resulting in unstable

product quality (Murooka and Yamshita 2008).

Halophilic aromatic yeast and lactic acid bacteria were

usually added to enhance the flavor and quality in the

production process of high-salt liquid soy sauce fermen-

tation (Suezawa et al. 2006). Common halophilic aromatic

yeast, such as Zygosaccharomyces rouxii, Torulopsis ver-

satile, Candida versatilis and C. etchellsii, have been used

in the production of high-salt soy sauce and the salt content

from 18 to 24% in Chinese soy sauce (Wanakhachornkrai

and Lertsiri 2003). Tetragenococcus halophilus strains

were isolated from soy sauce mash in southeast Asia,

J. Feng � X.-B. Zhan (&) � D. Wang � L.-M. Zhang � C.-C. Lin

The Key Laboratory of Carbohydrate Chemistry and Industrial

Biotechnology of Ministry of Education, School of

Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu

Province, People’s Republic of China

e-mail: xbzhan@yahoo.com

123

World J Microbiol Biotechnol (2012) 28:1451–1458

DOI 10.1007/s11274-011-0945-6

China, and Japan. These microorganisms caused a drop in

the acidity of the soy sauce mash (Ho et al. 1984; Robert

et al. 2000). T. halophilus, L. acidipiscis, L. farciminis,

L. pentosus, and L. plantarum strains were able to grow in a

high concentration of NaCl (10–14%) in Thai soy sauce

(Tanasupawat et al. 2002). The key factors for the produc-

tion of soy sauce with good flavor are: relevant and properly

formulated raw materials, suitable strains of specific

microorganisms, sound production technology and good

process control (O’Toole 1997). Understanding of the types

of soy sauce flavor and the mechanism for the production of

specific quality as well as consistency of the flavor generated

by the high-salt-tolerant halophilic aromatic yeast in the

production of soy sauce are absolutely essential.

During the soy sauce production process, ethanol,

higher-carbon alcohols, 4-hydroxy-furan ketones and other

substances are produced by Z. rouxii, which are important

to the formation of the flavor of the soy sauce. Researchers

found that threonine, cystathionine, branch-chain amino

acids significantly accelerated the growth of Z. rouxii and

increased the formation of glycerol and higher alcohols

(Hecquet et al. 1996). Further studies found in Z. rouxii

that, threonine, cystathionine significantly promoted the

production of a-keto acid metabolites and amino acid

metabolites but branch-chain amino acids inhibited their

production (Dahlen et al. 2001). However, a-keto acid was

not accumulated in Z. rouxii when ammonium was the sole

nitrogen source and a-keto acid was not secreted outside of

the yeast cells. If valine, leucine, threonine and methionine

were added to the culture medium, a-keto acids, especially

a-ketobutyric acid would accumulate in the extracellular

medium. Consequently, a-keto acids and higher alcohols

accumulation through the metabolism of amino acids by

Z. rouxii and Saccharomyces cerevisiae through the Ehr-

lich pathway are very similar. Except threonine which can

be synthesized either by the Ehrlich pathway or two other

channels of amino acid biosynthesis.

4-Ethyl-guaiacyl phenol and 4-ethyl phenol which

enhance soy sauce with clove and smoked favors, respec-

tively, are produced by the Candida sp. During the soy

sauce production process, a wheat-based ingredient is

broken down into ferulic acid and coumaric acid and then

converted into 4-ethyl-guaiacyl phenol and 4-ethyl phenol

by the biotransformation process of the Candida sp. The

Candida sp. can also biotransform maltose into ethanol

under high-salt condition. However, Z. rouxii is incapable

of both biotransformations.

The composition and content of organic acids, amino

acids and volatile flavor compounds serving as the index

for evaluation of the soy sauce flavor are determined by the

quality of the soy sauce fermentation raw material mash.

The interactions of various microorganisms, particularly

the halophilic aromatic yeast, transform the raw material

mash into the above metabolites to boost the flavor of the

resulting soy sauce (Lee et al. 1996; Yoshio et al. 1999;

Murooka and Yamshita 2008). Although it is well known

that such microorganisms play an important role to the

flavor of the soy sauce during the fermentation process, it is

still not well understood as the formation of the flavor is a

rather slow metabolic process. Much of the research work

was centered on how to screen and select the superior strain

of high-salt-tolerant halophilic aromatic yeast in order to

improve the flavor and quality of the soy sauce (Zhang

et al. 1997). However, little is known about the physio-

logical characteristics of the yeast (Yoshio et al. 1999).

We isolated a high-salt-tolerant halophilic aromatic yeast

from a raw soy sauce, used molecular biological methodology

to identify and characterize the yeast strain. The organic acids,

amino acids and volatile flavor were analyzed by HPLC and

SPME-GC/MS (Solid Phase Micro-Extraction Gas Chroma-

tography Mass Spectrum) to obtain important physiologic and

metabolic data of this yeast strain during the process of soy

sauce fermentation process in order to improve and stan-

dardize soy sauce production and quality.

Materials and methods

Strains and culture conditions

Strain: the strain used in this study was Candida etchellsii

CICIM Y0600 which was isolated in our laboratory from

the raw soy sauce. It was preserved by the Culture and

Information Center of Industrial Microorganism of China

as well as our Laboratory of Biochemical Engineering of

Jiangnan University.

The slant medium contained (g/l): peptone 10, glucose

20, yeast extract 5, and agar 15 at pH 5.8–6.0.

The seed medium contained (g/l): peptone 10, glucose

20, and yeast extract 5 at pH 5.8–6.0. The seed culture was

prepared in flask on a reciprocal shaker at 150 rpm and

30�C for 18–20 h.

The fermentation medium contained (g/l): peptone 10,

corn syrup 40, and yeast extract 5, at pH 5.8–6.0. The

fermentation culture was prepared in a 7-l fermentor

(Bioton Company, Korea. Model LiFlus GX) mixed at

200 rpm, aeration at 1 vvm and 30�C until the stationary

growth phase was reached at 72 h.

Analytical methods

Rapid extraction of chromosomal DNA of the strain to be

identified

100 ll of sterile water was added into 1.5 ml centrifuge

tube followed by 3–4 loopful of yeast cells. The sample

1452 World J Microbiol Biotechnol (2012) 28:1451–1458

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was centrifuged at 6,0009g for 1 min after mixing. 150 ll

of cell lysis buffer for western blot and IP was added to the

cell pellet and resuspended in the 1.5 ml centrifuge tube. It

was placed in the constant temperature bath for 15 min at

90�C. The crude extracts of chromosomal DNA were

obtained.

Amplification of ITS sequences of the strain to be identified

The ITS region were amplified by PCR from yeast DNA

using the method as described by James et al. (1996).

Determination of ITS sequences

The nucleotide sequence of PCR products were determined

in accordance with the BigDye Terminator v3.1 kit

instructions (Applied Biosystems, Forster City, California,

USA). 10 ll Hi-Di formamide was added to each tube to

dissolve the DNA after alcohol was completely evaporated.

Denaturation using the Model PTC-200 PCR instrument

(Bio-Rad Company, Hercules, California, USA) was con-

ducted for 4 min at 95�C and 4 min at 94�C followed by

electrophoresis.

Characterization of various cell growth parameters

of the C. etchellsii yeast

Biomass was determined by drying the cells at 80�C to a

constant weight. The reducing sugar was determined by the

dinitrosalicylic acid (DNS) assay method (Zhang and Tao

2008). Volatile esters flavor were determined by colori-

metric method (Ma 2001).

Determination of organic acids

Organic acids were determined with the Agilent 1100

HPLC under the following condition: Agilent ZORBAX

SB-Aq column (150 9 4.6 mm, 5 lm). Column tempera-

ture at 30�C; the injection volume was 10 ll and the

detection wavelength 210 nm. Flow rate was 0.5 ml/min

for the mobile phase containing 0.5% acetonitrile, 99.5%

0.02 mol/l KH2PO4 (pH adjusted to 2.0 with phosphoric

acid).

Determination of amino acids

Amino acids were determined with the Agilent Technolo-

gies amino acid analyzer under the following conditions:

Column at 40�C; the injection volume was 20 ll, detection

wavelength was 338 nm. Flow rate of mobile phase was

1 ml/min. Composition of the mobile phase was: A phase:

8.0 g sodium acetate crystals was added in the 1,000 ml

beaker, then 1,000 ml water was added and stirred until all

the crystals were dissolved in water. Thereafter, 225 ll

triethylamine was added, stirred with pH adjusted to

7.20 ± 0.05 with 5% acetic acid and 5 ml tetrahydrofuran

was subsequently added. The resulting system was ready

for use. B phase: 8.0 g sodium acetate crystals was added

in the 800 ml beaker with 400 ml water and stirred until all

the crystals were dissolved in water. The pH was adjusted

to 7.20 ± 0.05 with 5% acetic acid. This solution was

added to 800 ml methanol and 800 ml acetonitrile and used

after mixing. The gradient elution methodology was used.

Determination of the volatile flavor components

Gas chromatography tandem mass spectrometry 1200 L

GC/MS-MS (Varian Company of USA) was used. Chro-

matographic conditions: the column was DB-WAX,

30 m 9 0.25 mm 9 0.25 lm capillary column; Carrier

gas: helium gas; 0.8 ml/min for flow rate. Temperature

program: initial temperature at 40�C, maintained 4 min,

used 6�C min rate to 160�C, then used at 10�C/min rate

rose to 220�C and maintained for 6 min. Mass spectrom-

etry conditions: interface temperature, 250�C, ion source

temperature, 200�C; ionization mode, EI; electron energy,

70 eV; detection voltage, 350 V; emission current,

200 lA.

Results and discussion

Identification of the high-salt-tolerant halophilic

aromatic yeast

ITS nucleotide sequence of strains to be identified and to

upload the sequences were obtained from GenBank with

accession number JN703314 for this strain. The sequences

were submitted to the NCBI-BLAST for comparison. The

ITS nucleotide sequence of our yeast strain isolated from

raw soy sauce was identified and compared with that of the

C. etchellsii strain Miso 0208 (a typical high-salt-tolerant

halophilic aromatic yeast strain for miso production and

GenBank accession number is AB196222) have the highest

degree of homology of 99.74%. Therefore, this strain has

been identified as C. etchellsii.

Characterization of the physiological parameters

of the C. etchellsii yeast strain for volatile ester flavor

fermentation production

Various physiological parameters of the C. etchellsii yeast

strain were characterized in the 7-l fermentor during vol-

atile ester flavor production using the fermentation medium

with and without 18% NaCl. As shown in Fig. 1a, cells

grown in medium without NaCl grew faster, reached

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stationary phase at 30 h and maximum cell density of

10.497 ± 0.400 g/l, consumed less glucose and produced

less volatile ester flavor (maximum 0.394 ± 0.028 g/l)

than that grown in medium with 18% NaCl. In contrast,

cells grown in medium with 18% NaCl reached stationary

phase at 40 h but produced significantly more volatile ester

flavor (maximum 0.565 ± 0.015 g/l) (Fig. 1b). It is con-

ceivable that since this strain of C. etchellsii yeast was

isolated from raw soy sauce, it would be adaptable to high-

salt condition with superior metabolic functionality. Con-

sequently, this high-salt-tolerant halophilic aromatic yeast

produced higher level of volatile ester flavor enhancers in

the 18% NaCl medium than that without salt and thus

making it particularly suitable for use to enhance the flavor

of soy sauce in the production process.

Based on the volatile ester flavor fermentation kinetics

in medium with and without NaCl (Fig. 1a, b), the flavor

fermentation process was completed at 72 h. Comparison

of various fermentation data for both conditions at 72 h is

shown in Table 1.

To compare the metabolic functions of C. etchellsii, its

fermentation data and kinetic parameters at the stationary

growth phase at 72 h is selected. At 18% NaCl concen-

tration higher levels of cell density and volatile esters were

detected (Table 1). In addition, its maximum specific

growth rate was slightly lower and its maximum specific

glucose consumption rate and maximum specific produc-

tion rate were higher (Table 2). Conceivably, as this strain

of C. etchellsii is more adaptable to higher salinity, max-

imum specific glucose consumption rate and maximum

specific production rate were relatively high and thus

enabling it to better utilize glucose and produce more

volatile esters under this condition.

Analysis of organic acid production by C. etchellsii

To better understand the fermentation production of flavor

organic acids by C. etchellsii, HPLC analysis of six organic

acids were undertaken in media with 18% NaCl and without

NaCl. Figure 2 shows that tartaric acid production levels were

quite similar which reached maximum levels of 0.997 ±

0.040 and 1.145 ± 0.050 g/l, respectively at 40 h of fer-

mentation. Formic acid, lactic acid and citric acid production

yields were 0.636 ± 0.030, 2.800 ± 0.060 and 2.590 ±

0.040 g/l, respectively, at 60 h in 18% NaCl. a-Ketoglutaric

acid and succinic acid accumulated at the early stage of fer-

mentation but their production levels decreased at later fer-

mentation time and reached 0.267 ± 0.010 and 1.400 ±

0.020 g/l, respectively, at 80 h.

Without the presence of NaCl, C. etchellsii only pro-

duced low levels of citric acid and succinic acid. Formic

acid was produced after 70 h of fermentation. Conse-

quently, this strain of C. etchellsii was more conducive to

the fermentation production of flavor organic acids at 18%

NaCl concentration.

Analysis of amino acid production by C. etchellsii

The fermentation production of combined essential amino

acids levels (free and acid hydrolyzed) were 1.9495 ±

0.0011 and 4.1437 ± 0.0017 g/l, respectively (Table 3). The

conversion rate was 47.05% for free essential amino acids.

A

B

Fig. 1 Volatile ester flavor fermentation kinetics of the C. etchellsiiyeast in the fermentation medium without NaCl (a) and with 18%

NaCl (b) in 7-l fermentor

Table 1 Comparison of the 7-l fermentor data at 72 h using media with and without 18% NaCl for the high-salt-tolerant aromatic yeast

C. etchellsii

NaCl level (%) Time (h) pH Cell density (g/l) Yeast count

(9108 cell/ml)

Glucose (g/l) Ethanol (%) Volatile ester (g/l)

0 72 4.01 ± 0.01 10.43 ± 0.84 6.92 ± 0.56 13.36 ± 0.17 3.203 ± 0.076 0.497 ± 0.015

18 72 4.09 ± 0.01 9.25 ± 0.04 8.30 ± 0.69 5.44 ± 0.61 0.970 ± 0.039 0.57 ± 0.028

1454 World J Microbiol Biotechnol (2012) 28:1451–1458

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The autolysed essential amino acids levels (free and hydro-

lyzed) were 0.0137 ± 0.0009 and 0.0694 ± 0.0011 g/l

respectively, with conversion rate of 19.74% of free essential

amino acids.

The production of combined non-essential amino acids

levels (free and acid hydrolyzed) were 1.7859 ± 0.0016 and

7.4167 ± 0.0020 g/l, respectively (Table 4). The conversion

rate was 24.08% for free non-essential amino acids. The au-

tolysed non-essential amino acids levels (free and hydrolyzed)

were 0.0506 ± 0.0009 and 0.3144 ± 0.0016 g/l, respectively.

The conversion rate was 16.09% for free non-essential amino

acids.

Analysis of volatile flavor compounds production

by C. etchellsii

Volatile flavor compounds produced by C. etchellsii were

analyzed by solid phase micro extraction-mass spectrom-

etry technique and the spectrum is show in Fig. 3.

Table 2 Comparison of kinetic

parameters of C. etchellsii in

two different salt levels

NaCl level (%) Maximum specific

growth rate (h-1)

Maximum specific glucose

consumption rate (h-1)

Maximum specific

production rate (h-1)

0 0.18777 ± 0.00218 0.01217 ± 0.00097 0.00167 ± 0.00040

18 0.16980 ± 0.00029 0.01615 ± 0.00049 0.00260 ± 0.00028

Fig. 2 Comparison of organic acids by C. etchellsii in different salinity

Table 3 Comparison of essential amino acids produced by C. etchellsii

Essential amino acid 18% NaCl level (g/l) Autolysate (g/l) Odor Odor value

Free Acid hydrolysis Free Acid hydrolysis

Ile 0.1454 ± 0.0001 0.5519 ± 0.0002 0.0014 ± 0.0001 0.0152 ± 0.0001 Bitter -2

Leu 0.3288 ± 0.0002 0.7035 ± 0.0002 0.0025 ± 0.0002 0.0144 ± 0.0001 Slight bitter -1

Lys 0.0758 ± 0.0002 0.2490 ± 0.0002 0.0000 ± 0.0000 0.0014 ± 0.0002 Bitter -2

Met 0.3172 ± 0.0002 0.5037 ± 0.0003 0.0033 ± 0.0001 0.0070 ± 0.0001 Bitter -2

Phe 0.2512 ± 0.0001 0.5772 ± 0.0002 0.0014 ± 0.0001 0.0072 ± 0.0001 Bitter -2

Thr 0.2912 ± 0.0002 0.6012 ± 0.0003 0.0028 ± 0.0002 0.0103 ± 0.0003 Slight sweet 1

Val 0.5400 ± 0.0001 0.9572 ± 0.0003 0.0024 ± 0.0002 0.0139 ± 0.0002 Bitter -2

Total (g/l) 1.9495 ± 0.0011 4.1437 ± 0.0017 0.0137 ± 0.0009 0.0694 ± 0.0011 – –

World J Microbiol Biotechnol (2012) 28:1451–1458 1455

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64 types of compounds were identified by the above

spectrometry which includes 16 types of alcohols, 2 types of

phenols, 9 types of esters, 6 types of aldehydes, 8 types of

ketones, 11 types of acids, 8 types of heterocyclic compounds

and 4 types of alkanes. 22 types of important compounds were

quantitatively analyzed among these compounds (Table 5).

Some important compounds of alcohol, 3-methylthio-1-pro-

panol, 2-methoxy-phenol/guaiacol and 4-ethyl-phenol were

quantitatively detected at levels of 3.6237 ± 0.0300,

2.5152 ± 0.0200, 0.1257 ± 0.0050 and 0.0169 ± 0.0020

g/l, respectively. 3-Methylthio propanol has a strong irritating

odor which is a strong fragrance of the meat or broth-like

aroma and taste when the concentration was very low. 4-Ethyl

guaiacol (4-EG) has the typical smell of soy sauce and smoke

and it has unique taste of soy sauce fermentation. Fragrance

characteristic of the compound was apparent and fragrance

activity was strong. It contributed to the flavor and has bigger

contribution to the full flavor of soy sauce. It was important to

improve the aroma characteristics of the soy sauce. It can be

used as one of representative flavor compounds for soy sauce.

It also contributes to the flavor of the soy sauce.

Conclusions

During the fermentation of soy sauce, yeasts, micrococcus,

streptococcus, bacillus, lactic acid bacteria and other rela-

ted bacteria have appeared spontaneously in the soy sauce

Table 4 Comparison of non-essential amino acids produced by C. etchellsii

Non-essential amino acid 18% NaCl level (g/l) Autolysate (g/l) Odor Odor value

Free Acid hydrolysis Free Acid hydrolysis

Asp 0.1452 ± 0.0002 1.3763 ± 0.0003 0.0160 ± 0.0001 0.0526 ± 0.0002 Slight sweet 1

Glu 0.3713 ± 0.0003 2.0952 ± 0.0003 0.0127 ± 0.0001 0.0458 ± 0.0002 Umami 2

Ser 0.0319 ± 0.0001 0.5420 ± 0.0002 0.0000 ± 0.0000 0.0304 ± 0.0002 Sweet –

His 0.0863 ± 0.0001 0.2483 ± 0.0002 0.0006 ± 0.0001 0.0178 ± 0.0001 Slight bitter -1

Gly 0.0865 ± 0.0001 0.8096 ± 0.0002 0.0091 ± 0.0001 0.0550 ± 0.0002 Sweet 2

Arg 0.4920 ± 0.0002 0.6799 ± 0.0002 0.0054 ± 0.0001 0.0326 ± 0.0001 Bitter -2

Ala 0.4038 ± 0.0002 0.8387 ± 0.0002 0.0039 ± 0.0001 0.0317 ± 0.0002 Sweet –

Tyr 0.1103 ± 0.0002 0.1655 ± 0.0001 0.0017 ± 0.0001 0.0044 ± 0.0001 Slight bitter -2

Cys 0.0059 ± 0.0001 0.0370 ± 0.0001 0.0006 ± 0.0001 0.0054 ± 0.0001 Bitter -2

Pro 0.0526 ± 0.0001 0.6242 ± 0.0002 0.0007 ± 0.0001 0.0387 ± 0.0002 Slight sweet 1

Total (g/l) 1.7859 ± 0.0016 7.4167 ± 0.0020 0.0506 ± 0.0009 0.3144 ± 0.0016 – –

Fig. 3 Total ion

chromatograms of volatile

flavor components

of C. etchellsii

1456 World J Microbiol Biotechnol (2012) 28:1451–1458

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mash (Ho et al. 1984; Robert et al. 2000; Tanasupawat

et al. 2002). In the soy sauce mash the Aspergillus enzymes

continue to hydrolyze the soybeans and wheat and as a

result, a surplus of different types of sugars and amino

acids increases. These sugars and amino acids are con-

sumed by salt tolerant yeasts (Z. rouxii and C. versatilis).

Ethanol and higher alcohols, such as isobutyl alcohol,

isoamyl alcohol, methionol and 2-phenylethanol are syn-

thesized by Z. rouxii from the sugars, which are ample and

in wide variety present during the soy sauce mash fer-

mentation (Sluis et al. 2001).

We make important contribution to the identification

and understanding of the molecular biological and meta-

bolic functions of our newly isolated strain of halophilic

aromatic yeast C. etchellsii summarized as follows:

1. The ITS nucleotide sequence alignment method was

used to detect the sequence. This new strain which we

isolated from raw soy sauce was identified as C. etch-

ellsii by subjecting the sequence to NCBI-BLAST in

comparison with that of the C. etchellsii strain Miso

0208 (a typical high-salt-tolerant halophilic aromatic

yeast strain).

2. This yeast strain produced organic acids, amino acids

and volatile flavor compound which were analyzed by

the HPLC and 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), a-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) at 72 h, respectively,

were detected. Free and acid hydrolyzed amino acids at

levels of 3.7355 ± 0.0027 and 11.5604 ± 0.0037 g/l,

respectively, as well as 4-ethyl guaiacols and other

typical volatile flavor compounds were also detected.

Acknowledgments This work was supported by the National Key

Technology Research and Development Program during the 11th and

12th Five-Year Plan Period (No. 2008BAI63B06, No. 2007BAK36B03

and No. 2011BAD23B04) as well as Program of Introducing Talents of

Discipline to Universities (111-2-06).

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Table 5 Quantitative results of volatile flavor compounds of

C. etchellsii

Sort Name Content (g/l)

Alcohols Alcohol 3.6237 ± 0.0300

Isobutylalcohol 0.1406 ± 0.0040

Acetol 0.3173 ± 0.0050

Hexanol 0.0569 ± 0.0010

Octanol 0.0087 ± 0.0001

3-Methylthio-1-propanol 2.5152 ± 0.0200

Total (g/l) 6.6623 ± 0.0601

Esters Butyl formate 1.2333 ± 0.0100

Phenethylacetate 0.0270 ± 0.0030

Tetradecanoic acid ethylester 0.0016 ± 0.0004

Ethyl palmitate 0.0049 ± 0.0005

Isobutyl phthalate 0.3150 ± 0.0020

Total (g/l) 1.5817 ± 0.0159

Aldehydes 2-Methyl-2-butene aldehyde 1.9138 ± 0.0200

Caprylaldehyde 0.0385 ± 0.0030

Nonana 0.1440 ± 0.0030

Furfural 0.0140 ± 0.0010

Decanal 0.0081 ± 0.0001

Benzaldehyde 0.4315 ± 0.0050

Total (g/l) 2.5500 ± 0.0121

Phenols 2-Methoxy-phenol/guaiacol 0.1257 ± 0.0050

4-Ethyl-phenol 0.0169 ± 0.0020

Total (g/l) 0.1426 ± 0.0070

Heterocycles Methylpyrazine 0.1218 ± 0.0040

Ethylpyrazine 0.1533 ± 0.0040

2-Acetylfuran 1.6503 ± 0.0200

Total (g/l) 1.9254 ± 0.0280

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