Aroma-Active Compounds in Salt-Fermented Anchovy - ACS

Chapter 13

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 27, 2015 | http://pubs.acs.org Publication Date: July 1, 1997 | doi: 10.1021/bk-1997-0674.ch013

Aroma-Active Compounds in Salt-Fermented Anchovy 1

2

2,3

Y. J. Cha , G. H. Lee , and Keith R. Cadwallader 1

Department of Food Science and Nutrition, Changwon National University, Changwon, 641-773, Korea Department of Food Science and Technology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Box 9805, Mississippi State, MS 39762-5953 2

Volatile flavor compounds in salt-fermented anchovy with (koji) and without (control) added koji were analyzed by simultaneous steam distillation-solvent extraction/gas chromatography/mass spectrometry and gas chromatography/olfactometry. Ninety-eight volatile compounds were detected in control and 96 in koji. These included 59 odor-active compounds, such as 16 aldehydes, 8 esters, 4 ketones, 1 sulfur, 1 alcohol, 1 acid and 28 unknowns. Aldehydes and esters were found in the highest abundance in both samples. Alkylpyrazines (5) were found in koji only. Furthermore, the alcohols 3-methyl-1-butanol, 1-octen-3-ol and 2phenylethanol were generally at higher levels in koji than in control. Based on odor intensity and odor values of volatile compounds in both samples, the most potent odorants were 1-octen-3-one (mushroom, earthy), (Z)-4-heptenal (rancid, boiled potato), (E,Z)-2,6-nonadienal (cucumber, melon), 3-methylbutanal (dark chocolate), 3(methylthio)propanal (nutty, baked potato), ethyl 2-methylbutanoate (fruity, ripe apple), and ethyl 3-methylbutanoate (fruity, green apple). Other odorants, such as 1-penten-3-one (plastic bottle) and two unknowns (RI 1092 and RT 5.1 min), were more intense in control than koji treated samples. Salt-fermented fish, having a unique and desirable aroma and taste, has been favored by Koreans for centuries. These products are important protein and nitrogen sources because they contain about 10% (w/w) nitrogen, of which 80% is in the form of amino acids (1). The characteristic aroma and taste of these products are primarily formed during fermentation through protein and lipid degradation by autolytic and bacterial enzymes (2). Fermented fish products have been consumed as a condiment or as a seasoning added during the manufacture of Kimchi, a typical Korean pickled vegetable. Most fermented fish contain 25-30% salt and are matured for over 6 months by traditional methods. Economically, it would be more advantageous if fermentation time could be shortened 3

Corresponding author © 1997 American Chemical Society In Flavor and Lipid Chemistry of Seafoods; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

131


132

FLAVOR AND LIPID CHEMISTRY OF SEAFOODS

without causing undesirable spoilage or off-flavors. Furthermore, the common ripening agent in fermented fish products, sodium chloride, is known to be one factor that increases the potential of hypertension and renal disease (3). High salt content has effectively lessened the intake of fermented fish products (4,5). Therefore, attempts to reduce salt content and to reduce fermentation time of fermented fish products have been made (5-8). In certain areas of Korea addition of malt powder or cooked cereals to fermented fish is thought to enhance its flavor quality (7). Kunimoto et al. (9) reported that Aspergillus oryzae, which is useful in making koji, reduced fishy odor. Application of koji to make sardine meal was attempted by K i m et al. (10). As a consequence of consumer demands for natural seasonings rather than synthetic flavoring agents in processed foods, the Korean seafood industry has recently focused on developing natural seasoning agents from traditional salt-fermented fish products. This trend has in rum initiated additional studies of fermented fish products to improve their flavor quality. However, except for a few reports (11 -13), the volatile flavor of fermented fish has not been fully investigated. A n investigation of volatile flavor is essential if modified fermented fish products are to be successfully developed. The objective of this study was to identify and to compare odor-active components in low salt-fermented anchovy with (koji) and without (control) added koji during fermentation. Materials & Methods Sample Preparation. Fresh anchovy (Engraulis japonica) was purchased from a fish market in Chungmu, Korea and transported on ice in polyethylene bags to the laboratory within 1 h. A medium for koji production was prepared with 1:1:0.3 ratio (w/w) of boiled soybean, roasted barley and powdered dried anchovy, respectively. Fifty g of sterilzed mixture (121°C, 15 min) was inoculated with a culture of Aspergillus oryzae var. oryzae Murakami [made from about O.lg freeze dried A. oryzae (American Type Culture Collection, A T C C No. 22788) dissolved into 0.1 mL of sterilized saline water] and then incubated at 25°C for 3 days until mold growth was observed throughout the material. This procedure was repeated several times to attain the required koji feedstock. Salt-fermented anchovy was prepared as follows: control was produced by adding various additives to fresh anchovy, namely, 15% (w/w) salt, 6% (w/w) sorbitol, 0.5% (v/w) lactic acid and 5% (v/w) ethanol as preservatives and flavor enhancers; and koji was prepared as control except 5% (w/w) glucose and 10% (w/w) koji were added. These samples were ripened at 30°C for up to 4 weeks. Samples were homogenized using a Waring blender (Waring Products Co., Winsted, CT) before flavor analysis. Simultaneous steam distillation-solvent extraction (SDE). Homogenized fish paste (500g), distilled water (1.5 L) and 90.8jig of internal standard 2,4,6-trimethylpyridine (TMP; Aldrich Chemical Co., Milwaukee, WI) were placed in a Lickens-Nickerson (14) type SDE apparatus (Cat. No. K-523010-0000, Kontes, Vineland, NJ) to extract volatile flavor compounds into redistilled diethyl ether (100 mL). The procedure has been described elsewhere (15). Extracts were concentrated to 1.5 mL under a gentle stream of nitrogen. Each sample was extracted in duplicate.

In Flavor and Lipid Chemistry of Seafoods; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


13. CHA ET AL.

Aroma-Active Compounds in Salt-Fermented Anchovy

133

Gas chromatography/mass spectrometry (GC/MS). A 4uL aliquot from each SDE extract was injected into an HP 5790GC/5970B mass selective detector (MSD) (Hewlett Packard Co., Palo Alto, CA) by splitless mode (155°C injector temperature; 30s valve delay). Separation of volatile components was achieved on a fused silica open tubular (FSOT) column (Supelcowax 10; 60 m long x 0.25 mm i.d. x 0.25um d^; Supelco Inc., Bellefonte, PA). The linear velocity of the helium carrier gas was 25.7 cm/s. Oven temperature was programmed from 40°C to 175°C at a rate of 2°C/min with initial and final hold times at 5 and 30 min, respectively, then further increased to 195°C at a rate of 5°C and maintained for 25 min. Electron ionization energy was 70 eV, mass range was 33-300 a.m.u., electron multiplier voltage was 2000 V , and scan rate was 1.6/s. Other details of GC/MSD procedure have been described elsewhere (16). Duplicate analyses were performed on each SDE extract. Gas chromatography/olfactometry (GC/O). GC/O system consisted of a Varian series 3300 GC (Varian, Walnut Creek, CA) equipped with a flame ionization detector (FID) and a sniffing port. One uL of each extract was injected (splitless mode) into a 30 m x 0.32 mm i.d. x 0.25(im df Supelcowax 10 columm. Effluent from the end of the GC column was split 1:1 between FID and sniffing port. Further details of procedure have been reported elsewhere (17). Oven temperature was programmed from 40°C to 200°C at a rate of 6°C/min with initial and final hold times at 5 and 30 min, respectively. FID temperature was 250°C. Injector, sniffer port, and transfer line temperatures were maintained at 200°C. GC/O was performed on each extract by two trained panelists. Panelists were asked to assign odor properties and rate odor intensity of each compound using an 8-point scale (where 0 = no odor detected, 7 = very strong). Compound identification. Volatile compounds were identified by matching retention indices (RI) (18) and mass spectra of samples with those of authentic standards (Aldrich Chemical Co.). Tentative identifications were based on standard M S library data (Hewlett-Packard Co, 1988). The relative abundance of each compound was expressed by the ratio of its total ion peak area to that of the internal standard. Results & Discussion Volatile flavor components and odor intensity in salt-fermented anchovies made with (koji) and without (control) added koji were examined during fermentation. One hundred and fifteen volatile compounds were detected (Table I), including 34 aldehydes, 11 ketones, 19 alcohols, 24 esters, 6 nitrogen-contairiing compounds, 5 aromatic hydrocarbons, and 16 miscellaneous compounds (88 compounds were positively identified). As shown in Table I, 98 compounds were detected in control and 96 compounds in koji. Fifty-nine odor-active compounds, including 16 aldehydes, 8 esters, 4 ketones, 1 sulfur, 1 alcohol, 1 acid and 28 unknown compounds, were detected in both samples. Identification and odor description of these compounds are presented in Table n. Thirty-four aldehydes were identified in the control and koji (Table I). Levels of aldehydes increased with fermentation time in both koji and control. The aldehydes, 3-methylbutanal, hexanal, heptanal, (Z)-4-heptenal, (E,E)-2,4-heptadienal,


FLAVOR AND LIPID CHEMISTRY OF SEAFOODS

§3 p 5S

13

385 32558 3253383 333 3333s


33111 3s 3

325 !s33s

3S5§333

83 3

g 2 33111 222S3SS

SS I

fi 2 s S S i l BSSgS23

S

3333 332322s 2255335

3

§323 ! l s 2 S ? 2 53355JJS

333*33133 3^S2g5SS33 § § 5 ssigasgsi

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3395

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3233

I S 393§S s s l s s 3

jj8K8EssiI§S53iSSSi91IS9S



12 15 21 33 44 55 72 76 79 98 107

77 78 84 85 93 95 96 103 104 108 110

No.

6

e

e

e

e

e

e

(E,Z)-2,6-Nonadienal (E,E)-2,4-Octadienal (E)-2-Decenal Phenylacetaldehyde 2-Undecenal (E,E)-2,4-Decadienal 2-Chlorobenzaldehyde TetradecanaT a-Ethylidenephenylacetaldehyde Pentadecanal HexadecanaT Ketones 2,3-Butanedione l-Penten-3-one 2,3-Pentanedione 2-Heptanone l-Octen-3-one 2-Nonanone 1 -(2-Furany l)-ethanone (E,E)-3,5-Octadien-2-one 2-Undecanone Geranylacetone Pentadecanone*

Compound name by class

Table I. Continued

961 1015 1057 1177 1296 1389 1534 1567 1593 1850 2012

1582 1586 1639 1642 1746 1806 1813 1918 1928 2021 2128

RI* (RT)

1.93 0.29 3.97

3.13

6.63 0.95

10.81 2.38 3.88 2.25

1.89 0.41 0.39

3.83 40.15

31.84

2.71 1.78 1.00 0.62 1.11 2.60

C

S.D.

4.13 0.39 0.45

7.00 145.75

31.75

6.00 3.00 2.50 0.99 1.63 4.25

b

MAR

Oday

6.98 5.62

11.75 6.53

3.50

3.38 86.87

11.53

17.93 7.34 17.65 7.34 12.13 11.91

MAR

2.52 0.87

4.31 2.99

3.10

1.61 73.16

8.53

7.31 2.53 5.39 2.01 4.25 4.06

S.D.

13 day

Control

3.52 1.50 6.13 3.31 1.44

1.00 24.94

4.94

7.69 4.31 1.88 6.25 0.94 4.69

MAR

1.74 0.58 10.59 0.85 1.05

0.00 14.14

2.07

2.44 1.21 0.63 3.23 0.13 1.38

S.D.

30 day

1.74 1.34 0.71 0.51

0.87 10.42 1.52

2.05

0.98 0.55 0.20 0.16

0.46 8.74 0.42

1.02

5.22

1.33 2.25 2.13 2.13

14.92 1.33

4.50

38.96

7.00 2.00 3.96 1.58 12.29

0.81 0.50 1.77 0.49 3.00 0.94

2.09 1.14 2.55 0.64 6.03 1.08 14.19

3.96 2.42

MAR

1.41 0.71

S.D.

32.50 1.75

3.75

1.50 45.25

8.50 1.50 6.50 1.50 11.75

6.00 3.75

MAR

13.38 0.50

0.50

0.58 26.00

0.58 0.58 1.29 0.58 3.30

0.00 1.26

S.D.

30 day

0.47 3.00 0.82 1.26 2.00 0.82 1.31 2.50 1.29 1.31 1.00 0.00 Continued on next page

13.06 0.47

4.04

15.37

1.83 0.82 2.06 0.50 2.58

2.06 1.26

S.D.

13 day

Koji

2.05 1.39

MAR

Oday



5 10 11

30 32 36 42 45 46 51 61 64 68 75 81 86 92 99 102 106 113 115

No.

Alcohols Butanol l-Penten-3-ol 3-Methyl-l-butanol Pentanol (Z)-2-Penten-l-ol (E)-2-Penten-l-ol Hexanol l-Octen-3-ol Heptanol 2-Ethyl-l-hexanol Octanol (E>2-Octen-l-ol 2-Furanmethanol Decanol Benzylalcohol 2-Phenylethanol Dodecanol Pentadecanol Hexadecanol Esters Ethyl acetate Ethyl propanoate Ethyl 2-methyl propanoate


Table I. Continued

867 951 956

1139 1156 1204 1246 1310 1318 1351 1447 1454 1484 1552 1608 1656 1744 1874 1907 1971 2299 2376

a

RI (RT)

2.72 5.68

5.27 3.84 0.41 1.08

5.75 8.25

11.75 4.25 1.00 2.5

358.75 287.26 3.16 4.09

11.44 2.79 7.93

0.48 0.82 3.69

1.38 2 5.75

59.93 9.50

1.80 1.28 1.70

76.74 3.63 4.83 1.28 6.21

MAR

12.94

C

S.D. S.D.

47.85 7.32

0.40 0.53 1.01

3.44 0.85 2.45

16.49 2.30 3.54 0.53 1.81

13 day

27.00

b

MAR

Oday

Control

3.00 2.97

2.41 0.25 1.55

2.32 2.89 2.50 0.25

10.50 0.25 0.55 1.03 1.48

S.D.

219.56 140.22 8.25 2.50

2.50 5.00

6.81 1.13 1.69

5.88 3.50 4.63 0.88

31.25 0.88 1.19 1.69 2.44

MAR

30 day

125.51 8.23 3.48

108.38 2.50 2.18

1.92 0.23 0.43 0.09

1.18 0.57 0.73 0.50 0.67

2.54 1.30 5.15 1.75 2.83

2.24 0.52 0.48 0.39

0.16 10.97 4.46 0.36

S.D.

0.49 14.54 6.43 0.81

MAR

Oday

13 day

Koji

0.47

1.33

0.50

0.00 6.19 0.50

1.00 49.50 1.75 1.25

0.58 3.86 3.86 2.06

1.73 6.27 1.89

S.D.

1.50 14.25 4.25 6.75

21.50 23.00 2.25

MAR

30 day

302.88 172.30 313.00 121.58 4.50 3.57 15.50 9.85 30.79 20.99 39.50 5.69 Continued on next page

13.48 0.96

0.25 3.70 1.07 3.68

6.22 4.86 0.50

S.D.

29.46 1.58

1.21 9.50 2.08 5.67

15.00 14.54 1.25

MAR


1

i

8

H

o

i

I LVOR


e

6

6

e

0

6

e

6

e

6

c

e

1031 1047 1062 1130 1230 1333 1344 1435 1535 1633 1674 1736 1784 1840 1892 1939 2049 2143 2255 2357 2456

6

Ethyl butanoate Ethyl 2-methyl butanoate Ethyl 3-methyl butanoate* Ethyl pentanoate Ethyl hexanoate Ethyl heptanoate Ethyl 2-hydroxypropanoate Ethyl octanoate Ethyl nonanoate Ethyl decanoate Ethyl 3-(2-furyl)propanoate Ethyl undecanoate Ethyl phenylacetate Ethyl dodecanoate Methyl 2,8-dimethylundecanoate Ethyl tridecanoate Ethyl tetradecanoate Ethyl pentadecanoate Ethyl hexadecanoate Ethyl heptadecanoate Ethyl octadecanoate

17 20 22 27 40 49 50 60 73 83 88 91 94 97 100 105 109 111 112 114 116

a

RI (RT)


No.

Table I. Continued

65.12 4.53 54.26

2.00 1.19 4.63 2.07 0.75 0.41 50.73 0.75 1.58 1.19

C

S.D.

3.21 24.87 101.78

2.54 2.22 3.86 0.72 3.54 0.47 36.96 1.60 6.97 0.99 2.64 0.47 1.88 33.76

S.D.

7.75 2.87 653.63 360.60 32.13 9.75 326.88 271.93 11.69 9.13 22.56 6.36

1.76

3.14 1.31 8.88 32.21

2.19 3.75 4.25 1.44 4.50 1.31 48.94 2.31 4.56 3.69 11.19 1.31 6.06 69.50

2.34 1.37 6.97 1.50 3.32 1.09 7.35 2.55

4.78 3.00 10.38 2.50 10.02 1.84 22.73 4.68

MAR

MAR

30 day

S.D.

13 day

5.72 10.00 7.93 11.73 252.75 282.57 1360.67 411.40 48.75 16.46 115.58 79.22 104.63 72.35 1432.64 700.63 19.13 11.01 16.70 10.68 29.00 13.34 35.83 35.06

37.88 9.00 75.63

2.21 2.53 8.07 2.13 1.63 1.00 102.00 1.63 2.00 3.75

b

MAR

Oday

Control

0.71 4.09 6.76 0.65 1.46 75.72 5.80 77.94 0.62 2.46

1.50 2.58 7.51 1.20 0.47 0.53 19.22 0.46 0.56 1.92

2.82 3.80 9.75 1.65 1.59 1.03 29.73 3.66 1.18 3.03 1.28 6.70 23.54 0.88 3.54 221.38 14.13 219.30 2.24 4.69

S.D.

MAR

Oday

3.82 2.63 2.18 1.55 3.52 0.42 23.61 2.71 0.50 5.10

11.56 4.19 1.93 0.50 7.27 0.50 4.57 0.96 0.96 2.06

S.D.

4.50 0.58 70.75 11.70 146.00 31.84 3.20 4.25 12.75 9.46 1194.25 363.60 85.50 53.31 215.75 60.29 3.70 21.50 62.00 41.86

40.50 19.50 10.10 2.25 11.25 2.25 28.75 11.25 2.75 12.25

MAR

30 day

Continued on next page

5.88 3.12 38.46 18.39 121.58 49.48 4.96 2.08 15.83 6.69 643.71 274.33 58.04 12.49 469.42 172.43 16.38 9.91 25.54 12.85

15.38 7.45 4.33 2.88 4.46 1.46 33.38 7.00 1.58 8.42

S.D.

13 day

Koji

MAR


2 3 6 9 14 16 39 53

18 25 28 29 34

38 47 52 54 57 65 67

STo.

e

d

6

N-Containing compounds 2,4-Dimethylpyridine 2,6-Dimethylpyrazine 2,4,6-Trimethylpyridine (I.S.) 2-Ethyl-6-methylpyrazine Trimethylpyrazine 2-Ethyl-3,5-dimethylpyrazine Tetramethylpyrazine Aromatic hydrocarbons Toluene Ethylbenzene p -Xylene tn -Xylene o -Xylene Miscellaneous compounds Octane 1,2-Dimethy lcyclohexane Nonane 2-Ethylfuran Decane 2-Propylfuran 2-Pentylfuran 2,4,5-Trimethylthiazole


Table I. Continued

812 827 891 940 1002 1024 1227 1374

1037 1119 1132 1134 1178

1216 1320 1363 1384 1400 1461 1471

RT (RT)

2.25 0.46

1.00

b

MAR

Oday

1.19 0.45

0.00

C

S.D.

48.79 4.49 15.83 130.75 5.43 9.69 26.83 0.88

13.31 2.20 2.61 4.58 2.77

1.00

5.70

MAR

4.80 1.05 1.34 2.73 1.70

0.00

3.16

S.D.

23.68 2.48 14.53 51.17 2.03 3.30 9.29 0.25

13 day

Control

2.42 10.56 6.04 1.97 0.13 2.17 0.00 3.19 1.06 5.69 1.00

1.55

1.69 4.00 9.44 9.06

2.19 0.55 0.71

0.00

S.D.

3.81 1.19 1.50

1.00

MAR

30 day

6.19

6.73

3.18 0.18

1.29

6.25 0.83

10.88

5.13

2.25 2.38

1.33

2.00 1.00 1.13 5.50 6.96 25.42

MAR

In Flavor and Lipid Chemistry of Seafoods; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997. 6.84

2.59

1.26 1.11

0.47

0.82 0.00 0.25 1.73 3.33 8.63

S.D.

25.00 50.00 3.50

17.00

2.50 1.25

3.00 1.00

3.00 1.00 1.00 6.50 8.50 36.00

MAR

30 day

21.40 43.42 1.73

4.97

1.29 0.50

1.8 0.00

0.82 0.00 0.00 1.73 4.80 4.97

S.D.

5.19 6.25 1.50 1.00 0.00 0.33 Continued on next page

13 day

Koji

B

>

S

i

i

LIPID CH]

4.02 0.38

2.30

0.15

0.80

2.53

0.53

0.00

S.D.

1.00

MAR

Oday


/OR

Aroma-Active Compounds in Salt-Fermented Anchovy - ACS

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