Volatile Compounds Analysis and Off-Flavors Removing of Porcupine Liver

Abstract

Porcupine liver was an important source of protein, fat, and vitamins, and was of high economic and food value. But its stronger off-flavor which occurred during cooking was an impediment to consumption of liver products. In this study, the volatile compounds of porcupine liver were analyzed by headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). Results showed that hexanal (grassy), (Z)-2-heptenal (fishy), (E)-2-octenal (cardboard-like), 1-octen-3-one (metallic), and 1-octen-3-ol (mushroom-like) were responsible for the off-flavors of porcupine liver. The effects of removing off-flavors were compared by embedding with β-cyclodextrin (β-CD) and fermentation with yeast. Results indicated that fermentation with yeast was better and both the number and area ratio of volatile compounds in porcupine liver decreased with the addition of yeast. Taken together, the off-flavors of porcupine liver were removed by fermentation with the yeast effectively.

Introduction

Porcupines, raised in Hunan Province of China, are of high economic and food value. The porcupine meat, a kind of high-protein low-fat meat, is favored by more and more people. However, with porcupine meat-products produced, a large amount of porcupine by-products are left. Among the by-products, liver is the main. The waste of porcupine liver causes farmers an economic loss and leads to environmental pollution. Animal liver is an important source of protein, fat, and vitamins, particularly vitamins A, D, E and vitamin B complex (Martin-Sanchez et al., 2013). Porcupine liver has higher mineral content, particularly iron, than most other animal liver. In addition, the animal liver is often used in high-price food (Straßer and Schieberle, 2013). Therefore, porcupine liver products should be produced and consumed.

Volatile compounds of liver play an important role in the quality of liver products. Liver flavor has been one of the crucial factors through sensory studies based on consumer preference (Sunarharum et al., 2014). The contribution of compounds to off-flavors of other animal liver is taken into consideration (Lorenz et al., 1983; Mussinan and Walradt, 1974; Straβer and Schieberle, 2013), but investigations on the volatiles of porcupine liver are rather scarce. Mussinan and Walradt (1974) identified a total of 179 volatile compounds in cooked pork liver. The largest class of compounds detected were pyrazines, followed by furans and aldehydes. However, the contribution of single compounds to the overall off-flavors was not investigated. Im and Kurata (2003) found that the volatile compounds identified in porcine liver was acids, esters, aldehydes, ketones, alcohols, phenols, thiazoles, pyrazines and furans. 1-octen-3-one and hexanol were considered to be responsible for the fishy odor and other identified major volatiles with their off-flavors were as follows: (E,E)-2,4-heptadienal, (E)-2-octenal, (E)-2-nonenal, (Z)-4-decenal and (E,E)-2,4-decadienal.

However, the stronger characteristic off-flavors of porcupine liver which occur during cooking is an impediment to consumption of liver products. This is also the reason why animal liver constitutes a small portion of meat products and meat by-products consumed in Asia. In the study of Im and Kurata (2003), it was confirmed that the fishy flavor in liver was not completely removed even after subjecting to heat treatment. No detailed information was yet available for the better method on the off-flavors removed in liver. The feasibility of improving the acceptability of liver by employing different cooking methods to eliminate undesirable flavor is reported (Kimura et al., Kimura et al., 1990; 1995), but the result is unpleasant.

The methods of removing off-flavors consist of physical, chemical and biological deodorization. The major physical deodorization is embedding with β-cyclodextrin (β-CD) which is hydrolyzed by gastric acid and digested easily (Shieh and Hedges, 1996). The fact that β-CD has been approved as a safe ingredient for food applications makes β-CD a promising candidate for removing off-flavors (Arora and Damodaran, 2010). β-CD, containing peculiar hydrophobic cavum, can form inclusion complexes with various off-flavor molecules. Previously, β-CD was used to remove volatile beany flavor compounds from soy milk (Arora and Damodaran, 2010). The chemical deodorization is taken no consideration due to its chemical residues. By adopting biological deodorization, odor can be eliminated at a low monetary cost (Yan et al., 2013). Therefore, biological deodorization (fermentation) has become a hot topic in the study of odor control. A bacterium, identified as Staphylococcus xylosus, could change notes of an odor in fish sauce made in Thailand (Fukami et al., 2004). As a result of sensory evaluation, fishy, sweaty and fecal notes of the fish sauce treated with the bacterium were all weaker than those of the untreated fish sauce.

The objective of this study was to evaluate the volatile compounds contributing characterizing off-flavors in porcupine liver, using head space solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) analysis. Furthermore, the ability of embedding with β-CD and fermentation with yeast reducing or removing off-flavors of porcupine liver was also performed to avoid economic loss.

Materials and Methods

Material preparation    Porcupine liver was obtained from the porcupine farm (Changde, China) and kept frozen until used. Porcupine liver (50 g) was homogenized with 100 mL of distilled water through an organization blender (5000 r/min, 3 min) and transferred to a 500-mL flask until required. The porcupine liver had a proximate composition of 70.5% moisture, 15.8% crude protein, 6.9% fat and 0.8% ash, as determined by standard procedures.

Off-flavors removal    Two different methods, embedding and fermentation were respectively performed to remove off-flavors of porcupine liver. The embedding method was as follow: the complex (50 g) of porcupine liver and distilled water was heated at a bath temperature of 60°C with addition of β-CD (2.0%); sample vial was placed into the bath for 60 min with stirring constantly. The fermentation method was as follow: high active dry yeast (1.0%) was added to the complex (50 g) of porcupine liver and distilled water, heating at the bath temperature of 35°C for 45 min with stirring constantly. The reduction in odor strength by embedding and fermentation was investigated by attribution using a relative amount for each compound identified.

HS-SPME analysis    A SPME fiber (75 µm thickness, PDMS), purchased from Supelco, was used to extract volatile compounds. Fiber coated with PDMS was better in recognizing the contribution/concentration of single dominant volatile compounds in the previous experiment (Milosavljević et al., 2012). Before using for the extraction of the samples studied, the fiber was preconditioned according to the instruction in the GC injection port at 260°C for 20 min. The sampling procedure involved placing porcupine liver (3 g) into a glass vial (15 mL) and sealing with a screw-top septum-containing cap. Sample vial was placed into an ultrasonic bath for 20 min. After the equilibration between the matrix and headspace, the fiber was inserted into the sample vial through the septum and exposed to the headspace for 30 min at 50°C. Afterwards, the SPME fiber was inserted into the injector of a GC and the volatile compounds were desorbed for 3 min at 250°C.

GC-MS conditions    All determinations were carried out on a GC-MS system constituted by a SCION SQ 456 Series (BRUKER, USA) gas chromatograph interfaced with mass spectrometer detector. Volatile compounds were separated using a DB-WAX capillary column (30 m × 0.25 mm × 0.25 µm; J&W Scientific Inc., Folsom, CA, USA), consisting of 100% polyethylene glycol. Helium was the carrier gas at a constant flow of 0.9 mL·min−1. The column was submitted to the following temperature program: Initial temperature was 40°C, held for 3 min, ramped at 5°C·min−1 to 90°C, ramped at 10°C·min−1 to 230°C, held at 230°C for 7 min. Mass spectrometer was operated in electron ionization mode (70 eV), with a multiplier voltage of 1000 V. It was carried on in the mass range of m/z 33 – 450 to detect the ions at 200°C ion source temperature. The volatile compounds were identified by comparing the NIST and WILEY library and Kovats retention indices (RI). The RI of compounds was calculated based on a homologous series of alkanes (C6-C30) (Sigma-Aldrich, St. Louis, MO).

Results and Discussion

Volatile compounds of porcupine liver    A typical chromatogram of volatile compounds associated with untreated porcupine liver was obtained by the HS-SPME collection on GC-MS. Table 1 listed the identified flavor compounds in porcupine liver. The results of quantitative analysis of the volatile compounds were also shown in Table 1. Results of the area ratio of major classes of compounds in volatiles of porcupine liver were as follows: acids (21.688%), alcohols (20.779%), ketones (18.707%), aldehydes (16.719%), hydrocarbons (6.025%), furans (1.306%) and esters (0.702%).

Table 1. Volatile compounds in porcupine liver collected by HS-SPME and identified using GC-MS.

No.	RIa	Compounds	Chemical formula	Area ratiob/%
1	803	Octane	C8H18	1.185
2	843	Acetone	C3H6O	11.655
3	878	1-Octene	C8H16	0.645
4	890	2-Octene	C8H16	0.253
5	912	2-Butanone	C4H8O	0.228
6	936	Benzene	C6H6	0.366
7	970	2,4-Hexadienal	C6H8O	0.206
8	975	Pentanal	C5H10O	0.713
9	980	methyl-Benzene	C7H8	0.626
10	1023	2,3-Pentanedione	C5H8O2	0.189
11	1066	Hexanal	C6H12O	11.405
12	1089	ethyl-Benzene	C8H10	0.293
13	1123	2-n-butyl-Furan	C8H12O	0.271
14	1134	p-Xylene	C8H10	0.214
15	1165	2-Heptanone	C7H14O	1.195
16	1194	1-Penten-3-ol	C5H10O	0.245
17	1230	2-pentyl-Furan	C9H14O	1.036
18	1234	6-methyl-2-Heptanone	C8H16O	1.112
19	1240	Bicyclo[4.2.0]octa-1,3,5-triene	C8H8	2.066
20	1245	1,2,4-trimethyl-Benzene	C9H12	0.140
21	1254	1-Pentanol	C5H12O	1.912
22	1272	Octanal	C8H16O	0.798
23	1281	1-Octen-3-one	C8H14O	0.248
24	1305	2,3-Octanedione	C8H14O2	3.402
25	1320	(Z)-2-Heptenal	C7H12O	0.504
26	1331	(Z)-2-Penten-1-ol	C5H10O	0.747
27	1338	1-Hexanol	C6H14O	1.197
28	1372	Nonanal	C9H18O	2.085
29	1379	(E)-3-Octen-2-one	C8H14O	0.115
30	1406	(E)-2-Octenal	C8H14O	0.349
31	1428	1-Octen-3-ol	C8H16O	13.389
32	1430	Acetic acid	C2H4O2	8.356
33	1443	(5Z)-Octa-1,5-dien-3-ol	C8H14O	0.405
34	1449	1-Hexanol, 2-ethyl-	C8H18O	0.281
35	1452	(E,E)-3,5-Octadien-2-one	C8H12O	0.152
36	1516	(E)-2-Nonenal	C9H16O	0.209
37	1529	2,4-dimethyl-Cyclohexanol	C8H16O	0.394
38	1532	Propanoic acid	C3H6O2	0.676
39	1553	1-Octanol	C8H18O	0.360
40	1575	1,2-Propanediol	C3H8O2	0.185
41	1581	1-Methylcycloheptanol	C13H11O	0.856
42	1585	(E)-2-Octen-1-ol	C8H16O	0.811
43	1595	beta.-Cyclocitral	C10H16O	0.239
44	1610	dihydro-2(3H)-Furanone	C4H6O2	0.352
45	1616	1-phenyl-Ethanone	C8H8O	0.199
46	1628	2-methyl-Butanoic acid	C5H10O2	0.406
47	1645	.gamma. Hexalactone	C6H10O2	0.216
48	1701	(E)-2,2-dimethyl-4-Decene	C12H24	0.453
49	1721	2-methyl-Pentanoic acid	C6H12O2	0.529
50	1818	Hexanoic acid	C6H12O2	11.415
51	1841	trans-.beta.-Ionone	C7H14O2	0.214
52	1933	Heptanoic acid	C7H14O2	0.307
53	1967	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro	C11H16O2	0.134

^a  RI: Retention indices were determined by using a series of hydrocarbons on the DB-WAX column described under “Materials and Methods” section.

^b  Area ratio: The area ratio was the average of two measurement.

The unsaturated aldehydes had the lowest sensory thresholds and were usually considered as the primary sources of oxidized off-flavors (Refsgaard et al., 1999). Yoshiwa et al. (1997) believed 2,4-hexadienal and 2-heptenal were the main volatile compounds of sardine for fishy odor. Furthermore, (E)-2-octenal also had impact on off-flavors due to its cardboard-like odor. 2,4-hexadienal, (Z)-2-heptenal, (E)-2-octenal, 2-nonenal and β-cyclocitral were detected as unsaturated aldehydes in porcupine liver. Hexanal, which was responsible for grassy and fatty odor, contributed to painty flavor of oxidized fish oils (Im et al., 2004; Karahadian and Lindsay 1989; Wang et al., 2012). As reported in earlier publications, hexanal was found in drinking water distribution system and played a major role for the taste and odor (Yiqi and Zijian, 2006). Therefore, aldehydes (16.719%) were thought to be primarily responsible for the off-flavors in porcupine liver and (E)-2-octenal (0.349%), hexanal (11.41%) and (Z)-2-heptenal (0.504%) were the major off-flavor compounds (Table 1).

The ketones in volatile compounds, fatty and burning flavor, played an enhanced role for off-flavors. 1-octen-3-one was detected to be the compound responsible for the metallic flavor in oxidized butter oil and fresh whitefish (Josephson et al., 1983; Widder and Grosch R 1994). Many compounds, such as 1-octen-3-one, that contributed to meat smell and flavor were lipid breakdown products (Calkins and Hodgen, 2007). 1-octen-3-one (0.248%) was characterized as off-flavor compounds responsible for a metallic and mushroom-like aroma in porcupine liver (Table 1).

The unsaturated alcohols with lower thresholds made a greater contribution to the off-flavors (Refsgaard et al., 1999). 1-octen-3-ol and 1,5-octadien-3-ol appeared to have the most potential impact on fresh fish aromas. 1-octen-3-ol, exhibiting a distinct mushroom-like, was a product of linoleic acid hydroperoxide degraded (Im et al., 2004; Josephson et al., 1983). Publications (Wang et al., 2012) reported that 1-octen-3-ol contributed to the earthy-musty odor. The saturated alcohols, such as 1-pentanol and 1-octanol, were irresponsible for porcupine liver flavors. Therefore, 1-octen-3-ol (13.39%) was the major contributor to the off-flavors of porcupine liver (Table 1).

The hydrocarbons were also identified in volatile compounds of porcupine liver and alkanes of these, associated with off-flavors in porcupine liver, was not determined. However, some olefins, such as 1-octene and 2-octene, might form aldehydes or ketones under certain conditions. Therefore, the olefins were thought to have potential to impact the off-flavors. Many acids, detected in the experiment, were products of aldehydes oxidized. The acids had little impact on off-flavors of porcupine liver, to some extent. Acetic acid, hexanoic acid and propanoic acid were descripted as vinegar, fatty and unpleasant (Im et al., 2004). Twenty three furan derivatives in pressured cooked pork liver (Mussinan and Walradt, 1974), as contributors to some cooked flavor, were identified. 2-butylfuran and 2-pentylfuran were detected in this study and might be important flavor materials for porcupine liver.

Removal of off-flavor compounds    The number and area ratio analysis of major classes of compounds in volatiles of porcupine liver deodorized by embedding and fermentation was respectively shown in Figure 1 and Figure 2. The average number of aldehydes, alcohols and acids increased in porcupine liver processed by embedding with β-CD (Figure 1) and the average area ratio also increased (Figure 2). Only the area ratio of ketones, acids, hydrocarbons and furans decreased (Figure 2). The reason for increase in embedding was that the embedding temperature led to some other volatile compounds produced. However, after deodorization by fermentation, the average area ratio of aldehydes, ketones, acids, hydrocarbons and furans decreased 14.998%, 14.205%, 19.225%, 5.462% and 0.689% (Figure 2). Because the major contributor to the off-flavors was decreased, fermentation had a positive impact on removing off-flavors in porcupine liver.

Fig. 1.

The number analysis of major classes of compounds in volatiles of porcupine liver untreated or treated by embedding and fermentation.

Fig. 2.

The area ratio analysis of major classes of compounds in volatiles of porcupine liver untreated or treated by embedding and fermentation.

The major off-flavor compounds with strong odors in porcupine liver were shown in Table 2. The fermentation treatment decreased the area ratio of hexanal, (Z)-2-heptenal, (E)-2-octenal, 1-octen-3-ol and acids (Table 2). However, the area ratio of alcohols and esters increased, especially ethanol (Table 2). The reason why ethanol increased greatly was that yeast could break sugar in the liver to ethanol. The fermentation significantly decreased levels of the off-flavor compounds, particularly 1-octen-3-one and (Z)-2-heptenal, the main sources of off-flavors in porcupine liver, were undetectable after fermentation (Table 2). The current study demonstrated that fermentation could also reduce unwanted odor compounds from the liver, as Seo et al. (2012) found in sea tangle. Therefore, this process could contribute to the development of more easily accepted food and raise the value of raw-food material.

Table 2. The area ratio analysis of major off-flavor compounds with strong odors in porcupine liver untreated or treated by embedding and fermentation.

Compounds	Untreateda	Embeddinga	Fermentationa
Hexanal	11.405	20.000	0.924
Nonanal	2.085	6.161	0.581
Octanal	0.798	1.423	0.183
(Z)-2-Heptenal	0.504	2.023	0.132
(E)-2-Octenal	0.349	2.297	0.117
1-Octen-3-one	0.248	1.112	ND
1-Octen-3-ol	13.389	13.000	8.092
Hexanoic acid	11.415	8.374	1.972
Acetic acid	8.356	3.240	ND
2-Pentylfuran	1.036	1.250	0.518

ND means area ratio not detected.

^a  The area ratio of untreated, embedding and fermentation was the average of two measurement

The reason for affecting the embedment with β-CD might be the fact that hydrophobic cavity of β-CD was not large enough to form inclusion complexes with various off-flavor molecules. Besides, the temperature extracting volatile compounds might affect the result of deodorization. The embedding temperature led to some other compounds produced by origin volatile compounds conversion. According to the effect, the fermentation method showed an optimal deodorizing effect. Fermentation technology could remove off-flavor volatile compounds, and further flavor. However, the mechanism of yeast deodorization was not clear, the possible mechanism was: the major off-flavor compounds such as aldehydes and ketones participated in metabolism of yeast and were converted to some compounds without off-flavors; the yeast had a set of intracellular and extracellular enzyme system and some enzymes produced by yeast modified the molecular structure of off-flavor compounds to remove the off-flavors. Some studies had shown that enzymes produced by microbes could change the food flavor and remove the odor. Schroeder et al. (2008) indicated that the enzymatic treatment of apple juice using lactase resulted in the removal of off-flavors that was mainly from oxidizing phenolic compounds. It has been also reported that the removal of off-flavors in the sea tangle extract was associated with the degradation of odor compounds by enzymes produced by A. oryzae (Seo et al., 2012).

Conclusions

In summary, a total of 53 compounds were identified by comparing the NIST and WILEY library and retention indices. According to GC-MS analysis, hexanal, (Z)-2-heptenal, (E)-2-octenal, 1-octen-3-ol and 1-octen-3-one were the main contributors to the off-flavors of porcupine liver. This study also indicated the off-flavor compounds were not completely removed by embedding or fermentation, but fermentation with yeast had a more positive effect than embedding with β-CD. Further study was needed to demonstrate the mechanism of speculation about the yeast how to remove the off-flavors.

Practical Application

Volatile compounds analysis and off-flavors removing of porcupine liver were studied, thus the porcupine liver could be used for eating. What's more, the waste of porcupine liver and the economic loss could be avoided.

References

•  Arora,  A. and  Damodaran,  S. (2010). Competitive binding of off-flavor compounds with soy protein and β-cyclodextrin in a ternary system: a model study. J. Am. Oil Chem. Soc., 87, 673-679.
•  Calkins,  C.R. and  Hodgen,  J.M. (2007). A fresh look at meat flavor. Meat Sci., 77, 63-80.
•  Fukami,  K.,  Funatsu,  Y.,  Kwasaki,  K., and  Watabe,  S. (2004). Improvement of fish-sauce odor by treatment with bacteria isolated from the fish-sauce mush (Moromi) made from frigate mackerel. J. Food Sci., 69, M45-M49.
•  Im,  S.,  Hayakawa,  F., and  Kurata,  T. (2004). Identification and sensory evaluation of volatile compounds in oxidized porcine liver. J. Agric. Food Chem., 52, 300-305.
•  Im,  S. and  Kurata,  T. (2003). Characterization of off-flavors in porcine liver collected by SDE. Food Sci. Technol. Res., 9, 338-341.
•  Josephson,  D.B.,  Lindsay,  R.C., and  Stuiber  D.A. (1983). Identification of compounds characterizing the aroma of fresh whitefish. J. Agric. Food Chem., 31, 326-330.
•  Karahadian,  C. and  Lindsay,  R.C. (1989). Evaluation of compounds contributing characterizing fishy flavors in fish oils. J. Am. Oil Chem. Soc., 66, 953-960.
•  Kimura,  T.,  Fukuya,  Y., and  Kagaya,  M. (1990). Changes in properties of chicken liver cured in aged sake lees. Journal of Cookery Science of Japan, 23, 267-274 (in Japanese).
•  Kimura,  T.,  Fukuya,  Y.,  Kagaya,  M., and  Sugahara,  T. (1995). Effect of addition of sodium chloride on chicken liver cured in aged sake lees. Journal of Cookery Science of Japan, 28, 218-223 (in Japanese).
•  Lorenz,  G.,  Stern,  D.J.,  Flath,  R.A.,  Haddon,  W.F.,  Tillin,  S.J., and  Teranishi,  R. (1983). Identification of sheep liver volatiles. J. Agric. Food Chem., 31, 1052-1057.
•  Martin-Sanchez,  A.M.,  Ciro-Gomez,  G.,  Sayas,  E.,  Vilella-Espla,  J.,  Ben-Abda,  J., and  Perez-Alvarez,  J.A. (2013). Date palm by-products as a new ingredient for the meat industry: application to pork liver pâté. Meat Sci., 93, 880-887.
•  Milosavljević,  N.P.,  Blagojević,  P.D.,  Savić,  D.S., and  Radulović,  N.S. (2012). Application of HS-SPME–GC-MS-derived variables for monitoring ripening-induced changes in cheese volatile compounds. Dairy Sci. Technol., 92, 321-333.
•  Mussinan,  C.J. and  Walradt,  J.P. (1974). Volatile constituents of pressure cooked pork liver. J. Agric. Food Chem., 22, 827-831.
•  Refsgaard,  H.H.F.,  Haahr,  A.M., and  Jensen,  B. (1999). Isolation and quantification of volatiles in fish by dynamic headspace sampling and mass spectrometry. J. Agric. Food Chem., 47, 1114-1118.
•  Schroeder,  M.,  Pollinger-Zierler,  B.,  Aichernig,  N.,  Siegmund,  B., and  Guebitz,  G.M. (2008). Enzymatic removal of off-flavors from apple juice. J. Agric. Food Chem., 56, 2485-2489.
•  Seo,  Y.S.,  Bae,  H.N.,  Eom,  S.H.,  Lim,  K.S.,  Yun,  I.H.,  Chung,  Y.H.,  Jeon,  J.M.,  Kim,  H.W.,  Lee,  M.S.,  Lee,  Y.B., and  Kim,  Y.M. (2012). Removal of off-flavors from sea tangle (Laminaria japonica) extract by fermentation with Aspergillus oryzae. Bioresour. Technol., 121, 475-479.
•  Shieh,  W.J. and  Hedges,  A.R. (1996). Properties and applications of cyclodextrins. J. Macromolecular Sci., 33, 673-683.
•  Straβer,  S. and  Schieberle,  P. (2013). Characterization of the key aroma compounds in roasted duck liver by means of aroma extract dilution analysis: comparison with beef and pork livers. Eur. Food Res. Technol., 238, 307-313.
•  Sunarharum,  W.B.,  Williams,  D.J., and  Smyth,  H.E. (2014). Complexity of coffee flavor: A compositional and sensory perspective. Food Res. Int., 62, 315-325.
•  Wang,  Y.,  Zhang,  M,.  Mujumdar,  A.S., and  Mothibe,  K.J. (2012). Quality changes of dehydrated restructured fish product from silver carp (hypophthalmichthys molitrix) as affected by drying methods. Food Bioprocess Technol., 6, 1664-1680.
•  Widder,  S. and  Grosch  R, W. (1994). Study on the cardboard off-flavor formed in butter oil. Zeitschrify Fur Lebensmittel-untersuchung Und-forschung, 198, 297-301.
•  Yan,  Z.,  Liu,  X.,  Yuan,  Y.,  Liao,  Y., and  Li,  X. (2013). Deodorization study of the swine manure with two yeast strains. Biotechnol. Bioproc. E., 18, 135-143.
•  Yiqi,  Z. and  Zijian,  W. (2006). Extraction and analysis on fishy odor-causing compounds in the different part of carp. Chinese J. Anal. Chem., 34, S165-S167.
•  Yoshiwa,  T.,  Morimoto,  K.,  Sakamoto,  K.,  Ishikawa,  Y.,  Tokita,  M., and  Morita,  M. (1997). Volatile compounds of fishy odor in sardine bysimultaneous distillation and extraction under reduced pressure. Nippon Suisan Gakkaishi., 63, 222-230.