2.2. The Effect of SPME Fiber Coating on Analytical Results
From the point of view of the number of identified compounds, it is clear that the amount of identified compounds is the largest using Carboxen/PDMS for extracting volatile flavor constituents in FST1 and FST2. The result is agreement with Yu’s, who analyzed the volatile compounds in traditional smoke-cured bacon with different fiber coatings using SPME and found that Carboxen/PDMS showed the best results [11
]. Maybe Carboxen/PDMS is the most suitable fiber for extracting volatile flavor compounds in food among the four fibres.
The effect of PDMS/DVB is moderate. It can extract alcohol, phenol, carboxylic acids, ester, sulfide and heterocycle compounds. However, alkene compounds are not identified; the reasons are in two aspects. One is that the contents of alkenes are lower; the other is that PDMS/DVB may be not suitable for extracting alkene compounds.
The effect of DVB/CAR/PDMS is poorer than that of PDMS/DVB, and organic acids are not identified. At the same time, the number of aldehyde and ketone compounds identified is also much less.
Of the four fibers used in this work, when PDMS was used for extracting volatile compounds, the number of identified compounds was the least. Aldehyde and ketone compounds and alkene compounds are not identified; perhaps PDMS is not fit for extracting these three kinds of organic compounds. However, the three organic acids are all identified by using PDMS.
From the point of view of the relative peak area of identified compounds, the extract efficiency of four fibers on alcohols and phenol in FST1 and FST2 is close. The sum of relative peak area of alcohols and phenol in FST2 is bigger than that in FST1. It is 12.82–24.94% in FST2, but it is 9.31–17.93% in FST1. The results showed that the content of alcohols and phenol in FST2 might be indeed higher than that in FST1.
Identification of organic volatile flavor compounds in FST1 and FST2 using SPME.
Identification of organic volatile flavor compounds in FST1 and FST2 using SPME.
|Volatiles||CAS#||RI/RI *a||Qual b||I method c||Carboxen/PDMS||PDMS/DVB||DVB/CAR/PDMS||PDMS|
|Peak area (%)||Peak area (%)||Peak area (%)||Peak area (%)|
|60 min||60 min||30 min||30 min||60 min||60 min||75 min||75 min|
|Alcohols and phenol|
|Butyl propanoate ||590-01-2||909/910||83||MS,RI||0.44||ND||0.47||0.11||0.15||ND||0.40||ND|
|Aldehyde and ketone|| || || || || || || || || || || || |
|Methyl (methylthio) methyl disulfide||42474-44-2||1129/1139||86||MS,RI||0.19||0.11||0.13||0.21||ND||ND||ND||ND|
The extract efficiency of Carboxen/PDMS and PDMS/DVB on carboxylic acids is close; the sum of relative peak area of fatty acids in FST2 is larger than that in FST1. However, when PDMS was used, the sum of relative peak area of fatty acids in FST2 is smaller than that in FST1. We think there may be two reasons. One is that PDMS is the best fiber for extracting carboxylic acids; the other is that the value of peak area is relative. When more constituents are identified, the peak area of every constituent is smaller. Among the four fibers, the number of constituents identified in FST1 is the least by using PDMS.
The extraction efficiencies of Carboxen/PDMS and DVB/CAR/PDMS on esters and PDMS/DVB and PDMS on esters are close. When Carboxen/PDMS and DVB/CAR/PDMS were used, the peak area of esters in FST1 (9.96% and 5.27%) is bigger than that in FST2 (5.44% and 3.90%). However, when PDMS/DVB and PDMS were used, the peak area of esters in FST1 (5.63% and 5.04%) is smaller than that in FST2 (10.41% and 12.49%). The reason may be that the peak area is a relative value. It is affected by a number of factors, such as the kind of fiber, the thickness of fiber, the number of identified constituents, the polarity of constituents, etc.
The extraction efficiency of Carboxen/PDMS on aldehydes and ketone is best among the four fibers. The total peak areas in FST1 and FST2 were 1.54% and 0.59%, respectively. When PDMS/DVB and DVB/CAR/PDMS were used, aldehydes and ketone were identified only in FST1. When PDMS was used, aldehydes and ketone were not identified in FST1 and FST2. The results indicated that the total content of aldehydes and ketone was lower in FST, especially in FST2.
The extraction efficiencies of Carboxen/PDMS and PDMS/DVB on sulfide and DVB/CAR/PDMS and PDMS on sulfide are close. When these fibers were used, the peak area of sulfide in FST1 (22.79% and 38.39%) is bigger than that in FST2 (21.66% and 22.49%). However, when DVB/CAR/PDMS and PDMS were used, the peak area of sulfide in FST1 (15.13% and 23.05%) is smaller than that in FST2 (16.34% and 33.40%). The main reason may be that the peak area is a relative value, too.
The relative peak areas of indole in FST1 and FST2 were close when using Carboxen/PDMS, PDMS/DVB and PDMS, but the value by PDMS was smaller than those by Carboxen/PDMS and PDMS/DVB. Maybe PDMS was better for extracting fatty acids, and this made the relative peak areas of indole become smaller. Among the four fibers, the relative peak areas of indole in FST1 (64.79%) and FST2(45.55%) were biggest by using DVB/CAR/PDMS. Maybe the reason was that indole was adsorbed easier by DVB/CAR/PDMS and the total number of identified compounds was less.
2.3. Volatile Compounds of FST
From Table 2
, it could be seen that total of 39 volatile compounds are identified in FST samples, including nine esters, seven alcohols, five alkenes, four sulfides, three heterocycles, three carboxylic acids, three ketones, two aldehydes, one phenol, one amine and one ether (eucalyptol). The predominant volatile compound in FST is indole, followed by dimethyl trisulfide, phenol, dimethyl disulfide and dimethyl tetrasulfide.
The number of ester compounds is the largest among the detected compounds. The esters identified are acetate, propanoate and butanoate. They are formed by the esterification reaction of organic acids with alcohols under catalysis of enzymes, which were produced by molds. The three kinds of organic acids and most of alcohols forming these esters are also identified in the FST sample. These ester compounds can impart FST with fruity notes and make the odor of FST lifting and diffusive. Ethyl acetate has pleasant ethereal-fruity like aroma; 3-methyl-1-butyl acetate has sweet, banana, fruity with a ripe ester nuance; hexyl acetate has green, fruity, sweet, fatty odor; ethyl propanoate has fruity, rum, fermented and pineapple aroma; ethyl butanoate has sweet, fruity and tutti frutti odor [39
]. Carboxen/PDMS and PDMS/DVB are more suitable for the extraction of ester compounds. When they were used, nine esters were all identified.
Alcohol compounds are also isolated from FST, and their formations may be due to the fermentation of carbohydrates from soybean during the ripening step. 1-Butanol is the main linear aliphatic alcohol. The detected alkenes, mainly including limonene, copaene, α-caryophyllene, aromadendrene and α-panasinsen, may be from materials used in the manufacture process of stinky tofu, like pepper, which contains these alkene compounds [40
]. Carboxen/PDMS and PDMS/DVB are also suitable for the extraction of alcohol compounds
Phenol is the only phenol compound identified in FST. Maybe it comes from the decomposition of tyrosine. The reason is that structural formula of tyrosine contains the structure of phenol, and the content of tyrosine fluctuates and even sometimes cannot be detected during the ripening process [41
]. Phenol is not only a flavor compound but also a kind of bactericide. As a flavor compound, it has phenolic, plastic and rubber odor [39
]; as a kind of bactericide, it can extend the shelf life of stinky tofu.
The detected organic acids are acetic acid, propanoic acid and butanoic acid. They are considered from the hydrolysis of soybean lipids [3
] and from the action of deaminase of amino acids. In the course of deaminase, ammonia compounds are also formed. The isolation of dimethylamine in the experiments can prove this statement. PDMS was more suitable for the extraction of carboxylic acids. When it was used, three carboxylic acids were all identified in FST 1 and FST 2.
Aldehydes and ketones might be formed by beta-oxidation of fatty acids, which generated a few of important flavor compounds [11
]. Two aromatic aldehydes, two methylketones and a cyclopentenone are observed in FST. Benzaldehyde is described as almond-like aroma and can give FST a nutty aroma, benzeneacetaldehyde is described as floral, sweet, sauce and soy sauce odor and can impart FST savory odor, 2-pentanone and 2-heptanone have sweet, fruity and ethereal aroma and make FST have sweet nuance, and 2-methyl-2-cyclopenten-1-one gives FST wood smoke notes. Among the four fiber used, Carboxen/PDMS is the most suitable for the extraction of these compounds
Furans and their derivatives are considered derived from Maillard reactions [11
]. 2-Pentylfuran and 2-pentylthiophene are detected in FST. Furan derivatives possess caramel, sweet, roasted, burnt and sugar notes.
Four sulfides, including dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide and methyl (methylthio) methyl disulfide, are isolated from FST. They arise from the degradation of amino acids containing sulfur. Stinky tofu is made of soybean which is rich in proteins; the protein in the tofu is hydrolyzed by the microbial proteases to form amino acids, among which cysteine and methionine are sulfur-containing amino acids. During the ripening process of stinky tofu, the contents of the two sulfur-containing amino acids change, especially the content of methionine. When the ripening time is 80 day, methionine is not detected [42
]. Dimethyl disulfide is with sulfurous, cabbage and onion odor; dimethyl trisulfide has sulfureous, alliaceous, cooked, savory, meaty, eggy and onion note; dimethyl tetrasulfide is described as sulfureous, galic and meaty odor; methyl (methylthio) methyl disulfide has strong sulfureous and onion odor [43
]. Although most of sulfides are identified by using the four fibers, the number of identified compounds is more with Carboxen/PDMS and PDMS/DVB.
Indole is identified in FST and its content is the highest in the volatile flavor constituents of FST. It might rise from the degration of tryptophan. The reason is that the structure of tryptophan contains the structure of indole, and the content of tryptophan decreases during the ripening process [42
]. Indole has animal and fecal odor [39
], it gives FST an unpleasant odor.
From Table 2
, it can be seen that FST 1 have more volatile flavor compounds than FST 2. Maybe there are some differences between their manufacturing processes. Esters, alcohols, aldehydes and ketones can give FST fruity and sweet odors, but the aroma intensities of indole and sulfides exceed their aroma intensities. The reason is that indole and four sulfides all have very low odor threshold values and relatively higher contents. Indole and four sulfides are characteristic volatile flavor constituents of FST and they give FST its very strong unpleasant odor.