1. Introduction
Grass carp (Ctenopharyngodon idellus), one of the four largest freshwater fish in China, is very popular among consumers because of its rapid growth, high yield, and low price [1] . Grass carp is mainly freshly sold due to the high cost of storage [2] . Moreover, the production of fresh fish is greatly influenced by seasonal and regional weather conditions. To prevent fresh fish from deterioration, it is commonplace for the fish to be processed into salted fish. However, the traditional salted fish has high salt content and poor taste, which does not meet the dietary habits of modern people.
In recent years, a kind of low salt semi-dried fish has come into the markets. It has been welcomed by many people because of low salt content and good taste [3] . The grass carp processed into the low salt semi-dried fish has the broad market prospect. However, it suffers from having fewer flavor substances than traditional salted fish as a result of lower salt content, shorter drying time and the lack of natural inoculation and fermentation process. This problem paves the need for improvement in the flavor of fish meat. For instance, You gang et al. [4] inoculated compounded Lactobacillus on ornate threadfin bream. The results showed that a lot of carbonyl and alcohol compounds present in the flavor substances of fermented fish, which added the unique flavor of fermented fish on the basis of traditional salted fish flavor. Furthermore, Wu haiyan et al. [5] made use of Lactic acid bacteria and Staphylococcus to ferment the white cloud mountain minnows. Compared with the salted fish marinated by traditional techniques, the content of small molecules in the fermented fish meat was significantly increased, and the flavor of the final fermentation product was improved. To add on, Udomsil et al. [6] utilized Staphylococcus isolated from the traditional fish sauce as a starter culture to prepare fish sauce, which induced sauce black chocolate aroma and improved the flavor of fish sauce.
Monascus belongs to Eurotiales, Euascomycetes, Ascomycota, and Eumycophyta. It is one of the traditional fermentation moulds used by human beings and is mainly used in fermented food in some Asian countries. Monascus has been used for more than 1000 years in the production of wine, vinegar, and soy sauce in China. GDMCC3.439 Monascus purpureus is a kind of Monascus. It can produce Monascus red pigment in large quantities and has strong inhibition effect on alcohol, salinity and bacteriostasis [7] .
In order to further enhance the flavor of semi-dried fish, Monascus was inoculated to ferment the semi-dried fish. The aroma-producing effect of Monascus fermentation on low salt semi-dried grass carp was analyzed. The present study attempted to provide some new ideas for the traditional salted fish technology.
2. Materials and Methods
2.1. Materials and Reagents
Grass carp was purchased from China Resources Vanguard Supermarket. Monascus purpureus GDMCC3.439 was purchased from Guangdong Culture Collectioncenter of Microbiology. C7-C30 normal paraffins were purchased from the American Supelco Company. Potato dextrose agar (PDA) medium was purchased from Guangdong Huankai Microbiology Co., Ltd. Soy protein isolate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. All other chemicals and solvents used in the study were of analytical grade.
2.2. Main Instruments
7890A-5975C Gas Chromatography-Mass Spectrometer was purchased from Agilent Company. Chromatographic column was HP-INNOWAX (60 m × 0.25 mm × 0.25 um). Simultaneous distillation and extraction (SDE) was obtained from Guangzhou Dongju Experimental Instrument Co., Ltd. Vigreux column was purchased from Guangzhou Congyuan Experimental Instrument Co., Ltd.
2.3. Processing Craft
2.3.1. Preparation of Starter Cultures
The activated Monascus were inoculated into the slope composed of PDA medium in a test tube and cultured at 30˚C for 7 - 10 days. Then 5 mL of sterile water was pumped into the tube, shaken for 15 minutes, and the red-brown spores were washed into the Monascus culture medium with the 6 g of rice powder as a carbon source, 2.5 g of soybean protein as nitrogen source, and 0.05 g of MgSO4 and 0.1 g KH2PO4 as the mineral elements to incubate Monascus in the shaker for 5 days under 35˚C, shaking at 150 r/min. after which Monascus starter cultures were prepared. The concentration of Monascus was 109 CFU/mL [7] .
2.3.2. Craft of Low Salt Semi-Dried Fish
The Scale, head, tail, fin and viscera of grass carp were removed and cut into small pieces. They were marinated at 4˚C for 2 hours after 3% of salt (W/W) was added to them, then put into a heat pump drier, and dried at 27˚C and 20% humidity until the moisture content of the fish was about 50%. Finally, after vacuum packaging, they were unfermented group samples.
2.3.3. Craft of Low Salt Semi-Dried Fish Fermented by Monascus
The Scale, head, tail, fin and viscera of grass carp were removed and cut into small pieces. They were marinated at 4˚C for 2 hours after 3% (W/W) of salt was added to them. Monascus starter cultures were evenly applied to ferment them, and the inoculation amount was 5 mL/100g. Then, they were fermented at 30˚C for 18 hours, and transferred to a heat pump dryer. Next，they were dried at 27˚C and 20% humidity until the moisture content of the fish was about 50%. Finally, after vacuum packaging, they were fermented group samples.
2.4. Collection of Volatile Compounds
30 g of fish meat was placed in a 1000 mL round-bottom flask with 400 mL of purified water and the flask attached to the side of the SDE device. A 500 mL round-bottom flask containing 50 mL of dichloromethane was linked to the other side of the SDE device. The steams were cooled due to the circulation of water at 4˚C, the content of two round-bottom flasks were heated to a boil. The temperature of the dichloromethane flask was maintained by a water bath at 55˚C. The extraction was continued for 3 h, the extracts were collected and refrigerated overnight with 10 g of anhydrous sodium sulfate. The volume of the extract was then concentrated to 3 mL with a Vigreux column. 600 uL of sample concentrates were accurately absorbed into a 1.5 mL of sample bottle with 300 uL of butanol (the concentration was 400 ug/mL) as the internal standard. The sample bottle was frozen in the refrigerator for GC-MS analysis.
2.5. GC-MS Analysis Condition
Chromatographic columns used HP-INNOWAX (60 mm × 0.25 mm × 0.25 um). Helium was used as the carrier gas, the gas at a flow rate of 1.0 mL/min. The column temperature was maintained at 50˚C for 2 min, programmed at 5˚C/min to 220˚C and maintained for 5 min. The mass spectrometer was operated in electron impact (EI) ionization mode with electron energy of 70 eV and temperature at 230˚C. The four-stage bar temperature was 150˚C. Scan range was 30 - 550 m/z.
2.6. Qualitative Analysis
Unknown compounds were matched with the NIST08 library. Only compounds with mass greater than 75 were reported. Using the same heating program and taking saturated alkanes from C7 to C30 as the standard, the Retention Index (RI) of the corresponding compounds was calculated and qualitatively analyzed with the database retrieval results [8] .
(1)
tx: the retention time of the volatiles to be tested (min).
tn, tn+1: the retention time of saturated alkanes containing n and n + 1 carbon atoms (min)
2.7. Quantitative Analysis
Butanol was used as an internal standard, and the peak area of each test substance and internal standard were compared. The concentration of the volatile substance in the sample was calculated.
(2)
S: the ratio of peak area of volatile matter measured to that of internal standard butanol.
C: the concentration of the volatile substance (μg/kg).
2.8. Evaluation of Volatile Flavor Components by ROAV Method
Relative odor activity value (ROAV) was used to evaluate the contribution of volatile flavor components to the flavor of Monascus fermented semi-dried fish [9] [10] . ROAV of each compound was calculated according to the following formula:
(3)
ROAVi: the relative odor activity value of a volatile component.
Ci: the concentration of a volatile component (ug/kg).
Ti: the sensory threshold of a volatile component (ug/kg).
Cstan: the concentration of the component with maximum contribution to odors (ug/kg).
Tstan: the sensory threshold of the component with maximum contribution to odors. (ug/kg)
3. Results and Analysis
3.1. Detection Results of SDE-GC-MS
The total ion chromatograms of the fresh fish group, unfermented semi-dried fish group and the fermented semi-dried fish group were determined by SDE-GC-MS, respectively, as shown in Figure 1, Figure 2 and Figure 3.
Specific substances were identified by searching the NIST08 spectral library and the retention index was calculated according to Equation (1). Compared with the concentration and peak area of the internal standard, the concentration of other substances was calculated by Equation (2), as shown in Table 1. The types and relative contents of volatile substances in fish meat under different processing methods were further studied, as shown in Table 2.
From Table 1, compared with the fresh fish group, the contents of hexanal, Heptanal, nonanal, (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal, (E,E)-2,4-decadienal, (E,E)-2,4-heptadienal, 1-pentene-3-ol and 1-octen-3-ol significantly decreased in the fermentation group. Based on the literature analysis, these low molecular
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Figure 1. The total ion count (TIC) chromatogram of fresh fish sample.
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Figure 2. The total ion count (TIC) chromatogram of unfermented semi-dried fish sample.
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Figure 3. The total ion count (TIC) chromatogram of semi-dried fish sample fermented by Monascus.
weight aldehydes and alcohols, especially hexanal, heptaldehyde, nonanal, (E,E)-2,4-decadienal, (E,E)-2,4-heptadienal were typical fishy odor-causing compounds [11] [12] [13] [14] [15] . Moreover, 1-octene-3-alcohol had mushroom aroma and earthy odor [16] . The results showed that the processing of semi-dry grass carp could effectively improve the fishy smell.
Compared with the non-fermented group, the contents of 3-methyl butanol, pentanol, hexanol, heptanol, benzaldehyde and 3-Methyl-1-butanal increased in the fermented group. It had been reported that 3-methyl butanol, 1-pentene-3-alcohol, 1-octen-3-ol, pentanol, hexanol, hexanal, heptanal, benzaldehyde and 3-Methyl-1-butanal were the main flavor substances of salted fish [17] [18] [19] [20] . The results showed that the Monascus fermentation could enhance the
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Table 1. Volatile components and contents of fish meat processed in different ways.
“-”: The corresponding substance was not detected by the instrument. “RT”: Retention time.
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Table 2. Species and relative contents of volatile substances in fish meat under different processing methods.
aroma of salted fish and make up for the shortage of pickled flavor of semi-dried fish.
According to Table 1 and Table 2, 55 and 64 of volatile compounds were detected in fresh fish, non-fermented fish and fermented fish, most of which were alcohols, aldehydes and ketones, accounting for 89.23% and 88.51% respectively. As a result, alcohols and carbonyl compounds were the main volatile components of grass carp fish flavor. The total contents of alcohols and aldehydes in semi-dried grass carp after fermentation were 9.21% lower than those in non-fermented fish. Moreover the contents of ketones, hydrocarbons and esters were significantly increased, indicating that the fermentation of Monascus had a significant effect on the flavor of the semi-dried grass carp.
3.2. Identification of Key Aroma Substances
Why did fresh fish, unfermented fish and fermented fish have similar flavor components while the overall flavor was significantly different? Some data showed that the flavor contribution of volatile flavor substances was determined by their concentration and flavor threshold. The flavor characteristics of fermented fish could not be accurately described by the content of flavor substances alone [21] [22] [23] .
The volatile substance of which the concentration was highest in the fermentation group was the nonanal with 2767.5 ug/kg, and the sensory threshold of it was 1 ug/kg [24] , which contributed the most to the flavor of fermentation group. Thus, the nonanal was defined as the component with maximum contribution to odors. The sensory thresholds of some substances were not found in this paper. Most of them were hydrocarbons, esters and heteroatoms with high boiling point or high threshold, which had little effect on the overall flavor of fish. Volatile compounds of ROAV > 0.1 were counted in Table 3. Some researches suggested that the components of ROAV > 1 were key flavor compounds, those of 0.1 < ROAV < 1 were modified flavor compounds, and those of ROAV < 0.1 were potential flavor compounds [9] .
Table 3 showed that the main flavor substances (ROAV > 1) of Monascus fermented semi-dried grass carp were as follows: 1-octene-3-ol, phenylethanol, hexanal, heptanal, nonanal, (E)-2-octenal, 3-Methyl-1-butanal, benzaldehyde, (E)-2-nonenal, (E,E)-2,4-heptadienal, (E)-2-Decenal, phenylacetaldehyde, (E,E)-2.4-decadienal, tetradecanal, 2,3-butanone, 2,3-octanedione, alpha-pinene, 2-pentane furan. The content of saturated straight-chain alcohols in alcohols was higher, which contributed little to the flavor of fermented fish meat because of their high sensory threshold. However, some unsaturated alcohols had low threshold, such
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Table 3. Aroma characteristics, threshold and relative activity value of volatile components in semi-dried fish fermented by Monascus.
as 1-octene-3-alcohol and phenylethanol, which gave fermented fish meat mushroom and rose fragrance. Aldehydes, due to their low sensory threshold, accounted for the largest proportion of the main flavor substances in fermented fish meat, giving fish a variety of flavors. Ketones gave fermented fish fragrance, creamy fragrance and ester fragrance. Alpha-pinene and 2-pentylfuran gave the fermented fish oil and bean fragrance.
4. Conclusion
The volatile components of fresh fish, semi-dried fish and semi-dried fish fermented by Monascus were analyzed by SDE combined with GC-MS, and 43, 56 and 64 of volatile substances were detected respectively. The results showed that alcohols, aldehydes and ketones were the main components of the flavor of grass crap. The semi-dried fish fermented by Monascus could significantly affect the flavor of semi-dried grass carp. Furthermore, the processing could not only effectively improve the fishy smell of fresh fish, but also enhance the aroma of salted fish, and make up for the shortcomings of the pickled flavor of semi-dried fish. 18 of the main flavor components of semi-dried fish fermented by Monascus were identified by relative odor activity value (ROAV). They were as follows: 1-octene-3-ol, phenylethanol, hexanal, heptanal, nonanal, (E)-2-octenal, 3-Methyl-1-butanal, benzaldehyde, (E)-2-nonenal, (E,E)-2,4-heptadienal, (E)-2-decenal, phenylacetaldehyde, (E,E)-2.4-decadienal, tetradecanoaldehyde, 2,3-butanone, 2,3-octanedione, a-pinene, 2-pentane furan. The main aroma characteristics were mushroom, rose, gramineous, citrus taste, Vegetable-flavored, malt, bitter almond-flavored, cherry and nut-flavored, the fragrance of a flower, cream-flavored, fruit-flavored and wine-flavored.