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.

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.

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.

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.

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] .

RI = 100 n + 100 × t x − t n t n + 1 − t n (1)

t_x: the retention time of the volatiles to be tested (min).

t_n, t_n+1: the retention time of saturated alkanes containing n and n + 1 carbon atoms (min)

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.

C = S × 133.3   ug / mL × 3   mL 3   kg × 1.5 (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).

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:

ROAVi ≈ C i C s t a n × T s t a n T i × 100 (3)

ROAVi: the relative odor activity value of a volatile component.

C_i: the concentration of a volatile component (ug/kg).

T_i: the sensory threshold of a volatile component (ug/kg).

C_stan: the concentration of the component with maximum contribution to odors (ug/kg).

T_stan: the sensory threshold of the component with maximum contribution to odors. (ug/kg)

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

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

“-”: The corresponding substance was not detected by the instrument. “RT”: Retention time.

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.

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

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.