Discussion on the Differences in Aroma Components in Different Fragrant Rice Varieties during Storage

Rice (Oryza sativa L.) is an important food crop in Taiwan, among which fragrant rice is highly regarded for its special aroma when cooked. During the storage of fragrant rice, the aroma components will change, which will affect the aroma quality of fragrant rice. Therefore, headspace solid-phase microextraction (HS-SPME) was used in this study, combined with gas chromatography and gas chromatography-mass spectrometry, to analyze the difference in the aroma components of Taikeng No. 4 (TK4), Tainung No. 71 (TN71), Kaohsiung No. 147 (KH147), and Taichung No. 194 (TC194) fragrant rice. A total of 28 aroma components were identified in the four varieties of fragrant rice, and the main components were all Nonanal. Among them, TK4 contains a very high content of hydrocarbons, including Tridecane and Dodecane; TN71, KH147, and TC194 contain mainly aldehydes such as Nonanal and Hexanal. During different storage times, the contents of alcohols, monoterpenes, aromatic aldehydes, and furans increased with storage time, while the content of aliphatic aldehydes decreased with storage time. After storage, the fragrant rice samples showed a tendency for the total volatile component content to decrease, with the most pronounced reduction observed in Nonanal content.


Introduction
Rice is a one-to two-year-old herbaceous plant belonging to the Oryza genus of Poaceae, mainly divided into two subspecies: indica (Oryza sativa subsp.indica) and japonica (O.sativa subsp.japonica) [1].Rice is one of the most important food crops in the world [2].It can be divided into two types: ordinary rice and fragrant rice based on the presence of aroma.There are various fragrant rice varieties around the world, and they have different aroma characteristics.For example, jasmine rice from Thailand has a grass and citrus aroma [3], while Taiwanese fragrant rice (Tainung 71) has a taro aroma (Chen and Chiu, 2011) [4].However, the aroma composition of fragrant rice with different aroma types is quite complex (Lu et al. 2023) [5].
At present, studies have proven that the key volatile compound 2-Acetyl-1-pyrroline (2-AP), which makes fragrant rice produce a special aroma, is also the main factor affecting the smell of fragrant rice [6,7].2-AP is produced during rice growth and can usually be detected The method used was modified from those of Dias et al. [13]: 1.

2.
Comparisons of the extraction times: The above-mentioned optimal extraction fiber was used in the comparison of the extraction times.The extraction times were 14, 18, 22, 26, and 30 min to determine the optimal extraction conditions.After sampling, each fiber was immediately inserted into the GC and GC-MS.

3.
Analysis of aroma components in different fragrant rice varieties: Take TK4, TN71, KH147, and TC194 fragrant rice as samples.Crush them with a homogenizer, weigh 5 g of powder, and use the above-mentioned HS-SPME under optimal experimental conditions.4.
Analysis of the aroma components of fragrant rice at different storage times: This experimental method is referred to and modified from Choi et al. [14].TK4, TN71, KH147, and TC194 were used as samples, stored for 0, 1, 2, 4, and 6 months in sequence, and then extracted and analyzed using the above-mentioned HS-SPME optimal condition experimental method.

Gas Chromatography (GC)
The instrument used was the model 7890A GC of the Agilent Company (Santa Clara, CA, USA), and it was matched with the company's DB-1 (60 m × 0.25 mm × 0.25 mm) separation column; the heating condition was an initial temperature of 40 • C, maintained for 1 min, then raised to 150 • C at 5 • C/min, maintained for 1 min, and finally raised to 200 • C at 10 • C/min, which was maintained for 11 min.The detector used was the Flame Ionization Detector (FID), the injection mode was the splitless mode, the temperature of the injection port was 250 • C, and the temperature of the detector was 300 • C. The carrier gas used was nitrogen, and the gas flow rate was 1.0 mL/min.

Gas Chromatography-Mass Spectrometry (GC-MS)
The instrument used was an Agilent Company (Santa Clara, CA, USA) model 7890B GC connected in series with a 5977A quadrupole column mass spectrometer (Mass Selective Detector, MSD; Santa Clara, CA, USA).The operating conditions and columns of the GC were the same as those of the aforementioned GC, the electron energy was 70 eV at 230 • C, and the temperature of the quadrupole column was 150 • C. The carrier gas used was changed to helium.The mass spectrum data were compared with Wiley 7N mass spectrum library.

Retention Index (RI)
The GC-retention index of the volatile compounds in this experiment was based on a mixture of C 5 -C 25 n-alkanes (Sigma-Aldrich, St. Louis, MO, USA) and the GC retention time under the same conditions as a reference, and calculated according to the method of Van Den Dool and Kratz [15].
Its retention index formula is as follows: where t n and t n+1 represent the residence time of n-alkanes before and after compound elution n = carbon number of n-alkanes t x = residence time of the compound x = indicates an unknown compound

Statistical Analysis
The data were subjected to a mono-factorial variance analysis, with Duncan's multiple range method used by a significance of differences of p < 0.05 (SPSS Base 12.0).

HS-SPME Fiber Selection
Before extracting volatile compounds, the fiber coating type will be selected according to the properties and requirements of the volatile compounds [13].Therefore, to obtain the best extraction fibers, this experiment used five different extraction fibers to absorb fragrant rice for 18 min at a water bath temperature of 80 • C. The results showed that 100 µm PDMS and 85 µm PA were less suitable for extracting volatile compounds from fragrant rice, while 65 µm PDMS/DVB had the best extraction effect compared with 75 µm CAR/PDMS and 50/30 µm DVB/CAR/PDMS, and had a higher total volatile compound content and component quantity (Table 2).Peng et al. [16] used three different extraction fibers (100 µm PDMS, 75 µm CAR/PDMS, and 65 µm PDMS/DVB) to analyze the volatile compounds of four fragrant rice spices, and the results showed that 65 µm PDMS/DVB fibers had the best extraction performance.According to the literature, PDMS is a nonpolar coating.If it is combined with polar coating DVB or CAR, it can not only increase the extraction effect, but also adsorb a wider range of volatile compounds with different polarities [17].Dias et al. [13] noted that 50/30 µm DVB/CAR/PDMS fibers are more widely used to extract volatile compounds in rice because they can absorb more volatile compounds of different polarities.Although 50/30 µm DVB/CAR/PDMS fibers outperformed 65 µm PDMS/DVB fibers, 65 µm PDMS/DVB fibers were preferred because of the observed residual problem.However, when using 65 µm PDMS/DVB fibers, no residual problems were shown [18].
According to the preliminary experimental results and the discussion of the above literature, 65 µm PDMS/DVB fiber was selected as the extraction fiber for HS-SPME in this experiment.Compared with other extraction fibers, this fiber has the best extraction effect and can be used to analyze more fragrant rice aroma components.

HS-SPME Extraction Time
Based on the test results of the HS-SPME fiber, in this experiment, the 65 µm PDMS/DVB extraction fiber with the best extraction effect was used to extract fragrant rice for 14, 18, 22, 26, and 30 min in a water bath at a temperature of 80 • C. The results (Figure 1) showed that the content of total volatile compounds had a tendency to increase significantly from 14 to 18 min of extraction time, but not after 22 to 30 min, and there was no significant difference (p < 0.05).
Lim et al. [19] analyzed the volatile compounds of white rice and extracted the samples for 10, 20, 30, 40, and 50 min.The research results showed that as the extraction time increased, the number of peaks decreased.According to the literature, shorter extraction times are more suitable for volatile compounds, while longer extraction times are more favorable for less volatile compounds [20].In addition, the water molecules present in the headspace are also adsorbed by the fibers during extraction, especially at higher temperatures, so longer extraction times may cause more water to accumulate on the fibers and thus reduce extraction efficiency [16].Choi et al. [21] analyzed the volatile compounds of black rice with 50/30 µm DVB/CAR/PDMS and adsorbed them in the headspace at 80 • C for 18 min.Therefore, the experimental results of this experiment are the same as the experimental results of Choi et al. [21].The optimal extraction condition for fragrant rice is 80 • C for 18 min, which can absorb more aroma components and meet the analysis efficiency in actual operation.
for 18 min.Therefore, the experimental results of this experiment are the same as the experimental results of Choi et al. [21].The optimal extraction condition for fragrant rice is 80 °C for 18 min, which can absorb more aroma components and meet the analysis efficiency in actual operation.

Volatile Compounds of Different Varieties of Fragrant Rice
In this experiment, HS-SPME was used to extract the volatile compounds of TK4, TN71, KH147, and TC194 fragrant rice, and GC-MS was used to identify and compare the composition differences (Figure 2).In Taiwan, TK4, TN71, and KH147 are generally considered to have a taro aroma, while TC194 is considered to have a popcorn aroma.The results of this experiment show that among the white rice samples of the four fragrant rice varieties, the highest content of all samples is Nonanal (1293.32-3521.70),which is one of the most important components in many rice varieties (Table 3).
The content of 2-AP was the highest in TK4 (14.36), followed by TC194 (11.32),KH147 (8.46), and TN71 (7.52).Champagne [22] pointed out that there is no single compound that can represent the characteristic aroma of fragrant rice, except for 2-AP, which could be produced by the interaction of multiple volatile compounds.
Moreover, 2-AP has the aroma of popcorn [23], and exists in some other foods, such as corn, wheat, barley, sorghum, and even taro.Therefore, 2-AP is sometimes also classified as one of the components of the taro aroma [24], while in Taiwan, some farmers believe that TK4, TN71, and KH147 have a taro aroma, and TC194 is considered to have a popcorn aroma, but in the study, all four samples contained 2-AP with li le difference in content.Therefore, the speculation is only the subjective aroma perception of farmers.

Volatile Compounds of Different Varieties of Fragrant Rice
In this experiment, HS-SPME was used to extract the volatile compounds of TK4, TN71, KH147, and TC194 fragrant rice, and GC-MS was used to identify and compare the composition differences (Figure 2).In Taiwan, TK4, TN71, and KH147 are generally considered to have a taro aroma, while TC194 is considered to have a popcorn aroma.The results of this experiment show that among the white rice samples of the four fragrant rice varieties, the highest content of all samples is Nonanal (1293.32-3521.70),which is one of the most important components in many rice varieties (Table 3).
The content of 2-AP was the highest in TK4 (14.36), followed by TC194 (11.32),KH147 (8.46), and TN71 (7.52).Champagne [22] pointed out that there is no single compound that can represent the characteristic aroma of fragrant rice, except for 2-AP, which could be produced by the interaction of multiple volatile compounds.
Moreover, 2-AP has the aroma of popcorn [23], and exists in some other foods, such as corn, wheat, barley, sorghum, and even taro.Therefore, 2-AP is sometimes also classified as one of the components of the taro aroma [24], while in Taiwan, some farmers believe that TK4, TN71, and KH147 have a taro aroma, and TC194 is considered to have a popcorn aroma, but in the study, all four samples contained 2-AP with little difference in content.Therefore, the speculation is only the subjective aroma perception of farmers.
Zhao et al. [3] identified jasmine rice by the odor activity value (OAV) and detection frequency analysis (DFA), and indicated that Hexanal, Octanal, Nonanal, (E)-2-Octenal, and Decanal were the odor-active components of jasmine rice.Liu and Yang [25] used HS-SPME combined with GC-MS to investigate the influence of Yihchuan fragrant rice on volatile compounds under different milling conditions, and pointed out that Hexanal, Nonanal, Benzaldehyde, 1-Octen-3-ol, Geranylacetone, 2-Pentylfuran, and 2-AP are commonly found in the aroma profiles of fragrant rice and taro, and suggested that these volatile compounds may contribute to the taro-like flavor in fragrant rice, and the results are similar to the ingredients in this experiment (Figure 3).Hexanal and Decanal are aldehydes, which are the decomposition products of oleic acid and linolenic acid [26].Among them, TN71 has the highest content of Hexanal, followed by TC194, KH147, and TK4.In addition, Life 2023, 13, 2063 6 of 14 Hexanal has a grassy taste [12].Decantal has a citrus peel-like smell [27].Benzaldehyde has an almond fragrance [28].In addition, 1-Octen-3-ol is a product of lipid oxidation and has an earthy and mushroomy smell [17], while 2-Pentylfuran is the reaction product of lipoxygenase (Lipoxygenase) [29], and has bean and nutty flavors [30].Geranylacetone has a floral and fruity aroma [31], and among the four varieties, TC194 has the lowest Geranylacetone content.In addition, TK4 contains a fairly high content of hydrocarbons, including Tridecane and Dodecane, and most of the hydrocarbons have a high threshold and have no significant impact on the overall aroma characteristics [32].Based on the above and the experimental results, TN71, KH147, and TC194 are dominated by aldehydes, followed by hydrocarbons; TK4 is contrary to this result as it is dominated by hydrocarbons, followed by aldehydes.

Effects of Storage Time of Different Fragrant Rice Varieties on Aroma Components
HS-SPME was used to analyze and compare the aroma components of four fragrant rice varieties with different storage times.The results (Tables 4-8, Figure 4) showed that a total of 30 aroma components were identified, including 11 aliphatic hydrocarbons, 5 aliphatic alcohols, 5 aliphatic aldehydes, 1 aromatic aldehyde, 1 ketone, 1 lactone, 1 furan, 1 pyridine, 3 monoterpenes, and 1 sesquiterpene.Among them, aldehydes are the most important volatile compounds, and the content is the highest at the 0th month, but the aldehydes in the white rice of the four fragrant rice varieties all have a tendency to decrease significantly after storage.The aldehydes reached their lowest in the 6th month, among which the decline trend of Nonanal was the most obvious for this kind of compound, and TK4 decreased from 1293.32 to 261.11 in the 6th month; TN71 decreased from 3521.70 to 620.06 in the 6th month; KH147 decreased from 2218.19 to 375.99 in the 6th month; and TC194 decreased from 2968.41 to 866.89 in the 6th month.The content of hydrocarbons TK4 and TN71 was the highest at the 0th month, and there was an obvious trend of decline after storage, while that of TC194 showed a trend of increase.The aliphatic aldehydes tended to decrease after storage.According to Choi et al. [14], storage at higher temperatures can lead to an increase in the content of aldehydes (such as Nonanal), while lower temperatures can retard lipid oxidation.In addition, alcohols are considered to be the by-products of unsaturated fatty acid oxidation and formed by the further decomposition of aldehydes [9].At the same time, alcohols are also minor volatile compounds in rice (second only to aldehydes) [33].In this experiment, the alcohol compounds of all samples were significantly increased after storage, among which the content of 2-Ethyl-1-hexanol was the highest, and TK4 increased from 193.42 to 694.02 in the 6th month; TN71 rose from 99.02 to 323.87 in the 6th month; KH147 rose from 156.85 to 700.13 in the 6th month; and TC194 rose from 296.93 to 566.54 in the 6th month.

Effects of Storage Time of Different Fragrant Rice Varieties on Aroma Components
HS-SPME was used to analyze and compare the aroma components of four fragrant rice varieties with different storage times.The results (Tables 4-8, Figure 4) showed that a total of 30 aroma components were identified, including 11 aliphatic hydrocarbons, 5 aliphatic alcohols, 5 aliphatic aldehydes, 1 aromatic aldehyde, 1 ketone, 1 lactone, 1 furan, 1 pyridine, 3 monoterpenes, and 1 sesquiterpene.Among them, aldehydes are the most important volatile compounds, and the content is the highest at the 0th month, but the aldehydes in the white rice of the four fragrant rice varieties all have a tendency to decrease significantly after storage.The aldehydes reached their lowest in the 6th month, among which the decline trend of Nonanal was the most obvious for this kind of compound, and TK4 decreased from 1293.32 to 261.11 in the 6th month; TN71 decreased from 3521.70 to 620.06 in the 6th month; KH147 decreased from 2218.19 to 375.99 in the 6th month; and TC194 decreased from 2968.41 to 866.89 in the 6th month.The content of hydrocarbons TK4 and TN71 was the highest at the 0th month, and there was an obvious trend of decline after storage, while that of TC194 showed a trend of increase.The aliphatic aldehydes tended to decrease after storage.According to Choi et al. [14], storage at higher temperatures can lead to an increase in the content of aldehydes (such as Nonanal), while lower temperatures can retard lipid oxidation.In addition, alcohols are considered to be the byproducts of unsaturated fa y acid oxidation and formed by the further decomposition of aldehydes [9].At the same time, alcohols are also minor volatile compounds in rice (second only to aldehydes) [33].In this experiment, the alcohol compounds of all samples were significantly increased after storage, among which the content of 2-Ethyl-1-hexanol       However, the content of ketones, lactones, furans, monoterpenes, and sesquiterpenes increased after storage, and reached the highest at the 6th month.After the storage of monoterpenes, β-Pinene was formed in the 1st month and α-Pinene was formed in the 2nd month, and its content rose to the highest in the 6th month.In the study of Bao et al. [34], monoterpene compounds such as α-pinene, β-pinene, myrcene, and limonene were indicated as biogenic volatile organic compounds emitted from rice.The results of this experiment also detected α-pinene, β-pinene, and limonene, among which the content of limonene was the highest.
In addition, with the increase in storage time, the content of 2-AP, TK4 content was the highest before storage, and the lowest after storage in the 6th month, TN71 and TC194 had the highest content in the 2nd month of storage, and gradually decreased in the 3rd month, while the content of KH147 was the highest in the 1st month, and gradually decreased in the 2nd month.Compared with the results of Tananuwong and Lertsiri [10], it was found that except for the 2-AP content of TK4, which gradually decreased with the increasing storage time, the 2-AP content of other varieties did not change significantly during different storage periods.

Conclusions
In this study, the optimal adsorption condition for the HS-SPME analysis of fragrant rice aroma compounds was extraction using a 65 µm PDMS/DVB fiber at 80 • C for 18 min.Among the TK4, TN71, KH147, and TC194 samples, the common main component is Nonanal.TN71, KH147, and TC194 are dominated by aldehydes, followed by hydrocarbons; TK4 is dominated by hydrocarbons, followed by aldehydes.Aldehydes are the most

Figure 1 .
Figure 1.Total peak areas of fragrant rice with the 65 µm PDMS/DVB fiber at different adsorption times.Values are means ± SD of triplicates.Values with different superscripts are significantly (p < 0.05) different.

Figure 1 .
Figure 1.Total peak areas of fragrant rice with the 65 µm PDMS/DVB fiber at different adsorption times.Values are means ± SD of triplicates.Values with different superscripts are significantly (p < 0.05) different.

Figure 4 .
Figure 4. Analysis of volatile compounds in TK4, TN71, KH147, and TC194 with different storage times by HS-SPME.Values are means ± SD of triplicates.Values with different superscripts are significantly (p < 0.05) different.

Figure 4 .
Figure 4. Analysis of volatile compounds in TK4, TN71, KH147, and TC194 with different storage times by HS-SPME.Values are means ± SD of triplicates.Values with different superscripts are significantly (p < 0.05) different.

Table 1 .
Planting information for the fragrant rice used in this study.

Table 2 .
Comparisons of the total peak areas of total volatile compounds detected in the headspace of TC194 using different HS-SPME fibers.

Table 4 .
Analysis of volatile compounds in TK4 with different storage times by HS-SPME.

Table 5 .
Analysis of volatile compounds in TN71 with different storage times by HS-SPME.

Table 6 .
Analysis of volatile compounds in KH147 with different storage times by HS-SPME.

Table 7 .
Analysis of volatile compounds in TC194 with different storage times by HS-SPME.

Table 8 .
Concentration of functional groups in TK4, TN71, KH147, and TC194 with different storage times.