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Influence of gene regulation on rice quality: Impact of storage temperature and humidity on flavor profile
Accepted Manuscript Influence of Gene Regulation on Rice Quality: Impact of Storage Temperature and Humidity on Flavor Profile Yuan Biao, Zhao Chanjuan, Yan Ming, Huang Dechun, David Julian McClements, Huang Zhigang, Cao Chongjiang PII: DOI: Reference:
S0308-8146(19)30110-4 https://doi.org/10.1016/j.foodchem.2019.01.042 FOCH 24137
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
23 July 2018 5 November 2018 3 January 2019
Please cite this article as: Biao, Y., Chanjuan, Z., Ming, Y., Dechun, H., McClements, D.J., Zhigang, H., Chongjiang, C., Influence of Gene Regulation on Rice Quality: Impact of Storage Temperature and Humidity on Flavor Profile, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.01.042
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Influence of Gene Regulation on Rice Quality: Impact of Storage Temperature and Humidity on Flavor Profile
Yuan Biaoa,b, Zhao Chanjuanc, Yan Minga, Huang Dechuna, David Julian McClementsd, Huang Zhigangb, *,Cao Chongjianga, *
a
Department of Food Quality and Safety/ National R&D Center For Chinese Herbal Medicine
Processing, College of Engineering, China Pharmaceutical University, Nanjing, Jiangsu 211198, China b
Beijing Key Laboratory of Quality Evaluation Technology for Hygiene and Safety of Plastics, Beijing
Technology and Business University, Beijing 100048, China c
Collaborative Innovation Center for Modern Grain Circulation and Safety, College of Food Science
and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu 210023, China d
Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
Running title: Environmental Factors on Rice Flavor… *Corresponding Author: Cao Chongjiang, College of Engineering, China Pharmaceutical University, Nanjing, Jiangsu, 211198: Tel +86 13770625999; email [email protected]
Huang Zhigang, School of Materials Science and Mechanical Engineering, Beijing Technology and Business University, Beijing, 100048: Tel +86 13611259926; email [email protected]
Abstract: Effects of high temperature-high humidity (HT-HH) storage on the flavor profile of rice were investigated. Volatile compounds such as aldehydes, ketones, and furans increased when rice was stored under HT-HH conditions, leading to a pronounced deterioration in rice quality. Correspondingly, the fatty acid content of the rice significantly increased during storage. Lipid oxidation was also accelerated under HT-HH conditions leading to the formation of peroxides. However, catalase activity was reduced under these conditions promoting the accumulation of peroxides. For the first time, insights into the genetic mechanisms responsible for these changes were obtained using RNA-sequencing to establish the flavor metabolic pathways in rice. Under HT-HH conditions, gene expression of lipase increased while that of catalase decreased, leading to faster hydrolysis and oxidation of the rice lipids. As a result, a series of lipid degradation products was formed (such as fatty acids, aldehydes, and ketones) that decreased the rice flavor quality. Keywords: rice; storage; RNA-sequencing; flavor; metabolic pathway; transcriptomics.
1. Introduction Paddy rice (Oryza sativa L.) is an important stable food in many countries, providing essential nutrients and micronutrients to their diet (Londo, Chiang, Hung, Chiang, & Schaal, 2006). In 2014, China’s rice production was approximately 172 million tons, ranking first in the world (Nie & Peng, 2017). According to the Food and Agriculture Organization of the United Nations (FAO), 25% of global paddy rice is contaminated with mold, and at least 2% of paddy rice is lost due to mildew every year. Indeed, the average annual loss of grain in China due to mildew is reported to be more than 2.5 billion kilograms. This problem becomes even more pronounced when rice is processed by shelling and peeling, since then its nutrients are directly exposed to the environment. Many of the key constituents in rice chemically degrade during storage at a rate depending on the temperature, humidity, and oxygen level of their surroundings. Moreover, water absorption by rice during storage increases the likelihood of microbial contamination (Chinma, Anuonye, Simon, Ohiare, & Danbaba, 2015; Tananuwong & Malila, 2011; Zhou, Robards, Helliwell, & Blanchard, 2007). Thus, undesirable storage conditions accelerate the deterioration of rice quality (Pomeranz & Zeleny, 1971). To satisfy consumer demand throughout the year, harvested rice must be stored for extended periods under conditions that maintain its quality (Hu, Tang, Liu, Zhu, & Shao, 2017). Storage temperature and humidity are the most important environmental factors affecting rice quality during storage (Chen et al., 2015). Typically, rice is
stored under low-temperature and controlled atmosphere conditions to protect it from fungi and insects, as well as to inhibit undesirable chemical and enzymatic degradation reactions (Ahmad et al., 2017). But, the temperature can exceed 38-41°C in some rice producing areas in southern Asia during summer, while the relative humidity (RH) is around 85-98% (Krishnan & Ramakrishnan, 2011). Furthermore, the stored paddy rice can lead to a temperature around 10°C higher than that of the granary temperature due to its high physiological activity (Jagadish, Murty, & Quick, 2015). Consequently, there is strong interest in identifying the impact of elevated temperature and humidity conditions on the flavor quality of rice during storage (Lin et al., 2010). Previously, our group used RNA sequencing to study the effects of temperature and humidity on rice quality. This study showed that partial downregulation of the expression of genes encoding for sucrose transferases and hydrolases led to a decrease in the starch and sucrose contents of the rice (Zhao, Xie, Li, & Cao, 2017). In the current study, transcriptomic techniques were used to study the impact of storage conditions on the chemical degradation of rice lipids, as this phenomenon is known to have a major impact on rice quality. Rice lipids are hydrolyzed and oxidized during storage, which generates free fatty acids, aldehydes, ketones and other substances that cause off-flavors (Holman, 1954). Consequently, it is important to understand and control these undesirable reactions so as to improve the quality of commercial rice (Tran, Suzuki, Okadome, Ikezaki, Homma, & Ohtsubo, 2005). In the current, the
impact of high temperature-high humidity (HT-HH) storage on flavor degradation in rice was investigated using a variety of analytical methods. An electronic nose, gas chromatography-mass spectrometer (GC-MS), and lipid oxidation markers were used to qualitatively and quantitatively analyze the volatile substances generated during rice storage (Chumpolsri et al., 2015). A novel transcriptomics approach was used to examine the genetic basis of flavor formation based on high-throughput RNA sequencing. This technique enabled us to establish the specific genes involved in the flavor degradation pathway. This study provides valuable new insights into the origin of the flavor changes in rice during storage and lays the foundation for optimizing rice storage conditions.
2. Methods 2.1. Paddy rice samples Preparation Newly harvested paddy rice (cultivar Nanjing 9108) was grown in Nanjing (Jiangsu, China). The paddy rice was treated by sun-drying for 48 hours to reduce the moisture content. Then, a control group (intact rice) (Group D1) was stored at normal temperature (25 °C/50% Relative humidity (RH)) and a test group (Group D2) was stored at the environment of 37 °C and 70% RH. After 30 days’ storage, rice samples were frozen in liquid nitrogen and stored at -80℃ for further studies. According to our previous findings ( Zhao, Xie, Li, & Cao, 2017), paddy rice stored at the 37 °C and 70% RH for 30 days showed a significant reduction in quality.
2.2. Chemicals Analytical standards of 2,4,6-trimethylpyridine, isopropanol, potassium iodide, chloroform, glacial acetic acids were purchased from Sigma-Aldrich (St. Louis, MO). Ultra-pure water was obtained from a water purification system (18.2 MΩ, E-PureTM, Thermo Fisher Scientific, USA). 2.3. Analysis of volatile compounds by GC/MS A weighed mass (6 g) of rice sample was placed into a 20-mL vial and then 1 µL of 2,4,6-trimethylpyridine was added as an internal standard solution (Pei et al., 2016). The vial was then sealed with a silicon cap and placed in a water bath set at 95 o
C for 1 hour. Then The cap was then perforated with a
divinylbenzene/carboxen/polydimethylsiloxane needle (DVB/CAR/PDMS, 50/30 M). At the same time, the SPME extraction fiber head was inserted into the GC-MS inlet to pre-condition it for 1 hour at a temperature of 230 oC, as recommended by the manufacturer to remove any residual volatiles from previous experiments. Then, the extracted fiber was inserted into the sample vial and volatiles were allowed to absorb for 1 h. The collected analytes were finally desorbed for 5 min at 250 oC in the GC injector. GC-MS analysis was performed on an Agilent 7890A/5975C GC-MS instrument (Agilent Technologies Inc., Santa Clara, CA, USA). Samples were separated using a HP-5MS capillary column (30 m × 0.25 mm i.d. and 0.25 µm film thickness). The column temperature was initially held at 40 °C for 5 minutes. The temperature
increased to 200 °C at 5 °C min-1, then increased to 230 °C at 10 °C min-1, and was then held for 5 min. The injector was maintained at 230 °C and 1 µL was injected in splitless mode. Ultra-high purity helium (99.9999%) was employed as carrier gas at a constant flow of 1 mL min-1, The EI energy was 70 eV, transfer line and EI source temperature was set at 210 °C, and quadrupole mass analyzer temperature was set at 150 °C. Data acquisition was performed in full scan mode. All experiments were performed in triplicate. The identification of the volatile components was performed: (1) by comparing the mass spectra with those recorded in the National Institute of Standards and Technology (NIST 14) spectral database (matching score > 70); (2) by comparing their retention index and generated for each reference compound analyzed using a series of n-alkanes (C5–C26) with the theoretical value provided by NIST 14 library and published literature. The contents of volatiles components were semi-quantified by measuring the peak areas in the total ion chromatogram (TIC). 2.4. Electronic nose analysis A metal-oxide semiconductor (MOS)-based gas analyzer array electronic nose detector combined with a headspace auto-sampler was used (Fox 3000, Alpha M.O.S., Toulouse, France). This device contained an array of 12 sensors (LY2/LG (Oxidizing gas), LY2/G (Ketones and Alcohols), LY2/AA (Ketone, Ammonia), LY2/GH (Organic amines), LY2/gCTL (Sulfur compound), LY2/gCT (Alcohols), T30/1(Acids), P10/1 (Ammonia, Acid), P10/2 (Hydrocarbon), P40/1 (Fluorine), T70/2 (Aromatic
compounds), PA2(Ketones, Alcohols, and Sulfur compounds)) to discriminate odor patterns of different aroma compounds. The test conditions used were as follows: 6 g of rice samples were weighed into a 20 mL septa sealed screw cap vial and equilibrated for 5 min at 35 oC for headspace volatile generation. The aroma headspace above the sample was then introduced into the E-nose at a flow rate of 150 mL/min and sensors were exposed to the volatiles for 40 s. Each analysis was performed in triplicate. 2.5. Determination of fatty acid content in rice The fatty acid value of the samples was measured according to the process of benzene extraction using the Chinese National Standard method (GB/T 5510–2011). Briefly, paddy rice powder (10 g) was placed in an Erlenmeyer flask, 5-fold volume of benzene solution was added, the lid was closed, the sample was shaken for 30 min, and then filtered. Phenolphthalein (0.04%, w/v, 25 mL) was added to the filtrate (25 mL) and immediately titrated with sodium hydroxide (0.01 M) to determine the end-point. 2.6. Determination of lipase activity in rice Lipase activity was defined as the amount of sodium hydroxide (in mg) needed to neutralize the free fatty acids liberated from pure lipid by the lipase in 1 g sample of brown rice grain (on dry basis) at pH 7.4, according to the China National Standard GB/T 5523-2008 (AQSIQ-SA, 2008). The activity of lipase was calculated according to a previously reported method (Qian, Gu, Jiang, & Chen, 2014).
2.7. Determination of rice catalase activity A catalase kit was obtained from the Nanjing Research Institute (China). The catalase activity (U/mg protein) was defined as the mass of 1 mol H2O2 required to breakdown 1 mg of protein at 37 oC. 2.8. Determination of rice peroxide content The Soxhlet method was used to extract the lipids from the rice. After the ethyl
ether in the extraction bottle was volatilized, 10 mL of chloroform and glacial acetic acid solution (2:3 v/v) was added, and then added 1 mL of a saturated potassium iodide solution was added. The mixture was then left to stand for 3 min. Then 25 mL of distilled water was added and the mixture was left to stand for around 30 min. Finally, the supernatant was collected and the absorbance was measured at a wavelength of 410 nm using a UV-visible spectrophotometer. 2.9. RNA-sequencing Frozen paddy rice samples were ground in liquid nitrogen. The total RNA of each sample was extracted according to the manufacturer’s instructions and mixed in equal quantities (Waryong Company, China). A total of 2 mg RNA was reversetranscribed using the PrimeScript RT reagent kit (Takara, China). Sequencing libraries were constructed on Illumina sequencing platform using NEBNext Ultra RNA library Prep Kit. The sequencing Assessment and annotation were conducted according to the method described previously (Zhao, Xie, Li, & Cao, 2017).
2.10 Validation of Gene Expression by qRT-PCR. The qRT-PCR was performed to confirm the differentially expressed gene by RNA-sequencing. Total RNA samples were reverse-transcribed into cDNA using SYBR green-based PCR assay. The q-PCR reaction system was formulated to include diluted cDNAs (6 μL), of GoTaq qPCR Master Mix (10 μL) (Promega, Madison, WI, U.S.A.), and each primer (0.25 μM) to obtain the final volume of 20 μL. The melting curves were analyzed at 65-95 °C after 39 cycles. as previously reported (Zhao, Xie, Li, & Cao, 2017). We selected UBQ5 as the internal reference gene according to previous studies (Jain, Nijhawan, Tyagi, & Khurana, 2006; Hong, Seo, Yang, Xiang, &Park, 2008). The qRT-PCR reactions were normalized using the Ct value methods corresponding to the housekeeping gene. The experimental data was calculated using the 2−ΔΔCt method. 2.11. Statistical Analysis Data were presented as Means ± Standard Deviation (SD) for the indicated number of independently performed experiments. One-way factorial analysis of variance (ANOVA) (more than two groups) or Student's t-test (two groups) was used to assess the mean difference between treatment groups and the control group. A pvalue of < 0.05 was considered statistically significant. For the further classification of rice samples, cluster analysis according to the squared Euclidean distance methods was made to identify the similarity of the samples taken at different conditions. In addition, the output data from the E-nose was analyzed using the instrument software
of the electronic nose (AlphaSoft version 3.0.0, Toulouse, France).
3. Results and Discussion 3.1 Determination of Metabolism of Flavor during Storage of Rice As shown in Table 1, 29 kinds of volatile compounds were detected after storing rice under high temperature-high humidity (HT-HH) conditions for 30 days, including 2 aldehydes, 1 alcohol, 2 ketones, 2 phenols, 3 acids, 6 esters, 9 hydrocarbons and 4 other types. This result is in agreement with other studies that have focused on the aroma profile of paddy rice (Champagne, Thompson, Bettgarber, Mutters, Miller, & Tan, 2004; Jang, Lim, & Kim, 2009; M. Zeng et al., 2012). Aldehydes are the most important contributor to the flavor profile of rice during storage, which are mainly formed by the oxidation of certain amino acids and unsaturated fatty acids (Xie, Sun, & Wang, 2008). Aldehydes have a pronounced fatty odor, but excessive levels lead to an off flavor. Nonanal has the odor of citrus and rose, while benzaldehyde, which results from phenylalanine degradation, has a bitter taste (Xie, Sun, & Wang, 2008). The level of aldehydes in the rice was the lowest before storage and the highest after HT-HH storage (Table 1). The relatively high content of aldehydes in the rice produced a detectable rancid odor. The higher level of aldehydes in the HT-HH group (D2) was probably due to faster oxidative decomposition of the rice lipids at the elevated temperature, whereas the higher level of acids was probably due to the faster hydrolysis and oxidation of the rice lipids. The presence of phenolic substances in rice products adversely impacts
their taste (Tsugita, 1986). After storage for 30 days, the total phenolic content of the rice increased by about 0.79% for ambient storage (D1) and about 1.22% for HT-HH storage (D2), which was significantly different (p < 0.05). The formation of furans and terpenes in rice can produce pleasant odors (Cesarettin Alasalvar, Fereidoon Shahidi, & Cadwallader, 2003; An, Capucine, Fien, Bruno, & Norbert, 2011; An, Fien, Bruno, Agnieszka, & Norbert, 2012). In particular, 2-pentylfuran and 2,3-dihydrobenzofuran have a nutty and sweet aroma. Moreover, at lower concentrations they produce a beany aroma, but at high concentrations they produce a soybean odor (Z. Zeng, Zhang, Chen, Zhang, & Matsunaga, 2007; Fien, An, Agnieszka, Bruno, & Norbert, 2009). In our study, the furan-like substances increased significantly after storage (p < 0.05). The content of 2, 3-dihydrobenzofuran increased from 5.66 in the HT-HH samples (D1) to 6.17 in the ambient samples (D2). However, the amount of indoles decreased and disappeared during the storage period, which may be the result of the effect of light and temperature during the long storage of rice. 3.2 Electronic nose analysis of flavor changes in rice during storage The volatile profile of rice samples subjected to the two different storage conditions was also characterized using an electronic nose. This device contains an array of sensors that respond to different types of volatile compounds in a sample, leading to the production of a radar graph. Generally, the more similar the shape of the radar graphs of two samples, the more similar are their aroma profiles.
Radar graphs were acquired for samples stored under ambient conditions (D1) and HT-HH conditions (D2) for 30 days, as well as for fresh rice (0), which was used as a reference (Fig. 1). The radar graphs of the stored rice samples had a fairly similar shape (Fig.1a), indicating that the volatile gas components produced by rice metabolism were fairly similar for the two storage conditions. Compared with the radar graphs of the fresh rice, the responses of the stored rice to sensors PA/2, P40/1, P10/1, and T30/1 for both HT-HH and ambient groups increased significantly (p < 0.05). Based on the different sensitivities and responses of the E-nose sensors, LY/AA (positive correlated with ketones), LY/G (positive correlated with ketones and alcohols), T30/1 (positive correlated with acids) and PA/2 (positive correlated with sulfur compounds and acids), our results indicated that the flavor profiles generated by the E-nose were in accordance with those determined by GC-MS. The E-nose results indicated that during storage, the acids and ketones in the rice increased, which would adversely alter rice quality by producing off flavors. Under HT-HH conditions, this increase was significantly higher than under ambient conditions, which suggests that hot humid conditions accelerated the oxidation and decomposition of the rice lipids and proteins. Principal component analysis (PCA) was used to identify differences in the flavor profiles of the test samples (D1 and D2) and initial sample (0). The abscissa is the contribution of the first principal component after PCA conversion while the ordinate is the contribution of the second principal component (Fig. 1b). The bigger the
numerical value of the axis, the better the principal component reflects the sample information. In our study, the first principal component contribution was 95.2%, the second principal component contribution was 4.5%, and the total contribution was 99.7%. Therefore, the PCA method can be used to accurately reflect differences in the flavor profiles of the different samples. Because the contribution of the first principal component was much larger than that of the second principal component, the numerical difference between the sample and control on the abscissa is most representative. This difference was much greater for the HT-HH sample (D2) than for the ambient sample (D1), indicating that the change in flavor was greatest for the rice stored under hot humid conditions. 3.3 Fatty acid generation increased during storage Lipases, which are naturally present in rice, can hydrolyze rice lipids and produce free fatty acids during storage, which negatively impacts rice quality (Zhou, Robards, Helliwell, & Blanchard, 2002). The measured free fatty acid values of the two rice samples increased during storage (Fig. 2). The value for the HT-HH sample (D2) increased about 2.5-fold after storage, while that of the ambient sample (D1) only increased about 0.8-fold, which was significantly different (p < 0.05). This result suggested that hot humid conditions promoted the hydrolysis of rice lipids during storage. 3.4 Rice lipase activity increased during storage Additional insights into the role of lipase on free fatty acid formation during
storage was obtained by measuring changes in enzyme activity (Fig. 2b). For the HTHH sample (D2), lipase activity increased throughout the first 3 weeks of storage but then decreased, whereas for the ambient sample, the lipase continued to increase throughout storage. During the initial stages of storage, the lipase activity was appreciably higher for the sample stored under HT-HH conditions, which would account for the faster generation of free fatty acids observed in this sample. Previous studies have also reported that the activity of lipase can decline after a certain storage period (Aloulou, Rodriguez, Fernandez, Van, Puccinelli, & Carrière, 2006). This effect may be partly due to the impact of pH on lipase activity. The optimum pH value for rice lipase is 7.5-8.0. When rice is stored, the fatty acid content increases and so the pH decreases, resulting in a reduction in lipase activity (Zhou, Robards, Helliwell, & Blanchard, 2002). 3.5 Catalase activity in rice decreased during storage Catalase is an enzyme capable of decomposing H2O2 into H2O and O2, which can reduce the ability of peroxides to promote lipid oxidation. In our study, catalase activity decreased during storage in both samples, with the activity being significantly less for the rice stored under HT-HH conditions (Fig. 2d). Indeed, the activity of rice catalase decreased from 3.45 U/mg for the initial sample to 1.51 U/mg for the HT-HH sample (D2) and 2.61 U/mg for the ambient sample (D1). This suggested that hot humid conditions inhibited the activity of rice catalase, which would accelerate the formation of off flavors in rice during storage.
3.6 Peroxide value of rice increased during storage The susceptibility of the rice samples to lipid oxidation during storage was determined by measuring peroxide formation (Sowbhagya & Bhattacharya, 2010). The peroxide values increased appreciably during storage in both samples (Fig. 2c), indicating that lipid oxidation occurred. Indeed, the peroxide value increased from 0.2 meq/kg in the initial sample to 0.45 meq/kg in the ambient group (D1) and 0.71 meq/kg in the HT-HH group (D2). These results suggest that hot humid conditions promoted lipid oxidation in the rice, thereby adversely affecting rice quality by producing rancid off odors (Frankel, 1984). This result is consistent with those of the electronic nose and GC-MS experiments, which showed that a variety of volatile off odors were produced during storage. 3.7 Differential genes related to rice flavor metabolic pathways were identified Based on the results of our previous study using RNA-sequencing, the impact of nine genes involved in rice flavor metabolic pathways on the flavor changes occurring during rice storage were studied (Table 2). OS11G0605500 and OS05G0132100 are genes involved in the lipid degradation and heterocyclic compound synthesis pathways. OS08G0508800, OS03G0738600, and OS08G0509100 are genes involved in the linoleic acid oxidative degradation pathway. OS06G0604400, OS02G0676000, OS03G0826600, and OS09G0543100 are genes involved in lipase hydrolysis and phospholipase degradation.
3.8 Genes involved in genes related to the metabolic pathway Fig. 3 shows changes in the expression of the genes involved in the rice lipid metabolism pathway under ambient and HT-HH storage conditions. The OS11G0605500, OS05G0132100, OS08G0508800, OS03G0738600, and OS08G0509100 showed increased gene expression, with the expression being higher under HT-HH than ambient conditions (p < 0.05). These five genes act on the fatty acid synthesis and linoleic acid pathways (Lee et al., 2016). This shows that hot humid storage conditions stimulate the expression of genes that promote lipase activity, thereby producing free fatty acids that reduce rice quality. The OS11G0605500 and OS05G0132100 genes are also capable of acting on lipase to produce heterocyclic compounds, thereby further negatively impacting rice flavor. The expression of the OS06G0604400, OS02G0676000, OS03G0826600 and OS09G0543100 genes decreased during rice storage. Under HT-HH conditions, the decrease in gene expression was significantly more than under ambient conditions (p < 0.05). These genes participate in the hydrolysis of lipases and degradation of phospholipases. Consequently, the decrease in the expression of these genes under hot humid conditions would have reduced the ability of rice to control the activities of lipase and phospholipase, thereby promoting the formation of free fatty acids from triacylglycerols and phospholipids. In summary, storage of rice under high temperature-high humidity conditions led to faster lipid degradation, resulting in the formation of free fatty acids and
volatile compounds (such as aldehydes, ketones, and furans) that adversely impact rice quality. Transcriptomic analysis indicated that the origin of this effect was partly due to increased lipase activity and decreased catalase activity under hot humid conditions. To the authors knowledge, this is the first time that changes in gene expression activity have been related to the flavor metabolic pathway in rice during storage. These results suggested that transcriptomic methods may be useful in establishing the optimum storage conditions for rice.
Conflict of Interest The authors declare that there are no conflicts of interest.
Acknowledgements This work was supported by National Key Research and Development Program of China (2016YFD0400901), the National Natural Science Foundation of China (No. 31571901), the Key Research and Development Program of Jiangsu Province (BE2016386) and the fund of Beijing Key Laboratory of Quality Evaluation Technology for Hygiene and Safety of Plastics, Beijing Technology and Business University (PQETGP2018001).
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High Temperature during Grain Filling on the Accumulation of Storage Proteins and Grain Quality in Rice (Oryza sativa L.). Journal of Agricultural and Food Chemistry, 58(19), 1054510552. Londo, J. P., Chiang, Y. C., Hung, K. H., Chiang, T. Y., & Schaal, B. A. (2006). Phylogeography of Asian wild rice, Oryza rufipogon, reveals multiple independent domestications of cultivated rice, Oryza sativa. Proceedings of the National Academy of Sciences of the United States of America, 103(25), 9578-9583. Nie, L., & Peng, S. (2017). Rice Production in China. In B. S. Chauhan, K. Jabran & G. Mahajan (Eds.), Rice Production Worldwide, (pp. 33-52). Cham: Springer International Publishing. Pei, F., Yang, W.J., Ma, N., Fang Y., Zhao, L.Y., An X.X., Xin Z.H., & Hu, Q.H. Effect of the two drying approaches on the volatile profiles of button mushroom (Agaricus bisporus) by headspace GC-MS and electronic nose. LWT-Food Science and Technology, 72, 343-350. Pomeranz, Y., & Zeleny, L. (1971). Biochemical and functional changes in stored cereal grains. C R C Critical Reviews in Food Technology, 2(1), 45-80. Qian, J.-Y., Gu, Y.-P., Jiang, W., & Chen, W. (2014). Inactivating effect of pulsed electric field on lipase in brown rice. Innovative Food Science & Emerging Technologies, 22(4), 89-94. Sowbhagya, C. M., & Bhattacharya, K. R. (2010). LIPID AUTOXIDATION IN RICE. Journal of Food Science, 41(5), 1018-1023. Hong, S-Y., Seo, P. J., Yang, M-K., Xiang, F., & Park, C-M. (2008). Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biolgoy, 8(1), 112. Tananuwong, K., & Malila, Y. (2011). Changes in physicochemical properties of organic hulled rice during storage under different conditions. Food Chemistry, 125(1), 179-185. Tran, T. U., Suzuki, K., Okadome, H., Ikezaki, H., Homma, S., & Ohtsubo, K. i. (2005). Detection of Changes in Taste of japonica and indica Brown and Milled Rice (Oryza sativa L.) during Storage Using Physicochemical Analyses and a Taste Sensing System. Journal of Agricultural and Food Chemistry, 53(4), 1108-1118. Tsugita, T. (1986). Aroma of cooked rice. Food Reviews International, 1(3), 497-520.
Xie, J. C., Sun, B. G., & Wang, S. B. (2008). Aromatic constituents from chinese traditional smokecured bacon of mini-pig. Food Science & Technology International, 14(4), 329-340. Zeng, M., Zhang, L., He, Z., Qin, F., Tang, X., Huang, X., Qu, H., & Chen, J. (2012). Determination of flavor components of rice bran by GC-MS and chemometrics. Analytical Methods, 4(2), 539545. Zeng, Z., Zhang, H., Chen, J. Y., Zhang, T., & Matsunaga, R. (2007). Direct Extraction of Volatiles of Rice During Cooking Using Solid-Phase Microextraction. Cereal Chemistry, 84(5), 423-427. Zhao, C., Xie, J., Li, L., & Cao, C. (2017). Comparative transcriptomic analysis in the paddy rice under storage and identification of differentially regulated genes in response to high temperature and humidity. Journal of Agricultural & Food Chemistry, 65(37), 8145-8153. Zhou, Z., Robards, K., Helliwell, S., & Blanchard, C. (2002). Ageing of Stored Rice: Changes in Chemical and Physical Attributes. Journal of Cereal Science, 35(1), 65-78. Zhou, Z., Robards, K., Helliwell, S., & Blanchard, C. (2007). Effect of storage temperature on cooking behaviour of rice. Food Chemistry, 105(2), 491-497.
FIGURE LEGENDS Figure 1. a) Radar map of Paddy rice during different storage condition. D0 fresh rice, D1, Paddy rice stored Group D1 was stored at normal temperature (25 °C/50% Relative humidity (RH)) as a control group, and Group D2 was stored at 37 °C and RH of 70%. b) Principal component analysis of paddy rice during different storage time. Figure 2. a) Effect of different storage conditions on (a) fatty acid, (b) lipase activity, (c) catalase activity, (d) peroxide activity. Figure 3. Expression analysis of 9 different expressed genes mRNAs in the sample D1 and D2. Total RNA was isolated from fresh paddy rice and paddy rice stored at high temperature and humidity. 8BQ5 gene (ubiquitin 5) was used as an internal reference. Expression levels of each gene are shown as a ratio relative to the treatment time (0 day) that was set to 1. Each value represents the means of three biological replicates from three different samples,
Figures: Fig. 1 a)
LY2/LG
PA/2 PA/2 T70/2 T70/2
LY2/LG 0.3 0.2
LY2/G
LY2/G
LY2/AA LY2/AA
0.1 0
P40/1 P40/1
-0.1
LY2/GH LY2/GH
P10/2 P10/2
D0 0d 新鲜 温 28d 常D1 温高湿 28d 高D2
LY2/gCTL LY2/gCTL
P10/1 P10/1
LY2/gCT
LY2/gCT
T30/1
T30/1
0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 00 -0.05 -0.05 -0.1 -0.1 -0.15 -0.15 -0.2 -0.2
PC2-4.529%
PC2-4.53%
b)
D2
D1 D0
-1 -0.8 -0.8 -0.6 -0.6 -0.4 -0.4 -0.2 -0.2 00 0.2 -1 0.2 0.4 0.4 0.6 0.6 0.8 0.8 PC1-95.192% PC1-95.19%
11
D1 D2
**
4.5
*
*
b)
*
4.0 3.5 3.0
0
7
14
21
28
储藏时间/天 Storage time/d
d)
20
D2 D1
**
**
15
* 10
5 0
7
14
21
储藏时间（天）
Storage time/d
28
prot) (U/mg activity Catalase Prot) 过氧化 氢 酶活度(U/mg
5.0
化物值（meq/kg） 过氧activity (meq/kg) Peroxide
c)
脂肪酸（KOHmg/100g干基） mg/100g DW) Fatty acid(KOH
a)
(mg/g) activity Lipase 动度（mg/g） 脂肪酶活
Fig. 2
4
*
D1 D2
*
*
3
**
2 1 0
0
7
14
21
28
储藏时间(天)
Storage time/d 0.8 D1 D2
0.6
*
** **
0.4
0.2 0
7
14
21
储藏时间（天）
Storage time/d
28
Fig. 3
Table 1. Relative contents of volatile compounds in paddy rice under different storage conditions. Key: initial sample (D0); sample stored under high temperature-high moisture (D2) conditions for 30 days; sample stored at ambient conditions (D1) for 30 days.
Sampleb
Retention Index
Kovats
Compound name Indicesa
Calculated
Literature
D0
D2
D1
9.9
Nonanal
1091.7
1089-1105
3.9±0.3
4.8±0.2
5.2±0.4
17.1
Vanillin
1386.2
1399
0.6±0.1
0.1±0.1
0.2±0.1
9.2
1-octanol
1063.2
1064-1073
0.9±0.2
-
0.5±0.1
1457.0
1451-1456
2.0±0.3
0.4±0.1
0.6±0.1
1996.4
1838
4.0±0.5
-
1.6±0.3
1279.2
1345
2.0±0.1
2.5±0.1
2.8±0.2
Aldehydes
Alcohols
6,10-dimethyl-5,918.8
undecadien-2-one
Ketone 6,10,14-trimethyl-231.8
pentadecanone 4-hydroxy-314.5
Phenols
methoxystyrene 20.5
2,6-di-tert-butyl-p-cresol
1529.2
-
1.2±0.1
1.5±0.1
1.6±0.1
11.2
Acetic acid
1144.2
1013
1.4±0.1
-
-
13.2
Nonanoic acid
1227.6
1278
-
0.3±0.1
-
32.9
Phthalic acid
2037.9
1873
-
42.8±1.2
42.8±1.5
28.0
Methyl myristate
1837.4
1679
-
0.6±0.3
-
Acids
Esters
31.5
Isopropyl myristate
1983.9
1829
2.0±0.2
-
-
34.7
Dibutyl phthalate
2115.9
1969
-
4.1±0.1
4.3±0.2
36.7
Ethyl palmitate
2196.2
1990
-
2.8±0.3
3.5±0.4
38.1
Hexyl acetate
2253.4
1283
12.7±1.3
3.8±0.3
7.5±0.3
39.0
Isopropyl palmitate
2290.8
2025
17.1±1.2
8.6±0.7
8.7±0.4
11.6
Methylbenzene
1159.4
1100
1.1±0.4
1.0±0.2
1.1±0.1
11.8
n-dodecane
1167.8
1150
1.7±0.2
-
-
16.1
1-dodecene
1348.8
1300
1.3±0.2
-
-
17.3
n-tetradecane
1396.7
1400
3.8±0.6
0.3±0.1
1.7±0.2
22.7
n-pentadecane
1621.0
1600
1.2±0.2
0.3±0.1
0.5±0.1
23.5
Styrene
1650.0
1237
0.4±0.1
0.5±0.1
1.0±0.2
23.7
n-hexadecane
1660.5
1600
0.8±0.1
3.6±0.2
3.4±0.2
24.1
1,2-epoxyhexadecane
1674.8
-
0.4±0.1
-
-
31.0
Octadecane
1962.7
1800
-
2.0±0.5
6.3±0.9
1179.6
1006
3.8±0.2
5.7±0.8
6.2±0.4
Hydro carbons
2,3-dihydro-112.1
benzofuran
Others
a
14.0
Indole
1257.9
1116
0.4±0.1
0.1±0.1
-
21.2
Naphthalene
1555.2
1305
0.8±0.1
-
-
27.6
2-pentylfuran
1822.4
1241
1.6±0.2
1.9±0.2
2.7±0.3
Kovats indices were calculated by using a series of n-alkanes (C5–C26) under the
same chromatographic conditions as the samples.
b
The relative contents of volatiles components were semi-quantified by peak areas in
the total ion chromatogram using 2,4,6-trimethylpyridine as an internal standard solution.
Table 2. Differential gene KEGG enrichment list involved in the paddy rice flavor metabolism pathways
Term
Gene ID
Functional description
oxidoreductase activity; molecular function; OS11G0605500 Fatty acid
heterocyclic compound binding
degradation
catalytic activity; fatty acid ligase activity; molecular OS05G0132100 function oxidoreductase activity; molecular function; OS08G0508800 dioxygenase activity Molecular function; oxidoreductase activity;
Linoleic acid metabolism
OS03G0738600
dioxygenase activity; catalytic activity dioxygenase activity; molecular function;
OS08G0509100 oxidoreductase activity
phosphoric diester hydrolase activity; phospholipase OS06G0604400
activity; hydrolase activity; molecular function; lipase activity
Ether lipid metabolism
transferase activity; catalytic activity; molecular OS02G0676000 function hydrolase activity; molecular function; catalytic OS03G0826600 activity phosphoric ester hydrolase activity; phosphoric OS09G0543100 diester hydrolase activity; phospholipase activity;
hydrolase activity
Highlights
The impact of high temperature-high humidity (HT-HH) conditions on rice storage were examined.
Volatile aldehydes, ketones, and furans increased when rice was stored under HT-HH conditions.
The formation of these volatile compounds negatively impacted the aroma profile of the rice.
Gene expression of lipase increased under HT-HH conditions, leading to faster hydrolysis and oxidation of rice lipids.
S0308-8146(19)30110-4 https://doi.org/10.1016/j.foodchem.2019.01.042 FOCH 24137
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
23 July 2018 5 November 2018 3 January 2019
Please cite this article as: Biao, Y., Chanjuan, Z., Ming, Y., Dechun, H., McClements, D.J., Zhigang, H., Chongjiang, C., Influence of Gene Regulation on Rice Quality: Impact of Storage Temperature and Humidity on Flavor Profile, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.01.042
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Influence of Gene Regulation on Rice Quality: Impact of Storage Temperature and Humidity on Flavor Profile
Yuan Biaoa,b, Zhao Chanjuanc, Yan Minga, Huang Dechuna, David Julian McClementsd, Huang Zhigangb, *,Cao Chongjianga, *
a
Department of Food Quality and Safety/ National R&D Center For Chinese Herbal Medicine
Processing, College of Engineering, China Pharmaceutical University, Nanjing, Jiangsu 211198, China b
Beijing Key Laboratory of Quality Evaluation Technology for Hygiene and Safety of Plastics, Beijing
Technology and Business University, Beijing 100048, China c
Collaborative Innovation Center for Modern Grain Circulation and Safety, College of Food Science
and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu 210023, China d
Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
Running title: Environmental Factors on Rice Flavor… *Corresponding Author: Cao Chongjiang, College of Engineering, China Pharmaceutical University, Nanjing, Jiangsu, 211198: Tel +86 13770625999; email [email protected]
Huang Zhigang, School of Materials Science and Mechanical Engineering, Beijing Technology and Business University, Beijing, 100048: Tel +86 13611259926; email [email protected]
Abstract: Effects of high temperature-high humidity (HT-HH) storage on the flavor profile of rice were investigated. Volatile compounds such as aldehydes, ketones, and furans increased when rice was stored under HT-HH conditions, leading to a pronounced deterioration in rice quality. Correspondingly, the fatty acid content of the rice significantly increased during storage. Lipid oxidation was also accelerated under HT-HH conditions leading to the formation of peroxides. However, catalase activity was reduced under these conditions promoting the accumulation of peroxides. For the first time, insights into the genetic mechanisms responsible for these changes were obtained using RNA-sequencing to establish the flavor metabolic pathways in rice. Under HT-HH conditions, gene expression of lipase increased while that of catalase decreased, leading to faster hydrolysis and oxidation of the rice lipids. As a result, a series of lipid degradation products was formed (such as fatty acids, aldehydes, and ketones) that decreased the rice flavor quality. Keywords: rice; storage; RNA-sequencing; flavor; metabolic pathway; transcriptomics.
1. Introduction Paddy rice (Oryza sativa L.) is an important stable food in many countries, providing essential nutrients and micronutrients to their diet (Londo, Chiang, Hung, Chiang, & Schaal, 2006). In 2014, China’s rice production was approximately 172 million tons, ranking first in the world (Nie & Peng, 2017). According to the Food and Agriculture Organization of the United Nations (FAO), 25% of global paddy rice is contaminated with mold, and at least 2% of paddy rice is lost due to mildew every year. Indeed, the average annual loss of grain in China due to mildew is reported to be more than 2.5 billion kilograms. This problem becomes even more pronounced when rice is processed by shelling and peeling, since then its nutrients are directly exposed to the environment. Many of the key constituents in rice chemically degrade during storage at a rate depending on the temperature, humidity, and oxygen level of their surroundings. Moreover, water absorption by rice during storage increases the likelihood of microbial contamination (Chinma, Anuonye, Simon, Ohiare, & Danbaba, 2015; Tananuwong & Malila, 2011; Zhou, Robards, Helliwell, & Blanchard, 2007). Thus, undesirable storage conditions accelerate the deterioration of rice quality (Pomeranz & Zeleny, 1971). To satisfy consumer demand throughout the year, harvested rice must be stored for extended periods under conditions that maintain its quality (Hu, Tang, Liu, Zhu, & Shao, 2017). Storage temperature and humidity are the most important environmental factors affecting rice quality during storage (Chen et al., 2015). Typically, rice is
stored under low-temperature and controlled atmosphere conditions to protect it from fungi and insects, as well as to inhibit undesirable chemical and enzymatic degradation reactions (Ahmad et al., 2017). But, the temperature can exceed 38-41°C in some rice producing areas in southern Asia during summer, while the relative humidity (RH) is around 85-98% (Krishnan & Ramakrishnan, 2011). Furthermore, the stored paddy rice can lead to a temperature around 10°C higher than that of the granary temperature due to its high physiological activity (Jagadish, Murty, & Quick, 2015). Consequently, there is strong interest in identifying the impact of elevated temperature and humidity conditions on the flavor quality of rice during storage (Lin et al., 2010). Previously, our group used RNA sequencing to study the effects of temperature and humidity on rice quality. This study showed that partial downregulation of the expression of genes encoding for sucrose transferases and hydrolases led to a decrease in the starch and sucrose contents of the rice (Zhao, Xie, Li, & Cao, 2017). In the current study, transcriptomic techniques were used to study the impact of storage conditions on the chemical degradation of rice lipids, as this phenomenon is known to have a major impact on rice quality. Rice lipids are hydrolyzed and oxidized during storage, which generates free fatty acids, aldehydes, ketones and other substances that cause off-flavors (Holman, 1954). Consequently, it is important to understand and control these undesirable reactions so as to improve the quality of commercial rice (Tran, Suzuki, Okadome, Ikezaki, Homma, & Ohtsubo, 2005). In the current, the
impact of high temperature-high humidity (HT-HH) storage on flavor degradation in rice was investigated using a variety of analytical methods. An electronic nose, gas chromatography-mass spectrometer (GC-MS), and lipid oxidation markers were used to qualitatively and quantitatively analyze the volatile substances generated during rice storage (Chumpolsri et al., 2015). A novel transcriptomics approach was used to examine the genetic basis of flavor formation based on high-throughput RNA sequencing. This technique enabled us to establish the specific genes involved in the flavor degradation pathway. This study provides valuable new insights into the origin of the flavor changes in rice during storage and lays the foundation for optimizing rice storage conditions.
2. Methods 2.1. Paddy rice samples Preparation Newly harvested paddy rice (cultivar Nanjing 9108) was grown in Nanjing (Jiangsu, China). The paddy rice was treated by sun-drying for 48 hours to reduce the moisture content. Then, a control group (intact rice) (Group D1) was stored at normal temperature (25 °C/50% Relative humidity (RH)) and a test group (Group D2) was stored at the environment of 37 °C and 70% RH. After 30 days’ storage, rice samples were frozen in liquid nitrogen and stored at -80℃ for further studies. According to our previous findings ( Zhao, Xie, Li, & Cao, 2017), paddy rice stored at the 37 °C and 70% RH for 30 days showed a significant reduction in quality.
2.2. Chemicals Analytical standards of 2,4,6-trimethylpyridine, isopropanol, potassium iodide, chloroform, glacial acetic acids were purchased from Sigma-Aldrich (St. Louis, MO). Ultra-pure water was obtained from a water purification system (18.2 MΩ, E-PureTM, Thermo Fisher Scientific, USA). 2.3. Analysis of volatile compounds by GC/MS A weighed mass (6 g) of rice sample was placed into a 20-mL vial and then 1 µL of 2,4,6-trimethylpyridine was added as an internal standard solution (Pei et al., 2016). The vial was then sealed with a silicon cap and placed in a water bath set at 95 o
C for 1 hour. Then The cap was then perforated with a
divinylbenzene/carboxen/polydimethylsiloxane needle (DVB/CAR/PDMS, 50/30 M). At the same time, the SPME extraction fiber head was inserted into the GC-MS inlet to pre-condition it for 1 hour at a temperature of 230 oC, as recommended by the manufacturer to remove any residual volatiles from previous experiments. Then, the extracted fiber was inserted into the sample vial and volatiles were allowed to absorb for 1 h. The collected analytes were finally desorbed for 5 min at 250 oC in the GC injector. GC-MS analysis was performed on an Agilent 7890A/5975C GC-MS instrument (Agilent Technologies Inc., Santa Clara, CA, USA). Samples were separated using a HP-5MS capillary column (30 m × 0.25 mm i.d. and 0.25 µm film thickness). The column temperature was initially held at 40 °C for 5 minutes. The temperature
increased to 200 °C at 5 °C min-1, then increased to 230 °C at 10 °C min-1, and was then held for 5 min. The injector was maintained at 230 °C and 1 µL was injected in splitless mode. Ultra-high purity helium (99.9999%) was employed as carrier gas at a constant flow of 1 mL min-1, The EI energy was 70 eV, transfer line and EI source temperature was set at 210 °C, and quadrupole mass analyzer temperature was set at 150 °C. Data acquisition was performed in full scan mode. All experiments were performed in triplicate. The identification of the volatile components was performed: (1) by comparing the mass spectra with those recorded in the National Institute of Standards and Technology (NIST 14) spectral database (matching score > 70); (2) by comparing their retention index and generated for each reference compound analyzed using a series of n-alkanes (C5–C26) with the theoretical value provided by NIST 14 library and published literature. The contents of volatiles components were semi-quantified by measuring the peak areas in the total ion chromatogram (TIC). 2.4. Electronic nose analysis A metal-oxide semiconductor (MOS)-based gas analyzer array electronic nose detector combined with a headspace auto-sampler was used (Fox 3000, Alpha M.O.S., Toulouse, France). This device contained an array of 12 sensors (LY2/LG (Oxidizing gas), LY2/G (Ketones and Alcohols), LY2/AA (Ketone, Ammonia), LY2/GH (Organic amines), LY2/gCTL (Sulfur compound), LY2/gCT (Alcohols), T30/1(Acids), P10/1 (Ammonia, Acid), P10/2 (Hydrocarbon), P40/1 (Fluorine), T70/2 (Aromatic
compounds), PA2(Ketones, Alcohols, and Sulfur compounds)) to discriminate odor patterns of different aroma compounds. The test conditions used were as follows: 6 g of rice samples were weighed into a 20 mL septa sealed screw cap vial and equilibrated for 5 min at 35 oC for headspace volatile generation. The aroma headspace above the sample was then introduced into the E-nose at a flow rate of 150 mL/min and sensors were exposed to the volatiles for 40 s. Each analysis was performed in triplicate. 2.5. Determination of fatty acid content in rice The fatty acid value of the samples was measured according to the process of benzene extraction using the Chinese National Standard method (GB/T 5510–2011). Briefly, paddy rice powder (10 g) was placed in an Erlenmeyer flask, 5-fold volume of benzene solution was added, the lid was closed, the sample was shaken for 30 min, and then filtered. Phenolphthalein (0.04%, w/v, 25 mL) was added to the filtrate (25 mL) and immediately titrated with sodium hydroxide (0.01 M) to determine the end-point. 2.6. Determination of lipase activity in rice Lipase activity was defined as the amount of sodium hydroxide (in mg) needed to neutralize the free fatty acids liberated from pure lipid by the lipase in 1 g sample of brown rice grain (on dry basis) at pH 7.4, according to the China National Standard GB/T 5523-2008 (AQSIQ-SA, 2008). The activity of lipase was calculated according to a previously reported method (Qian, Gu, Jiang, & Chen, 2014).
2.7. Determination of rice catalase activity A catalase kit was obtained from the Nanjing Research Institute (China). The catalase activity (U/mg protein) was defined as the mass of 1 mol H2O2 required to breakdown 1 mg of protein at 37 oC. 2.8. Determination of rice peroxide content The Soxhlet method was used to extract the lipids from the rice. After the ethyl
ether in the extraction bottle was volatilized, 10 mL of chloroform and glacial acetic acid solution (2:3 v/v) was added, and then added 1 mL of a saturated potassium iodide solution was added. The mixture was then left to stand for 3 min. Then 25 mL of distilled water was added and the mixture was left to stand for around 30 min. Finally, the supernatant was collected and the absorbance was measured at a wavelength of 410 nm using a UV-visible spectrophotometer. 2.9. RNA-sequencing Frozen paddy rice samples were ground in liquid nitrogen. The total RNA of each sample was extracted according to the manufacturer’s instructions and mixed in equal quantities (Waryong Company, China). A total of 2 mg RNA was reversetranscribed using the PrimeScript RT reagent kit (Takara, China). Sequencing libraries were constructed on Illumina sequencing platform using NEBNext Ultra RNA library Prep Kit. The sequencing Assessment and annotation were conducted according to the method described previously (Zhao, Xie, Li, & Cao, 2017).
2.10 Validation of Gene Expression by qRT-PCR. The qRT-PCR was performed to confirm the differentially expressed gene by RNA-sequencing. Total RNA samples were reverse-transcribed into cDNA using SYBR green-based PCR assay. The q-PCR reaction system was formulated to include diluted cDNAs (6 μL), of GoTaq qPCR Master Mix (10 μL) (Promega, Madison, WI, U.S.A.), and each primer (0.25 μM) to obtain the final volume of 20 μL. The melting curves were analyzed at 65-95 °C after 39 cycles. as previously reported (Zhao, Xie, Li, & Cao, 2017). We selected UBQ5 as the internal reference gene according to previous studies (Jain, Nijhawan, Tyagi, & Khurana, 2006; Hong, Seo, Yang, Xiang, &Park, 2008). The qRT-PCR reactions were normalized using the Ct value methods corresponding to the housekeeping gene. The experimental data was calculated using the 2−ΔΔCt method. 2.11. Statistical Analysis Data were presented as Means ± Standard Deviation (SD) for the indicated number of independently performed experiments. One-way factorial analysis of variance (ANOVA) (more than two groups) or Student's t-test (two groups) was used to assess the mean difference between treatment groups and the control group. A pvalue of < 0.05 was considered statistically significant. For the further classification of rice samples, cluster analysis according to the squared Euclidean distance methods was made to identify the similarity of the samples taken at different conditions. In addition, the output data from the E-nose was analyzed using the instrument software
of the electronic nose (AlphaSoft version 3.0.0, Toulouse, France).
3. Results and Discussion 3.1 Determination of Metabolism of Flavor during Storage of Rice As shown in Table 1, 29 kinds of volatile compounds were detected after storing rice under high temperature-high humidity (HT-HH) conditions for 30 days, including 2 aldehydes, 1 alcohol, 2 ketones, 2 phenols, 3 acids, 6 esters, 9 hydrocarbons and 4 other types. This result is in agreement with other studies that have focused on the aroma profile of paddy rice (Champagne, Thompson, Bettgarber, Mutters, Miller, & Tan, 2004; Jang, Lim, & Kim, 2009; M. Zeng et al., 2012). Aldehydes are the most important contributor to the flavor profile of rice during storage, which are mainly formed by the oxidation of certain amino acids and unsaturated fatty acids (Xie, Sun, & Wang, 2008). Aldehydes have a pronounced fatty odor, but excessive levels lead to an off flavor. Nonanal has the odor of citrus and rose, while benzaldehyde, which results from phenylalanine degradation, has a bitter taste (Xie, Sun, & Wang, 2008). The level of aldehydes in the rice was the lowest before storage and the highest after HT-HH storage (Table 1). The relatively high content of aldehydes in the rice produced a detectable rancid odor. The higher level of aldehydes in the HT-HH group (D2) was probably due to faster oxidative decomposition of the rice lipids at the elevated temperature, whereas the higher level of acids was probably due to the faster hydrolysis and oxidation of the rice lipids. The presence of phenolic substances in rice products adversely impacts
their taste (Tsugita, 1986). After storage for 30 days, the total phenolic content of the rice increased by about 0.79% for ambient storage (D1) and about 1.22% for HT-HH storage (D2), which was significantly different (p < 0.05). The formation of furans and terpenes in rice can produce pleasant odors (Cesarettin Alasalvar, Fereidoon Shahidi, & Cadwallader, 2003; An, Capucine, Fien, Bruno, & Norbert, 2011; An, Fien, Bruno, Agnieszka, & Norbert, 2012). In particular, 2-pentylfuran and 2,3-dihydrobenzofuran have a nutty and sweet aroma. Moreover, at lower concentrations they produce a beany aroma, but at high concentrations they produce a soybean odor (Z. Zeng, Zhang, Chen, Zhang, & Matsunaga, 2007; Fien, An, Agnieszka, Bruno, & Norbert, 2009). In our study, the furan-like substances increased significantly after storage (p < 0.05). The content of 2, 3-dihydrobenzofuran increased from 5.66 in the HT-HH samples (D1) to 6.17 in the ambient samples (D2). However, the amount of indoles decreased and disappeared during the storage period, which may be the result of the effect of light and temperature during the long storage of rice. 3.2 Electronic nose analysis of flavor changes in rice during storage The volatile profile of rice samples subjected to the two different storage conditions was also characterized using an electronic nose. This device contains an array of sensors that respond to different types of volatile compounds in a sample, leading to the production of a radar graph. Generally, the more similar the shape of the radar graphs of two samples, the more similar are their aroma profiles.
Radar graphs were acquired for samples stored under ambient conditions (D1) and HT-HH conditions (D2) for 30 days, as well as for fresh rice (0), which was used as a reference (Fig. 1). The radar graphs of the stored rice samples had a fairly similar shape (Fig.1a), indicating that the volatile gas components produced by rice metabolism were fairly similar for the two storage conditions. Compared with the radar graphs of the fresh rice, the responses of the stored rice to sensors PA/2, P40/1, P10/1, and T30/1 for both HT-HH and ambient groups increased significantly (p < 0.05). Based on the different sensitivities and responses of the E-nose sensors, LY/AA (positive correlated with ketones), LY/G (positive correlated with ketones and alcohols), T30/1 (positive correlated with acids) and PA/2 (positive correlated with sulfur compounds and acids), our results indicated that the flavor profiles generated by the E-nose were in accordance with those determined by GC-MS. The E-nose results indicated that during storage, the acids and ketones in the rice increased, which would adversely alter rice quality by producing off flavors. Under HT-HH conditions, this increase was significantly higher than under ambient conditions, which suggests that hot humid conditions accelerated the oxidation and decomposition of the rice lipids and proteins. Principal component analysis (PCA) was used to identify differences in the flavor profiles of the test samples (D1 and D2) and initial sample (0). The abscissa is the contribution of the first principal component after PCA conversion while the ordinate is the contribution of the second principal component (Fig. 1b). The bigger the
numerical value of the axis, the better the principal component reflects the sample information. In our study, the first principal component contribution was 95.2%, the second principal component contribution was 4.5%, and the total contribution was 99.7%. Therefore, the PCA method can be used to accurately reflect differences in the flavor profiles of the different samples. Because the contribution of the first principal component was much larger than that of the second principal component, the numerical difference between the sample and control on the abscissa is most representative. This difference was much greater for the HT-HH sample (D2) than for the ambient sample (D1), indicating that the change in flavor was greatest for the rice stored under hot humid conditions. 3.3 Fatty acid generation increased during storage Lipases, which are naturally present in rice, can hydrolyze rice lipids and produce free fatty acids during storage, which negatively impacts rice quality (Zhou, Robards, Helliwell, & Blanchard, 2002). The measured free fatty acid values of the two rice samples increased during storage (Fig. 2). The value for the HT-HH sample (D2) increased about 2.5-fold after storage, while that of the ambient sample (D1) only increased about 0.8-fold, which was significantly different (p < 0.05). This result suggested that hot humid conditions promoted the hydrolysis of rice lipids during storage. 3.4 Rice lipase activity increased during storage Additional insights into the role of lipase on free fatty acid formation during
storage was obtained by measuring changes in enzyme activity (Fig. 2b). For the HTHH sample (D2), lipase activity increased throughout the first 3 weeks of storage but then decreased, whereas for the ambient sample, the lipase continued to increase throughout storage. During the initial stages of storage, the lipase activity was appreciably higher for the sample stored under HT-HH conditions, which would account for the faster generation of free fatty acids observed in this sample. Previous studies have also reported that the activity of lipase can decline after a certain storage period (Aloulou, Rodriguez, Fernandez, Van, Puccinelli, & Carrière, 2006). This effect may be partly due to the impact of pH on lipase activity. The optimum pH value for rice lipase is 7.5-8.0. When rice is stored, the fatty acid content increases and so the pH decreases, resulting in a reduction in lipase activity (Zhou, Robards, Helliwell, & Blanchard, 2002). 3.5 Catalase activity in rice decreased during storage Catalase is an enzyme capable of decomposing H2O2 into H2O and O2, which can reduce the ability of peroxides to promote lipid oxidation. In our study, catalase activity decreased during storage in both samples, with the activity being significantly less for the rice stored under HT-HH conditions (Fig. 2d). Indeed, the activity of rice catalase decreased from 3.45 U/mg for the initial sample to 1.51 U/mg for the HT-HH sample (D2) and 2.61 U/mg for the ambient sample (D1). This suggested that hot humid conditions inhibited the activity of rice catalase, which would accelerate the formation of off flavors in rice during storage.
3.6 Peroxide value of rice increased during storage The susceptibility of the rice samples to lipid oxidation during storage was determined by measuring peroxide formation (Sowbhagya & Bhattacharya, 2010). The peroxide values increased appreciably during storage in both samples (Fig. 2c), indicating that lipid oxidation occurred. Indeed, the peroxide value increased from 0.2 meq/kg in the initial sample to 0.45 meq/kg in the ambient group (D1) and 0.71 meq/kg in the HT-HH group (D2). These results suggest that hot humid conditions promoted lipid oxidation in the rice, thereby adversely affecting rice quality by producing rancid off odors (Frankel, 1984). This result is consistent with those of the electronic nose and GC-MS experiments, which showed that a variety of volatile off odors were produced during storage. 3.7 Differential genes related to rice flavor metabolic pathways were identified Based on the results of our previous study using RNA-sequencing, the impact of nine genes involved in rice flavor metabolic pathways on the flavor changes occurring during rice storage were studied (Table 2). OS11G0605500 and OS05G0132100 are genes involved in the lipid degradation and heterocyclic compound synthesis pathways. OS08G0508800, OS03G0738600, and OS08G0509100 are genes involved in the linoleic acid oxidative degradation pathway. OS06G0604400, OS02G0676000, OS03G0826600, and OS09G0543100 are genes involved in lipase hydrolysis and phospholipase degradation.
3.8 Genes involved in genes related to the metabolic pathway Fig. 3 shows changes in the expression of the genes involved in the rice lipid metabolism pathway under ambient and HT-HH storage conditions. The OS11G0605500, OS05G0132100, OS08G0508800, OS03G0738600, and OS08G0509100 showed increased gene expression, with the expression being higher under HT-HH than ambient conditions (p < 0.05). These five genes act on the fatty acid synthesis and linoleic acid pathways (Lee et al., 2016). This shows that hot humid storage conditions stimulate the expression of genes that promote lipase activity, thereby producing free fatty acids that reduce rice quality. The OS11G0605500 and OS05G0132100 genes are also capable of acting on lipase to produce heterocyclic compounds, thereby further negatively impacting rice flavor. The expression of the OS06G0604400, OS02G0676000, OS03G0826600 and OS09G0543100 genes decreased during rice storage. Under HT-HH conditions, the decrease in gene expression was significantly more than under ambient conditions (p < 0.05). These genes participate in the hydrolysis of lipases and degradation of phospholipases. Consequently, the decrease in the expression of these genes under hot humid conditions would have reduced the ability of rice to control the activities of lipase and phospholipase, thereby promoting the formation of free fatty acids from triacylglycerols and phospholipids. In summary, storage of rice under high temperature-high humidity conditions led to faster lipid degradation, resulting in the formation of free fatty acids and
volatile compounds (such as aldehydes, ketones, and furans) that adversely impact rice quality. Transcriptomic analysis indicated that the origin of this effect was partly due to increased lipase activity and decreased catalase activity under hot humid conditions. To the authors knowledge, this is the first time that changes in gene expression activity have been related to the flavor metabolic pathway in rice during storage. These results suggested that transcriptomic methods may be useful in establishing the optimum storage conditions for rice.
Conflict of Interest The authors declare that there are no conflicts of interest.
Acknowledgements This work was supported by National Key Research and Development Program of China (2016YFD0400901), the National Natural Science Foundation of China (No. 31571901), the Key Research and Development Program of Jiangsu Province (BE2016386) and the fund of Beijing Key Laboratory of Quality Evaluation Technology for Hygiene and Safety of Plastics, Beijing Technology and Business University (PQETGP2018001).
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FIGURE LEGENDS Figure 1. a) Radar map of Paddy rice during different storage condition. D0 fresh rice, D1, Paddy rice stored Group D1 was stored at normal temperature (25 °C/50% Relative humidity (RH)) as a control group, and Group D2 was stored at 37 °C and RH of 70%. b) Principal component analysis of paddy rice during different storage time. Figure 2. a) Effect of different storage conditions on (a) fatty acid, (b) lipase activity, (c) catalase activity, (d) peroxide activity. Figure 3. Expression analysis of 9 different expressed genes mRNAs in the sample D1 and D2. Total RNA was isolated from fresh paddy rice and paddy rice stored at high temperature and humidity. 8BQ5 gene (ubiquitin 5) was used as an internal reference. Expression levels of each gene are shown as a ratio relative to the treatment time (0 day) that was set to 1. Each value represents the means of three biological replicates from three different samples,
Figures: Fig. 1 a)
LY2/LG
PA/2 PA/2 T70/2 T70/2
LY2/LG 0.3 0.2
LY2/G
LY2/G
LY2/AA LY2/AA
0.1 0
P40/1 P40/1
-0.1
LY2/GH LY2/GH
P10/2 P10/2
D0 0d 新鲜 温 28d 常D1 温高湿 28d 高D2
LY2/gCTL LY2/gCTL
P10/1 P10/1
LY2/gCT
LY2/gCT
T30/1
T30/1
0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 00 -0.05 -0.05 -0.1 -0.1 -0.15 -0.15 -0.2 -0.2
PC2-4.529%
PC2-4.53%
b)
D2
D1 D0
-1 -0.8 -0.8 -0.6 -0.6 -0.4 -0.4 -0.2 -0.2 00 0.2 -1 0.2 0.4 0.4 0.6 0.6 0.8 0.8 PC1-95.192% PC1-95.19%
11
D1 D2
**
4.5
*
*
b)
*
4.0 3.5 3.0
0
7
14
21
28
储藏时间/天 Storage time/d
d)
20
D2 D1
**
**
15
* 10
5 0
7
14
21
储藏时间（天）
Storage time/d
28
prot) (U/mg activity Catalase Prot) 过氧化 氢 酶活度(U/mg
5.0
化物值（meq/kg） 过氧activity (meq/kg) Peroxide
c)
脂肪酸（KOHmg/100g干基） mg/100g DW) Fatty acid(KOH
a)
(mg/g) activity Lipase 动度（mg/g） 脂肪酶活
Fig. 2
4
*
D1 D2
*
*
3
**
2 1 0
0
7
14
21
28
储藏时间(天)
Storage time/d 0.8 D1 D2
0.6
*
** **
0.4
0.2 0
7
14
21
储藏时间（天）
Storage time/d
28
Fig. 3
Table 1. Relative contents of volatile compounds in paddy rice under different storage conditions. Key: initial sample (D0); sample stored under high temperature-high moisture (D2) conditions for 30 days; sample stored at ambient conditions (D1) for 30 days.
Sampleb
Retention Index
Kovats
Compound name Indicesa
Calculated
Literature
D0
D2
D1
9.9
Nonanal
1091.7
1089-1105
3.9±0.3
4.8±0.2
5.2±0.4
17.1
Vanillin
1386.2
1399
0.6±0.1
0.1±0.1
0.2±0.1
9.2
1-octanol
1063.2
1064-1073
0.9±0.2
-
0.5±0.1
1457.0
1451-1456
2.0±0.3
0.4±0.1
0.6±0.1
1996.4
1838
4.0±0.5
-
1.6±0.3
1279.2
1345
2.0±0.1
2.5±0.1
2.8±0.2
Aldehydes
Alcohols
6,10-dimethyl-5,918.8
undecadien-2-one
Ketone 6,10,14-trimethyl-231.8
pentadecanone 4-hydroxy-314.5
Phenols
methoxystyrene 20.5
2,6-di-tert-butyl-p-cresol
1529.2
-
1.2±0.1
1.5±0.1
1.6±0.1
11.2
Acetic acid
1144.2
1013
1.4±0.1
-
-
13.2
Nonanoic acid
1227.6
1278
-
0.3±0.1
-
32.9
Phthalic acid
2037.9
1873
-
42.8±1.2
42.8±1.5
28.0
Methyl myristate
1837.4
1679
-
0.6±0.3
-
Acids
Esters
31.5
Isopropyl myristate
1983.9
1829
2.0±0.2
-
-
34.7
Dibutyl phthalate
2115.9
1969
-
4.1±0.1
4.3±0.2
36.7
Ethyl palmitate
2196.2
1990
-
2.8±0.3
3.5±0.4
38.1
Hexyl acetate
2253.4
1283
12.7±1.3
3.8±0.3
7.5±0.3
39.0
Isopropyl palmitate
2290.8
2025
17.1±1.2
8.6±0.7
8.7±0.4
11.6
Methylbenzene
1159.4
1100
1.1±0.4
1.0±0.2
1.1±0.1
11.8
n-dodecane
1167.8
1150
1.7±0.2
-
-
16.1
1-dodecene
1348.8
1300
1.3±0.2
-
-
17.3
n-tetradecane
1396.7
1400
3.8±0.6
0.3±0.1
1.7±0.2
22.7
n-pentadecane
1621.0
1600
1.2±0.2
0.3±0.1
0.5±0.1
23.5
Styrene
1650.0
1237
0.4±0.1
0.5±0.1
1.0±0.2
23.7
n-hexadecane
1660.5
1600
0.8±0.1
3.6±0.2
3.4±0.2
24.1
1,2-epoxyhexadecane
1674.8
-
0.4±0.1
-
-
31.0
Octadecane
1962.7
1800
-
2.0±0.5
6.3±0.9
1179.6
1006
3.8±0.2
5.7±0.8
6.2±0.4
Hydro carbons
2,3-dihydro-112.1
benzofuran
Others
a
14.0
Indole
1257.9
1116
0.4±0.1
0.1±0.1
-
21.2
Naphthalene
1555.2
1305
0.8±0.1
-
-
27.6
2-pentylfuran
1822.4
1241
1.6±0.2
1.9±0.2
2.7±0.3
Kovats indices were calculated by using a series of n-alkanes (C5–C26) under the
same chromatographic conditions as the samples.
b
The relative contents of volatiles components were semi-quantified by peak areas in
the total ion chromatogram using 2,4,6-trimethylpyridine as an internal standard solution.
Table 2. Differential gene KEGG enrichment list involved in the paddy rice flavor metabolism pathways
Term
Gene ID
Functional description
oxidoreductase activity; molecular function; OS11G0605500 Fatty acid
heterocyclic compound binding
degradation
catalytic activity; fatty acid ligase activity; molecular OS05G0132100 function oxidoreductase activity; molecular function; OS08G0508800 dioxygenase activity Molecular function; oxidoreductase activity;
Linoleic acid metabolism
OS03G0738600
dioxygenase activity; catalytic activity dioxygenase activity; molecular function;
OS08G0509100 oxidoreductase activity
phosphoric diester hydrolase activity; phospholipase OS06G0604400
activity; hydrolase activity; molecular function; lipase activity
Ether lipid metabolism
transferase activity; catalytic activity; molecular OS02G0676000 function hydrolase activity; molecular function; catalytic OS03G0826600 activity phosphoric ester hydrolase activity; phosphoric OS09G0543100 diester hydrolase activity; phospholipase activity;
hydrolase activity
Highlights
The impact of high temperature-high humidity (HT-HH) conditions on rice storage were examined.
Volatile aldehydes, ketones, and furans increased when rice was stored under HT-HH conditions.
The formation of these volatile compounds negatively impacted the aroma profile of the rice.
Gene expression of lipase increased under HT-HH conditions, leading to faster hydrolysis and oxidation of rice lipids.