Effect of storage on the nutritional and antioxidant properties of brown Basmati rice

Abstract The purpose of the present study is to evaluate the effect of storage time and temperature on the nutritional and antioxidant values of different varieties of brown rice. PARB approved indigenous Basmati varieties (Basmati 86, Basmati 515, Basmati super, Basmati super fine and Basmati kainat) were procured and initially tested for physicochemical parameters, including moisture, ash, lipids, proteins, carbohydrates, and fibers from the brown rice powder. Similarly, antioxidant capacity of these brown rice samples was assessed in terms of total phenolic content and 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging potential. Samples of brown rice were stored for 3 and 6 months at 25 and 5°C. On increasing the storage time and temperature, antioxidant activity of rice decreases up to 50%. Nutritional parameters, such as minerals, carbohydrates, and fatty acids were characterized using UV/Vis spectrophotometer, ICP‐OES, GC–MS, and HPLC, revealing significant changes in the chemical composition of brown rice. Observation indicates that storage at high temperatures leads to a rapid decrease in carbohydrate and moisture content than at lower temperatures. The protein and ash content remains controlled and integrate with the mineral composition found. Decrease in the glucose and fructose amount was observed in brown rice varieties except for Basmati super fine and Basmati kainat at 5°C. Regarding fatty acids, oleic and linoleic acids were prominent in oils extracted from the different brown rice varieties, and their content was reduced during the storage due to conversion to behenic, and erucic acids, respectively. From the present study, it can be concluded that low storage temperatures reduce the loss of nutrients, offering better nutritional quality for the consumer.

nese (Mn), potassium (K), magnesium (Mg), calcium (Ca), and sodium (Na) are also present in rice grains (Kalpanadevi et al., 2019). In addition, many heavy metals, such as iron (Fe), aluminum (Al), sodium (Na), silicon (Si), and zinc (Zn) are found in the rice bran (Lin et al., 2020;Zhou et al., 2015). Phytochemicals, for example, homologues of vitamin-E (tocopherols and tocotrienols) and gamma oryzanol are major antioxidants in rice (Bhattacharya, 2017). The rice bran is also a rich source of phenolic acids; rice has the highest concentration of free phenolic acids after corn, whereas the antioxidant capacity of rice is less than the other grains due to its lower concentration of bound phenolic acids. Therefore, consumption of brown rice is more beneficial than that of milled rice due to its nutritional superiority (Van Hung, 2016). Consumers of different countries have various preferences toward freshly harvested and stored rice. Freshly harvested rice is preferred in China, Korea, Vietnam, and Japan due to their stickiness while stored rice is liked in subcontinent and Middle East countries (Pratiwi & Purwestri, 2017). Rice grains can be stored in paddy or milled forms. Storage of paddy (rough rice with husk) reduces loss of nutrients and grain quality while storage of milled rice is more convenient economically and requires less space, making it more attractive for grocery stores and supermarkets in cities (Ahmad et al., 2017). Appropriate storage conditions are important to avoid spoilage, degradation, and germination of dry rice. However, the long journey of rice "from farm to table" involves changes in the physical and chemical properties of brown rice, leading to modifications in the nutritional value. Thus, the commercial value of the rice grains is influenced by aging process and storage conditions (Devraj et al., 2020). Protein, starch, and lipid contents could drop at considerable rates at higher temperatures storage. Increase in bitterness and considerable worsening in the taste and flavor of rice occur due to production of free fatty acids or peroxides from hydrolysis and oxidation of fatty acids and carbohydrates. As the albumin decreases, the solubility of protein could be reduced even if protein contents remain unchanged. Storage of rice results into the loss of free amino acids of outer layer of rice grain which causes the browning of the rice. Furthermore, enzymatic activities could occur in stored milled rice including decreases in amylase and increases in protease and lipase (Devraj et al., 2020). Storage temperature and duration play significant role in variations of nutrients of rice. Storage temperature could affect enzymatic activities, influencing protein, starch, and lipid contents of stored rice (Ojha et al., 2018). A conceptual model gave information about interaction between starch and non-starch components (lipids and proteins) in the endosperm cells of the old rice (Zhou et al., 2015). This mechanism suggested that protein molecules undergo chemical reactions altering structure and thus properties of proteins (Kraithong et al., 2018). These days' researchers prefer to maintain nutrients quality in whole grains during stor- Gujranwala, Pakistan. Samples of paddy rice were dried in the sun to make it suitable for storing to prevent microbial contamination. Each sample (of 5.0 kg) rice of each variety was equally divided into five parts. One part was used for the initial physiochemical study and the other four were packed in polythene bags and were stored in two groups of storage (3 month and 6 month) as well as temperature (5 and 25°C) as shown in Table 1.

| Husk removal and grinding of brown rice
Husk was removed from all types of dry paddy rice samples at each stage (initial, 3 months and 6 months) and each temperature using Stake Rice Huller (model THO35A) to obtain brown rice. For analysis purposes, these brown rice samples were processed into a fine powder (80 mesh size) in a coffee grinder. Ground rice samples were stored in polythene bags in a refrigerator to avoid lipase activity and other physicochemical changes.

| Determination of Physicochemical parameters of brown rice
In the present work, proximate composition is determined by a series of methods given by AOAC-1990 and expressed as percentage (g/100 g) of brown rice. Determination of the physicochemical composition values of dehulled and pulverized brown rice samples, including moisture, ash, crude (protein, fiber, and lipid) and total carbohydrates of each phase (initial, after 3 months, and after 6 months) and each temperature was performed by using the Standard methods of analysis, such as ultraviolet-visible (UV/Vis) spectrophotometer, inductively coupled plasma optical emission spectrophotometer (ICP-OES), gas chromatography mass spectrometry (GC-MS), and high performance liquid chromatography (HPLC) (Katsa et al., 2021;Tai et al., 2021).

| Determination of Total phenolic contents of brown rice
Antioxidants were extracted from rice flour with pure methanol and acetone as well as 80: 20 methanol: water and 80:20 acetone: water on orbital shaker (Hu et al.,1996;Duvernay et al.,2005). For this purpose, 10 g of rice flour was extracted by applying 100 ml (1:10) 100%, 80% methanol, and 100%, 80% acetone at 40°C and 120 rpm for 1 day. After filtration, the extract was concentrated on a rotary vacuum evaporator. These extracts were kept in the freezer for further analysis. Samples of the control and stored selected varieties of brown rice were analyzed for TPCs using the modified method of Mir et al. (2020). Briefly, 0.1 ml of sample solution was poured into a test tube having 2.0 ml sodium carbonate (7.5%) then mixed for 3 min. 1.0 ml of Folin-Ciocalteau reagent (10%) was added followed by the addition of 6.9 ml distilled water to make final volume 10 ml.
This mixture was kept for half an hour to complete the reaction.
Then absorbance at 760 nm was recorded by UV-Vis spectrophotometer. Gallic acid was used as reference standard and concentrations are reported as GAE/kg.

| DPPH radical inhibition potential of the brown rice
Following the procedure used by Zubair et al. (2012) extracts from brown rice samples were tested for DPPH radical-scanning activity.
A 0.1 ml of sample solution was poured in a test tube having 4 ml of pure methanol. Then 1.0 ml DPPH solution (0.001 M in methanol) was added. This mixture was allowed to stand for half an hour then the absorbance was noted at 515 nm using UV-spectrophotometer (Park et al., 2020). Calibration curve of butylhydroxytoluene (BHT) was developed for the calculation of results of scavenging activity.
Absorbance of each mixture was noted thrice.

| Statistics
All the samples analyzed in triplicate and mean were calculated.
Repeated measures ANOVA was used to identify the significant variations (5%) in sugar contents of rice varieties with different storage conditions. The Tukey HSD test was then applied on those groups where a significant difference was observed.

| Effect of storage on the Physicochemical composition of the brown rice
Physiochemical analysis of brown rice of different varieties describes the content of wet (moisture) and dry matter having organic (protein, fibers, carbohydrates, and fats) and inorganic composition (ash). Fresh moisture contents measurement reveals Basmati kainat has the lowest (9.20%) moisture than all varieties which is found in close agreement with the research work in Pakistani on the rice varieties resulting Basmati kainat has the lowest moisture among all the selected varieties (Jamal et al., 2016) while the highest moisture contents observed in Basmati super (10.90%) as depicted in Figure 1. Generally, less than 12% moisture content is recommended for storage of rice to avoid attack of microorganisms and insects while about 9.19-11.1% moisture is reported in the literature for different Pakistani rice varieties (Chavan et al., 2018). The results of our study indicated that storage at the higher temperature at 25°C reduced moisture contents more rapidly as compared with the lower temperature 5°C which is earlier observed and in close agreement with other findings (Ahmad et al., 2017b). Loss of moisture contents during the storage of 3 months was found to be less than that of the second storage for 6 months which is probably due to comparatively cold weather during first storage than that of second stage. Variations in moisture content may be the cause of changes in physicochemical parameters responsible for nutritional quality of rice. Standard deviation among the samples of fresh and stored brown rice depicts more change at longer storage at higher temperature.
Fresh brown rice samples were evaluated for ash contents which found to be varied from 1.22-2.08%, is higher than the reported earlier in the range of 0.52-1.15% in white Basmati rice (Kraithong et al., 2018). Evaluation of crude protein contents of different brown rice varieties in fresh and stored samples is 7.98-9.33% as shown in Figure 3, which is slightly higher than reported values (7.1-8.9%) in white Basmati rice (Kraithong et al., 2018). Present research findings are in accordance with the previous research (Park et al., 2020) where 7.95-9.52% protein contents were reported in brown rice which is slightly higher than our results of brown Basmati rice. The results show that Basmati 515 variety has the lowest protein content, while Basmati super fine has the highest. Comparative values of crude protein are shown in Figure 3, indicate no significant variation after storage, in accordance with earlier findings (Shi et al., 2017;Yu et al., 2017 Figure 4 shows that the loss of lipids is linked to the increase in storage time as well as storage temperature. In a similar research work, higher lipid contents were reported in rice stored at 4°C than at 37°C (Kumar et al., 2018). More than 50% loss of lipid contents occurred during storage of rice for 1 month at 60°C while 1/3 lipid contents were lost during storage at 30°C (Ahmad et al., 2017). In another similar work, it was concluded that brown rice flour produced at low-temperature has excellent physicochemical properties Zakarya et al., 2018).

| Effect of storage on simple sugars of brown rice
Three different sugars, namely, sucrose, fructose, and glucose were analyzed in the selected aromatic brown rice varieties of fresh and stored at different conditions (  Table S1).
These results are in close agreement with other research studies (Chmiel et al., 2018;Lim et al., 2017). A similar research study found that total reducing sugar contents increased sharply in the 16month storage period and thereafter, increased steadily.   Free fatty acids formed may be further oxidized into hydroperoxides and related products . reported in literature is found in the range of 10-50 mg/100 g which is however lower than the present work, while the amount of sodium (Na) of our study is in line with the previous finding (Kraithong et al., 2018;Somaratne et al., 2017 (Liu, Zheng, & Chen, 2017).

| Effect of storage on the minerals concentrations of the brown rice
Concentrations of the other minerals were higher than those cited in earlier research probably due to increase in industrialization and metals pollution in water and soils (Ahmad et al., 2021). Our results are in close agreement with those previously reported by other researchers (Ahmed et al., 2017;Al-Zoreky & Saleh, 2019).

| Effect of storage on TPCs and antioxidant potential of the brown rice
Antioxidants have protective functions in the body, by controlling the free radicals through reducing or scavenging them. Free radicals are known to attack proteins, lipids, and DNA. In addition, these can    Table 3.
Elongation in storage duration results into decrease of TPC's in varieties of brown rice. A significant decline up to 70% in TPCs after second stage of storage at 25°C among the brown rice varieties in fresh and stored samples was observed. Total phenolics ranging from 0.81 to 1.64 mg GAE/g (810-1640 mg GAE/kg) were reported earlier for brown rice (Mir et al., 2016). Maximum decrease occurred during the first stage of storage (i.e., the first 3 months), especially in samples stored at 25°C as shown in Table 3. Decrease in the concentration of total phenolics after storage of brown rice was also investigated in earlier research suggesting involvement of phenolics in aging. Aging process is faster at higher temperatures leading to a more phenolics loss. Lowing in phenolic contents after storage is in accordance with a previous work (Mir et al., 2020) in which a decrease in phenolic contents was noted during 7 months from 17.17 to 6.07 and 7.29 mg/g at 25 and 37°C, respectively. There lies a significant difference ( Means with different superscript letters with in the same row indicate significant differences (p < .05).

TA B L E 3
Effect of storage on the brown rice total phenolic contents (mg GAE/kg of rice) elicitation of rice grains and the results obtained were in close agreement with our results (Ampofo et al., 2020).

| Effect of storage on DPPH radicalscavenging potential
Bioavailability and activity of phenolics is essential for their health claims; therefore, this research study further investigated and demonstrated the antioxidant properties of phenolic extracts obtained from stored brown rice. The results obtained in our study were in close agreement with those reported by Ampofo and co-authors (Ampofo et al., 2020). Brown rice antioxidants are beneficial for health because they have the ability to scavenge the free radicals and reactive oxygen species. Percentage inhibition of selected rice varieties ranged from 41 to 63% in methanol, 27-47% in methanol: water, 41-47% in acetone and 24-35.75% in acetone: water, which are in close agreement with previously cited research (Somaratne et al., 2017). Highest DPPH radical-scavenging ability was exhibited by Basmati super and lowest for Basmati kainat (

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
No data is available for Data Availability Statement.