Effect of Maturing Stages on Bioactive Properties, Fatty Acid Compositions, and Phenolic Compounds of Peanut ( Arachis hypogaea L.) Kernels Harvested at Different Harvest Times

: The present study investigated the effects of harvesting time on the physicochemical properties, antioxidant activity, fatty acid composition, and phenolic compounds of peanut kernels. The moisture content (air-dried basis) of peanut kernels was determined between 4.47% (September 15, 2019) and 7.93% (October 6, 2019), whereas the oil contents changed from 45.95% (October 6, 2019) to 49.25% (September 22, 2019). The total carotenoid, chlorophyll

are the biggest peanut-producing countries. The total production of these countries constitutes approximately 69 of worldwide peanut production 1, 19 . Peanut seeds are directly used or processed, as cookies, biscuits, confectionery, chocolate products, pistachio-added ice cream, peanut butter, and ready-made breakfast packages 20 . About 53 of peanut production in the world is allocated for cooking oil manufacture, 32 for peanut butter and cookies, and 15 for animal feed 21 . Moreover, oil composition is affected by the production location, cultivar, and climate soil humidity and temperature 5, 22 . Variations in climatic conditions affect the oleic/linoleic acid ratio 2 . The aim of present study was to investigate the effects of harvest time on moisture, oil, carotenoid, chlorophyll, total phenol, and total flavonoid contents, antioxidant activity, fatty acid composition, and phenolic components of peanut Arachis hypogaea L. kernels harvested at different periods were investigated.

Materials
The peanut was obtained from the field cultivated on May 15, 2019 in Osmaniye City, Turkey. Harvesting dates were scheduled on August 25, September 1, September 8, September 15, September 22, September 29, and finally on October 6, at 1-week intervals. Peanut samples were airdried after harvesting.

Moisture content
The moisture content of the samples were measured at 105 in an oven Nüve FN055 Ankara, Turkey until reaching a constant weight.

Oil content
The dried peanut material was extracted using petroleum ether in a Soxhlet apparatus for 6 h at 50 to determine the oil content Harwood, 1984 . After drying the extract in a rotary evaporator, oil content was determined as the difference in weight between the dried peanut sample before and after extraction 23 .

Fatty acid composition
The method of ISO-5509 24 was used for fatty acid methylation. Methyl esters were analyzed through gas chromatography-flame ionization detection GC-FID using a Shimadzu GC 2010 chromatograph equipped with a flame ionization detector FID on a capillary column coated with Teknokroma TR CN100, P/N TR 882162 fused silica column 60 mm length; 0.25 mm id; 0.2 mm film thickness . The temperature of the injection block and detector was 260 . Nitrogen was used as the mobile phase, with 1.51 mL/min flow rate. The total flow rate was 80 mL/min, and split rate was also 1/40 mL/min. Column temperature was pro-grammed at 120 for 5 min and increased to 240 at 4 / min and held for 25 min at 240 .

Extraction procedure
The samples were extracted according to the method previously described by Iacopini et al. 14 , with slight modifications. The ground samples 5 g were added to 15 mL of methanol. The mixture was kept in an ultrasonic waterbath for 1 h, followed by centrifugation at 6,000 rpm for 10 min, and then the supernatant was filtered using 0.45-µm membrane. Then, n-hexane 15 mL was added and mixed using a vortex apparatus. The methanol and n-hexane layer were separated using separating funnel. This step was carried out twice with 10 mL of n-hexane. In each step, the methanol phases were collected and then evaporated at 40 . The dried extracts were dissolved in 25 mL of methanol.

Chlorophyll analysis
The chlorophyll content of the peanut oil samples were measured at 670 nm using a spectrophotometer 25 .
Chlorophyll mg/kg A 670 106/613 100 d where, A is the absorbance, and d is the bathtub thickness.

Total phenolic content
The total phenolic content of the extracts were determined using Folin-Ciocalteu FC reagent as previously described by Yoo et al. 26 , with slight modifications. For the extraction, 20 mL of methanol water 80:20 v/v was added to approximately 2 g of the sample and shaken for 3 h at room temperature in a shaking water bath. Then, 20 mL of n-hexane was added to the remaining extract from the filtered samples, and after phase separation was achieved in the separation funnel, the underlying methanol phase was transferred into the tubes and used for further analysis. FC reagent 1 mL was added and mixed for 5 min after 10 mL of 7.5 Na 2 CO 3 was added. The solution in the tubes was mixed again, and the final volume was adjusted to 25 mL using deionized water. At the end of 1 h, the total phenolic content was determined at a wavelength of 750 nm using a spectrophotometer. The results were given as mg gallic acid equivalent GAE / L of fresh weight.

Total flavonoid content
Total flavonoid content was determined according to the method previously described by Dewanto et al. 27 . Methanol extracts were appropriately diluted with distilled water. Then, 0.3 mL of 5 NaNO 2 solution was added to each test tube. After 5 min, 0.3 mL of 10 AlCl 3 solution was added, and after 6 min, 2 mL of 1.0 M NaOH was added. At the end of this period, the total volume was adjusted to 5 mL using water, and the solutions in the test tubes were thoroughly mixed. The absorbance of the pink solution obtained was measured at 510 nm. The calibration curve was prepared using catechol as a standard. Flavonoid content was expressed as mg catechol equivalents CE per dry weight mg CE/L DW .

Antioxidant activity
The 2,2-diphenyl-1-picrylhydrazyl DPPH radical-scavenging ability of the peanut kernel extracts was measured according to the methods described by Lee et al. 28 . The mixture was vigorously shaken and allowed to stand at room temperature for 30 min. After which, absorbance was recorded at 517 nm using a spectrophotometer. DPPH radical-scavenging ability was calculated using the following equation: where, A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. All samples were analyzed in triplicate. 2.2.9 Determination of phenolic compounds HPLC analysis of phenolic compounds were performed using a Shimadzu HPLC equipped with a PDA detector and an Inertsil ODS-3 5 µm; 4.6 250 mm column. The mobile phase was a mixture of 0.05 acetic acid in water A and acetonitrile B . The flow rate of the mobile phase was 1 mL/min at 30 , and the injection volume was 20 µL. The peaks were recorded at 280 and 330 nm using a PDA detector. The gradient program was as follows: 0-0. Extraction of carotenoids was performed according to the method previously described by Silva da Rocha et al. 29 . The ground sample 2 g was added to 25 mL of acetone. The mixture was shaken by vortex for 10 min and filtered using filter paper Whatman No. 1 and passed through a separation funnel. The filtrate was fractionated with 20 mL of petroleum ether and washed with 100 mL of distilled water to remove the acetone. These steps were repeated twice. Whatman No. 1 covered with anhydrous sodium sulfate 5 g for removing residual water was used to filter the petroleum ether layer. The volume of the extracts was adjusted to 25 mL using petroleum ether. Then, absorbance was measured at 450 nm.

Statistical analyses
A complete randomized split-plot block design was used, and analysis of variance ANOVA one way was performed using the JMP software version 9.0 SAS Inst. Inc., Cary, N.C., USA . All analyses were done in triplicate, and the results are presented as mean standard deviation MSTAT-C of independent harvest times 30 .

Results and Discussion
3.1 Effects of harvest time on the physicochemical properties, bioactive compound content, and antioxidant activity of peanut kernels Table 1 presents the physicochemical properties, bioactive compound content, and antioxidant activity of peanut kernels harvested at 1-week intervals starting from August 25 to October 6, 2019. Results showed several variations, depending on the harvest time. Statistically significant differences p 0.05 were observed among the moisture, oil, total carotenoid, chlorophyll, total phenol, and total flavonoid contents, as well as antioxidant activity of peanut kernels harvested at different times. The moisture content air-dried basis of peanut kernels ranged from 4.47 September 15, 2019 to 7.93 October 6, 2019 , whereas the oil content of peanut kernels ranged from 45.95 October 6, 2019 to 49.25 September 22, 2019 . With the progress of the harvest time, a certain decrease was observed in the moisture content of peanut kernels; however, a partial increase was observed in the last harvest time. With the progress of the harvest time until September 29, 2019, the oil content of peanut kernels was increased. Rosales-Martinez et al. 31 reported that raw and roasted peanut kernels contained 2.91 and 2.1 moisture, and 47.14 and 51.87 lipids, respectively. The moisture and oil contents of peanut cultivars grown in Tunisia ranged from 7.3 to 8.48 to 45.32 to 48.53 , respectively 4 . Mora-Escobedo et al. 5 reported that the oil content of eight peanut varieties grown in Mexico ranged from 37.9 to 56.3 . Chukwumah et al. 32 reported that peanut kernels  32 reported that the total phenolic and flavonoid contents of raw peanut kernels were 25.71 mg catechin equivalent CE /g and 0.01 mg GAE/g, respectively. Although the carotenoid, chlorophyll, and total phenolic contents of peanut kernel samples were found at low levels throughout the harvest time, they showed partial differences, depending on the harvest time. The antioxidant activity of the peanut oil extracts ranged from 4.42 first harvest time on August 25, 2019 to 4.70 September 1, 2019 . Additionally, it was observed that the antioxidant activity of mature peanut kernels was increased with the progress of the harvest time. There were positive relations among the total phenol content, flavonoid content, and antioxidant activity of peanut oil extracts. The total phenolic content and antioxidant activity of Tunisian peanut cultivars ranged from 1.0 and 2.1 mg GAE/g DW to 550 and 1550 IC 50 µg/mL, respectively 4 . The total phenol content and antioxidant activity of raw peanut kernels were determined as 370 mg GAE/100 g DW and 6 µmol Trolox equivalent/g DW, respectively 31 . Gimeno et al. 34 reported that olive oil phenol content decreased during rip-ening. When results were compared with the previous studies, partial differences were observed. These differences may be attributed to variety, the place of origin, climatic factors, maturation, cultivation factors; such as irrigation, fertilization, harvest time, and extraction; and quantification methods used.
3.2 Effects of harvest time on the fatty acid content of peanut kernels Table 2 shows the fatty acid composition of oil extracted from peanut kernels harvested at 1-week intervals August 25-October 6, 2019 . The dominant fatty acids were palmitic, oleic, and linoleic acids, depending on the harvest time, followed by stearic, behenic, arachidic, and linolenic acids. October 6, 2019 to 3.17 September 1, 2019 . Other fatty acids identified in peanut kernel oils obtained at different harvest times were found at low levels 0.08 . Myristic acid was only identified in peanut kernel oil harvested on September 29, 2019. Statistically significant differences were observed among the fatty acid composition of peanut kernel oils, depending on the harvest times p 0.05 . With the advancement of harvest time, stearic acid, oleic acid, and linoleic acid were increased until a certain harvest time, whereas polyunsaturated fatty acids, such as linolenic acid, were decreased. Their amounts were increased until a certain harvest time,  33 . The fatty acid composition of the oil from peanut kernels exhibited greater variation at different harvesting dates. Although changes were clearly observed between total saturated and unsaturated acids, these differences were quite pronounced in palmitic, stearic, oleic, and linoleic acids. Results showed several differences, compared with the results of previous studies. These differences can be probably due to agricultural factors, genetic structure, locations, climatic factors, maturation time, several analytical conditions, and solvent types. Table 3 illustrates the amounts of phenolic compounds of peanut kernels harvested at a 1-week interval. Depend-ing on the harvest time, the amount of phenolic compounds of peanut kernels exhibited partial variations. Statistically significant differences were observed among the amount of phenolic compounds of peanut kernels harvested at different maturity stages p 0.05 . The key phenolic compounds of peanut kernels were gallic acid, 3,4-dihydroxybenzoic acid, -catechin, and 1,2-dihydroxybenzene. The gallic acid content of peanut kernels ranged from 1.  -catechin and 1,2-dihydroxybenzene contents as they were increased until a certain harvest time and then were decreased. Rosales-Martinez et al. 31 determined that raw and roasted peanut kernels contained 5.84 and 8.24 µg/g resveratrol, 114.35 and 122.14 µg/g catechin, 262.23 and 238.04 µg/g epicatechin, and 29.42 and 49.62 µg/g quercetin, respectively. Ballistreri et al. 39 determined the effects of the degree of maturity on the total polyphenol content in pistachio kernels, and they found that polyphenol content decreased with the maturity stage of pistachios. Persic et al. 40 reported an increase in polyphenol content with the ripening of hazelnuts. When the results were compared with those of previous studies, our findings showed partial differences. Moreover, the phenolic compound content of peanut kernels showed fluctuations, depending on the harvest time. These variations may be attributed to genetic factors, variety, agricultural factors, growing conditions, and ecological and climatic factors.

Conclusion
Depending on the maturity stage, all the peanut kernel samples that were analyzed exhibited differences in their bioactive compounds, antioxidant activity, fatty acid composition, and phenolic compounds. With the progress of the harvest time, a certain decrease was observed in the moisture content of peanut kernels; however, a partial increase was observed in the last harvest time. With the progress of the harvest time until September 29, 2019, the oil content of peanut kernels was increased. With the advancement of harvest time, stearic acid, oleic acid, and linoleic acid were increased until a certain harvest time, whereas polyunsaturated fatty acids, such as linolenic acid, were decreased. Their amounts were increased until a certain harvest time, and then they were decreased. The amounts of other phenolic compounds of peanut kernels were found at low concentrations, depending on the maturity stages of the peanut kernels. It was determined that the amount of gallic acid decreased until a certain harvest time and then increased with the progress of the harvest time. The results showed that peanut kernel and oil had distinctive phenolic profiles and fatty acid contents. The findings of the present study may provide information for the best time to harvest peanut to achieve its maximum health benefits.