Comparative Evaluation of Fatty Acid Composition, Tocopherols, and Volatile Compounds of Walnut Oil between Juglans mandshurica Maxim. var. sachalinensis (Komatsu) Kitam and J. regia L.

: We investigated the fatty acid composition and regiospecific distribution of triacylglycerol in Juglans mandshurica Maxim. var. sachalinensis (Komatsu) Kitam and Juglans regia L. oils. Significant differences are observed in the fatty acid compositions and regiospecific distribution of triacylglycerol in both oils. In addition, we measured volatile compounds and tocopherol content in two walnut oils. In results of volatile compound analysis, vanillin is specifically detected from J. mandshurica var. sachalinensis oil, and was not detected in J. regia L. oil. Notably, γ-tocopherol content in the J. mandshurica var. sachalinensis oil was significantly higher than J. regia L. oil.


Introduction
Juglans regia L. is one of the most popular walnuts all over the world, and it has well been investigated its function and nutrition 1,2 . In particular, unsaturated fatty acids UFAs , including oleic acid, linoleic acid, and α-linolenic acid, are abundant in J. regia L. oil 3 9 . The intake of UFAs including α-linolenic acid from J. regia L. oil can decrease total plasma cholesterol and low-density-lipoprotein LDL cholesterol, thereby reducing the risk of coronary heart disease 10 12 .
Triacylglycerol TAG is a major component of edible oils and consists of three fatty acids and one glycerol moiety. The binding positions of fatty acids on TAG are the sn-1,3 and sn-2, according to the primary and secondary alcohol groups on glycerol, respectively. The TAG molecular species from J. regia L. oil are mainly composed of trilinolein LLL , followed by dilinoleoyl-linolenoyl-glycerol LLLn and dilinoleoyl-oleoyl-glycerol OLL 7,9,13,14 . Recent studies have revealed that the binding position of fatty acids on TAG affects the functions of fats and oils, such as absorption and oxidative stability. Martin et al. revealed that the TAG containing UFAs at the sn-2 position was less oxidized than that at the sn-1,3 position 15 . Therefore, the effect of the regiospecific distribution of fatty acids on oxidative stability needs to be assessed in order to estimate the quality of walnut oils. In addition to UFAs, walnut oil contains tocopherols, which inhibit the oxidation of UFAs. Tocopherol isomers of J. regia L. oil is mainly composed of α-, γ-, and δ-tocopherol 3, 6, 8, 9, 16 18 . Among them, αand γ-tocopherol are the most abundant natural antioxidants in vegetable fats 19 . Tocopherols have numerous beneficial properties, such as anti-inflammatory and antiproliferative effects in human cancers 20 . In this way, walnut oils display many benefits to human health and are good sources of UFAs and tocopherols. To present, few studies have investigated tocopherol content in walnut species other than J. regia L.
J. mandshurica Maxim. var. sachalinensis Komatsu Kitam Onigurumi in Japanese is endemic to Japan, with only a few studies reporting on the species. Chaudhary et al. reported that extracts from leaves and immature fruits of J. mandshurica var. sachalinensis and J. mandshurica var. cordiformis have a transthyretin amyloid fibril disruption ability 21 . Machida et al. isolated four enantiomerically pure α-tetralones and three phenolic glycoside syringates from the fruits and bark of J. mandshurica var. sachalinensis 22,23 . However, fatty acid composition and regiospecific distribution of fatty acids in Japanese walnut oil are not well understood.
This study aimed at comparing the composition and regiospecific distribution of fatty acids between J. mandshurica var. sachalinensis and J. regia L oils. In addition, volatile compounds and tocopherol content were also investigated between two walnuts oils.

Chemicals and materials
J. mandshurica var. sachalinensis were collected from Aizu-Wakamatsu City, Fukushima, Japan. J. regia L., imported from the USA, were purchased from Assist Food Inc. Gunma, Japan . CHIRAZYME L-2 C4 and other reagents were purchased from FUJIFILM Wako Pure Chemical Corporation Osaka, Japan .

Preparation of walnut oil
Walnut oil from J. mandshurica var. sachalinensis and J. regia L. were extracted respectively, according to the method of Folch et al. with slightly modification 24 . In brief, the walnut sample 100 g was homogenized in 10 volumes of a mixture of chloroform/methanol 2/1, v/v . The homogenate was vacuum filtered through a filter paper using a Buchner funnel. The filtrates were evaporated in vacuo to dryness and used as walnut oil for analysis.

Enzymatic reaction of TAG in walnut oil
The regiospecific distribution of fatty acids in walnut oil was evaluated using CHIRAZYME L-2 C4 25 . Walnut oil 0.5 g was mixed with 5 g of 99.5 ethanol and 0.33 g of CHI-RAZYME L-2 C4. The mixture was incubated in a 30 water bath for 3 h with shaking at 150 rpm and dried in a rotary evaporator. The resulting oil 0.1 mL was applied to a Sep-Pak Silica cartridge 0.65 g, Waters Co., Milford, MA, USA , which was pre-equilibrated with a solvent mixture of hexane and diethyl ether 8:2, v/v , and eluted with nhexane and diethyl ether to remove the free fatty acid and diacylglycerol fractions. Then, 2-monoacylglycerol 2-MAG was eluted with diethyl ether and dried in a rotary evaporator.

Analysis of fatty acid composition in walnut oil and 2-MAG fraction
The walnut oil and 2-MAG fractions prepared enzymati-cally were methyl esterified according to a modified version of the American Oil Chemist s Society AOCS official method, Ce 1b-89 26 . Walnut oil 50 mg was mixed with 1.0 mL of 0.5 N sodium hydroxide in methanol and heated at 100 for 5 min. The resulting solution was added to 1.5 mL of 14 boron trifluoride solution in methanol and reheated at 100 for 5 min. After cooling the solution to room temperature, 1.5 mL of hexane and 3.0 mL of 20 saline solution were added and mixed. The supernatant was transferred to a new screw-capped sample tube and used as a sample for analysis of fatty acid composition.
The fatty acid methyl esters were subjected to a GCflame ionization detector GC-FID system Nexis GC-2030, Shimadzu Corporation, Kyoto, Japan equipped with an In-ertCap Pure-WAX capillary column 30 m 0.25 mm ID, 0.25 μm, GL Sciences Ltd., Tokyo, Japan . The temperature of the injection port and detector was set at 250 . The column temperature, was initially set at 40 for 5 min and increased to 250 at a rate of 10 /min and held for 15 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The split ratio was 50/1 v/v , and the injection volume was 1 μL. The fatty acid species were identified using the retention time of a standard fatty acid methyl ester Supelco 37 Component FAME Mix, Merck, Darmstadt, Germany . The content of each fatty acid was calculated using the GC-FID chromatogram.

Quantifications of tocopherols in walnut oil
Tocopherol content was quantified using an HPLC system that included a PU-2080 plus pump JASCO Corporation, Tokyo, Japan , a CO-2065 Plus column oven at 40 JASCO , and an FP-2020 Plus fluorescence detector JASCO . The mobile phase was hexane/acetic acid 85:15, v/v and the flow rate was 1.0 mL/min with monitoring at 298 nm and 325 nm for excitation Ex and emission Em , respectively. One gram of walnut oil was dissolved in 10 mL of hexane and add 2,2,5,7,8-pentamethyl-6-hydroxychroman as an internal standard, and 10 μL of the prepared samples were injected into an Inertsil NH 2 column 250 mm 4.6 mm, 5 μm particle size, GL Sciences Ltd. . The amounts of each tocopherol were calculated using Vitamin E Reference Standard FUJIFILM Wako Pure Chemical Corporation .

Analysis of volatile compounds in walnut oil
A solid phase microextraction SPME fiber length 10 mm coated with 50/30 mm divinylbenzene/carbon WR/ polydimethylsiloxane DVB/CAR/PDMS phase Restek, Tokyo, Japan was used to extract volatile compounds. The fibers were conditioned before use and thermally cleaned between analyses by inserting them into the injector port of the gas chromatography system set at 270 for 30 min in a stream of helium.
Headspace SPME was used to extract the headspace volatiles from the samples. The walnut oils 500 mg were placed in a 20 mL headspace vial fitted with a silicone septum. As an internal standard, 10 μL of cyclohexanone 1,000 μg/mL in methanol was added. After an equilibration time of at least 10 min, SPME sampling was performed by exposing the fiber for 60 min in the headspace of the sample at 50 . After sampling, the SPME device was placed into a GC system equipped with a mass spectrometer GC:

Statistical analysis
Across all the experiments, our analyses considered the average of triplicate measurements from two species of walnut oils. A student s t-test was used to estimate differences between a 33.3 mol/100 mol as a theoretical value with the experimental regiospecific value of each fatty acid at the sn-2 position. To determine the tocopherol content, student s t-test was also performed to assess differences between oils. Statistical significance was set at p 0.05.

Results and Discussion
This is the first report to investigate the composition and regiospecific distribution of fatty acids in oil extracted from J. mandshurica var. sachalinensis. and J. regia L. contained 41.7 and 46.5 g of oil per 100 g of kernels, respectively. Table 1 shows the total fatty acid composition and fatty acid composition at the sn-2 position of TAG in J. mandshurica var. sachalinensis and J. regia L. Total fatty acid composition was similar between the J. mandshurica var. sachalinensis and J. regia L. oils. Linoleic acid was the most abundant 71.7 and 60.8 g/100 g oil, respectively , followed by oleic acid 13.0 and 15.0 g/100 g oil, respectively , and α-linolenic acid 11.1 and 15.2 g/100 g oil, respectively . In contrast, palmitic acid was a major component of the saturated fatty acids in both J. mandshurica var. sachalinensis and J. regia L. oils, with values of 2. was linoleic acid 75.3 and 69.9 g/100 g oil , followed by oleic acid 16.8 and 15.6 g/100 g oil, respectively , and α-linolenic acid 7.5 and 13.9 g/100 g oil, respectively . The regiospecificity of each fatty acid at the sn-2 position of TAG in the J. mandshurica var. sachalinensis and J. regia L. oil is shown in Fig. 1. In the regiospecific distribution of fatty acids, palmitic, stearic, and α-linolenic acids Table 1 Regiospecific analysis of fatty acid composition g/100 g oil in J. mandshurica var. sachalinensis and J. regia.

Fatty acid
Total (g/100 g oil) sn-2 (g/100 g oil)  13 . Further research is required to elucidate the effects of genetic and environmental factors on the fatty acid composition in J. mandshurica var. sachalinensis oil. According to previous reports, palmitic, stearic, and α-linolenic acids were selectively esterified at the sn-1,3 position, and oleic and linoleic acids were selectively esterified at the sn-2 position of TAG in J. regia L. oil 7,8,13,14 . This tendency supports our results for the regiospecific distribution of fatty acids in J. regia L. oil Fig. 1 . We previously investigated the effect of the binding position of fatty acids in TAG on the catabolic rates of fatty acids, and observed that the catabolic rate of palmitic, oleic, and α-linolenic acids bound to the sn-2 position of TAG is slowly catabolized for long duration compared with each was bound to the sn-1,3 position 28,29 . We proposed that saturated fatty acids bound to the sn-1,3 position in walnut oils can more rapidly be catabolized and converted to energy. In addition, high concentrations of UFAs in J. regia L. oils reduce LDL concentrations and enhance the level of high-density lipoprotein HDL and are associated with a decreased risk of cardio-vascular diseases 2,15 . In particular, α-linolenic acid in the walnut oil has been reported to decrease the amount of LDL cholesterol 12,30 . Accordingly, J. mandshurica var. sachalinensis oil is a good source of α-linolenic acid, similar to that of J. regia L. oils. The volatile components were detected in the J. mandshurica var. sachalinensis and J. regia L. oil and are detailed in Table 2. 1-Hexanol, heptanal, hexanal, and nonanal were detected in J. regia L. oil Table 2 , which is consistent with previous reports 4,6,7,31 . On the other hand, in J. mandshurica var. sachalinensis, the major aldehydes were hexanal, 2,4-heptadienal, nonanal, 2,4-decadienal, and benzaldehyde. Other volatile components were 1-pentanol, 1-hexanol, 3,5-octadien-2-one, and vanillin. In this study, the secondary oxidation products such as 2,4-heptadienal, 2,4-decadienal, and benzaldehyde were only detected in J. mandshurica var. sachalinensis oil Table 2 . The secondary oxidation products, such as aldehyde, alcohol, and ketone, are typical lipid hydroperoxide derivatives derived from oleic acid, linoleic acid, and α-linolenic acid 4,8,16 . More interestingly, vanillin, which contributes to sweet and vanilla aromas, was only detected in J. mandshurica var. sachalinensis oil. This suggests that vanillin is specific to J. mandshurica var. sachalinensis oil, and was not detected in J. regia L. oil in this study and previous studies 4, 6,7,31 . Table 3 shows the tocopherol content of the J. mandshurica var. sachalinensis and J. regia L. oils. There were no significant differences in δ-tocopherol content between J. mandshurica var. sachalinensis and J. regia L oil 30  The data are represented as mean±SD (n = 3). N.D.: Not detected.

Comparative of Characterization in Two Walnut Oils
and 15 μg/g, respectively . No αor β-tocopherol was detected in both oils 4.73 μg/g . In this study, J. regia L. oil consisted of γ-tocopherol 374 μg/g and δ-tocopherol 15 μg/g , which are consistent with previous reports where γ-tocopherol was the main component 197-375 μg/g , followed by α-12.3-38.0 μg/g and δ-tocopherol 5.38-62.1 μg/ g 3,8,9,13 . On the other hand, the γ-tocopherol content in J. mandshurica var. sachalinensis oil 745 μg/g was significantly higher than that of J. regia L. oil Table 3 . Tocopherols are important antioxidant components of J. regia L. oil 8,16 . The distribution of tocopherol isomers can also affect the formation and composition of volatile oxidation compounds 32 . Based on this, it is assumed that content of tocopherols plays a key role as antioxidation agent in walnut oils. Our results suggest that the abundant γ-tocopherol in J. mandshurica var. sachalinensis suppresses UFA oxidation, when the walnut oil stored at long term. Additionally, γ-tocopherol can effectively reduce platelet aggregation, LDL oxidation, and delay intra-arterial thrombus formation 33 . We therefore propose that the high γ-tocopherol content in J. mandshurica var. sachalinensis identifies it as a potentially important source of natural antioxidants in the maintenance of healthy bodily function.