Indian Journal of Experimental Biology Vol. 55, October 2017, pp. 734-739

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Fatty and amino acid compositions of Argentina and Paraguay are the countries where it spread widely followed by , as well as fordii: a source of 3 α-eleostearic acid and methionine southern regions of . In 2012, China produced about 80% of the world production of tung 4 Sandeep Kumar1*, MK Dhillon2, M Singh3, RS Rathi4, AK oil . The word ‘Tung’ is an ancient Chinese term for Misra5 & JC Rana1 heart. Today, the term is also used to refer the heart 1 1Division of Germplasm Evaluation; 4Division of shaped, large and dark green of this . Exploration and Germplasm Collection, Tung plant is of great economic importance due to its ICAR-National Bureau of Plant Genetic Resources, novel high value oil. Tung oil, derived from , is non- New Delhi-110 012, India edible and classifies as a conjugated polyunsaturated 2 Division of Entomology, ICAR-Indian Agricultural Research drying oil as it contains high content of cis, trans, trans- Institute, New Delhi-110 012, India 9, 11, 13-octa-decatrienoic acid5. It is commonly used in 3ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla-171 004, India formulations of coatings, resins, dyes and inks due to its 6,7 5ICAR-National Bureau of Plant Genetic Resources unique property to dry to a clear and hard finish . Dried Regional Station, Shillong-793 103, India tung oil does not allow many chemicals to pass through Received 05 September 2016; revised 29 November 2016 and also remains unaffected by environmental conditions such as moisture and temperature. Tung oil, unlike other Tung [ (Hemsl.) Airy Shaw, Fam. drying oils, does not darken with the passage of time. It is ] was analysed for α-eleostearic acid content along because of these properties that tung oil makes a widely with seed meal amino acid composition to determine its industrial used drying ingredient in , , coatings and applications. Tung seed contains high contents of oil and protein 8,9 with 54.4 and 18.55%, respectively. Saturated, monounsaturated finishes . Tung oil has been explored as a raw material 10-12 and polyunsaturated fatty acids accounted for 4.98, 6.50 and to produce biodiesel , polyurethane and flour 88.51%, respectively. α-Eleostearic acid was the major fatty acid composites13, thermosetting polymer14 and repairing with 81.02%, along with substantial amounts of oleic (6.50%) and agent for self-healing epoxy coatings15. It is also used in linoleic (7.49%) acids. Similarly, tung defatted seed meal was found rich in sulphur containing amino acid i.e., methionine large scale for production of linoleum, oil cloth, brake (614.59 mg/100 g), and arginine (244.64 mg/100 g). The higher linings, soap, leather dressings, inks, insulating concentration of α-eleostearic acid makes tung seed oil suitable for compounds and fibreboard. Its oil cake is a good fertilizer use as drying ingredient in paints, varnishes and coatings. The but is unsuitable as an animal feed1. But after extracted amino acids/proteins or detoxified tung seed meal can be detoxification, it can be used as a feed ingredient for used as nutritional additive in feedstuffs. Further, the determined cetane index, iodine value and saponification number were not in cattle. accordance with the European Standard Organization. However, Despite some studies carried out on tung in China, it these biodiesel production related properties can be improved after is still an under-utilized plant. No such studies have mixing with other oils. been conducted in India. Earlier studies although Keywords: fordii, Biodiesel, Drying ingredient, Mizoram, reported fatty acid composition, but their results are 4,16 Nutritional additive, Shillong, Sulphur containing amino highly variable . Information available on amino acid acids, Tung seed oil extraction from tung seed meal as an additive in feedstuffs is also scarce17. Therefore, present study was Vernicia fordii (Hemsl.) Airy Shaw (previously classified undertaken to determine the fatty acid and amino acid as Aleurites fordii) belongs to the family Euphorbiaceae compositions in tung seeds collected from northeastern and its common names are tung tree, tung oil tree, tung part of India. nut tree, China wood tree, China wood oil tree, Aceite 1 Materials and Methods chino, Lumbang oil, etc. . Originally, it was reported in southeast Asian region, but later on, it was also grown Seed material in Malawi, Argentina and the USA2. Western-China, The seed material of tung germplasm IC278136 was initially collected from Champhai (Mizoram), India. ————— *Correspondence: The seedlings were planted in 2003 in the field gene E-mail: [email protected] bank of National Bureau of Plant Genetic Resources KUMAR et al.: FATTY AND AMINO ACID COMPOSITIONS OF VERNICIA FORDII 735

Regional Station, Shillong. The plant takes 6-7 years for maturity and yields about 4-5 kg per annum. The tree reaches maximum productivity at around 10-12 years of age. The biochemical analysis of the seeds received from field genebank, ICAR-NBPGR Shillong station, was carried out at Division of Germplasm Evaluation, ICAR-NBPGR, New Delhi. The material is also conserved at National Genebank, ICAR-NBPGR, New Delhi. Fig. 1 depicts the seed (A) and its kernel (B). Fig. 1 — Seed (A); and kernel (B) of tung (Vernicia fordii) tree. Oil, protein and moisture content Oil content of seeds was determined by petroleum reagent kit (WAT052880-Waters Corporation, USA). ether extraction in a soxhlet apparatus for 16 h The vials were heated for 10 min in a water bath at according to the AOAC procedure 7.04818. Protein 55C before analysis with high performance liquid content was estimated by conventional Micro-Kjeldahl chromatography (HPLC). The AccQ-Fluor amino acid method 984.13 of AOAC19. Moisture content was derivatives were separated on a Waters 2707 Module determined by oven dried method. HPLC System attached to a PDA 2998 model. The

Fatty acid analysis using gas liquid chromatography column temperature was set at 37C. The amino acids Seed kernels were grounded and 100 mg test sample were detected at 254 nm. The peaks were acquired was extracted with 10 mL of the extraction solution using Empower Pro Software® by Waters Corporation consisting of chloroform: hexane:methanol (8:5:2 v/v/v). (2005-08) and quantification was done based on The extracts were evaporated in nitrogen gas at 60C for standard amino acids calibration (Thermo Scientific 30 min. Fatty acids were converted to respective methyl Amino Acid Standard H, Prod # NCI0180) run at five esters as per method of Neff et al.20. One µL of the methyl concentrations viz., 10, 20, 30, 40 and ester solution was injected into a highly polar HP 50 µL. Amino acid peaks were visually checked to Innowax capillary column (30 m length, 0.32 m diameter, verify the results. The proportional molar 0.5 µm thickness) with a split ratio of 1:80. A Hewlett concentration for each amino acid was calculated based Packard gas chromatograph equipped with flame on the concentration of standard amino acids and ionization detector (model 6890) was used for the expressed as mg amino acid/100 g sample on dry analysis. Injector and detector temperatures were 260 and weight basis.

275C, respectively. The temperature of the oven was Statistical analysis programmed from 150C holding at 1 min to 210C at the Fatty acid composition was analysed in three rate of 15C/min, followed by 210 to 250 ⁰C at the rate replications whereas all other observations were taken of 5C/min for 12 min. Peaks were identified by in two replications. The data was analysed using comparing their retention time with that of standards. Microsoft Excel 200724. All data is expressed as Peak integration was performed by applying HP3398A mean±Standard error of replicates. software. Results Iodine value (IV), Saponification number (SN) and Cetane index (CI) Oil content and Fatty acid composition Iodine value, saponification number and cetane Seed oil, protein and moisture contents obtained index of tung oil were calculated empirically from its were 54.4, 18.55 and 10.01%, respectively. The oil fatty acid constituents21,22. quality is determined by its fatty acid composition. Amino acid analysis Analysis of tung oil composition showed the presence The 20 mg seed was lyophilized and the vacuum- of palmitic acid (2.39±0.04%), stearic acid dried samples were hydrolyzed as described by Dhillon (2.59±0.05%), oleic acid (6.50±0.08%), linoleic acid et al.23. The samples were dried in an oven at 114 ± 2C (7.49±0.09), and α-eleostearic acid (81.02±0.19%). for 24 h. After hydrolysis, excess HCl was removed Fig. 2 shows the different fatty acids separated using and the samples were vacuum dried for gas chromatograph along with respective retention 90 min. The seed samples were reconstituted with times. α-Eleostearic acid (18:3 Δ9cis,11trans,13trans) was 200 µL of 20 mM boiling HCl. The reconstituted found to be the major fatty acid accounting for 81.02% 20 µL samples were derivatized with AccQ-Fluor of the total fatty acids. Tung seed oil contains 4.98% 736 INDIAN J EXP BIOL, OCTOBER 2017

Fig. 2 — Chromatogram showing separated fatty acids of tung seed oil using GC.

saturated fatty acids as palmitic and stearic fatty acids, Table 1— Amino acid composition of defatted tung seed meal 6.50% monounsaturated fat as oleic acid, and 88.51% polyunsaturated fatty acids as linoleic and Amino acids Tung cake (mg/100g) octadecatrienoic acid (α-eleostearic acid). The SFA: Asp 68.39 ±0.01 MUFA:PUFA ratio of oil was 1:1.30:17.77. Ser 100.85 ± 0.06 Glu 93.32 ± 0.03 Fuel related properties Gly 155.04 ± 0.05 Cetane index provides an idea about the ignition His 70.77 ± 0.01 quality of the fuel. Lower value of the cetane index Arg 244.64 ± 0.06 reflected the dominance of polyunsaturated fatty acids. Thr 87.08 ± 0.07 Higher the CI value of fuel, more quick it will ignite. Ala 85.68 ± 0.02 Pro 98.79 ± 0.06 Iodine value is an indicator of degree of unsaturation. Cys 16.78 ± 0.03 Iodine value, saponification number and cetane index Tyr 95.89 ± 0.07 of the tung seed oil were determined empirically and Val 92.90 ± 0.01 found to be 241.17, 201.13 and 19.17, respectively. Met 614.59 ± 0.10 Lys 56.49 ± 0.03 Seed cake protein content and amino acid composition Ile 53.41 ± 0.01 After extraction of oil, the remaining part of seed Leu 119.82 ± 0.05 known as seed cake or meal was analysed for protein Phe 130.57 ± 0.05 content and amino acid profile. Seed meal contained phenylalanine and valine were found present in tung 23.22% protein content. seed cake except tryptophan which could be detected A total of 17 amino acids were detected in the present by the method used in the present study. Seed meal study as shown in Fig. 3. Different amino acids along contained highest content of sulphur containing amino with respective concentrations are listed in Table 1. In acid methionine with 614.59 mg/100 g and minimum general, concentration of all the amino acids except content of cystine i.e. 16.78 mg/100 g. All other amino cystine was high, especially of methionine and arginine. acids were present in the concentration higher than 50 All the essential amino acids i.e. histidine, arginine, mg/100 g. Lysine which is generally found deficient in threonine, methionine, lysine, leucine, isoleucine, cereals was found in high concentration i.e. 56.49 KUMAR et al.: FATTY AND AMINO ACID COMPOSITIONS OF VERNICIA FORDII 737

Fig. 3 — Chromatogram showing amino acid profile of defatted tung seed cake. mg/100 g. Other amino acids such as arginine, glycine, Table 2—Amino acid scores of defatted tung seed meal phenylalanine, serine and leucine were present in more Amino acid Tung EAAC Tung AAS than 100 mg/100 g. (mg/g protein) The quantity of total essential and non-essential Isoleucine 2.3 5.7 amino acids was 1470.26 and 714.74 mg/100 g, Leucine 5.2 7.4 respectively. Sulphur containing amino acids Lysine 2.4 4.4 were present in higher concentration with a total of Methionine+Cystine 27.2 77.7 631.37 mg/100 g, whereas aromatic amino acids Phenylalanine+Tyrosine 9.7 16.2 constitute a total of 226.46 mg/100 g. The protein Threonine 3.7 9.4 content was basic in nature as total basic amino acid Tryptophan ND ND content (371.90 mg/100 g) was significantly higher than Valine 4.0 8.0 total acidic amino acids (161.70 mg/100 g). Amino acids Total 54.5 8.8 score of the tung seed meal essential amino acids are EAAC = Essential amino acid composition; AAS = Amino acid listed in Table 2. scores; ND = Not determined

Discussion acids with 2.4 and 2.3 %, respectively, followed by Differences between fatty acid profiles among oil of linolenic acid (0.4%). But in the present study, tung (Aleurites fordii) samples collected from linolenic acid was below detectable levels. This may be Guangxi, Chongqing, Hunan and Guizhou provinces of because conjugated trienoic fatty acids China were studied by Li et al.4. The variation (α-eleostearic acid in case of tung oil) are generally observed for octadecatrienoic acid and poly- prepared by an alkaline isomerization of α-linolenic unsaturated fatty acids ranged from 72.56-88.59 and acid25. Similar results were reported by Zhan et al.26 80.26-92.39 percent, respectively. Their results who measured the tung oil composition at three suggested that the concentration of octadecatrienoic different developmental stages and found eleostearic acid and polyunsaturated fatty acids in tung oil vary acid as the most abundant fatty acid which increased between different regions and among varieties. Zhang from 72.28 to 77.5% whereas linolenic acid was et al.16 analysed seed oil from 41 tung tree accessions undetectable at the two later developmental stages. So, collected from different places in China for fatty acid the results of the above mentioned studies indicate that composition. The relative abundances of fatty acids the fatty acid profile varies with region and included α-eleostrearic acid (77.2%), linoleic acid variety/genotypes, justify the fatty acid analysis of tung (7.6%), oleic acid (5.9%), β-eleostearic acid (4.2%), seed oil collected from Champhai (Mizoram), India in saturated fatty acids including palmitic and stearic the present study. Further, the presence of high content 738 INDIAN J EXP BIOL, OCTOBER 2017

of α-eleostearic acid (81.02%) makes the tung In case of seed cake, which is a by-product of the accession IC278136 of higher industrial importance as tung crop, the essential amino acid scores were low the fatty acids with additional or unusual functional because it is a oilseed crop. It contains good quantities groups in chain represent excellent feed stocks for of sulphur containing amino acids and arginine. All industry27. essential amino acids except sulphur containing amino Poor agronomic features of Vernicia species, makes acids scored as limiting amino acids. Further, tung seed it impossible the large-scale production of its oil contains some toxic components, which also makes its through traditional farming. Furthermore, limited oil cake unsuitable as an animal feed. So, its protein success has been achieved in the efforts to transfer can be used after removal or detoxification of such genes involved in the biosynthesis of these unusual components or in the form of extracted purified fatty acids to higher yielding through proteins or amino acids, as additive in feedstuffs. transgenics, providing only 15-30% of desired fatty Further, to add more values for industrial applications, acid28-30. These studies clearly indicate that additional there is need to evaluate tung seed for phytochemical genes and more knowledge of seed oil biosynthesis are profile and, for antioxidant and antimicrobial required before plants are engineered to produce these activities36,37. Zheng et al.17 extracted high yield of industrially important fatty acids at a large scale. amino acids from tung meal exhibiting low cytotoxic and high antioxidant activity and highlighted its According to European Standard Organization, potential as a functional ingredient. fuels with CI>51 and IV<115 can be used as 31 biodiesel . Although, our values for CI and IV did not Conclusion fulfil these standards, but efforts have been made For this study, it is concluded that tung accession 10- towards development of biodiesel from tung seed oil IC278136 from Indian region contains high seed oil 12 . Tung seed oil contains high degree of poly- content (54.4%) as reported by earlier studies, unsaturation (88.51% in the present study) which however, it varies in its fatty acid composition. Higher makes it unstable for a long period due to oxidation. content of α-eleostrearic acid (81.02%) and linoleic It’s fatty acid composition and so the oxidation acid (7.49%), makes it important from industrial point stability can be improved by blending with other oils. of view. The values for IV and CN were not found The IV, SN and CI required as per the ESO can be desirable as per ESO standards, but can be improved achieved by blending tung oil biodiesel with other by mixing with oil from other sources. Similarly, its biodiesels. Therefore, tung biodiesel finds application seed meal is a good source of protein (23.22%) and as a blend component of an alternative engine fuel, amino acids particularly, sulphur containing amino although tung biodiesel is not good enough as a fuel for acid methionine, and arginine. The extracted 10 diesel engines by itself . The properties of the tung oil protein/amino acids or detoxified tung seed meal can biodiesel can be improved by blending with palm or be used as nutritional additive in feedstuffs. canola oil biodiesels to meet the biodiesel specifications11. References 1 Kureel RS, Kishor R, Dutt D & Pandey A, Tung: A potential 17 Zheng et al. reported 40.20 (in tunnelling microscopy) tree borne oilseed. (NOVOD, Ministry of Agriculture, and 17.30 (in ultrafast scanning tunnelling microscopy) per Government of India), 2009. cent crude protein in tung meal. Similarly, in the present study, 2 Derksen JTP, Cuperus FP & Kolster P, Renewable resources in coatings technology: a review. Prog Org Coat, 27 (1996) the tung seed cake was found rich source of the methionine, a 45. sulphur containing amino acid and arginine, whereas, cystine 3 Sharma V, Das L, Pradhan RC, Naik SN, Bhatnagar N & content was lowest among amino acids. 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