Aqueous enzymatic extraction of camellia seed oil (camellia oleifera) from Viet Nam

JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0069 Natural Sci. 2016, Vol. 61, No. 9, pp. 162-168 This paper is available online at 162 AQUEOUS ENZYMATIC EXTRACTION OF CAMELLIA SEED OIL (Camellia oleifera) FROM VIETNAM Hoang Than Hoai Thu 1 , Dao Van Hoang 1 , Doan Lan Phuong 2 , Hoang Thi Bich 2 and Pham Quoc Long 2 1 National Key Laboratory for Petrochemical and Refinery Technology 2 Institute of natural products chemistry Abstract. Aqueous enzymatic extraction method was applied to extract oil from camellia seed (Camellia oleifera). Effects of individual enzymes, pH, reaction time and temperature were studied. The effects of hexane and aqueous enzymatic extraction on the oil„s physico-chemical properties (fatty acid compositions, free fatty acid) were compared. Using protease yielded significantly more oil than others did. Under the optimal conditions of reaction such as enzyme concentration 0.6 %; pH neutral; reaction temperature 50 o C; reaction time 3 hours, a free-oil 75.9% yield was obtained. Compared with hexane - extracted oil, aqueous enzymatic extracted oil was more acidic, and had a higher percentage of mono-unsaturated fatty acid, lower percentage of total saturated fatty acid, and poly-unsaturated acid. Keywords: Enzymes, Aqueous enzymatic oil extraction, Camellia seed. 1. Introduction The shrub Camellia oleifera originates from China. In Vietnam, it is distributed in many provinces in the north and the central regions including Lang Son, Quang Ninh, Phu Tho, Yen Bai, Ha Giang, Nghe An, Thanh Hoa provinces, etc. C. oleifera seed oil (COS oil) is a high-quality edible oil that has been proved to protect liver against CCl4 – an induced oxidative damage [1]. Long and Wang [2] labeled COS oil as “Eastern olive oil” for its containing of approximately 77% mono-unsaturated fatty acids and several compounds that helped to reduce the risk of cancer [3]. Due to its high oleic content and levels of natural anti-oxidants (phenol and tocopherol), COS oil is highly resistant to peroxidation [3]. There are three traditional techniques to extract oil from seeds: hydraulic pressing, expeller pressing and organic solvent (mostly hexane) extraction [4]. Most commercial COS oil is obtained by either hexane extraction or combining expeller pressing with hexane extraction. However, hexane extraction has some unwanted effects, such as oil is more flammable and contaminated by solvent residues and air pollution. Taking into account environmental and safety concerns, more rigorous methods have been introduced to utilize hexane world-widely [5]. Received April 6, 2016. Accepted November 30, 2016. Contact Hoang Than Hoai Thu, e-mail address: Thuhth@gmail.com Aqueous enzymatic extraction of camellia seed oil (Camellia oleifera) from Vietnam 163 In 2001, the U.S. Environmental Protection Agency (EPA) issued more stringent guidelines for hexane emissions by vegetable oil extraction facilities [6]. For pilot studies, aqueous enzymatic extraction (AEE) is a rising method that has been developed and practiced in the laboratory and at the industrial-scale level [7]. The process usually includes grinding and milling seed or oil-rich material, enzymatic hydrolysis and centrifugation. In comparison with conventional methods, AEE of oil has many advantages. For example, it eliminates solvent consumption and requires lower energy. It also enables simultaneous recovery of good-quality oil and protein from the most oil seeds. One disadvantage associated with this method is low-oil yeld which limits industrial application of AEE [8]. In order to effectively increase the oil yeld, some pre-treatment approaches have been utilized. Sharma and Gupta [9] reported that the ultra-sonic pre-irradiation enhanced the oil yield from 77% to 95% w/w in the AEE of almond. Proteases improved oil extraction from both soybean flour and extruded flakes, however once combining extraction processing with enzyme treatment, yielded more free oil than treating flour with enzyme alone [10]. In this paper, we describe a novel process for extraction of COS oil. An important step in the process development has been the pre-treatment of the C.oleifera seed powders with microware puffing before enzyme treatment leading to very high yield of oil. 2. Content 2.1. Material and methods * Materials C.oleifera seeds were purchased from local markets in Quang Ninh province. All other chemicals and solvents were analytically graded. Enzymes were purchased from China and stored in plastic bag at -18 o C and thawed in a refrigerator at 4 oC a day before use. Four enzymes: pectinase, protease, cellulase and α-amylase were obtained from Nanning Pangbo Biological Engineering Co., Ltd and Imperial Jade Bio- technology Co., Ltd (China). Pectinase was produced from a selected strain of Aspergillus niger with an activity of > 6,000.000 units/gram; protease was produced from Bacillus subtilis, with an activity of > 60.000 units/gram; cellulase was produced from Trichoderma reesei, with an activity of 2,000 U/g and α-amylase was produced from Bacillus subtilis, with an activity of > 9,500 units/g. The temperature and pH for an optimal activity of pectinase, protease, cellulase and α-amylase were 45 - 55 o C at pH 4.5 - 5.5; 40 - 60 o C at pH 7 - 7.5; 40 - 60 o C at pH 4.5 - 5 and 50 - 55 o C at pH 6 - 7, respectively. * Microwave treatment C.oleifera seeds were cracked, shells were carefully removed, thus the kernels obtained were used for oil saponin extraction. Later, the kernels were dispersed in 70% ethanol aqueous solution (1:3 w/w), then milled by crusher. Saponins were subsequently extracted within a specified period of time (30s) in a home microwave oven with an adjustable power setting ranging from 100 - 800 W. After centrifugation of the slurry at 5000 rpm for 5 minutes, the saponin content was detected, then, added with another 70% ethanol aqueous solution (1:2 w/w) in to COS kernels for the Hoang Than Hoai Thu, Dao Van Hoang, Doan Lan Phuong, Hoang Thi Bich and Pham Quoc Long 164 second microwave-assisted extraction of saponin. The saponin yield was checked by its extraction quality for each run. * Oil extraction Hexane extraction A Soxhlet extractor was heated in a water bath. Each thimble was filled with 10 g of the COS flour and extracted for 8 hours. The micelle was evaporated in a rotary evaporator. The oil obtained was dried in a hot-air oven at 105 o for 30 minutes to eliminate residual hexane and collected in a sealed glass bottle. Aqueous enzyme extraction After extracting saponin, COS meal was collected and sieved through a 100-mesh sieve to obtain the fine powder meal. The basis procedure for aqueous enzymatic extraction of COS is as follows: (1) place powder into a plastic tube; (2) add distilled water at a ratio of 1:7 w/v (5 g powder for 35 mL distilled water); (3) adjust pH (1M H2SO4 and 0.5M NaOH), then add enzyme; (4) induce enzyme hydrolysis at 50 o C in a water bath with constant shaking; centrifuge at 10.000 rpm for 10 minutes in a centrifuge; (6) carefully remove the top oil into a glass tube. The free oil yield is calculated by the following equation, which assumes that 100% oil was recovered with the hexane extraction. Free oil yield (%) = oil in glass tube (g) ∕ total oil in camellia seed (g) × 100 For individual enzymes screening, four enzymes (cellulase, protease, amylase, pectinase) were selected and treated. * Analytical methods Moisture and volatile content were determined as weight loss accorded by TCVN 7035:2002 at 103 ± 2 o for 3 hours, and the crude oil in the COS kernel and residual oil in the defatted flour were extracted using Soxhlet method (AOAC, 920.85). Nitrogen content was analyzed by the Kjeldahl method (TCVN 8125:2009). The flour was burnt in a furnace at 600 o C for 2 hours to determine the ash content (TCVN 8124:2009). The oil obtained by AEE method was analyzed for free-fatty acid content, iodine value, saponification value by methods recommended by the EN 14103; TCVN 6122-07; TCVN 6126-96; free-fatty acid content is 0.5 × acid value * Fatty acid composition The fatty acid composition of COS oil was determined using its fatty acid methyl ester by ISO/FDIS 5590:1998 method. GC-MS analysis was performed on Agilent. The detector temperature was programmed for 370 o C in 1 minute, increased 150 o C with a rate of 100 o C /min and increased 270 o C within 5 minutes. The injector temperature was set at 270 o C with a flow rate of 20mL/min. 2.2. Result and discussion 2.2.1. Chemical composition of the COS Chemical composition of the COS is presented in Table 1. The COS has high oil content (60.4%), while its water content is about 8.22%. Aqueous enzymatic extraction of camellia seed oil (Camellia oleifera) from Vietnam 165 Table 1. Chemical composition of COS Composition Value Water (%) 8.22 Oil (%) 60.4 Protein (%) 6.8 Starch (%) 9.15 Ash (%) 2.56 2.2.2. Micro wave – assisted saponin extraction Tea saponins is one kind of glycoside compound, which dissolve in water to form a stable soapy froth [11]. Since it is generally extracted from camellia seed-oil cake at low quality, it is difficult to extract saponins from this source in industry [11]. We directly remove camellia saponins from kernels with alcohol as extraction solvent under the help of micro-wave heating. The saponins in camellia seed kernels can be completely removed by the second microwave- assisted extraction. Figure 1shows that the extraction efficiency obtained with AEE method is high, which the recovery yield is up to 80.2% in 30 seconds and the saponins left can be completely removed by the second AEE. 0 10 20 30 40 50 60 70 80 90 1 2 Extraction time Sa p o n in y ie ld ( % ) Figure 1. Micro wave – assisted extraction saponin from Camellia oleifera seed kernels 2.2.2. Optimization of aqueous enzymatic extraction * Screening enzymes The effect of four individual enzymes and a control with no enzyme on free-oil yield were investigated and the unchanged enzyme concentration is 5%. Protease caused the highest 73.27 % oil yields suggesting that the oleosins and others protein surrounding the oil-rich liposomes are the main barrier that hinders the liberation and coalescence of oil from its bodies. Treatment with pectinase, cellulase and α-amylase caused intermediate oil yields of 50.56%, 45.25 % and 31.92%, respectively (Figure 2). Camellia seed contained much starch when amylase was applied, the slurry became viscous and it was more difficult to obtain free oil by centrifugation than treatment with other enzymes. Hoang Than Hoai Thu, Dao Van Hoang, Doan Lan Phuong, Hoang Thi Bich and Pham Quoc Long 166 The cellular organelles called oleosomes in the oil seed cotyledons are the principal repository sites of lipid reserves in oil seeds [12]. The walls surrounding the cells are primarily composed of cellulose, hemicellulose and lignin in addition to pectin. The rupture of cell walls is a critical step in improving oil extraction yield during aqueous enzyme extraction oil. Using most hydrolytic enzyme such as cellulose, protease, pectinase, amylase in the usual AEE is to break the structure of the cotyledon cell walls, and to release oil from plant material [12]. Figure 2. Camellia oleifera seed kernel meal treatments were performed with neutral protease at pH 7 - 7.5, and 50 o C for 3 hours, at neutral pH and, 50 o C for 1 hour (in case of amylase), while at pH 4.8 and 50 o C for 3 hours (in case of cellulase and pectinase) Figure 3. Effect of enzyme concentration on extract yield obtained during enzyme-assisted aqueous extraction oil process. COS kernel meal treatments were performed with protease at pH 7 - 7.5, and 50 o C for 3 hours Figure 4. Effect of reaction time on extract yield obtained during enzyme-assisted aqueous extraction oil process. COS kernel meal treatments were performed with protease at pH 7 - 7.5, and 50 o C enzyme concentration 0.6 %; for different time Figure 5. Effect of temperature on yield obtained during enzyme-assisted aqueous extraction oil process. COS kernel meal treatments were performed with protease at pH 7 - 7.5, in 3 hours, enzyme concentration 0.6 % at different temperatures * Optimized reaction conditions Enzyme pre-treatment increased the yield of oil in compared with the control. In our experiments, further optimization was done by variety of single-factor protease concentration. As show in Figure 3, the oil yields increased from 54.4% to 75.2% with a rise of enzyme concentration from 0.2 to 1%. Similar results have been reported earlier in other cases of aqueous enzyme extraction oil [13]. Aqueous enzymatic extraction of camellia seed oil (Camellia oleifera) from Vietnam 167 The effect of reaction time on the yield of COS oil within several trials is given in Figure 4. As shown in the graph, for protease, 7 hours of reaction time do not result in a higher yield of oil as compared to shorter one which possibly relates to emulsion in AEE process. Therefore, the reaction time of 3 hours was suitable for oil extraction. The effect of extraction temperature on oil extractability is illustrated in Figure 5. Less substantial effect of temperature was observed when extraction temperature rised from 40 o C to 50 o C at the same condition. However, starting from 60 o C, oil extractability decreased, maybe due to water evaporation and activity of enzyme. Therefore, 50 o C seemed to be satisfactory for oil extraction. 2.2.3. Oil quality * Acid value (FFA%), iodine value and saponification value Table 1 shows the quality parameters for the oil, together with data for hexane - extracted oil. The free-fatty acid content, iodine, as well as saponification values of the oil obtained by AEE of oil extraction were clearly comparable with other samples. The acid value indicates the amount of free-fatty acid in the oil. The free-acid content of oil from AEE method (0.5%) was higher than that for hexane-extracted oil. Table 2. Acid value, iodine value and saponification value from AEE of oil and hexane extracted COS Hexane extraction AEE of oil Free fatty acid (%) 0.32 0.5 Iodine value 82 83 Saponification value 191 192 * Fatty acid compositions Table 3. Fatty acid composition of aqueous enzymatic - extracted and hexane - extracted camellia seed oil Fatty acid composition (%) Hexane-extraction AEE Acid palmitic (C16:0) 8.36 6.5 Acid Stearic (C18:0) 2.4 1.86 Acid Oleic (C18:1) 79.48 82.36 Acid Linoleic (C18:2) 9.77 9.29 Total saturated fatty acid 10.76 8.36 Total unsaturated fatty acid 89.24 91.64 Mono unsaturated fatty acid 79.48 82.36 Poly unsaturated fatty acid 9.77 9.29 Hoang Than Hoai Thu, Dao Van Hoang, Doan Lan Phuong, Hoang Thi Bich and Pham Quoc Long 168 The fatty acid compositions were analyzed between aqueous enzymatic and hexane extracted camellia seed oils (Table 3). The major fatty acid was oleic (82.36%), palmitic ( 6.5%), linoleic (9.29%), stearic ( 1.86%) acid. The high amount of oleic acid in the COS oil indicates that it is suitable for either cooking or salad oil [14]. A higher intake of mono-unsaturated/oleic acid is associated with a decreased risk of coronary heart disease [15]. 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