《现代食品工程高新技术》课程教学资源（文献资料）膨化食品技术 Aqueous Extraction and Nutraceuticals Content of Oil Using Industrial Enzymes from Microwave Puffing-pretreated Camellia oleifera Seed Powder

Food Science and Techmology Research.22 (1).31-38.2016 Copyright2016.Japanese Society for Food Science and Technology dot10.3136/tr22.31 http://wwwjsfst.or.jP Original paper Aqueous Extraction and Nutraceuticals Content of Oil Using Industrial Enzymes from Microwave Puffing-pretreated Camellia oleifera Seed Powder Wei-guo ZHaNG School of Chemistry and Chemical Engineering.Lingnan Normal University.Guangdong 524048.China Received April 30.2015:Accepted October 19 2015 The obiective of this study was to extract the oil from Camellia oleifera seeds by agueous enzymatic oil extraction (AEOE)A novel process for extraction of using industriale ymes from owave puffing d Th ed tha with mi he free to 55%(dry ave pre nent and phyt oreover,a very low acid value and peroxide value of oi superior to those of oi ained by previous works wa obtained.From the obtained results,this novel process may prove to be an environment-friendly alternative to solvent extraction Keywords:Camellia oleifera seed oil,aqueous extraction,enzyme.microwave puffing Introduction mainly due to the low oil vields (Hagenmaier etal 1975) Camellig oleifera (theaceae)is one of the widely cultivated Aqueous enzymatic oil extraction (AEOE)is becoming an and important oilseed crops in China c olerfera seed oil is the important alternative to hexane oil extraction which offers mam main cooking oil in China's southem provinces.especially Hunan advantages comnared to conventional oil extraction With this and Jangxi.C.is cmposed mainly process ony the use of hexane but som fatty a acid (C18-D bei the predominant fatt steps can also be omitted.Ma published ers have bee the effects of en zymes nts (pher and tocophero).C.lefer seed oil is very forming few free radicals 2013). vantage a (Ye,er al,2001 il yield and a high productior cost in this process,which limit Industrial processes for the extraction of edible oil from industrial application of AEOE.In order to effectively increase th Camellia oleifera seeds generally involve a solvent extraction step oil yield,some pretreatment approaches have been utilized base preceded by pressing.Mechanical pressing is often associated with on aqueous enzymatic oil extraction.Sharma er al.(2006)reporte lower yield and more energy consumption.while the use of hexane. that the ultrasonic pre-irradiation enhanced the oil yield from 77% generally employed for oilseed extraction is being questioned to 95%(w/w)using aqueous enzymatic oil extraction from almond because of its toxicity and flammability (Latif.er al.2009).Safety Proteases improved oil extraction from both sovbean flour and considerations on the use of organic solvents prompted attempts in extruded sovbean flakes.but combining extrusion with enzyme the past to develop aqueous extraction but these were unsuccessful treatments vielded more free oil than enzyme treating flour alone To whom correspondence should be addressed E-mail:sgxyzwg@126.com

Food Science and Technology Research, 22 (1), 31_ 38, 2016 Copyright © 2016, Japanese Society for Food Science and Technology doi: 10.3136/fstr.22.31 http://www.jsfst.or.jp *To whom correspondence should be addressed. E-mail: sgxyzwg@126.com Original paper Aqueous Extraction and Nutraceuticals Content of Oil Using Industrial Enzymes from Microwave Puffing-pretreated Camellia oleifera Seed Powder Wei-Guo Zhang* School of Chemistry and Chemical Engineering, Lingnan Normal University, Guangdong 524048, China Received April 30, 2015 ; Accepted October 19, 2015 The objective of this study was to extract the oil from Camellia oleifera seeds by aqueous enzymatic oil extraction (AEOE). A novel process for extraction of oil using industrial enzymes from microwave puffing￾pretreated Camellia oleifera seed powder was described. The results indicated that the free oil extraction efficiency obtained with microwave-assisted extraction was very high, which the recovery yield was up to 55% (dry weight of Camellia oleifera seed). Besides, microwave pretreatment of Camellia oleifera seed increased the tocopherols (by 22.2% _ 39.4%), squalene(by 6.3% _ 29.2%) and phytosterols (by 6.7% _ 14.8%) of the oils extracted by AEOE. Moreover, a very low acid value and peroxide value of oil superior to those of oils obtained by previous works was obtained. From the obtained results, this novel process may prove to be an environment-friendly alternative to solvent extraction. Keywords: Camellia oleifera seed oil, aqueous extraction, enzyme, microwave puffing Introduction Camellia oleifera (Theaceae) is one of the widely cultivated and important oilseed crops in China. C. oleifera seed oil is the main cooking oil in China’s southern provinces, especially Hunan and Jiangxi. C. oleifera seed oil is composed mainly of unsaturated fatty acids with oleic acid (C18:1) being the predominant fatty acid (Zhang, et al., 2011). Due to its high oleic acid content and to high levels of natural antioxidants (phenols and tocopherol), C. oleifera seed oil is very resistant to peroxidation, forming few free radicals (Ye, et al., 2001). Industrial processes for the extraction of edible oil from Camellia oleifera seeds generally involve a solvent extraction step preceded by pressing. Mechanical pressing is often associated with lower yield and more energy consumption, while the use of hexane, generally employed for oilseed extraction is being questioned because of its toxicity and flammability (Latif, et al., 2009). Safety considerations on the use of organic solvents prompted attempts in the past to develop aqueous extraction but these were unsuccessful mainly due to the low oil yields (Hagenmaier, et al., 1975). Aqueous enzymatic oil extraction (AEOE) is becoming an important alternative to hexane oil extraction, which offers many advantages compared to conventional oil extraction. With this process we can eliminate not only the use of hexane but some refining steps can also be omitted. Many papers have been published on the effects of enzymes on the extraction and characteristics of olive oil (Najafian, et al., 2009; Tabtabaei, et al., 2013). One disadvantage associated with AEOE is still a low free oil yield and a high production cost in this process,which limits industrial application of AEOE . In order to effectively increase the oil yield, some pretreatment approaches have been utilized based on aqueous enzymatic oil extraction. Sharma et al. (2006) reported that the ultrasonic pre-irradiation enhanced the oil yield from 77% to 95% (w/w) using aqueous enzymatic oil extraction from almond. Proteases improved oil extraction from both soybean flour and extruded soybean flakes, but combining extrusion with enzyme treatments yielded more free oil than enzyme treating flour alone

W-G ZHANC (Lamsal er al.2006)Enzyme-assisted aqueous extraction neasured by China official Method GB/T5530-2005/5. Determinaion f Tocopherols were analyse 96%6 for soybean (De Mou etal,2008:Wu et al HPLC follo Official Method GBTS009 82 2009).Aqueou ym ted by mic 2003.A20uL sphere ODS extraction of oil fr yellow horn seed kernels yielded the (4.cm.5um).The flow rate was kept at 1.7mL/min an the UV det was fixed at 300nm.Tocopherols were qualifie using standard UV spectrum analysis.Tocopherols were identifie The present study deals with a novel process for aqueous by comparing their retention times with those of pure standards o extraction of C.oleifera seed oil using industrial enzymes from a-,B-.y-,and 6-tocopherols and were quantified on the basis of microwave puffing-pretreated Camellia oleifera seed samples.We peak area of the unknowns with those of pure standards (Sigma also studied and compared the physico-chemical attributes of oil Aldrich Chemical Co).The tocopherol/oil ratio was expressed as and the nutraceuticals contents of the enzyme-extracted oils with mg/100g.Under the chromatographic conditions used.B-and those of the solvent-extracted oil To our knowledee this is the first y-tocopherols did not separate fully.so the sum of B-and report on the effect of microwave treatment for oilseed powder -toconherols was determined with a high moisture content on the quality of oil. Determination of squalene Quantification of the squalene was rmed on a agilent-7890A g Materials and methods Camellia oleifera seed samples were obtained from onducted by simply diluting 2.The the local market andards SA)Fou vere purchased ctly ed from Na ngin cering Co., column (30 m 0 mm,0.50μm)was ral pro and Imperial Jad onditions were Bio-technology Co.,Ltd.(cellulose and pectinase,China) s used as the carrier gas,and temperature programming wa respectively.All chemicals and solvents used were of analytical sothermal:the oven temperature was 250C,and the temperature grade. for injector and detector was 300C.Squalene was qualified using Microwave puffing-assisted aqucous enzymatic oil extraction standard spectrum analysis. C.oleifera seed kernels in 70%ethanol aqueous solution Plrvtosterol analysis For analysis of phytosterols.oil sample (1:I w/y)containing no or 1%sodium bicarbonate (w/v)were were first saponified The weighed oil sample (0 25 mg)was mixed milled by crusher c oleifera seed kernel meal was collected and horoughly with 5 mL of 0.5 M KOH in 95%ethanol in a glass sieved through a 120-m mesh sieve to obtain the fine powder meal tube and shaken in a water bath at 90 for 15 min After coolins subicct to microwave heating at 800 W power for specified period the tuhes 2 ml of water and of hexane were added an of time.The microwave puffing powder was mixed with distilled water at a ratio of :7w(10g der for 70 mL distilled w 3000 rpm cont was added and the p as incubated at 50-60 fo separated for further sis.An Agile 0.5-2hina water bath with constant shaking.Total free oil olumn (30m 0,32mm,0,50m） forming the llected and weighed.The a ed toa Agilent-7890A with a flame ionizati on dete or(FID nts were car d out on extracted oi s were:(a)injector 310℃；(b)oven60℃for1mi at a rate of 40C min to a final temperature of 310 Oil extraction by solvent Oil was extracted from C.ofeiferd neld for 27 min,(c)nitrogen as the carrier gas at a flow rate o seed samples by solvent.In brief,powdered C.oleifera seeds (50 g) 36 mL/min and (d)detector temperature of 310C.Quantification were extracted with 500 mL hexane at room temperature with of phytosterols was done relative to 5a-cholestane as an internal vigorous shaking for 48 h in an Erlenmever flask covered by standard. aluminium foil.Then.the mixture was filtrated through defatted Fatty acid compositions Fatty acids were derivatized to thei filter papers.using a Buchner funnel under vacuum.Solvent was corresponding methyl esters using BF.-MeOH.Fatty acids methy removed under reduced pressure at 60C Experiments were carried esters (Fame)were iniected into a agilent he8&capillary columr out on extracted oil samples as soon as nossihle after oil extraction (60m 025 mm inper diameter lum film thickness)installed in Ouality indices analysis of C.oleifera seed oil The pere Agilent 7820 A gas chromatography (Agilent Technologies) value of seed oil was dete nined according to china official edwith a flame ionization tor (FID).The sample(1uL) Method GB/T 5538-2005 and exp ed as imole of act iniected with a split tio of :and the inet tmperat ygen per kilog n of oil The (mg KOH/g 270℃.During ch graphy,initial ove

32 W.-G. Zhang (Lamsal, et al., 2006). Enzyme-assisted aqueous extraction processing (EAEP) of extruded flakes yielded the highest oil yield, which were 96% for soybean (De Moura, et al., 2008; Wu, et al., 2009). Aqueous enzymatic process assisted by microwave extraction of oil from yellow horn seed kernels yielded the maximal oil yield of 55.8% under optimal conditions (Li, et al., 2013). The present study deals with a novel process for aqueous extraction of C. oleifera seed oil using industrial enzymes from microwave puffing-pretreated Camellia oleifera seed samples. We also studied and compared the physico-chemical attributes of oil and the nutraceuticals contents of the enzyme-extracted oils with those of the solvent-extracted oil. To our knowledge, this is the first report on the effect of microwave treatment for oilseed powder with a high moisture content on the quality of oil. Materials and Methods Sample Camellia oleifera seed samples were obtained from the local market. The sample standards were purchased from Sigma–Aldrich Co. (USA). Four different types of enzymes were obtained from Nanning Pangbo Biological Engineering Co., Ltd. (neutral protease and alkaline proteinase, China) and Imperial Jade Bio-technology Co., Ltd. (cellulose and pectinase, China), respectively. All chemicals and solvents used were of analytical grade. Microwave puffing-assisted aqueous enzymatic oil extraction C. oleifera seed kernels in 70% ethanol aqueous solution (1:1 w/v) containing no or 1% sodium bicarbonate (w/v) were milled by crusher. C. oleifera seed kernel meal was collected and sieved through a 120-m mesh sieve to obtain the fine powder meal subject to microwave heating at 800 W power for specified period of time. The microwave puffing powder was mixed with distilled water at a ratio of 1:7 w/v (10 g powder for 70 mL distilled water). Enzyme was added and the mixture was incubated at 50 _ 60℃ for 0.5 _ 2 h in a water bath with constant shaking. Total free oil forming the upper phase was collected and weighed. The amounts of oil recovered were calculated as percentage of dry weight of C. oleifera seed kernel. Experiments were carried out on extracted oil samples as soon as possible after oil extraction. Oil extraction by solvent Oil was extracted from C. oleifera seed samples by solvent. In brief, powdered C. oleifera seeds (50 g) were extracted with 500 mL hexane at room temperature with vigorous shaking for 48 h in an Erlenmeyer flask covered by aluminium foil. Then, the mixture was filtrated through defatted filter papers, using a Buchner funnel under vacuum. Solvent was removed under reduced pressure at 60℃. Experiments were carried out on extracted oil samples as soon as possible after oil extraction. Quality indices analysis of C. oleifera seed oil The peroxide value of seed oil was determined according to China Official Method GB/T 5538-2005 and expressed as millimole of active oxygen per kilogram of oil. The acid value (mg KOH/g oil) was measured by China Official Method GB/T5530-2005/5. Determination of tocopherols Tocopherols were analysed using an HPLC following China Official Method GBT5009.82- 2003. A 20 μL sample was injected into a ultrasphere ODS column (4.6 mm × 25 cm, 5 μm). The flow rate was kept at 1.7 mL/min and the UV detector was fixed at 300 nm. Tocopherols were qualified using standard UV spectrum analysis. Tocopherols were identified by comparing their retention times with those of pure standards of α-, β-, γ-, and δ- tocopherols and were quantified on the basis of peak area of the unknowns with those of pure standards (Sigma￾Aldrich Chemical Co.). The tocopherol/oil ratio was expressed as mg/100 g. Under the chromatographic conditions used, β- and γ-tocopherols did not separate fully, so the sum of β- and γ-tocopherols was determined. Determination of squalene Quantification of the squalene was performed on a Agilent-7890A gas chromatograph equipped with a flame ionization detector (FID). The content of squalene was conducted by simply diluting 2 g of oil in 10 mL hexane. The samples were previously filtered. 1 µL of the mixture was injected directly into the gas chromatograph and a DB1701 capillary column (30 m × 0.32 mm, 0.50 μm) was used. The operating conditions were as follows: nitrogen at a flow rate of 2.5 mL/min was used as the carrier gas, and temperature programming was isothermal: the oven temperature was 250℃, and the temperature for injector and detector was 300℃. Squalene was qualified using standard spectrum analysis. Phytosterol analysis For analysis of phytosterols, oil samples were first saponified. The weighed oil sample (0.25 mg) was mixed thoroughly with 5 mL of 0.5 M KOH in 95% ethanol in a glass tube, and shaken in a water bath at 90℃ for 15 min. After cooling the tubes, 2 mL of water and 1.5 mL of hexane were added and mixed vigorously. Thereafter, the mixture was centrifuged at 3000 rpm for 5 min and the hexane layer containing unsaponifiables was separated for further analysis. An Agilent DB1701 capillary column (30 m × 0.32 mm, 0.50 μm) was used. The column was connected to a Agilent-7890A gas chromatograph equipped with a flame ionization detector (FID). The analysis conditions were: (a) injector 310℃; (b) oven 60℃ for 1 min, increased at a rate of 40℃ / min to a final temperature of 310℃ held for 27 min; (c) nitrogen as the carrier gas at a flow rate of 36 mL/min and (d) detector temperature of 310℃. Quantification of phytosterols was done relative to 5α-cholestane as an internal standard. Fatty acid compositions Fatty acids were derivatized to their corresponding methyl esters using BF3-MeOH. Fatty acids methyl esters (FAME) were injected into a Agilent HP88 capillary column (60 m, 0.25 mm inner diameter, 1 µm film thickness) installed in Agilent 7820 A gas chromatography (Agilent Technologies) equipped with a flame ionization detector (FID). The sample (1 µL) was injected with a split ratio of 50:1 and the inlet temperature was set at 270℃. During chromatography, initial oven temperature was

Aqucous Extraction and Nutraceuticals Content of Oil from Microwave Puffing-pretreated Camellia oleifera Seed Powder 33 Table 1.Effect of enzymes on oil extraction yield at 50C for 2h of incubation time Extraction vield for microwave pretreatment using Enzyme and solvent Ethanol(%) Sodium bicarbonate+ethanol(%) Neutral protease 546H028 54803 Alkaline proteinase 54.8±0.14 55.0=0.24 Cellulose 49.90.3 50.0出0.20 Pectinas 49.60.14 49.8±0.2 Control 47.7±0.8 48.1±0.3 Table 2.Effect of incubationt Enzyme Temperature n yield for microwav Neutral proteas 45℃ 546+02发 548+0 60 522H024 52.3t0.27 Alkaline proteinase 450 53.3±0.26 53.540.2 54.8±0.14 55.040.24 60℃ 52.90.32 53.00.29 ed with Cellulose and Pectinase.The highe mined car n be was troge with a flow breakdown in the FAME achieved by retentio times und the oi dropl wave p ng-pret of authentic c nds analyzed under the same condition. ellia oleifera ng the more compos FAME was expressed as relativearea percent of total FAMEs he emperature effect upon extraction are prese ted in lable The experiments were done in triplicate and the The results indicated the higher oil extraction yield and lower a values reported were the means standard deviation. 55C and 60C to be 54.8%and 52.2%for neutral protease and 55 and 52.9%for alkaline proteinase.On the whole,literature reports Results and Discussion do not agree with respect to the temperature impact.Although Selection of enyme species ofaqueous envmatic oil extraction thoughtfully reported as an essential parameter in some oilseeds A maior feature of the oil seed cotvledon is that it is made un of ases (Dominguez.er al.1994).it has also been shown to exhibit cells that contain discrete cellular organelles called lipid and no considerable effect in others (Sarkar etal 2004)In this work protein hodies (also known as spberosomes and oleosomes)which the decrease of oil extraction vield has heen found at 60c in are the principal repository sites of lipid reserves in oil seeds elation to others which reveals that some engymatic activity could (acks ).The walls thatsu have been lost under this condition.Since high temperatures will d of cellulose hemicellulose ssibility of thei The ratio and more energy the lowe ction oil. is pre hemicel incut pectinases in the usual aqueous enzymatic extraction on extraction is giv n in Ta able s.It can be se from Table 3 tha is to break the structure of the cotyledon cell walls and to rel for neutral protease and alkalin I-2 h of incubatio oil from the plant material cells (Gai,et al.,2013).In our prev time resulted in a higher yield of oil.These results are not in findings,microwave puffing as a pretreatment step ruptured the agreement with those reported by Passos et al.(2009)that it wa structure of oilseed cells where there were not the intact cells to be not effective to detect observable enhancements in extraction yield found and most of the oil was located on the surface of oilseed for shorter times and increments of 8.9.19.5.46.5.60.2 and 136% materials (Zhang.et al.2011).When Neutral protease.Alkalinc were measured for t=8.16.24.48 and 120 h of reaction In our proteinase Cellulose and Pectinase were added.54 6-54 8 ase.from I h to 2 h.only a small increment of 0 4%was achieved. 54 8-55 49 6 -49 8 and 49 9-50%of oil vield were obtained but the oil vield has reached 54 4-54 7%which clearly surnassed respectively (Table 1).The highest oil vield was obtained from most earlier studies.despite other parameters having not vet been Alkaline proteinase treated seed samples,whereas,the lower oil onsidered and optimised.This might be due to the complete

Aqueous Extraction and Nutraceuticals Content of Oil from Microwave Puffing-pretreated Camellia oleifera Seed Powder 33 170℃ and initial time was 14 min. Then the temperature increased to 250℃ at a rate of 10℃ /min and kept for 8min. The carrier gas was nitrogen with a flow rate of 1.5 mL/min. Identification of FAME was achieved by comparing their retention times with those of authentic compounds analyzed under the same conditions. The composition of FAME was expressed as relative area percent of total FAMEs. Data analysis The experiments were done in triplicate and the values reported were the means ± standard deviation. Results and Discussion Selection of enzyme species of aqueous enzymatic oil extraction A major feature of the oil seed cotyledon is that it is made up of cells that contain discrete cellular organelles called lipid and protein bodies (also known as spherosomes and oleosomes), which are the principal repository sites of lipid reserves in oil seeds (Jacks, et al., 1990). The walls that surround 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 enzymatic extraction oil. Using most hydrolytic enzymes such as cellulases, hemicellulases, and pectinases in the usual aqueous enzymatic extraction oil processes is to break the structure of the cotyledon cell walls and to release oil from the plant material cells (Gai, et al., 2013). In our previous findings, microwave puffing as a pretreatment step ruptured the structure of oilseed cells where there were not the intact cells to be found and most of the oil was located on the surface of oilseed materials (Zhang, et al., 2011). When Neutral protease, Alkaline proteinase, Cellulose and Pectinase were added, 54.6 _ 54.8, 54.8 _ 55,49.6 _ 49.8 and 49.9 _ 50% of oil yield were obtained, respectively (Table 1). The highest oil yield was obtained from Alkaline proteinase treated seed samples, whereas, the lower oil contents were observed with Cellulose and Pectinase. The higher oil contents determined can be explained by the hydrolysis of proteins, which possibly causes a breakdown in the protein network that surround the oil droplets in microwave puffing-pretreated Camellia oleifera seed samples, thereby liberating the more oils. Temperature effect The experiments carried out to evaluate the temperature effect upon extraction are presented in Table 2. The results indicated the higher oil extraction yield and lower at 55℃ and 60℃ to be 54.8% and 52.2% for neutral protease and 55 and 52.9% for alkaline proteinase. On the whole, literature reports do not agree with respect to the temperature impact. Although thoughtfully reported as an essential parameter in some oilseeds cases (Dominguez, et al., 1994), it has also been shown to exhibit no considerable effect in others (Sarkar, et al., 2004). In this work, the decrease of oil extraction yield has been found at 60℃ in relation to others, which reveals that some enzymatic activity could have been lost under this condition. Since high temperatures will inactivate the enzymes, thus excluding the possibility of their recuperation and recycling, and require more energy, the lower value is preferable in industrial application. Incubation time effect The effect of different incubation times on extraction is given in Table 3. It can be seen from Table 3 that, for neutral protease and alkaline proteinase, 1 _ 2 h of incubation time resulted in a higher yield of oil. These results are not in agreement with those reported by Passos et al. (2009) that it was not effective to detect observable enhancements in extraction yield for shorter times and increments of 8.9, 19.5, 46.5, 60.2 and 136% were measured for t = 8, 16, 24, 48 and 120 h of reaction. In our case, from 1 h to 2 h, only a small increment of 0.4% was achieved, but the oil yield has reached 54.4 _ 54.7%, which clearly surpassed most earlier studies, despite other parameters having not yet been considered and optimised. This might be due to the complete Table 1. Effect of enzymes on oil extraction yield at 50℃ for 2h of incubation time Table 2. Effect of incubation temperatures on oil extraction yield for 2h of incubation time

W-G ZHANC Table 3.Effect of incubation times on oil extraction vield at 50'c Enzyme Time(h) yield for microw ve pret tment using Neutral protease 0.5 517402 54.4+0.14 545+019 2 54.6士0.28 54.8±0.3 Alkaline proteinase 0.5 515±0.21 51.8±02 54.502 54.702 54,8±0.14 55.0H0.24 Table 4.Physicochemical analysis of oils extracted from Camellig oleifera seeds Extracted by Parameter Acid value me KOH/s oil 038±001 019000 021000s9 Peroxidevalue mmolkg 10610.04 ND ND. N.D.not determined upture puffing-pretreated seed detected,indicating that thereareo themal degradation of m wave trear with the (2008)where moisture content of the rapesc samples befor treatment was /and acid value and p de value by FFA content (Saad,et al.2006).The FFA limit in China oil extracted by pressing were 1.83 mg KOH/g oil and 0.92 meg O. Camellia oleifera seed oil mills is 2.5mg KOH/g oil(free fatty acid kg oil).respectively.It should be mentioned that the microwave 1.25%as oleic acid).In particular,extra virgin olive oil (EVOo) treated samples in our study have a high moisture content of more (free acidity <0.8 g/100g)is recognised by the EC Regulation as than 30%. the product with the highest quality among different commercial Tocopherol and squalene analysis Tocopherols are lipophili categories of olive oils (European Community.Commission antioxidants present in vegetable oils.Tocopherols occur in fou Regulation)(2003). related forms,designated alpha (a).beta (B),gamma (y)and delta The acid values determined for oils extracted by different (8)on the basis of their chromanol ring.It is reported tha methods from Camellia ofeifera seeds are presented in Table 4.A -tocopherol has the stronger vitamin E activity.whereas the significantly lower content of free fatty acid (0.10%-0.15%as -has better antioxidant ffcay than ith rols (Ozcan et al 2005)Too a2010 be due amnles nined by HPLC(Fig Table5 to the fact that the endogen s lipases in Camellia oleif ion ofa-Toconberol in nil samples m625 s used in /100g and othe study.Previ es ha is an en ed in this study.a-tocop in oil palm fruits,and nificantly higher in the enzym oil irst enzyme to be involved in the degradation triacylglyce 7.64-8 71 mg/100 g)than that of the solvent-extracte The action of lipase causes increment in free fatty acid levels in (6.25 mg/100 g)oil showing an enhancement of 22.2 to 39.4% palm oil once the disruption of cells occurs.On the other hand, which may be attributed to the fact that the microwave puffing Lorenzo Cerretani reported that lipolysis was significant only at the pretreatment damaged the intact cell structure of oilseed,increase longer microwave treatment times (more than 12 min),especially the release of tocopherols,and enhanced their amount in the in EVOo.and it seemed to be directly related to the water content extracted oils as shown in Table 5.Results obtained from thes of oils (Cerretani etal 2009) experiments are consistent with those presented in our previou The oxidative state of oil was determined using the peroxide oublication (Zhang etal 2011)Azadmard-Damirchietal (2010) value The peroxide yalue determines the formation of lso renorted that the pretreatment of comellia seeds hy hydroperoxides (primary oxidation products)(Gunstone.et al owave prior to oil extraction by press increased the tocopherols 2004)The peroxide values determined for oils extracted by nificantly in oils and improved the oxidative stability Similar its had also been published by(2003) Table4.The peroxide values of s acted by AEOE we alene,a CH hydr bon with six

34 W.-G. Zhang rupture of oleosomes in microwave puffing-pretreated oilseed samples. Quality indices analysis FFA (free fatty acid) is one of the most frequently determined quality indices during the production, storage, and marketing of oil products and the oil price is dictated by FFA content (Saad, et al., 2006). The FFA limit in China Camellia oleifera seed oil mills is 2.5 mg KOH/g oil(free fatty acid 1.25% as oleic acid). In particular, extra virgin olive oil (EVOo) (free acidity <0.8 g/100 g) is recognised by the EC Regulation as the product with the highest quality among different commercial categories of olive oils (European Community, Commission Regulation) (2003). The acid values determined for oils extracted by different methods from Camellia oleifera seeds are presented in Table 4. A significantly lower content of free fatty acid (0.10% _ 0.15% as oleic acid) compared with previously reported publications (Baboli, et al., 2010) was observed in the AEOE oils, which might be due to the fact that the endogenous lipases in Camellia oleifera seed were inactivated by ethanol and microwave treatments used in this study. Previous studies have shown that there is an endogenous lipase (triacylglycerol acylhydrolase) in oil palm fruits, and is the first enzyme to be involved in the degradation of triacylglycerols. The action of lipase causes increment in free fatty acid levels in palm oil once the disruption of cells occurs. On the other hand, Lorenzo Cerretani reported that lipolysis was significant only at the longer microwave treatment times (more than 12 min), especially in EVOo, and it seemed to be directly related to the water content of oils (Cerretani, et al., 2009). The oxidative state of oil was determined using the peroxide value. The peroxide value determines the formation of hydroperoxides (primary oxidation products) (Gunstone, et al., 2004). The peroxide values determined for oils extracted by different methods from Camellia oleifera seeds are also stated in Table 4. The peroxide values of oils extracted by AEOE were not detected, indicating that there are no thermal degradation of oil in the process of microwave treatment samples. These results were found to be not in agreement with the findings of Uquiche et al. (2008) where moisture content of the rapeseed samples before microwave treatment was 7% and acid value and peroxide value of oil extracted by pressing were 1.83 mg KOH/g oil and 0.92 meq O2/ kg oil), respectively. It should be mentioned that the microwave treated samples in our study have a high moisture content of more than 30%. Tocopherol and squalene analysis Tocopherols are lipophilic antioxidants present in vegetable oils. Tocopherols occur in four related forms, designated alpha (α), beta (β), gamma (γ) and delta (δ) on the basis of their chromanol ring. It is reported that α-tocopherol has the stronger vitamin E activity, whereas the δ-tocopherol has better antioxidant efficacy than either γ-, β- or α-tocopherols (Ozcan, et al., 2005). Tocopherol contents of all the extracted oil samples were determined by HPLC (Fig.2, Table 5). The concentration of α-Tocopherol in oil samples ranged from 6.25 to 8.71 mg/100 g and other tocopherols were not detected under the experimental conditions used in this study. α-tocopherol was found to be significantly higher in the enzyme-extracted oils (7.64 _ 8.71 mg/100 g) than that of the solvent-extracted (6.25 mg/100 g) oil showing an enhancement of 22.2 to 39.4%, which may be attributed to the fact that the microwave puffing pretreatment damaged the intact cell structure of oilseed, increased the release of tocopherols, and enhanced their amount in the extracted oils as shown in Table 5. Results obtained from these experiments are consistent with those presented in our previous publication (Zhang, et al., 2011). Azadmard-Damirchi et al. (2010) also reported that the pretreatment of Camellia oleifera seeds by microwave prior to oil extraction by press increased the tocopherols significantly in oils and improved the oxidative stability. Similar results had also been published by Ko et al. (2003). Squalene, a C30H50 triterpenic hydrocarbon with six Table 3. Effect of incubation times on oil extraction yield at 50℃ Table 4. Physicochemical analysis of oils extracted from Camellia oleifera seeds N.D. not determined

Aqucous Extraction and Nutraceuticals Content of il from Microwave Puffing-pretreated Powder 10.0 3 7.5 10.0 Fig1.High-perfomance liquid chromatogram of tocopherol (1X(B+y)-Ttocopherol:(2)8-tocopherol:(3)a-tocopherol. Table5.Tocopherol and Squaene from seeds Extracted by Extracted by aqucous enzymatic processes after microwave pretreatment using Tocopherol and Squalene Solvent 00 Ne um bica a-Tocopherol mg/100g 6.25±0.07 7640.02 76900 (B+)-Tocopherol mg/100g ND ND ND. ND 6-Tocopherol mg/100g ND N.D Squaleneμgmg 0.048±0000 0.06240.00 0.06240.002 0.051±0.002 7 20 10 20 10 Fig.3.GC-FID chromatogram of squalene standard atogram of squalene of nonconjugated double bonds,is usually found in the deep sea shkolineolandpinmolrsakng of oil for ding sod i-tmcti of chem herapeuti aents,inhibiting cance pretreatment destroyed squalene in alkaline experimenta growth and increasing the efficiency of the immune system (Ko, al.,2002).Consequently,the presence of a high content of squalenc Phytosterol analysis Phytosterols(plant sterols)are mino in Camellia oleifera seed oil would improve its nutraceutical value components of vegetable oils and form a major proportion of the (Xu,eral.,2011). unsaponifiables (Azadmard-Damirchi,et al.,2005).Phytosterols in The squalene content of the ois is shown in Fig4 and Table5 vegetable oils are important from a nutritional point of view and a mean of .048-0.062 ug/mg was detected.The oils from because they contribute to lowering serum cholesterol levels,and microwave-treated Camellia oleifera seeds were significantly are also considered to have anti-inflammatory.anti-bacterial.anti- richer in squalene (0.051-0.062 g/mg)as against the solvent- ulcerative and antitumour properties in humans,as well as extracted oil (0.048 ug/mg).indicating that microwave-treatment contributing to the oxidative and thermal stability and shelf life of improved the qulaty of oils.However,the squalene concentrations vegetable oils (Przvbviski et al 2006)The amount of individual under two microwaye treatment conditions were very different and total phytosterols varied significantly depending on the ranging/m of squalene/mg of oi for extraction method (Table6)Oil extracted from C.r seed by

Aqueous Extraction and Nutraceuticals Content of Oil from Microwave Puffing-pretreated Camellia oleifera Seed Powder 35 nonconjugated double bonds, is usually found in the deep sea shark, olive oil and palm oil. It is a known natural antioxidant that plays a important role in lowering blood cholesterol, enhancing the anti-tumor action of chemotherapeutic agents, inhibiting cancer growth and increasing the efficiency of the immune system (Ko, et al., 2002). Consequently, the presence of a high content of squalene in Camellia oleifera seed oil would improve its nutraceutical value (Xu, et al., 2011). The squalene content of the oils is shown in Fig.4 and Table 5 and a mean of 0.048 _ 0.062 µg/mg was detected. The oils from microwave-treated Camellia oleifera seeds were significantly richer in squalene (0.051 _ 0.062 µg/mg) as against the solvent￾extracted oil (0.048 µg/mg), indicating that microwave-treatment improved the qulaty of oils. However, the squalene concentrations under two microwave treatment conditions were very different, ranging from 0.062 to 0.062 µg/mg of squalene/mg of oil for microwave pretreatment only adding ethanol and 0.051 _ 0.052 µg of squalene/mg of oil for microwave pretreatment adding sodium bicarbonate+ethanol. This can be explained that microwave pretreatment destroyed squalene in alkaline experimental conditions. Phytosterol analysis Phytosterols (plant sterols) are minor components of vegetable oils and form a major proportion of the unsaponifiables (Azadmard-Damirchi, et al., 2005). Phytosterols in vegetable oils are important from a nutritional point of view because they contribute to lowering serum cholesterol levels, and are also considered to have anti-inflammatory, anti-bacterial, anti￾ulcerative and antitumour properties in humans, as well as contributing to the oxidative and thermal stability and shelf life of vegetable oils (Przybylski, et al., 2006). The amount of individual and total phytosterols varied significantly depending on the extraction method (Table 6). Oil extracted from C. oleifera seed by Fig. 1. High-performance liquid chromatogram of tocopherols of standards (1)(β+γ)-Ttocopherol; (2) δ-tocopherol;(3)α-tocopherol. Table 5. Tocopherol and Squalene contents of oils extracted from Camellia oleifera seeds Fig. 2. High-performance liquid chromatogram of tocopherols of Camellia oleifera seed oil Fig. 4. GC-FID chromatogram of squalene of Camellia oleifera seed oil Fig. 3. GC-FID chromatogram of squalene standard

W-G ZHANC Table 6.Phytosterol content of oils extracted from Camellig oleifera seeds Phvtosterol Solvent Neutral protease Alkaline proteinase Neutral protease Alkaline proteinase Total mg/kg 2366±21.21 2679,56361 2716,557.28 2612+1414 2524.5±40.31 30 40 Fig5.GC-FID chromatogram of fatty acid methyl esters of standards .ea et al From Table 7.no significant variation was observed for the fatty acid composition in the oils extracted by different methods suggesting no effect of microwave treatment on the fatty acid composition ofoil.No previous data were available on the effect of microwave treatment on the fatty acid composition of oil for 10 20 comparison.However.Yaqoobera reported that the effect of gamma irradiation on the fatty acid composition ofo extracted from irradiated sunflower seeds was significant (Yagoob,er al. 2010). solvent had the lowest phytosterol content (2366 mg/kg)among all Conclusions ess are the cost of AEOE from micr had higer amo t stud we p process for content in oil samples extracte d by press increa m mcrowave puffing-pretreated Ca microwave treatment time and total phytosterol From the obtained is ad able to treat Came extracted by press from untreated oil and oil treated for 2 and 4 min seed powder with microwave before oil extraction by AEOE with microwaves were 6601,7407,and 7813 ppm,respectively pecause it gives relatively good recovery of oils and improves (Azadmard-Damirchi,etal,2010). nutraceuticals content of oils extracted by AEOE.Furthermore,we Fatry acid composition analysis The fatty acid compositions firstly reported that there was a positive effect of microwave of the camellia oleifera seed oi are given in Fig6 and Table 7, pretreatment of Camellia oleifera seed powder with a high which shows the principal fatty acid components in the camellia moisture content on quality of oil,which gave a very low acid oleifera seed oil.The maior saturated fatty acids in oil samples value and peroxide value of oil superior to those of oils obtained by were palmitic and stearic acids which accounted for 8.9-9.0%and orevious works overall the quality of Comellia oleifera seed oil 3 6-3 8%of the total fatty acids respectively.The main obtained by the novel extraction ntocess is suneriot to that of oi unsaturated fatty acids were oleic acid (78.1-787%)and linoleic obtained by conventional methods Therefore.this novel proces acid (77-8 3%)Oleic acid being of mo turated fatty acids prove to be an environment-friendly alternative to solven (MUFA)was (appre mately 7%).As ompared with

36 W.-G. Zhang solvent had the lowest phytosterol content (2366 mg/kg) among all the analysed samples while phytosterol content of oil extracted by AEOE from microwave puffing-pretreated Camellia oleifera seed had higer amounts of phytosterol (2524.5 _ 2716.5 mg/kg). These results concur with previously published results where phytosterol content in oil samples extracted by press increased with increasing microwave treatment time and total phytosterol contents of oil extracted by press from untreated oil and oil treated for 2 and 4 min with microwaves were 6601, 7407, and 7813 ppm, respectively (Azadmard-Damirchi, et al., 2010). Fatty acid composition analysis The fatty acid compositions of the camellia oleifera seed oil are given in Fig.6 and Table 7, which shows the principal fatty acid components in the camellia oleifera seed oil. The major saturated fatty acids in oil samples were palmitic and stearic acids which accounted for 8.9 _ 9.0% and 3.6 _ 3.8% of the total fatty acids, respectively. The main unsaturated fatty acids were oleic acid (78.1 _ 78.7%) and linoleic acid (7.7 _ 8.3%). Oleic acid being of monounsaturated fatty acids (MUFA) was the most abundant in C. oleifera seed oil (approximately 78%). As compared with virgin olive and other edible oils already reported (Baboli, et al., 2010; Dıraman, et al., 2009; Mitra, et al., 2009), camellia oleifera seed oil has a higher content of oleic acid indicating a good nutritional value of seed oil. From Table 7, no significant variation was observed for the fatty acid composition in the oils extracted by different methods, suggesting no effect of microwave treatment on the fatty acid composition of oil. No previous data were available on the effect of microwave treatment on the fatty acid composition of oil for comparison. However, Yaqoob et al. reported that the effect of gamma irradiation on the fatty acid composition of oil extracted from irradiated sunflower seeds was significant (Yaqoob, et al., 2010). Conclusions The two major drawbacks in the AEOE process are the cost of the enzyme and the low oil yield, which limits its industrial adoption. In the present study, we provided a novel process for aqueous extraction of C. oleifera seed oil using industrial enzymes from microwave puffing-pretreated Camellia oleifera seed samples. From the obtained results, it is advisable to treat Camellia oleifera seed powder with microwave before oil extraction by AEOE, because it gives relatively good recovery of oils and improves nutraceuticals content of oils extracted by AEOE. Furthermore, we firstly reported that there was a positive effect of microwave pretreatment of Camellia oleifera seed powder with a high moisture content on quality of oil, which gave a very low acid value and peroxide value of oil superior to those of oils obtained by previous works. Overall, the quality of Camellia oleifera seed oil obtained by the novel extraction process is superior to that of oil obtained by conventional methods.Therefore, this novel process may prove to be an environment-friendly alternative to solvent extraction. Table 6. Phytosterol content of oils extracted from Camellia oleifera seeds Fig. 5. GC-FID chromatogram of fatty acid methyl esters of standards Fig. 6. GC-FID chromatogram of fatty acid methyl esters of Camellia oleifera seed oil

Aqucous Extraction and Nutraceuticals Content of il from Microwave Puffing-pretreated CmeSeed Powder 37 Table 7.Fatty acid composition of oils extracted from Camellia oleifera seeds Extracted by Extracted by aqueous enzymatic processes after microwave pretreatment using Fatty acid Ethanol Solvent Sodium bicarbonate+ethanol Neutral protease Alkaline proteinase Neut ralprotease Alkaline proteinase 0.046±0000 8.940.000 40.000 89-0000 Hexadecvlenic acid(16-1 0.09240.001 0.086±0.001 0.086±0.000 0.09=0.000 0.0890±0.001 Heptadecanoic acid(17:0 0.064±0.000 0.065±0.001 0.065±0.00 0.066±0.00 0.065±0.00 Hepa 180 d17:1 V. Oleie acid (18-1) 7870000 78240.000 78.1±0.000 78540000 7845+0071 Linoleic acid (18:2 7.7±0.000 8.3±0.00d 8340.000 79±0.00 7.9±0.000 Linolenic acid (3 0.165±0.00 .160.00 0,165+0.00 0.170.00 0.7±0.00 Eicosanpic acid (20:1) 0445+0007 0440000 044+0000 045+0000 0.45±0 references Hasen aier R D.Cater C.M.and Mattil K F.(1975).Aqueo processing Azadmard-Damirchi s Habibi-Nodeh F Hesari 1 Nemati M and of coconuts-economic analysis ut oil Chem Soe 52 5-9 Achachlouei B.F.(2010).Effect of pretreatment with microwaves on Jacks T Hensarling T.P,Neucere JN.Yatsu L.Y.and Barker R.H. oxidative stability and nutraceuticals content of oil from rapeseed.Food (1990).Isolation and physice hemical characterization of the half-unit Ch9.12L1211-1215. es of oilseed lipid Am Oul Chem Soc 67 353-361 Azadmard-Da irchi S.Savage G.Pand Dutta P.C.(2005).Sterol Ko S.N.Kim CJ.Kim C.T.Kim H.Chune S.H.Lee S.M.Yoon H.H. s and d 40- and Kim LH.(2003).Ch with narkers for detecti L5c,32717-725 Ko T.F.Weng YM.and Chiou R.Y.Y.(2002).Squ Baboli ZM.and Kordi A.A.S.(2010).Characteristics and composition o antioxidant activity of Terminalia catappa leavesand seeds.gric watermelon seed oil and solvent extraction parameters effects.Am.Oi Fo0 d Chem.,50,5343-5348 Chem.S0c.,87,667-67 amsal B.P,Murphy P.A_,and Johnson L.A.(2006).Flaking and extrusior Cerretani L.,Bendini A.,Rodriguez-Estrada M.T.,Vittadini E.,and as mechanical treatments for enzyme-assisted aqueous extraction of oil Chiavaro E.(2009).Microwave heating of different commercial from soybeans.J.Amt.Oil Chem.Soc.83,973-979. categories of olive oil:Part I.Effect on chemical oxidative stability Latif S.and Amwar F.(2009).Effect of aqucous enzymatic processes on indices and phenolic compounds.Food Chen.115.1381-1388. sunflower oil quality.J.Amt.Oil Chemt.Soc.86.393-400. De Moura JMLN Mahfuz A.Campbell K June S.Glatz C.E and Li J.,Zu Y.G.,Luo M.,Gu C.B.Zhao C.J.,Efferth T.,and Fu Y.J.(2013) Johnson L.(2008).Enzymatic aqucous extraction of soybean oil and Aqucous enzymatic process assisted hy microwave extraction of oil from protein and cream de-emulsification.J.Am.Oul Chemt.Soc.85.985-998 vellow hom (.Xamthoceras sorbifolia Bunge.)seed kemels and its quality Drraman H.and Dibcklioglu H.(00).Chara erizntion of Turkish virgin valuation.Food Chemt.138.2152-2158. olive oils produced from early harvest olives.n Oul Chem.Soc.86. Mitra P Ramaswamy HS and Chane K S (2009)Pumpkin ( 663-674. maxima)seed oil extraction using H.Nuncz MJ.and LemaJM.(1 20.213 fruits and o CheL.9.271-286 2009 EC November 2003 amending Regulation No. Food Res m,42.171-175 the characteristics of olive oil and olive pomace oil and on the relevar Ozcan M.,Haciseferogullari H.,Marakoglu T.,and Arslan D.(2005 mcthods of analysis.Oficial Journal of European Comunrfies,L295. Hawthom (.)physical and chemical properties 57-71 Food Eng.,69,409-413 GaiQ.Y.JiaoJ.MuP.S.Wang W.Luo M.LiC.Y.Zu Y.G.Wei F.Y. Passos C.P.Yilmaz S.Silva C.M.and Coimbra M.A.(2009) and Fu YJ.(2013).Microwa ssisted agueous enzymatic extraction of Enhancement of grape seed oil extraction using a cell wall degrading oil from /satis indigotica sceds and its evaluation of physicochemica cnzyme cocktail.Food Chem.115.48-53. properties,fatty acid compositions and antioxidant activities.Ind.Crop Przybylski R.and Eskin N.A.M.(2006).Minor components and the Pmd.45.303.311 stability of veectable oils /nforor 17 186-188 Gunstone F.D.(2004).Rapeseed and canola oil:production.processing Saad B.Ling C.W.Jab MS.Lim B.P.Ali A.S.M.Wai W.T.and Saleh MI.(06).Determination of free fatty ids n palm samles using

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