Ultrasonic-Assisted Aqueous Extraction and Physicochemical

Ultrasonic-assisted Aqueous Extraction and Physicochemical Characterization of Oil from Clanis bilineata Mingmei Sun, Xiao Xu, Qiuqin Zhang, XinRui, Junjun Wu and Mingsheng Dong＊ College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR CHINA

Abstract: Ultrasound-assisted aqueous extraction (UAAE) was used to extract oil from Clanis bilineata (CB), a traditional edible insect that can be reared on a large scale in China, and the physicochemical property and antioxidant capacity of the UAAE-derived oil (UAAEO) were investigated for the first time. UAAE conditions of CB oil was optimized using response surface methodology (RSM) and the highest oil yield (19.47%) was obtained under optimal conditions for ultrasonic power, extraction temperature, extraction time, and ultrasonic interval time at 400 W, 40℃, 50 min, and 2 s, respectively. Compared with Soxhlet extraction-derived oil (SEO), UAAEO had lower acid (AV), peroxide (PV) and p-anisidine values (PAV) as well as higher polyunsaturated fatty acids contents and thermal stability. Furthermore, UAAEO showed stronger antioxidant activities than those of SEO, according to DPPH radical scavenging and β-carotene bleaching tests. Therefore, UAAE is a promising process for the large-scale production of CB oil and CB has a developing potential as functional oil resource.

Key words: Clanis bilineata oil, ultrasonic-assisted aqueous extraction, fat acid composition, thermal stability, antioxidant activity

1 Introduction 23.68％（ w/w）and relatively high in the unsaturated fatty Clanis bilineata（Walker）, belonging to the family acids（64.17％）2）. The results indicate that CB larvae serve Sphingidae（Lepidoptera）, is a major agricultural pest as potential sources of oil for food and medicine. usually causing considerable damage to soybean produc- There are many methods for oil extraction from vegeta- tion in China. Interestingly, it is also regarded as an impor- ble and animal sources, such as conventional non-polar tant economic insect because its larvae contain a large per- solvent or aqueous extraction, Supercritical CO2 extraction, centage of protein and oil. In fact, the larvae of C. cold and hot pressing3－6）. The use of different extraction bilineata（CB）, also known as Dou-Dan, have been con- methods results in different yields7）and in different lipid sumed for centuries as a high-protein food and edible oil. profiles8）. Because of safety, quality, and environmental As early as 300 years ago,“ Nong Sang Ji”, an agricultural issues, aqueous extractions are industrially applied to treatise written by famous litterateur Pu Song Ling in 1705, extract animal fat and vegetable oils9）. However, one major recorded the extraction method of oil from CB larvae. Re- disadvantage associated with aqueous process is relatively cently, C. bilineata can be raised by artificial way on a Abbreviations: BHT, butyl-hydroxytoluene; DPPH, large- scale in different regions of China such as Jiangsu, 1,1-diphenyl-2-picrylhydrazyl; RE-HPLC, reversed-phase high Anhui, Henan, Sandong and Gangdong provinces1）, thereby performance liquid chromatography; UAE, ultrasound-assisted opening interesting possibilities with alimentary and indus- extraction; UAAE, ultrasound-assisted aqueous extraction; trial purposes. However, previous studies of C. bilineata UAAEO, ultrasound-assisted aqueous extraction-derived oil; have mainly focuses on its reproductive biology, protein SE, Soxhlet extraction; SEO, Soxhlet extraction-derived oil; CB, Clanis bilineata; RSM, response surface methodology; AV, contents and cooking methods. To date, Very few studies acid values; PV, peroxide values; PAV, p-anisidine values; have been carried out on the extraction, chemical composi- SAFA, saturated fatty acids; MUFA, monounsaturated fatty tion and physiochemical properties of C. bilineata oil. Wu acids; PUFA, polyunsaturated fatty acids; OCFA, odd carbon et al reported that the lipid content of CB larvae was fatty acid.

＊Correspondence to: Mingsheng Dong, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR CHINA E-mail: [email protected] Accepted September 15, 2017 (received for review May 5, 2017) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

151 M. Sun, X. Xu, Q. Zhang et al.

low efficiency10）. Ultrasonic treatment could be used to surements, calculated as shown in the Eq.15） enhance extraction yields and shorten extraction time by dT 11－13） P＝m. C ＊ mass transfer, heating and cavitation effect . Alterna- p dt tives to conventional non-polar solvent extraction, ultra- where Cp is the heat capacity of the solvent at constant sound-assisted processing using as“ green and innovative pressure（J g－1 k－1）, dT/dt is temperature rise according to technique”14）may also contribute to environmental preser- time and m is the mass of solvent（g）. The generator has an vation and increase production efficiency by reducing the output power of 650 W, while the power consumed in the use of solvents, elimination of wastewater, generation of medium was about 600 W. hazardous substances and energy14－16）. Ultrasonic-assist Frozen CB larvae（75％ water content）of 50g was mixed extraction（UAE）has been widely used to extract bioactive with 300 mL of demineralized water（solid/liquid ratio＝1:6, compounds and oil from plant sources17, 18）. However, there w/v）and blended for a 5 min. After ultrasonic treatment, is no information on the ultrasound-assist aqueous extract the mixture was boiled for 15 min. The obtained suspen- of oil from edible insects in the literature. sion was sieved through a stainless steel ﬁlter sieve with a In the present work, the UAAE of oil from CB larvae was pore size of 300 μm. Then, the sieved suspension was cen- investigated. Subsequently, the physicochemical proper- trifuged at 12,000g for 30 min at 4℃ and the anhydrous oil ties, fatty acid compositions and antioxidant activities of fraction was acquired. Subsequently, the CB oil was trans- the obtained oil were evaluated and compared with that of ferred to glass vial and stored under nitrogen atmosphere the organic solvent-extracted oil. Meanwhile, the perfor- at －20℃ for further analysis. Oil yield was determined per mances of UAAEO and SEO were compared in terms of CB larvae sample（100g）on a dry-weight basis using the fol- processing time, energy consumption, and quantity of lowing equation: solvent. To the best of our knowledge, this innovative insect oil extraction process and antioxidant activity of CB Oil yield（％）＝（ weight of extracted oil/weight of CB oil has not been reported. larvae）× 100％

The flow chart of UAAE from CB oil was shown in Fig. 1. 2.2.2 Single factor experiment design 2 Materials and methods The effect of ultrasonic power levels on CB oil yield was 2.1 Chemicals Butyl-hydroxytoluene（BHT）, 1,1-diphenyl-2-picrylhydrazyl（DPPH）, β-carotene, linoleic acid were purchased from Sigma-Aldrich. Other analytical or RE-HPLC grade reagents purchased from Shanghai, China.

2.2 Oil extraction from CB larvae The frozen CB larvae were obtained from the commercial supplier（Xinpu, Jiangsu Provice, China）. The larvae were freeze-dried until arriving at stable weight and its moisture content was determined. Subsequently, the freeze-dried CB larvae were milled into power and stored at －20℃ for subsequent operations. 2.2.1 Extraction of CB larvae oil using ultrasound-assisted aqueous extraction（UAAE） UAAE was performed in a BILON-650CT multi-purpose constant temperature ultrasonic extraction system（Shang- hai, China）with 20×20 cm internal dimensions and maximal capacity of 5 L, equipped with one powerful ultrasonic transducer（20 kHz, 650 W）and a versatile power meter which can change all important parameters during ultrasonic processing. The double-layered reaction tank allowed water to circulate using a heating/cooling system to control the temperature in the medium. Taking into account that the actual input power is converted to heat dissipated in the medium. Therefore, in this study, actual ultrasonic power was measured using calorimetric mea- Fig. 1 The flow chart of UAAE from CB oil. 152 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

detected at 300, 350, 400, 450, 500, and 550 W and an ex- Table 1 The levels of variables employed in the present traction time of 15 min, an extraction temperature of 30℃ study for the construction of Box-Behnken and ultrasonic interval time 2s. The effect of extraction design（BBD）. temperature on oil yield was studied at 20, 30, 40, 50, and Levels 60℃ and ultrasonic interval time 2s, an extraction time of Variables 15 min, and an ultrasonic power of 400 W. A range of ex- －1 0 ＋1

traction time（30, 40, 50, 60, and 70 min）was investigated Extraction temperature X1, 30 40 50 at 40℃ and ultrasonic interval time 2s using ultrasonic Extraction time X2, min 40 50 60 power 400 W. Finally, the effect of ultrasonic interval time Ultrasonic power X , W 200 400 600 on oil yield was determined at 0.5, 1, 1.5, 2, 2.5 and 3s and 3 an ultrasonic power of 400 W, an extraction temperature of Ultrasonic Interval timeX4, s 1 2 3 40℃, and an extraction time of 50 min. 2.2.3 Box-Behnken experimental design（BBD）for UAAE values of CB oils by UAAE and SE were determined by Box-Behnken experimental design which is popular in AOCS Standard Methods20）. Peroxide values（PV）and p-an- food processes due to its economical design applies three isidine values（PAV）were measured as indexes for the oxi- levels（－1, 0, ＋1）for each factor19）. The design includes a dative stability of CB oil using IUPAC methods21）. specific subset of the factorial combinations from the 3k factorial design19）. Four factors were selected as indepen- 2.5 Determination of antioxidant activity dent variables, while oil yield was defined as the dependent The 1, 1-diphenyl-2-picrylhydrazyl（DPPH）radical-scav- variable（Table 1）. The range of the values of each factor enging assay, and β-carotene bleaching test were used for was determined according to the results of single factor analysis of antioxidant activity. All the data were the aver- experiments. The experimental design of UAAE based on ages of triplicate determinations of three independent the preliminary single-factor tests was shown in（Table 2）. tests. Scavenging activities of CB oils toward DPPH radicals The responses were analyzed using Design Expert soft- were detected as described by Long et al.22）. β-carotene/ ware. linoleic acid bleaching test of CB oil was investigated ac- 2.2.4 Extraction of CB oil by Soxhlet extraction（SE） cording to the pervious method reported by Zhang et al.23）.

The freeze-dried CB larvae powder（10 g）was continu- Antioxidant activities of CB oils were reported as IC50 ously extracted with 120 mL petroleum ether as a solvent values, which were calculated using logarithmic regression for 6 h in a Soxhlet apparatus（SZF-06B, Shanghai, China）. curves for DPPH radical scavenging or β-Carotene bleach- After extraction was completed, petroleum ether was ing inhibition percentage（％）versus the concentration of removed at 40℃ under reduced pressure using a rotary tested oils. evaporator（2034c-02, Louisiana, USA）and temperature is further increased up to 60℃ until no presence of solvent. 2.6 Thermal stability The oil obtained was weighed and the yield was calculated. The thermal stability of CB oil was detected by thermo- Determination was done in triplicate. gravimetric analysis（TGA）using a NETZSCH STA 449C （NETZSCH Instruments, Germany）. About 5-6 mg of CB 2.3 Determination of fatty acid（FA）composition oil was weighed in solid fat index（SFI）aluminium oxide The FA composition of the oil extract from CB larvae crucible; an empty pan was used as a reference. The refer- was analyzed as fatty acid methyl esters prepared by sa- ence and sample pans were placed in the calorimeter. Ni- ponification and methyl esterification in accordance with trogen was used as an inert gas to purge at 40ml/min at ISO5509:2000（E）. The profile of FA composition was per- 10℃/min. From room temperature to 700℃, the decompo- formed by means of gas chromatography with ﬂame ioniza- sition temperatures and weight of the CB oil samples were tion detector（GC-FID）（ GC, Agilent 7890A, Agilent Tech- recorded. nologies, DE, USA）using SPTM – 2560 capillary column （100 m×0.25 mm×0.2 μm thickness, Supelco, Beelefonte, 2.7 Statistical analysis PA, USA）. The details carried out were as follows: inlet All statistical analyses in this study were done by SPSS temperature:250℃; carrier Gas and flow Rate: helium at 20 PASW statistical software（version 18.0）. One way ANOVA cm/s; make-up gas and flow rate: nitrogen at 30 mL/min; was performed to detect for significant differences column temperature profile: hold at 100℃ for five minutes, between the extraction methods, followed by pos-hoc LSD ramp to 240℃ at 4℃/min, hold at 240℃ for 30 min; sample test and Duncan test. By analysis of ANOVA, all data were concentration and solvent: 1.00uL. expressed as mean±standard deviation. Further, the means were analyzed using Origin Pro 8. Throughout the 2.4 Physicochemical characterization study, a signiﬁcance level of p＜0.05 was considered to be The density, acid values（AV）, saponification, and iodine statistically significant.

153 J. Oleo Sci. 67, (2) 151-165 (2018) M. Sun, X. Xu, Q. Zhang et al.

Table 2 Possible combinations of UAAE parameters based on the each parameter were obtained from Box-Behnken design. The oil yield/ freeze-dried insect powder（％ w/w）was made as the oil yield. Yield of oil (%) No X1 X2 X3 X4 Experimental Predicted 1 40 40 200 2 9.56 9.90 2 30 50 600 2 7.28 7.91 3 50 50 400 1 9.32 10.01 4 40 50 200 3 10.42 10.50 5 40 40 400 3 14.67 14.91 6 40 50 400 2 18.47 18.62 7 50 50 600 2 9.16 8.27 8 50 40 400 2 9.89 9.77 9 30 50 400 3 10.56 10.25 10 30 50 200 2 6.58 6.98 11 30 60 400 2 10.78 10.99 12 40 50 200 1 11.63 12.03 13 40 50 400 2 17.89 18.62 14 40 60 400 1 16.62 15.89 15 40 50 400 2 18.89 18.62 16 40 50 600 1 13.89 13.90 17 50 50 200 2 5.39 4.27 18 40 60 200 2 12.17 12.06 19 50 50 400 3 9.88 10.45 20 40 50 400 2 18.39 18.62 21 40 60 600 2 12.36 12.41 22 40 40 400 1 17.58 17.38 23 40 40 600 2 13.98 14.48 24 40 60 400 3 16.78 16.50 25 40 50 400 2 19.47 18.62 26 50 60 400 2 9.26 10.11 27 30 40 400 2 11.99 11.23 28 40 50 600 3 13.87 13.56 29 30 50 400 1 12.74 12.56

3 Results and discussion with findings obtained with perilla oil26）. In addition, at ul- 3.1 Effect of independent variables on CB oil yield trasonic power level higher 400 W, oil color deepened, Effect of ultrasonic power on CB oil yield was shown in which indicate a decrease in oil quality. Moreover, the Fig. 2A. The oil yield increased with increasing ultrasonic bubbles created by cavitation effect were collapsed and the power up to 400 W. At ultrasonic power levels higher 400 cavitation effect was destroyed when ultrasonic power was W, the oil yield decreased. The first effect may be attribut- too large. Therefore, the optimal UAAE power was 400W. ed to the fact that many bubbles created by ultrasonic sig- As shown in Fig. 2B, the oil yield increased with increas- nificantly and a fragmentation of the matrix occurred in ul- ing extraction temperatures up to 40℃. It is likely that trasound process accelerate the release of components temperature accelerated cycling substance27）. However, from cells into the distilled water and the penetration of when the temperature exceeded 40℃, the oil yield showed distilled water into cells5, 24, 25）. This result was consistent a decreasing trend. At temperatures higher than 40℃,

154 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

(A) (B)

Fig. 2 The effect ofultrasonic power（A）, extraction temperature（B）, extraction time（C）and ultrasonic interval time（D）on the yield of CBoil. Data are shown as mean±SD（n＝3）. some thermal labile constituents will decompose and impu- stroyed when the time was too long. The rate of oil extrac- rities dissolution will accelerate28）. And the oil was wrapped tion decreased. Therefore, the 50 min was chosen for by denatured protein when the temperature was too high. subsequent treatment optimization. At this point the oil was more difficult to extract. As a Effect of ultrasonic interval time on oil yield was shown result, the maximum yield of oil extraction was in 40℃. in Fig. 2D. The results showed that with the extension of From 30 to 50 min of extraction time, the oil yield in- the interval time, the yield of the oil increased When ultra- creased, reaching a maximum yield at 50 min（Fig. 2C）. sound was 3 seconds each time using pulsed ultrasonic However, with prolonged extraction time, oil yield de- （Fig. 2D）. However, when the ultrasonic interval time was creased. In the beginning of 50 min, insect cells were well 2 s, the oil yield was the maximum. This effect may be at- ruptured in an ultrasonic environment, leading to a good tributed to improve mechanical and cavitation effect of ul- penetration of the water into the insect27）. When the tem- trasound in interval time. The solvent permeates fully into perature and power were optimal, cavitation effect would the cell in the interval time29）. When the interval time was quickly produce. Therefore, the collapse of bubbles ob- 2.5 seconds, the oil yield significantly declined. Transient tained by cavitation effect occurred and cavitation was de- state of high temperature and high pressure as well as mi-

155 J. Oleo Sci. 67, (2) 151-165 (2018) M. Sun, X. Xu, Q. Zhang et al.

cro-jet can be produced when ultrasonic wave propagated Where Y is the oil yield（％, w/w）; X1 is extraction tempera- in liquid. This state will quickly disappear rapidly when the ture（℃）; X2 is the extraction time（min）; X3 is the ultra- interval time was prolonged. In addition, when the percent- sonic power（W）; and X4 is the interval time（s）. age of the total extraction time occupied by the interval The model low p value（＜0.0001）revealed that the time was very large, the extraction of the oil was disadvan- model was statistically significant. In this case X1, X3, X4, 2 2 2 2 tageous. Therefore, the optimal of interval time was 2 s. X1 , X2 , X3 , X4 , and X2X3 were significant model terms. The order of influence of each factor on oil yield was ultra- 3.2 Fitting the mathematical model sonic power＞extraction temperature＞ultrasonic interval As the results of ANOVA for the quadratic model shown time＞extraction time. in Table 3, the“ Lake of Fit F-value” of 1.77 implies the The obtained values corresponding to optimal conditions Lake of Fit is not significant relative to the pure error. Not- were as follows: X1＝40℃, X2＝50min, X3＝400W, X4＝2s significant Lake of Fit is good-we want the model to fit （Table 2）. Reproducibility experiments were studied by Table 3. Moreover, the“ Pred-Squared” of 0.9131 is in rea- using these optimal conditions to assess the predictive sonable agreement with the“ AdjR-Squared” of 0.9652. ability of the model. The experimental values for essential Therefore, this model can be used to navigate the design oils（18.91±0.20 g/100 g）was very close to the predicted space30）. The following second-order polynomial equation value（18.62 g/100 g）. It shows that there is a high degree applied to the test variables: of correlation between the predicted and experimental 2 2 data of the regression model（R ＝0.9826, R adj＝0.9652） 2 Y＝18.62 －0.59X1＋0.025X2＋1.23X3－0.47X4－6.72X1 （Table 3）. Therefore, the model could be applied effective- 2 2 2 －1.37X2 －5.04X3 －1.08X4 ＋0.15X1X2＋0.77X1X3＋ ly to predict the essential oil of Clanis bilineata by untra-

0.69X1X4－1.06X2X3＋0.77X2X4＋0.30X3X4 sound-assist aqueous extraction.

Table 3 Analysis of variance（ANOVA）for the fitted quadratic polynomial model. Sum of Mean Source DF F Value Prob> F Squares Square Model 430.49 14 30.75 56.42 <0.0001 **

X1 4.12 1 4.12 7.56 0.0157 *

X2 7.500E-003 1 7500E-003 0.014 0.9083

X3 18.23 1 18.23 33.45 <0.0001 **

X4 2.61 1 2.61 4.80 0.0460 * 2 X1 293.12 1 293.12 537.87 <0.0001 ** 2 X2 12.19 1 12.19 22.37 0.0003 ** 2 X3 164.75 1 164.75 302.32 <0.0001 ** 2 X4 7.58 1 7.58 13.91 0.0022 **

X1 X2 0.084 1 0.084 0.15 0.7004

X1 X3 2.36 1 2.36 4.32 0.0564

X1 X4 1.88 1 1.88 3.44 0.0846

X2 X3 4.47 1 4.47 8.21 0.0125 **

X2 X4 2.36 1 2.36 4.32 0.0564

X3 X4 0.35 1 0.35 0.65 0.4337 Residual 7.63 14 0.54 Lack of Fit 6.23 10 0.62 1.77 0.3053 Pure Error 1.40 4 0.35 Cor Total 438.11 28

2 2 R =0.9826 R adj=0.9652 * Significant at 0.05 level, ** Significant at 0.01 level.

156 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

3.3 Analysis of the response surface tained using temperature of 34.8-44.2℃and an extraction The interaction between these factors was studied by time of 40-60 min. At the extraction time 50 min and inter- using surface plots produced by the selected model31）. val time 2 s, the highest CB oil yield should be in the tem- Two-dimensional（2D）contour plots and three-dimensional perature ranging from 34.8℃ to 44.3℃ and the power （3D）response surface graphs were constructed to detect 314.8-535.4W（Fig. 3B）. Moreover, when the extraction the levels of the multiple factors to obtain the optimal CB time and the ultrasonic power were 50 min and 400 W, re- oil yield. Contour shapes represent the size of interactions. spectively, the optimal CB oil yield could be achieved at the Circles represent the interaction of two factors was not sig- interval time of 1.0-2.9 s and an extraction temperature nificant. Oval represent the interaction of two factors was range of 34.8 to 44.3℃（ Fig. 4A）. The effects of ultrasonic significant. power and time on CB oil yield at an extraction tempera- Figure 3A showed that with a treatment power of 400 W ture（40℃）and ultrasonic interval time（2s）were shown in and the interval time 2 s, the optimal CB oil yield was ob- Fig. 4B. As a result, the interaction between these two

Fig. 3A Response surface and contour plot for the oil yield as a function of temperature and time at power＝400 W, interval time＝2 s.

Fig. 3B Response surface and contour plot for the oil yield as a function of temperature and power at time＝50 min, interval time＝2 s.

157 J. Oleo Sci. 67, (2) 151-165 (2018) M. Sun, X. Xu, Q. Zhang et al.

Fig. 4A Response surface and contour plot for the oil yield as a function of temperature and interval time at time＝50 min, power ＝400 W.

Fig. 4B Response surface and contour plot for the oil yield as a function of power and time at interval time＝2 s, temperature＝40℃. variables had a significant effect on CB oil yield. The oil respectively. The interaction of two factors had a non-sig- yield of CB increased with increasing extraction time and nificant effect on oil yield. This result may be caused by ultrasonic power until it reached a peak and subsequently the non-significant extraction time factor and the signifi- declined. Moreover, the effect of ultrasonic power was cant of interval time was 0.046. Figure 5B showed that the more significant than that of time and temperature, although the effect of ultrasonic power and interval time because the increasing of power may be more quickly on CB oil yield were significant, interaction of ultrasonic change the energy state than temperature and ultrasonic power and interval time had non-significant effect on CB time, the system was likely to be a steady state with in- oil yield（p＞0.05）. It is likely that the system may have creasing power. Figure 5A showed that the effects of inter- already reached equilibrium at ultrasonic time 50 min. val time and extraction time on oil yield when the ultrason- Based on above data, the optimal experimental condi- ic power and temperature were 400 W and 40℃, tions for UAAE were as follows: extraction temperature

158 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

Fig. 5A Response surface and contour plot for the oil yield as a function of interval time and time at power＝400 W, temperature＝40℃.

Fig. 5B Res ponse surface and contour plot for the oil yield as a function of interval time and power at time＝50 min, temperature＝40℃.

40℃, extraction time 50 min, ultrasonic power 400 W and （p＜0.05）lower than those of SE-derived oil（SEO）（ 0.93± ultrasonic interval time 2s. Five replicates of the central 0.07 mg KOH/g, 2.76±0.34meq O2/kg and 1.42±0.43, re- point were constructed in order to ensure adequacy of ex- spectively）, which indicated this UAAEO possessed a perimental model. This model was adequate in predicting higher oxidative stability and could be stored long-term32, 33）. the optimal results. Under optimal conditions, the highest On the one hand, the lower oxidative stability of SEO is oil yield by UAAE was 19.47％. The oil yield by UAAE was probably attributed to the elevated operational tempera- significantly lower than that by SE（24.56％）but much ture and prolonged extraction duration of the conventional higher than that of aqueous extraction without ultrasonic SE process, which can accelerate the oil oxidation9）. More- treatment（10.17％）. over, the iodine value（155.34±4.76 g I2/100 g oil）of UAAEO was significantly（p＜0.05）higher than SEO

3.4 Physicochemical properties of CB oils （145.17±2.82 g I2/100 g oil）, which suggested that it pos- At room temperature, CB oils by UAAE and SE were sessed more contents of unsaturated fatty acids. In addi- yellow and clear. As shown in Table 5, AV（0.72±0.18 mg tion, UAAEO possessed the similar density as against SEO.

KOH/g）, PV（1.53±0.45meq O2/kg）and PAV（1.01±0.26）of Based on these data, the results of physicochemical prop- UAAE-derived oil（UAAEO）were found to be significantly erties demonstrated that UAAE produced an oil of higher

159 J. Oleo Sci. 67, (2) 151-165 (2018) M. Sun, X. Xu, Q. Zhang et al.

quality than the conventional organic solvent method. of SAFA, followed by stearic acid（C18: 0）; Oleic acid （C18:1）was the predominant MUFA. PUFA includedα- 3.5 Fatty acid composition of CB oils linolenic acid（C18:3）, γ-linolenic acid（C18:3）, linoleic acid As shown in Table 4, a total of 19 fatty acids from （C18:2）, DH-linolenic acid（C20:3）and arachidonic acid UAAEO and SEO were identified by GC-MS, which were （C20:4）, α-linolenic acid being the most dominant compo- comprised of eight saturated fatty acids（SAFA）, three nent. These values were similar to those reported by Wu et monounsaturated fatty acids（MUFA）, five polyunsaturated al.1）, however, SAFA（26.91±1.91％）were lower but USFA fatty acids（PUFA）and three odd carbon fatty acids were higher（72.37±1.20％）in our studies, which indicated （OCFA）. Similar to the proﬁles found in previous studies that UAAEO might be very useful to the food and pharma- on lipids, the FA proﬁles found in the CB oil has higher ceutical industries. Moreover, the proportions of two amounts of unsaturated FA（USFA）than saturated FA typical PUFA, linoleic acid（ω-6）and linolenic acid（ω-3）, （SFA）34, 35）Palmitic acid C16:0 was the principal component were significantly（p＜0.05）higher in UAAEO（10.24±

Table 4 Fatty acid composition（g/100 g）of CB oil UAAEO in comparison with SEOa.

Fatty acid composition UAAEO SEO Saturated fatty acid 26.91±1.91a 28.79±0.73a Butyric acid (C4:0) 0.09±0.01 nd Caprylic acid (C8 : 0) nd 0.20±0.03 Capric acid (C10 : 0) nd 0.08±0.02 Lauric acid (C12 : 0) nd 0.11±0.04 Myristic acid (C14:0) 0.34±0.03a 0.22±0.06b Palmitic acid (C16 : 0) 23.84±0.73a 22.54±0.21b Stearic acid (C18 : 0) 2.55±0.13a 5.55±0.29b Arachidic acid (C20:0) 0.09±0.01a 0.19±0.08b Monounsaturated fatty acid 15.48±0.72a 16.90±0.62b Palmitoleic acid (C16:1 cis9) 0.65±0.01a 0.52±0.06b Oleic acid (C18:1 cis9) 14.65±1.65a 16.38±0.56a Gadoleic acid (C20:1 cis11) 0.18±0.06 nd Polyunsaturated fatty acid 56.89±0.48a 53.71±0.71b Linoleic acid (C18:2 cis9,12) (ω-6) 9.24±0.18a 8.77±0.22b γ-linolenic acid (C18:3 cis6,9,12) (ω-6) 0.34±0.06a 0.26±0.02b α-linolenic acid C18:3 cis9,12,15 (ω-3) 46.57±0.74a 43.98±0.24b DH-linolenic acid (C20:3 cis11,14,17) (ω-3) 0.08±0.01a 0.07±0.03a Arachidonic acid (C20:4 cis5,8,11,14 ) (ω-6) 0.66±0.02a 0.53±0.07b Odd carbon fatty acid 0.57±0.11a 0.41±0.10b Pentadecanoicacid (C15:0) 0.09±0.01a 0.15±0.03b Heptadecanoicacid (C17:1 cis10) 0.05±0.01 nd Heptadecanoic acid (C17:0) 0.43±0.09a 0.26±0.07b Total ω-3 FA 46.65±0.75a 44.05±0.27b Total ω-6 FA 10.24±0.26a 9.56±0.31b ratio ω-3/ω-6 4.58±0.14a 4.56±0.22a * Mean ±SD (n = 3). Means within a row with the same letter are not significantly different as indicated. Different superscripts indicate significant differences (p < 0.05) according to pos-hoc LSD test and Duncan test. a Results expressed as percent over the total content (relative content); SEO, Soxhlet extraction- derived oil and UAAEO, ultrasonic-assisted extraction-derived oil.

160 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

Table 5 Physicochemical properties of CB oils obtained by UAAE and SE methods.

UAAEO SEO Density (15℃, g/mL) 0.94±0.09a 0.92±0.06a Iodine value (g/100 g oil) 155.34±4.76a 145.17±2.82b Acid value (mg KOH/g oil) 0.72±0.18a 0.93±0.07b a b Peroxide value (meq.O2/kg oil) 1.53±0.45 2.76±0.34 Saponification value (mg KOH/g oil) 209±4.95a 196±3.21b p-Anisidine value (PAV) 1.01±0.26a 1.42±0.43b UAAEO-Ultrasound-assisted aqueous extraction-derived oil; SEO-Soxhlet extraction-derived oil Different superscripts indicate significant differences (p < 0.05) according to pos-hoc LSD test and Duncan test.

0.26％ and 46.65±0.75％）as compared to those in SEO oil quality. Several studies have demonstrated that the an- （9.56±0.31％ and（44.05±0.27％）, respectively. Unlike tioxidant potentials of oils can be attributed to the PUFA, previous reports36）, the ratio of ω-3/ ω-6 was very higher tocopherols and phenolics9, 40）. UAAEO exhibited higher than other insects. The high amounts of C18:3 cis9, 12, 15 antioxidant activity than SEO, which can be partly ex- （ω-3）, which is the omega-3 FA, would be responsible for plained by the higher PUFA（56.89％）in UAAEO than that the high ω-3/ω-6 ratio. In addition, it is worthy of mention- in SEO（53.71％）. In addition, as the temperature increasing that odd carbon fatty acids（OCFA）, such as pentadeca- es, the viscosity of the oil decreases, thereby increasing the noic acid, heptadecanoic acid（C17:0）and heptadecanoic flow of biologically active compounds. Ultrasound extrac- acid（C17:1 cis 10）, were found in CB oils for the first time. tion will obtain more biologically active ingredients. There- Moreover, UAAEO contained more OCFA species and fore, the antioxidant properties of oils and fats are in- higher total contents as compared to SEO. Because OCFA creased due to the above reasons40）. These results are possess the favorable beneficial physiological effects37）the consistent with the data for aqueous enzymatic extraction health-beneficial value of CB oil obtained by UAAE was of sesame and flaxseed oils9－11）. preferable over that by the conventional SE. Therefore, UAAE was more efficient method to extract CB oil than the 3.7 Thermal stability traditional SE process. The weight and decomposition temperatures of the CB oil samples were shown in Fig. 7. In the initial stage（20℃ 3.6 Antioxidant capacity －100℃）, with the increase of temperature, the quality de- Antioxidant activities of UAAEO and SEO were assessed creased no matter what kind of extraction method. At the using DPPH radical-scavenging assay and β-carotene/lin- initial stage, the volatilization of the solvent was the reason oleic acid bleaching test. DPPH is commonly used to test of a decrease in weight. DTG curves showed solvent evapo- antioxidant capacity. As illustrated in Fig. 6A, DPPH radi- ration of SEO evaporation reaches maximum at 30.7℃. cal-scavenging activities rose with increasing concentration This is consistent with the boiling of petroleum ether（30℃ of CB oils. However, UAAEO exhibited superior efficacy in －60℃）. The vaporization temperature of little water in

scavenging the DPPH radicals（p＜0.05, IC50＝76.23 μg/ UAAEO was higher than that of petroleum ether. There-

mL）compared with SEO（IC50＝80.47 μg/mL）. β-carotene fore, the weight of UAAEO was decreased more slowly bleaching test is a convenient and accurate method used to than SEO in the initial stage. measure the oil peroxidation inhibitory activity of the TG curves showed thermal stability of UAAEO until a tested sample38）. As shown in Fig. 6B, inhibition of temperature of 303.82℃ and SEO until 208.42℃, with a β-carotene bleaching was dose-dependent. The lipid per- mass loss of 5％. Therefore, UAAEO was more thermos-

oxidation inhibitory activity of UAAEO（IC50＝1.56 mg/mL） stable than SEO. A 90％ mass loss occurred at 458.9℃ and

was significantly（p＜0.05）higher than SEO（IC50＝2.67 mg/ 456.2℃ for UAAEO and SEO, respectively. The number of mL）. In the current investigation of both tested systems, oil decomposition phases depends on the fatty acid compo- UAAEO exhibited superior antioxidant attributes than sition and chemical composition of the oil. Moreover, the SEO. length of the fatty acid chain, the degree of unsaturation, Oxidation reactions are a major reason of deterioration and the branching of the fatty acids decides the thermal in oil during storage or heat treatment39）. Therefore, anti- stability41）. Thermal decomposition of polyunsaturated oxidant capacity can be used as an important indicator of fatty acids decides the first stage of decomposition42）.

161 J. Oleo Sci. 67, (2) 151-165 (2018) M. Sun, X. Xu, Q. Zhang et al.

(A) (A)

(B)

Fig. 7 TG and DTG of UAAE and SE CB oil.

3.8 Evaluation of ultrasonic degradation The results showed that the fatty acid composition of UAAE was similar to that of soxhlet. And UAAEO contained more OCFA species and higher total contents as Fig. 6 Antioxidant activities of the CB oils assessed by compared to SEO. The results showed that fatty acids did DPPH radical scavenging assay（A）and β-carotene/ not degrade. Although volatile aromatics are more likely to linoleic acid bleaching test（B）. MAAEO: MAAE- diffuse into the cavitation bubble for pyrolysis44）, what we derived oil; SEO: SE-derived oil. need is the above oils that contain most of the medium and long chain fatty acids. Moreover, many studies have also UAAEO also decomposed in first stage with maxima at reported that bioactive substances such as polyphenols, 422.9℃, however, SEO decomposed in first stage with hydroxy alcohols, and tyramine45）, which enhance antioxi- maxima at 408.5℃. Therefore, it was likely that UAAEO dant activity, have not been degraded in ultrasonic-assisted contained more polyunsaturated fatty acids. This is consis- process. That’s why antioxidant capacity of UAAEO has tent with the results of fatty acids. SEO had the second de- improved. And in this study, we adopted moderate ultra- composition stage at 469.4℃. Figure 7 showed that there sonic power to extract Clanis bilineata fats and oils. was almost no second phase decomposition of UAAEO Therefore, in the experiment, no significant degradation compared with SEO. This should be related to the content was observed in the obtained fats and oils. From above of monounsaturated fatty acids and saturated fatty acids43）. results, UAAE can improve oil extraction yield, which This is also consistent with the results of fatty acids. could be due to a solubilization and degradation of macro- molecules, such as fibers46）, rather than degradation of other biologically active substances.

162 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

3.9 Up-scaling and Cost Reference UAAE compared to Soxhlet extraction provides numer- 1） Wu, S. Supercritical carbon dioxide extraction of oil ous advantages from an industrial point of view. With oil from Clanis bilineata（Lepidoptera）, an edible in- yield comparable to conventional extraction process, the sect. Afr. J. Biotechnol. 11, 4607-4610（2012）. Ultrasound-assisted aqueous extraction is performed in 50 2） Wu, S.J.; Men, X.; Chen, S.J. The analysis and evalua- min without adding solvent. From these results, it can be tion of main nutrient components for herse Bilineata concluded that UAAE is a rapid technique that consumes tsingtauica. J. Huaihai. Technol. 9, 58-61（2000）. less energy and is beneficial from an environmental point 3） Stanisavljevic, I.T.; Lazic, M.L.; Veljkovic, V.B. Ultra- of view. This technique can also improve product efficiency sonic extraction of oil fromtobacco（Nicotiana taba- and reduce maintenance cost. It has substantial commer- cum L.）seeds. Ultrason. Sonochem. 14, 646-652 cial characteristics as a green and innovative technology （2007）. and can provide high returns on capital investment. Large 4） Azmir, J.; Zaidul, I.S.M.; Rahman, M.M.; Sharif, F.; Sa- scale experiments in previous studies on this ultrasonic-as- hena, M.H.A. Optimization of oil yield of Phaleria sisted extraction process showed promising potential for macrocarpa seed using response surface methodolo- industrial applications, especially using water as solvent. gy and its fatty acids constituents. Ind. Crops. Prod. Moreover, an industrial up-scaling is currently being 52, 405-412（2014）. studied to use UAAE for Clanis bilineata oil. This is a 5） Toma, M.; Vinatoru, M.; Paniwnyk, L.; Mason, T.J. In- good opportunity for eco-friendly development and ratio- vestigation of the effects of ultrasound on vegetal tis- nalization of industrial practices. sues during solvent extraction. Ultrason. Sonochem. 8, 137－142（2001）. 6） Sun, Y.; Liu, Z.; Wang, J. Ultrasound-assisted extraction of five isoflavones from Iris tectorum Maxim. 4 Conclusions Sep. Purif. Technol. 78, 49-54（2011）. In the present study, the highest oil yield of 19.47％ was 7） Pérez-Palacios, T.; Ruiz, J.; Martín, D. Comparison of obtained under optimal conditions for ultrasonic power, different methods for total lipid quantification in meat extraction temperature, extraction time, and ultrasonic in- and meat products. Food Chem. 110, 1025-1029 terval time at 400 W, 40℃, 50 min, and 2 s, respectively. （2008）. Moreover, UAAEO exhibited stronger antioxidant activities 8） Christie, W.W. Preparation of ester derivatives of fatty as measured by scavenging the DPPH radical and inhibiting acids for chromatographic analysis. Advances in Lip- β-Carotene bleaching test as against SEO. In term of id Methodology 2, 69-111（1993）. energy saving, cleanliness and solvent free extraction such 9） Latif, S.; Anwar, F. Aqueous enzymatic sesame oil and as petroleum ether, UAAE is an environmental-friendly protein extraction. Food Chem. 125, 679-684（2011）. technique for the insect oil extraction. Meanwhile, the 10） Ricochon, G.; Muniglia, L. Influence of enzymes on the present investigations indicated that the CB oil obtained by oil extraction processes in aqueous media. Oléag- UAAE could be as functional edible oil for the pharmaceu- ineux, Corps gras, Lipides 17, 356-359（2010）. tical and healthy food fields. This is the first time that the 11） Cuoco, G.; Mathe, C.; Archier, P. A multivariate study odd carbon fatty acids of CB oil are reported. The objective of the performance of an ultrasound-assisted madder of this study was to appreciate the benefits of using ultra- dyes extraction and characterization by liquid chroma- sound-assisted aqueous processing method: reduction in tography-photodiode array detection. Ultrason. Sono- time, organic solvent consumption and energy. chem. 16, 75-82（2009）. 12） Vilkhu, K.; Mawson, R.; Simons, L. Applications and opportunities for ultrasound assisted extraction in the food industry-A review. Innov. Food Sci. Emerg. Conflict of interest Technol. 9, 161-169（2008）. The authors declare no conflict of interest. 13） Zhao, S.; Kwok, K.C.; Liang, H. Investigation on ultrasound assisted extraction of saikosaponins from Radix Bupleuri. Sep. Purif. Technol. 55, 307-312（2007）. 14） Chemat, F.; Rombaut, N.; Meullemiestre, A. Review of Acknowledgments Green Food Processing techniques. Preservation, This work was financial supported by Jiangsu Province transformation, and extraction. Innov. Food Sci. Collaborative Innovation Center and the Priority Academic Emerg. Technol. 41, 357-377（2017）. Program Development of Jiangsu Higher Education Institu- 15） Boukroufa, M.; Boutekedjiret, C.; Petigny, L. Bio-refin- tions. ery of orange peels waste: A new concept based on in- tegrated green and solvent free extraction processes

163 J. Oleo Sci. 67, (2) 151-165 (2018) M. Sun, X. Xu, Q. Zhang et al.

using ultrasound and microwave techniques to obtain sound-assisted extraction of carotenoids from pome- essential oil, polyphenols and pectin. Ultrason. Sono- granate wastes using vegetable oils. Ultrason. Sono- chem. 24, 72-79（2015）. chem. 34, 821-830（2017）. 16） Jalili, F.; Jafari, S.M.; Emam-djomeh, Z.; Malekjani, N. 30） Lee, M.H.; Behera, S.K.; Park, H.S. Optimization of op- Optimization of ultrasound-assisted extraction of oil erational parameters for ethanol production from Ko- from canola seeds with the use of response surface rean food waste leachate. Int. J. Environ. Sci. Tec. 7, methodology. Food Anal. Method. DOI 10.1007/ 157－164（2010）. s12161-017-1030-z（2017）. 31） Sarfarazi, M.; Jafari, S.M.; Rajabzadeh, G. Extraction 17） Wang, L.; Weller, C.L.; Meullemiestre, A. Recent ad- optimization of saffron nutraceuticals through re- vances in extraction of nutraceuticals from plants. sponse surface methodology. Food Anal. Method. 8, Trends Food Sci. Tech. 17, 300-312（2006）. 2273-2285（2015）. 18） González-Centeno, M.R.; Knoerzer, K.; Sabarez, H. Ef- 32） Złotek, U.; Michalak-Majewska, M.; Szymanowska, U. fect of acoustic frequency and power density on the Effect of jasmonic acid elicitation on the yield, chemi- aqueous ultrasonic-assisted extraction of grape pom- cal composition, and antioxidant and anti-inflammato- ace（Vitis vinifera L.）-A response surface approach. ry properties of essential oil of lettuce leaf basil（Oci- Ultrason. Sonochem. 21, 2176-2184（2014）. mum basilicum L.）. Food Chem. 213, 1-7（2016）. 19） Yolmeh, M.; Jafari, S.M. Applications of response sur- 33） Said, Z.B.O.S.; Haddadi-Guemghar, H.; Boulekbache- face methodology in the food industry processes. Food Makhlouf, L. Essential oils composition, antibacterial Bioproc. Tech. 10, 413-433（2017）. and antioxidant activities of hydrodistillated extract of 20） AOCS. AOCS Official Method Cd 3d-63,（n.d.）. https:// Eucalyptus globulus fruits. Ind. Crop. Prod. 89, 167- aocs. personifycloud.com/PersonifyEBusiness/Default. 175（2016）. aspx?TabID=251&productId=111545（2015）. 34） Rumpold, B.A.; Schlüter, O.K. Nutritional composition 21） Paquot, C.; Hautfenne, A. Standard methods for the and safety aspects of edible insects. Mol. Nutr. Food analysis of oils, fats, and derivatives. Blackwell Sci- Res. 57, 802-823（2013）. entific Publications（1987）. 35） Thompson, S.N. Review and comparative characteriza- 22） Long, J.; Fu, Y.; Zu, Y. Ultrasound-assisted extraction tion of fatty-acid compositions of 7 insect orders. of flaxseed oil using immobilized enzymes. Bioresour. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 45, Technol. 102, 9991-9996（2011）. 467-482（1973）. 23） Zhang, S.; Zu, Y.G.; Fu, Y.J. Supercritical carbon diox- 36） Tzompa-Sosa, D.A.; Yi, L.; Van Valenberg, H.J.F. Insect ide extraction of seed oil from yellow horn（Xanthoc- lipid profile: aqueous versus organic solvent-based ex- eras sorbifolia Bunge）and its anti-oxidant activity. traction methods. Food Res. Int. 62, 1087-1094 Bioresour. Technol. 101, 2537-2544（2010）. （2014）. 24） Tian, Y.; Xu, Z.; Zheng, B. Optimization of ultrasonic- 37） Jenkins, B.; West, J.A.; Koulman, A. A review of odd- assisted extraction of pomegranate（Punica grana- chain fatty acid metabolism and the role of pentadeca- tum L.）seed oil. Ultrason. Sonochem. 20, 202-208 noic acid（C15: 0）and heptadecanoic acid（C17: 0）in （2013）. health and disease. Molecules 20, 2425-2444（2015）. 25） Chemat, F.; Rombaut, N.; Sicaire, A.G. Ultrasound as- 38） Mitra, P.; Ramaswamy, H.S.; Chang, K.S. Pumpkin（Cu- sisted extraction of food and natural products. Mecha- curbita maxima）seed oil extraction using supercriti- nisms, techniques, combinations, protocols and appli- cal carbon dioxide and physicochemical properties of cations. A review. Ultrason. Sonochem. 34, 540-560 the oil. J. Food Eng. 95, 208-213（2009）. （2017）. 39） Taghvaei, M.; Jafari, S.M.; Assadpoor, E. Optimization 26） Li, H.Z.; Zhang, Z.J.; Hou, T.Y. Optimization of ultra- of microwave-assisted extraction of cottonseed oil and sound-assisted hexane extraction of perilla oil using evaluation of its oxidative stability and physicochemi- response surface methodology. Ind. Crops Prod. 76, cal properties. Food Chem. 160, 90-97（2014）. 18-24（2015）. 40） Bakhshabadi, H.; Mirzaei, H.; Ghodsvali, A. The effect 27） Hemwimol, S.; Pavasant, P.; Shotipruk, A. Ultrasound- of microwave pretreatment on some physico-chemical assisted extraction of anthraquinones from roots of properties and bioactivity of Black cumin seeds’ oil. Morinda citrifolia. Ultrason. Sonochem. 13, 543- Ind. Crops Prod. 97, 1-9（2017）. 548（2006）. 41） Garcia, P.M.; Adams, T.T.; Goodrum, J.W.; Das, K.C.; 28） Dong, J.E.; Liu, Y.B.; Liang, Z.S.; Wang, W.L. Investiga- Geller, D.P. DSC studies to evaluate the impact of bio- tion on ultrasound-assisted extraction of salvianolic oil on cold flow properties and oxidation stability of acid B from Salvia miltiorrhiza root. Ultrason. So- bio-diesel. Bioresour. Technol. 101, 6219-6224 nochem. 17, 61-65（2010）. （2010）. 29） Goula, A.M.; Verver, M.; Adamopoulou, A. Green ultra- 42） Santos, J.C.O.; Santos, I.M.G.; Conceicao, M.M.; Porto,

164 J. Oleo Sci. 67, (2) 151-165 (2018) Clanis bilineata

S.L.; Trinidade, M.F.S.; Souza,A, G.; Prasad, S.; Fer- compounds and organic dyes – a review. Sci. Total nandes Junior, V.J.; Araujo, A.S. Thermoanalyticalki- Environ. 407, 2474-2492（2009）. netic and rheological parameters of commercial edible 45） Achat, S.; Tomao, V.; Madani, K. Direct enrichment of vegetable oils. J. Therm. Anal. Calorim. 75, 419-428 olive oil in oleuropein by ultrasound-assisted macera- （2004）. tion at laboratory and pilot plant scale. Ultrason. So- 43） Sbihi, H.M.; Nehdi, I.A.; Tan, C.P.; Al-Resayes, S.I. Bit- nochem. 19, 777-786（2012）. ter and sweet lupin（Lupinus albus L.）seeds and seed 46） Jacotet-Navarro, M.; Rombaut, N.; Deslis, S. Towards a oils: a comparison study of their compositions and “dry” bio-refinery without solvents or added water us- physicochemical properties. Ind. Crops Prod. 49, ing microwaves and ultrasound for total valorization of 573-579（2013）. fruit and vegetable by-products. Green Chem. 18, 44） Chowdhury, P.; Viraraghavan, T. Sonochemical degra- 3106-3115（2016）. dation of chlorinated organic compounds, phenolic

165 J. Oleo Sci. 67, (2) 151-165 (2018)