Article pubs.acs.org/JAFC
Ultrasound-Enhanced Subcritical CO2 Extraction of Lutein from Chlorella pyrenoidosa Xiao-Dan Fan, Yan Hou, Xing-Xin Huang, Tai-Qiu Qiu,* and Jian-Guo Jiang* College of Food and Bioengineering, South China University of Technology, Guangzhou 510640, China ABSTRACT: Lutein is an important pigment of Chlorella pyrenoidosa with many beneficial functions in human health. The main purpose of this study was to extract lutein from C. pyrenoidosa using ultrasound-enhanced subcritical CO2 extraction (USCCE). Effects of operating conditions on the extraction, including extraction pretreatment, temperature, pressure, time, CO2 flow rate, and ultrasonic power, were investigated, and an orthogonal experiment was designed to study the effects of extraction pressure, temperature, cosolvent amount, and time on the extraction yields. The USCCE method was compared with other extraction methods in terms of the yields of lutein and the microstructure of C. pyrenoidosa powder by scanning electron microscopy. A maximal extraction yield of 124.01 mg lutein/100 g crude material was achieved under optimal conditions of extraction temperature at 27 °C, extraction pressure at 21 MPa, cosolvent amount at 1.5 mL/g ethanol, and ultrasound power at 1000 W. Compared to other methods, USCCE could significantly increase the lutein extraction yield at lower extraction temperature and pressure. Furthermore, the kinetic models of USCCE and subcritical CO2 extraction (SCCE) of lutein from C. pyrenoidosa were set as E = 130.64 × (1 − e−0.6599t) and E = 101.82 × (1 − e−0.5683t), respectively. The differences of parameters in the kinetic models indicate that ultrasound was able to enhance the extraction process of SCCE. KEYWORDS: subcritical CO2 fluid, extraction, kinetic model, ultrasound, lutein, Chlorella pyrenoidosa
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INTRODUCTION Lutein is widely used in food processing to impart color to a variety of foods because of its safe and strong coloring ability.1,2 Recent studies indicate that it also has antioxidant, vision protection, immune, cardiovascular protection, anticarcinogenic, and other functions.3−6 The sources of lutein are plentiful; it can be extracted from vegetables, flowers, fruits, and some kinds of algae. Lutein has three chiral centers and eight kinds of stereoisomers; therefore, it is better to extract it from natural plants than to employ chemical synthesis.7,8 In nature, lutein commonly presents in many plants such as marigolds, calendula, algae, kale, and alfalfa, among which marigold is used the most to extract lutein.9,10 Whereas lutein in marigold is mainly in the form of lutein ester, it needs saponification to convert into the free state before it can be absorbed by the human body. Compared to marigold, Chlorella pyrenoidosa not only contains an equal amount of lutein, its lutein being in free state also makes it an advantage in the extraction and separation for lutein. Furthermore, C. pyrenoidosa can be in high cell concentrations and heterotrophic culture and not restricted by seasonal, climatic, and geographical conditions. Its small footprint and stable product quality provide a broader outlook for industrial production.11,12 Many drawbacks in traditional extraction techniques such as low efficiency, pollution, and organic solvent residues hindered the further application of natural active ingredient.13−16 Supercritical fluid extraction attracted a lot of attention, but the high demands of pressure, energy, and investment impeded its large-scale application.17,18 Later, the subcritical CO2 extraction (SCCE) technique emerged, which is beneficial to the environment and satisfies today’s pursuit for “green” processing.19 As a modified method of the supercritical CO2 extraction (SCE) technique, SCCE requires lower temperature © 2015 American Chemical Society
and pressure, with higher density and extraction power. These advantages enable SCCE to be a better technique to extract the ingredient that was heat sensitive, easily oxidized, and decomposed.20 Furthermore, SCCE is able to obtain higher extraction yield with the enhancement of ultrasound.21 So far, there has been some research on the extraction of lutein from C. pyrenoidosa by SCE,22−24 but the use of ultrasound-enhanced subcritical CO2 extraction (USCCE) has not been reported. Therefore, in this research, the USCCE technique was introduced, and the aim was to acquire the optimum conditions of USCCE for lutein extraction from C. pyrenoidosa for potential application. The factors involved in the USCCE process were systematically investigated, and the optimal extraction condition was determined. The performance of USCCE was compared with Soxhlet extraction (SE), subcritical water extraction (SWE), SCE, and SCCE with and without pretreatment in terms of process conditions and extraction yield. Moreover, the kinetic model was set on the basis of the experimental results. The microstructure of the C. pyrenoidosa powder was also investigated by performing scanning electron microscopy.
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MATERIALS AND METHODS
Materials and Equipment. C. pyrenoidosa var. tumidus West was purchased from Dongtai Cibainian Bio-Engineering Co., Ltd. A voucher specimen was deposited in the Department of Natural Products Studies, School of Light Chemistry and Food Science, South China University of Technology. Cellulase powder, 15000 u/g, was purchased from Wuxi Xuemei enzyme Technology Co., Ltd. All
Received: Revised: Accepted: Published: 4597
January 25, 2015 March 30, 2015 April 2, 2015 April 2, 2015 DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
Article
Journal of Agricultural and Food Chemistry
and collected in the separator. For the determination of lutein, chromatographic conditions used a Dionex HPLC system determination, Dikma C-18 (250 × 4.6 × 5 μm) separation column; the mobile phase was methanol/acetonitrile (90:10 by volume), the column temperature was 30 °C, the flow rate was 0.8 mL/min, the injection volume was 20 μL, and the detection wavelength was 450 nm. The lutein yield was determined gravimetrically as displayed in
chemical reagents were of analytical grade and manufactured by Sinopharm Chemical Reagent Co., Ltd. Methanol and acetonitrile were of HPLC grade and purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. The extraction procedure was conducted in an apparatus of USCCE that was designed by our laboratory to be suitable for industrial production, and its operation is clarified in Figure 1. The model of ultrasound probe is JY92-IIDN, purchased
TLY (%) =
E × 100% FM
(1)
where TLY represents total lutein yield, E represents extracts (mg), and FM represents feed materials (g). Factors that affect ultrasoundenhanced subcritical CO2 extraction methods are mainly extraction temperature, extraction pressure, CO2 flow, cosolvent, extraction time, and ultrasonic power. To study the impact of various factors on the extraction yield of lutein, a single-factor experiment was designed, and then significant factors were chosen; orthogonal experiments were designed to optimize the extraction process parameters. The independent variables were pretreatment (absolute ethanol soaking and enzymatic pretreatment), temperature (18, 21, 24, 27, and 30 °C), pressure (9, 13, 17, 21, 25, and 29 MPa), flow rate (20, 25, 30, 35, and 40 kg/h), amount of cosolvent (0, 0.5, 1.0, 1.5, and 2.0 mL/g absolute ethanol), extraction time (0, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 h), and ultrasound power levels (0, 250, 500, 750, and 1000 W). Three pretreatment methods were investigated: no treatment, cosolvent was 2.0 mL/g absolute ethanol; ethanol soak, 100 mL of absolute ethanol was added, and soaked for 18 h, cosolvent was 1.0 mL/g absolute ethanol; enzymatic broken, 150 mL of absolute ethanol was added, 5% citric acid solution was used to adjust the pH value of the enzyme solution to 5.0, 2.0 g of cellulose was added to the enzyme solution and stirred for 3 h at 50 °C, and after completion of enzymatic hydrolysis, C. pyrenoidosa together with enzyme solution put into the kettle, cosolvent was 0.5 mL/g absolute ethanol. On the basis of single-factor results, four factors that had a significant impact on the lutein yield were determined: extraction pressure, temperature, amount of cosolvent ethanol, and ultrasonic power, L9 (33); orthogonal experimental design was used to determine their influence on the extraction of USCCE process of lutein C. pyrenoidosa experiments. Comparison with Other Extraction Methods. In Soxhlet extraction (SE), C. pyrenoidosa powder was wrapped with a paper tube and then added to the Soxhlet extractor; 100 mL of petroleum ether was added, extraction was at 43 °C reflux for 18 h, petroleum ether was removed by rotary evaporation, and the concentrated solution was dissolved with ethanol and then transferred to a brown volumetric flask and stored in a cold and no-light environment. The whole process is under weak light. In subcritical water extraction (SWE), 100.00 g of dried C. pyrenoidosa was extracted by deionized water in the extraction tank, the same as in USCCE; extraction at pressure 5 MPa and temperature of 150 °C for 20 min, collected when cooled to room temperature, and NaCl as emulsification. Dichloromethane was added to the extract at a 1:1 ratio and mixed thoroughly; the separated liquid was allowed to stand for 1 h and stratified. The dichloromethane part was added to anhydrous sodium sulfate and then filtered, and the filtrate was collected. In supercritical CO2 extraction (SCE), 100.0 g of dried C. pyrenoidosa was extracted in the extraction apparatus the same as in USCCE; extraction at pressure 25 MPa, flow of CO2 30 kg/h, temperature of 50 °C for 4 h, cosolvent dosage of 1.5 mL/g ethanol; separation kettle I temperature of 35 °C and pressure of 6.5 MPa; separation kettle II temperature of 30 °C and pressure of 5 MPa. In subcritical CO2 extraction (SCCE), 100.00 g of dried C. pyrenoidosa was extracted in the extraction apparatus the same as in USCCE. SCCE involved an extraction pressure of 21 MPa, a CO2 flow of 30 kg/h, a temperature 27 °C for 4 h, and a cosolvent dosage of 1.5 mL/g ethanol; separation kettle I temperature of 35 °C and pressure of 6.5 MPa; separation kettle II temperature of 30 °C and pressure 5 MPa. In the SCCE with pretreatment, the ultrasonic process was not used and the other conditions were in accordance with the optimal
Figure 1. Scheme of ultrasound-enhanced subcritical CO2 apparatus: 1, CO2 cylinder; 2, bus-bar; 3, purifier; 4, refrigerator; 5, condenser; 6, main pump; 7, auxiliary pump; 8, cosolvent cylinder; 9, mixer; 10, ultrasonic generator; 11, ultrasonic transducer; 12, extraction kettle; 13, separation kettle I; 14, separation kettle II; T, temperature probe; P, pressure gauge. from Ningbo Xinke Instrument Co., and its main technical parameters are operating frequency of 20−24 kHz and ultrasonic power of 20− 900 W (adjustable), and the ultrasonic system is composed of a piezoelectric transducer (ultrasonic intensity range of 0−19 W cm−2) and an ultrasonic generator. The generator comprises a power piezoelectric impedance-matching box and a power generator unit. The power generator unit consists of two parts: a power amplifier and a resonant frequency control system to maintain constant power to the transducer during the USCCE process. The transducer was placed in the extractor of the USCCE equipment, whereas the generator was located outside to control the ultrasonic intensity. The operation of the apparatus of USCCE is as follows: CO2 goes from the cylinder to the purifier to filter out possible liquid and particulate impurities in the gas, and then goes into the condenser and is cooled; after that, it is pumped into the mixer by a high-pressure pump and mixed with cosolvent pumped by the auxiliary pump; the mixture is pumped into the 5 L extraction kettle; under ultrasound, the mixture makes full contact with the material inside the extraction kettle; the extracted substances dissolve in supercritical CO2 and flow into separation kettles I and II, in which case CO2 goes back into the gaseous state and extracted substances sink to the bottom of separation kettle. Collected with plastic bottles, CO2 flows out to the purifier and recycles again. During the extraction process, the pressure of the extraction kettle and separation kettle is controlled by the valves; the temperature is adjusted by the water bath outside the reactor. Ultrasound-Enhanced Subcritical CO2 Extraction. Extraction was carried out in the self-designed apparatus shown in Figure 1. C. pyrenoidosa (100.00 g) in the size of 4.5−5.5 μm was dried by spraydrying and then soaked in absolute ethanol and extracted at the designated extraction temperature, extraction pressure, CO2 flow cosolvent, extraction time, and ultrasonic power conditions. Every experiment was in triplicate; 100.00 g was charged into the extract holder every time. The single-factor experiment can, on the one hand, investigate the effects of various factors on the law of USCCE and, on the other hand, lay the foundation for optimization design. The lutein dissolved in the subcritical CO2 was separated from the carbon dioxide 4598
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Figure 2. Effects of different factors on the ultrasound-enhanced subcritical CO2 extraction of lutein: (A) effect of temperature on extraction yield of lutein; (B) effect of pressure on extraction yield of lutein; (C) effect of CO2 flow rate on extraction yield of lutein; (D) effect of cosolvent amount on extraction yield of lutein; (E) effect of extraction time on extraction yield of lutein; (F) effect of ultrasound power on extraction yield of lutein.
Table 1. Orthogonal Experiment Results of Lutein Extraction Yield by USCCE
a
no.
temperature, A (°C)
pressure, B (MPa)
cosolvent, C(mL /g)
ultrasonic power, D (W)
yield (mg/100 g Chlorella)
1 2 3 4 5 6 7 8 9 k1 k2 k3 Δa
24 24 24 27 27 27 30 30 30 106.833 109.690 109.443 2.857
17 21 25 17 21 25 17 21 25 99.773 114.107 112.087 14.334
0.5 1.0 1.5 1.0 1.5 0.5 1.5 0.5 1.0 104.753 109.670 111.543 6.790
500 750 1000 750 500 750 750 1000 500 102.497 110.217 113.253 10.756
87.89 114.86 117.75 106.42 111.87 110.78 105.01 115.59 107.73
Δ, max−min values for each column. wherein E∞ is the lutein extraction yield when time tends to infinity, mg/100 g C. pyrenoidosa; k is the ratio of the solute concentration gradient descending rate; t is the extraction time, h; and E is lutein yield when the extraction time is t, mg/100 g C. pyrenoidosa. For different extraction conditions, values of E∞ are different, because the ultrasound, pretreatment, and entrainment agents will make a difference in equilibrium of the active component in solid particles.26 For example, adding of cosolvent will increase the solubility of polar substances in subcritical CO2, and ultrasound and pretreatment will destroy the cell structure and affect the absorption− desorption equilibrium in the solid particles. To obtain E∞ and k values with and without ultrasound conditions, appropriate experi-
process of orthogonal experiment. USCCE with pretreatment was based on the optimal process of the orthogonal experiment. Kinetic Model Validation. The kinetic model can not only help us explore the subcritical fluid extraction process and predict the experimental results but also help budget before the investment, therefore effectively reducing the investment risk. However, studies on the kinetic model are complex; to reduce the load of work, only the extraction kinetic model established by our group was studied.25 The ultrasound-enhanced subcritical CO2 extraction kinetics equation for C. pyrenoidosa lutein is established as E = E∞(1 − e−kt )
(2) 4599
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Journal of Agricultural and Food Chemistry ments were needed. The experimental data were simulated in order that the yields calculated from the mathematical model have the smallest deviation with the lutein yields obtained under different experimental conditions. The analysis of variance (ANOVA) was performed using origin 7.0, release 10 (OriginLab Corp., Northampton, MA, USA). Multiplerange tests were used to compare means of the estimated kinetic parameters. Evaluations were based on the p < 0.05 significance level. Scanning Electron Micrographs (SEM). To further reveal the mechanism of USCCE, the present study investigated the effects of various extraction methods on C. pyrenoidosa powder microstructure. C. pyrenoidosa powders obtained with different extraction methods (Soxhlet extraction, subcritical CO2 extraction, USCCE without pretreatment, USCCE with pretreatment) were observed by SEM. The control was dried in a vacuum oven at 45 °C for 12 h. The surface characteristics of dried cell were observed and recorded by using a Japanese Electronics Corp. JSM-6490LV scanning electron microscope. The samples were placed in a preparation chamber, and the morphology of the samples was magnified and digitally recorded.
cell can be better exposed to the solvent, thereby accelerating the release and extraction of the active ingredients and improving the yield of lutein. Effect of Temperature on Lutein Yield. The results are shown in Figure 2A, when the extraction pressure was 17 MPa and the optimum temperature 27 °C. When the temperature was 27 °C, the yield began to decrease. The effect of temperature on the extraction efficiency was mainly reflected in two aspects. On the one hand, when the temperature rises, the pressure of solute vapor increases, and the thermal motion of molecules becomes faster, the chance of colliding with each other increases, and the opportunity of subcritical fluid associating with lutein increases. On the other hand, an elevated temperature under a certain pressure will decrease the density of the CO2 and the ability to dissolve, resulting in the lutein yield reducing. This phenomenon has been reported elsewhere when some natural components were extracted by supercritical CO2.28,29 Lutein having the highest yield at 27 °C is a combined effect of these two aspects. Effect of Pressure on Lutein Yield. The results are shown in Figure 2B: with the extraction pressure increasing, lutein yield increases, and when the pressure exceeds 21 MPa, increasing the extraction pressure makes the yields decline, so the best extraction pressure is 21 MPa. The ability of subcritical CO2 to dissolve a substance is closely related to its density; changing the extraction pressure can make the subcritical CO2 density change, thereby increasing or reducing its ability to dissolve lutein. When the temperature remains constant, extraction pressure rising leads to an increase in density, so there will be two opposite effects: increasing the ability of CO2 to dissolve a substance, but reducing the diffusion of the substance.30 At relatively low temperature, the ability of CO2 to dissolve a substance is the dominant factor, so with pressure increasing, lutein yield increases; however, when the pressure exceeds 21 MPa, the diffusion of lutein was assumed to be inhibited, and this effect is greater than the positive effects brought about by the increased ability of CO2 to dissolve the substance, and therefore yields begin to decline. Effect of Flow Rate of CO2 on Lutein Yield. The CO2 flow rate also has some influence on the extraction capacity. It works mainly in two ways: when the fluid flow increases, CO2 goes through the material layer more quickly, the agitation and contact with the material are enhanced, and the mass transfer coefficient and contact area are increased accordingly and promote the solubility of the fluid, thereby increasing the extraction speed. On the contrary, excessive flow decreases the relative retention time of the fluid in the extractor, so the solute and fluid cannot work sufficiently and the yield is limited. In addition, excessive velocity will also lead to subcritical fluid
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RESULTS AND DISCUSSION Effect of Pretreatment on Lutein Yield. The parameters remained constant as the effects of the different factors on the Table 2. Variance Analysis between Factors Levels of Extraction Yield factor
dev sq
degrees of freedom (DOF)
A B C D error
15.033 361.143 73.787 184.526 15.03
2 2 2 2 2
F ratio (F)
critical value (α = 0.05)
24.023 4.908 12.275
19.000 19.000 19.000
significance *
“ultrasound enhanced subcritical CO2 extraction” of lutein were investigated. After enzymatic and soaking treatments of the C. pyrenoidosa, the yields of lutein were 85.05 and 69.02 mg/100 g C. pyrenoidosa, respectively. The better pretreatment method was enzymatic treatment; therefore, in subsequent experiments enzymatic treatment was adopted as the pretreatment method of C. pyrenoidosa. C. pyrenoidosa has a tough cellulose layer cell wall; it is not conducive for the solvent to penetrate into the cells to extract intracellular substances, so without any pretreatment, the extraction effect of lutein is poor. After soaking in high concentrations of polar solvents, its cells swelled, the structure loosened, and ingredients were released; the extraction became more conducive, so after soaking treatment the yield of lutein significantly increased. This effect has also been reported by the study conducted by Fang and others;27 they studied the supercritical CO2 extraction technology on the extraction of the colchicine roots. After enzymatic treatment, the cell wall of C. pyrenoidosa was degraded by cellulose, and the material in the
Table 3. Comparison between Different Extraction Technologies of Lutein extraction method
temperature (°C)
pressure (MPa)
ultrasound power (W)
time (h)
lutein yield (mg 100 g−1)
SE SWE SCE SCCE SCCE with pretreatment USCCE with pretreatment
43 150 50 27 27 27
0.1 5 25 21 21 21
0 0 0 0 0 1000
18 1/3 4 4 4 (+3 h pretreatment) 4 (+3 h pretreatment)
54.64 0 39.33 42.29 92.15 124.01
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Figure 3. HPLC of extraction by different methods: (A) lutein standard; (B) SE; (C) SWE; (D) SCE; (E) SCCE; (F) SCCE with pretreatment; (G) USCCE with pretreatment.
separated in separation kettles I and II, respectively, but after the addition of ethanol, most of the extracts were brought into separation vessel II, resulting in lutein not being separated out. Experimental results are shown in Figure 2D: the yield of lutein increases as cosolvent increases rapidly in the range of 0−1.0 mL/g, but when the amount is >1.0 mL/g, the effect of cosolvent started to slow; considering the cost, the amount of cosolvent was chosen as 1.0 mL/g. Effect of Extraction Time on Lutein Yield. The results are shown in Figure 2E: with the extension of the extraction time, yield also has a corresponding increase, but the slope of the yield curve is gradually reduced and eventually levels off. A similar result was presented in research by Deenu et al.,11 in which when the temperature was fixed, the lutein yield increased until a certain amount of time (approximately 5 h) and then decreased. This could be explained as the chemical decomposition of the bioactive compound present in the extract may occur as the extraction time prolongs, resulting in a decrease in the extraction yield. Considering the technology and production cost, the preferred time for the extraction of lutein is 4 h. Effect of Ultrasound Power on Lutein Yield. The results are shown in Figure 2F: with the introduction of ultrasound,
carrying the solute into the circulation system before complete separation in the separation kettle, thus reducing the yield rate. Experimental results are shown in Figure 2C: with the increase of the fluid flow, lutein yield first increased and then decreased. The optimum fluid flow rate was 30 kg/h. Effect of Cosolvent on Lutein Yield. Adding an appropriate amount of suitable cosolvent can significantly improve the solubility of certain extracted components in subcritical fluid and the selectivity of subcritical fluid. CO2 is a nonpolar substance, whereas lutein is a polar material, its dipole moment being 1.863.31 High pressure can increase lutein’s solubility in subcritical carbon dioxide, but this effect is limited. Ethanol is the only organic reagent that is permitted for use in functional food and pharmaceutical processing.32 As a cosolvent, ethanol can increase the density of a subcritical fluid, and its penetration ability is strong in the matrix diffusion, so after the addition of a cosolvent, the yield of lutein greatly improved. However, the positive effect of cosolvent on the extraction of lutein is limited; besides increasing the subcritical fluid solubility, it would also weaken the selective ability of an extraction system, resulting in an increase of total extracts. This phenomenon has been reported in a study by Wu;23 before the addition of ethanol, lutein and other substances could be well 4601
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Figure 5. Model fitting curve of lutein extraction yield for USCCE and SCCE. The k value of USCCE was 0.6599, and the k value of SCCE was 0.5683.
with the findings of many other researchers on natural product extraction.11,14,33−35 In research conducted by Riera et al.,33 mass transfer enhancement in supercritical fluids extraction (SFE) by means of power ultrasound was calculated; at the end of the extraction time (8.5 h), the yield of the oil was significantly increased when the SFE was ultrasonically assisted. The improvement was about 20%. At the same time the process was accelerated by using ultrasound. Similar extraction yields were obtained in about 30% shorter time. Optimization of Extraction Conditions of Lutein. Experiments were performed to determine the importance order of the factors influencing ultrasound-enhanced subcritical CO2 extraction of lutein and calculate the optimum process parameters; according to the result of the orthogonal test, range analysis and variance analysis of factors are made. The orthogonal experiment results and the analysis results are shown in Tables 1 and 2, respectively. Table 1 shows that in the four factors, extraction pressure had the maximum effect; extraction temperature had the minimum effect. According to Table 1, the importance order of each factor on lutein yield was B > D > C > A, and the optimization of process parameters of USCCE was A2B2C3D3 in which extraction temperature was 27 °C, extraction pressure was 21 MPa, dosage of ethanol was 1.5 mL/g, and ultrasonic power was 1000 W. Experiments were performed under the optimal conditions to verify the credibility of the best level combination, and results showed that the yield of lutein, 124.01 mg/100 g C. pyrenoidosa, was even higher than the highest yield in the orthogonal experiment, 117.75 mg/100 g C. pyrenoidosa (Table 1), so the best conditions were determined as A2B2C3D3. As can be seen from the results of the analysis variance in Table 2, when α = 0.05, the effects of extraction pressure (factor B) on the yield of lutein in broken C. pyrenoidosa reached a significant level, whereas the effects of the ultrasonic power (factor D) and cosolvent dosage (factor C) were not significant. The effect of ultrasonic power, by contrast, is more significant than that of cosolvent dosage, and with the increase of ultrasonic power, lutein yield increases. The extraction temperature (factor A) has a smaller effect on extraction; in the horizontal range of the experiments, the significance of factor A was the least, considering the production cost, and compared
Figure 4. Absorption spectrum of different components: (A, top) lutein standard (retention time, 8.35); (B, middle) substance retains at 10.06 min; (C, bottom) substance retains at 10.93 min.
Table 4. Extraction Yield of Lutein from Chlorella pyrenoidosa by SCCE and USCCE yield (mglutein/100gChlorella)
yield (mglutein/100gChlorella)
time (h)
USCCE
SCCE
time (h)
USCCE
SCCE
0 0.5 1 1.5 2 2.5 3
0 50.23 62.46 80.1 89.98 102.06 113.01
0 31.12 46.91 56.6 64.29 74.36 83.85
3.5 4 4.5 5 5.5 6
117.21 124.01 125.62 126.73 127.44 127.77
90.36 92.15 95.37 96.45 97.01 97.39
lutein yield rises rapidly. Because of the increase in sound power, sound intensity and the effect of ultrasonic vibration increases, leading to accelerated movement of solute molecules and the enhancement of the mass transfer inside the material, thereby improving the yield of lutein. This result is consistent 4602
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Figure 6. Scanning electron micrographs of seed powder from treatments of (A) control, (B) Soxhlet extraction (SD), (C) subcritical CO2 extraction (SCCE), (D) USCCE without pretreatment, and (E) USCCE with pretreatment.
with other research, the temperature could be chosen as room temperature.11 Comparison of USCCE with Other Extraction Methods. It can be seen from Table 3, in terms of the extraction temperature, the temperature of USCCE and SCCE with or without pretreatment was room temperature, which was lower than the other three methods. In terms of the extraction pressure, the pressure of USCCE and SCCE is lower than that of SCE; therefore, the former two methods have a relatively low requirement for the equipment. In terms of extraction time, supercritical and subcritical CO2 extraction needed at most 7 h, compared with 18 h for Soxhlet extraction. We assume efficiency as extraction rate per unit time; the least efficiency of supercritical and subcritical CO2 extraction was 9.8, whereas the efficiency of Soxhlet was 3.04. Extraction time could be greatly reduced and still achieve higher efficiency. Compared with SCCE without pretreatment, the extraction yield of lutein after pretreatment is greatly improved, pretreatment being more conducive to the extraction of effective constituents in C. pyrenoidosa. Because of the introduction of ultrasound and pretreatment, USCCE with pretreatment had a much higher yield than the other four methods; yield had a 100−200% increase. Compared with the
SCCE with pretreatment, the yield also had a 34.5% increase. To sum up, the USCCE with pretreatment is the best to extract lutein; the yield of SCCE was slightly higher than the SCE; Soxhlet extraction yield is medium, but it was so timeconsuming that its efficiency is not high. The HPLC conditions achieved baseline separation of lutein from the total extract, the retention time of lutein was in the vicinity of 8.35 min, and in Figure 3A lutein typical strong shoulder peak can be seen at 445 and 474 nm. Except for the subcritical water extracts, the other five extracts have the other two substances detected at retention times of 10.06 and 10.93 min; the two substances were full wavelength scanned, and the results are shown in Figure 4B,C. The main absorption band profile of these two substances was in the 400−500 nm range and has a shoulder peak or even three absorption peaks of typical carotenoid,36 so these two substances were inferred as carotenoids, too, but their specific structure needs more analysis. Extraction Kinetics. The results of USCCE and SCCE are shown in Table 4. According to the experimental data, we used the nonlinear fitting method to get the E∞ and k values in Origin7.0 software. As is shown in Figure 5 and Table 4, the kinetic model and experimental data have a rather good fitting 4603
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Journal of Agricultural and Food Chemistry
*(J.-G.J.) E-mail: [email protected]. Phone: +86-2087113849. Fax: +86-20-87113843.
degree. The k value of USCCE was 0.6599, larger than that of SCCE 0.5683, suggesting that ultrasound can increase the diffusion coefficient of internal solute and material; E∞ increased significantly after using ultrasound, suggesting that ultrasound had an effect on the adsorption/desorption balance of solute between the interface of solid and liquid phases, and it made the balance tilt in the desorption direction. This conclusion is consistent with research conducted by Romdhane.30 Overall, E∞ and k increase at the same time, suggesting that ultrasound can enhance subcritical CO2 extraction. Microstructure of C. pyrenoidosa Powder. The images of C. pyrenoidosa treated by ultrasound with or without enzymatic pretreatment and conventional extraction are shown in Figure 6. The microstructure of the microalgal cell by SD (Figure 6B) had a lot of holes and interspaces because of a long time soaking (18 h). Part of the microalgal cell surface by SCCE (Figure 6C) floated up and had stepped lamella, which was because 4 h is not enough for CO2 to penetrate into the interior. The microstructure of the microalgal cell by USCCE (Figure 6D) had more interspaces and holes than the microalgal cell by SCCE. This was the same as the results of Li et al.37 and Zhao et al.,38 in which ultrasound treatment disrupted tissues and cell walls. Moreover, the physical disruption of C. pyrenoidosa cells by ultrasound, together with enzymatic pretreatment, was the most efficient method to destroy microalgal cell walls (Figure 6E). Enzymatic pretreatment has recently been shown to be another alternative method, which opens up cell walls through biodegradation and releases bioactive compounds from microalga.39 The cell walls were damaged by the application of ultrasound and enzymatic pretreatment, which resulted in the greater penetration of solvent into the sample matrix and increased the contact surface area between the solid and liquid phase, and, as a result, the solute quickly diffused from the solid phase to the solvent. Hence, USCCE with pretreatment is much more efficient and rapid for the extraction of the bioactive compound. Therefore, the sonication played an important role in breaking up the microalgal cell walls to enhance the extraction yield. The extraction conditions for USCCE with enzyme pretreatment of lutein were evaluated and optimized with regard to the extraction efficiency. Results indicate that lutein was successfully extracted using USCCE with enzyme pretreatment due to its high efficiency, especially compared to other extraction methods. Among the four significant factors that affect USCCE with enzyme pretreatment (extraction temperature, extraction pressure, cosolvent dosage, and ultrasonic power), extraction pressure has maximum effect and extraction temperature has minimum effect. The optimal parameters of USCCE with enzyme pretreatment are extraction temperature of 27 °C, extraction pressure of 21 MPa, ethanol dosage of 1.5 mL/g, and ultrasonic power of 1000 W. Experiments performed under the optimal conditions reached 124.01 mg/100 g C. pyrenoidosa. Kinetic models of USCCE and SCCE in C. pyrenoidosa were established; different E∞ and k values illustrate that ultrasound could strengthen the subcritical CO2 extraction, and the model can also be a significant guide to the study of the amplification of the extraction process.
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Funding
This project was supported by the National Natural Foundation of China (Grant 21406074), Guangdong Province Science and Technology plan project (2013B020311006), and Guangdong Provincial Bureau of Ocean and Fishery Science and Technology to promote a special (A201301B04). Notes
The authors declare no competing financial interest.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*(T.-Q.Q.) E-mail: [email protected]. Phone: +86-2087113849. Fax: +86-20-87113843. 4604
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Ultrasound-Enhanced Subcritical CO2 Extraction of Lutein from Chlorella pyrenoidosa Xiao-Dan Fan, Yan Hou, Xing-Xin Huang, Tai-Qiu Qiu,* and Jian-Guo Jiang* College of Food and Bioengineering, South China University of Technology, Guangzhou 510640, China ABSTRACT: Lutein is an important pigment of Chlorella pyrenoidosa with many beneficial functions in human health. The main purpose of this study was to extract lutein from C. pyrenoidosa using ultrasound-enhanced subcritical CO2 extraction (USCCE). Effects of operating conditions on the extraction, including extraction pretreatment, temperature, pressure, time, CO2 flow rate, and ultrasonic power, were investigated, and an orthogonal experiment was designed to study the effects of extraction pressure, temperature, cosolvent amount, and time on the extraction yields. The USCCE method was compared with other extraction methods in terms of the yields of lutein and the microstructure of C. pyrenoidosa powder by scanning electron microscopy. A maximal extraction yield of 124.01 mg lutein/100 g crude material was achieved under optimal conditions of extraction temperature at 27 °C, extraction pressure at 21 MPa, cosolvent amount at 1.5 mL/g ethanol, and ultrasound power at 1000 W. Compared to other methods, USCCE could significantly increase the lutein extraction yield at lower extraction temperature and pressure. Furthermore, the kinetic models of USCCE and subcritical CO2 extraction (SCCE) of lutein from C. pyrenoidosa were set as E = 130.64 × (1 − e−0.6599t) and E = 101.82 × (1 − e−0.5683t), respectively. The differences of parameters in the kinetic models indicate that ultrasound was able to enhance the extraction process of SCCE. KEYWORDS: subcritical CO2 fluid, extraction, kinetic model, ultrasound, lutein, Chlorella pyrenoidosa
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INTRODUCTION Lutein is widely used in food processing to impart color to a variety of foods because of its safe and strong coloring ability.1,2 Recent studies indicate that it also has antioxidant, vision protection, immune, cardiovascular protection, anticarcinogenic, and other functions.3−6 The sources of lutein are plentiful; it can be extracted from vegetables, flowers, fruits, and some kinds of algae. Lutein has three chiral centers and eight kinds of stereoisomers; therefore, it is better to extract it from natural plants than to employ chemical synthesis.7,8 In nature, lutein commonly presents in many plants such as marigolds, calendula, algae, kale, and alfalfa, among which marigold is used the most to extract lutein.9,10 Whereas lutein in marigold is mainly in the form of lutein ester, it needs saponification to convert into the free state before it can be absorbed by the human body. Compared to marigold, Chlorella pyrenoidosa not only contains an equal amount of lutein, its lutein being in free state also makes it an advantage in the extraction and separation for lutein. Furthermore, C. pyrenoidosa can be in high cell concentrations and heterotrophic culture and not restricted by seasonal, climatic, and geographical conditions. Its small footprint and stable product quality provide a broader outlook for industrial production.11,12 Many drawbacks in traditional extraction techniques such as low efficiency, pollution, and organic solvent residues hindered the further application of natural active ingredient.13−16 Supercritical fluid extraction attracted a lot of attention, but the high demands of pressure, energy, and investment impeded its large-scale application.17,18 Later, the subcritical CO2 extraction (SCCE) technique emerged, which is beneficial to the environment and satisfies today’s pursuit for “green” processing.19 As a modified method of the supercritical CO2 extraction (SCE) technique, SCCE requires lower temperature © 2015 American Chemical Society
and pressure, with higher density and extraction power. These advantages enable SCCE to be a better technique to extract the ingredient that was heat sensitive, easily oxidized, and decomposed.20 Furthermore, SCCE is able to obtain higher extraction yield with the enhancement of ultrasound.21 So far, there has been some research on the extraction of lutein from C. pyrenoidosa by SCE,22−24 but the use of ultrasound-enhanced subcritical CO2 extraction (USCCE) has not been reported. Therefore, in this research, the USCCE technique was introduced, and the aim was to acquire the optimum conditions of USCCE for lutein extraction from C. pyrenoidosa for potential application. The factors involved in the USCCE process were systematically investigated, and the optimal extraction condition was determined. The performance of USCCE was compared with Soxhlet extraction (SE), subcritical water extraction (SWE), SCE, and SCCE with and without pretreatment in terms of process conditions and extraction yield. Moreover, the kinetic model was set on the basis of the experimental results. The microstructure of the C. pyrenoidosa powder was also investigated by performing scanning electron microscopy.
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MATERIALS AND METHODS
Materials and Equipment. C. pyrenoidosa var. tumidus West was purchased from Dongtai Cibainian Bio-Engineering Co., Ltd. A voucher specimen was deposited in the Department of Natural Products Studies, School of Light Chemistry and Food Science, South China University of Technology. Cellulase powder, 15000 u/g, was purchased from Wuxi Xuemei enzyme Technology Co., Ltd. All
Received: Revised: Accepted: Published: 4597
January 25, 2015 March 30, 2015 April 2, 2015 April 2, 2015 DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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and collected in the separator. For the determination of lutein, chromatographic conditions used a Dionex HPLC system determination, Dikma C-18 (250 × 4.6 × 5 μm) separation column; the mobile phase was methanol/acetonitrile (90:10 by volume), the column temperature was 30 °C, the flow rate was 0.8 mL/min, the injection volume was 20 μL, and the detection wavelength was 450 nm. The lutein yield was determined gravimetrically as displayed in
chemical reagents were of analytical grade and manufactured by Sinopharm Chemical Reagent Co., Ltd. Methanol and acetonitrile were of HPLC grade and purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. The extraction procedure was conducted in an apparatus of USCCE that was designed by our laboratory to be suitable for industrial production, and its operation is clarified in Figure 1. The model of ultrasound probe is JY92-IIDN, purchased
TLY (%) =
E × 100% FM
(1)
where TLY represents total lutein yield, E represents extracts (mg), and FM represents feed materials (g). Factors that affect ultrasoundenhanced subcritical CO2 extraction methods are mainly extraction temperature, extraction pressure, CO2 flow, cosolvent, extraction time, and ultrasonic power. To study the impact of various factors on the extraction yield of lutein, a single-factor experiment was designed, and then significant factors were chosen; orthogonal experiments were designed to optimize the extraction process parameters. The independent variables were pretreatment (absolute ethanol soaking and enzymatic pretreatment), temperature (18, 21, 24, 27, and 30 °C), pressure (9, 13, 17, 21, 25, and 29 MPa), flow rate (20, 25, 30, 35, and 40 kg/h), amount of cosolvent (0, 0.5, 1.0, 1.5, and 2.0 mL/g absolute ethanol), extraction time (0, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 h), and ultrasound power levels (0, 250, 500, 750, and 1000 W). Three pretreatment methods were investigated: no treatment, cosolvent was 2.0 mL/g absolute ethanol; ethanol soak, 100 mL of absolute ethanol was added, and soaked for 18 h, cosolvent was 1.0 mL/g absolute ethanol; enzymatic broken, 150 mL of absolute ethanol was added, 5% citric acid solution was used to adjust the pH value of the enzyme solution to 5.0, 2.0 g of cellulose was added to the enzyme solution and stirred for 3 h at 50 °C, and after completion of enzymatic hydrolysis, C. pyrenoidosa together with enzyme solution put into the kettle, cosolvent was 0.5 mL/g absolute ethanol. On the basis of single-factor results, four factors that had a significant impact on the lutein yield were determined: extraction pressure, temperature, amount of cosolvent ethanol, and ultrasonic power, L9 (33); orthogonal experimental design was used to determine their influence on the extraction of USCCE process of lutein C. pyrenoidosa experiments. Comparison with Other Extraction Methods. In Soxhlet extraction (SE), C. pyrenoidosa powder was wrapped with a paper tube and then added to the Soxhlet extractor; 100 mL of petroleum ether was added, extraction was at 43 °C reflux for 18 h, petroleum ether was removed by rotary evaporation, and the concentrated solution was dissolved with ethanol and then transferred to a brown volumetric flask and stored in a cold and no-light environment. The whole process is under weak light. In subcritical water extraction (SWE), 100.00 g of dried C. pyrenoidosa was extracted by deionized water in the extraction tank, the same as in USCCE; extraction at pressure 5 MPa and temperature of 150 °C for 20 min, collected when cooled to room temperature, and NaCl as emulsification. Dichloromethane was added to the extract at a 1:1 ratio and mixed thoroughly; the separated liquid was allowed to stand for 1 h and stratified. The dichloromethane part was added to anhydrous sodium sulfate and then filtered, and the filtrate was collected. In supercritical CO2 extraction (SCE), 100.0 g of dried C. pyrenoidosa was extracted in the extraction apparatus the same as in USCCE; extraction at pressure 25 MPa, flow of CO2 30 kg/h, temperature of 50 °C for 4 h, cosolvent dosage of 1.5 mL/g ethanol; separation kettle I temperature of 35 °C and pressure of 6.5 MPa; separation kettle II temperature of 30 °C and pressure of 5 MPa. In subcritical CO2 extraction (SCCE), 100.00 g of dried C. pyrenoidosa was extracted in the extraction apparatus the same as in USCCE. SCCE involved an extraction pressure of 21 MPa, a CO2 flow of 30 kg/h, a temperature 27 °C for 4 h, and a cosolvent dosage of 1.5 mL/g ethanol; separation kettle I temperature of 35 °C and pressure of 6.5 MPa; separation kettle II temperature of 30 °C and pressure 5 MPa. In the SCCE with pretreatment, the ultrasonic process was not used and the other conditions were in accordance with the optimal
Figure 1. Scheme of ultrasound-enhanced subcritical CO2 apparatus: 1, CO2 cylinder; 2, bus-bar; 3, purifier; 4, refrigerator; 5, condenser; 6, main pump; 7, auxiliary pump; 8, cosolvent cylinder; 9, mixer; 10, ultrasonic generator; 11, ultrasonic transducer; 12, extraction kettle; 13, separation kettle I; 14, separation kettle II; T, temperature probe; P, pressure gauge. from Ningbo Xinke Instrument Co., and its main technical parameters are operating frequency of 20−24 kHz and ultrasonic power of 20− 900 W (adjustable), and the ultrasonic system is composed of a piezoelectric transducer (ultrasonic intensity range of 0−19 W cm−2) and an ultrasonic generator. The generator comprises a power piezoelectric impedance-matching box and a power generator unit. The power generator unit consists of two parts: a power amplifier and a resonant frequency control system to maintain constant power to the transducer during the USCCE process. The transducer was placed in the extractor of the USCCE equipment, whereas the generator was located outside to control the ultrasonic intensity. The operation of the apparatus of USCCE is as follows: CO2 goes from the cylinder to the purifier to filter out possible liquid and particulate impurities in the gas, and then goes into the condenser and is cooled; after that, it is pumped into the mixer by a high-pressure pump and mixed with cosolvent pumped by the auxiliary pump; the mixture is pumped into the 5 L extraction kettle; under ultrasound, the mixture makes full contact with the material inside the extraction kettle; the extracted substances dissolve in supercritical CO2 and flow into separation kettles I and II, in which case CO2 goes back into the gaseous state and extracted substances sink to the bottom of separation kettle. Collected with plastic bottles, CO2 flows out to the purifier and recycles again. During the extraction process, the pressure of the extraction kettle and separation kettle is controlled by the valves; the temperature is adjusted by the water bath outside the reactor. Ultrasound-Enhanced Subcritical CO2 Extraction. Extraction was carried out in the self-designed apparatus shown in Figure 1. C. pyrenoidosa (100.00 g) in the size of 4.5−5.5 μm was dried by spraydrying and then soaked in absolute ethanol and extracted at the designated extraction temperature, extraction pressure, CO2 flow cosolvent, extraction time, and ultrasonic power conditions. Every experiment was in triplicate; 100.00 g was charged into the extract holder every time. The single-factor experiment can, on the one hand, investigate the effects of various factors on the law of USCCE and, on the other hand, lay the foundation for optimization design. The lutein dissolved in the subcritical CO2 was separated from the carbon dioxide 4598
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Figure 2. Effects of different factors on the ultrasound-enhanced subcritical CO2 extraction of lutein: (A) effect of temperature on extraction yield of lutein; (B) effect of pressure on extraction yield of lutein; (C) effect of CO2 flow rate on extraction yield of lutein; (D) effect of cosolvent amount on extraction yield of lutein; (E) effect of extraction time on extraction yield of lutein; (F) effect of ultrasound power on extraction yield of lutein.
Table 1. Orthogonal Experiment Results of Lutein Extraction Yield by USCCE
a
no.
temperature, A (°C)
pressure, B (MPa)
cosolvent, C(mL /g)
ultrasonic power, D (W)
yield (mg/100 g Chlorella)
1 2 3 4 5 6 7 8 9 k1 k2 k3 Δa
24 24 24 27 27 27 30 30 30 106.833 109.690 109.443 2.857
17 21 25 17 21 25 17 21 25 99.773 114.107 112.087 14.334
0.5 1.0 1.5 1.0 1.5 0.5 1.5 0.5 1.0 104.753 109.670 111.543 6.790
500 750 1000 750 500 750 750 1000 500 102.497 110.217 113.253 10.756
87.89 114.86 117.75 106.42 111.87 110.78 105.01 115.59 107.73
Δ, max−min values for each column. wherein E∞ is the lutein extraction yield when time tends to infinity, mg/100 g C. pyrenoidosa; k is the ratio of the solute concentration gradient descending rate; t is the extraction time, h; and E is lutein yield when the extraction time is t, mg/100 g C. pyrenoidosa. For different extraction conditions, values of E∞ are different, because the ultrasound, pretreatment, and entrainment agents will make a difference in equilibrium of the active component in solid particles.26 For example, adding of cosolvent will increase the solubility of polar substances in subcritical CO2, and ultrasound and pretreatment will destroy the cell structure and affect the absorption− desorption equilibrium in the solid particles. To obtain E∞ and k values with and without ultrasound conditions, appropriate experi-
process of orthogonal experiment. USCCE with pretreatment was based on the optimal process of the orthogonal experiment. Kinetic Model Validation. The kinetic model can not only help us explore the subcritical fluid extraction process and predict the experimental results but also help budget before the investment, therefore effectively reducing the investment risk. However, studies on the kinetic model are complex; to reduce the load of work, only the extraction kinetic model established by our group was studied.25 The ultrasound-enhanced subcritical CO2 extraction kinetics equation for C. pyrenoidosa lutein is established as E = E∞(1 − e−kt )
(2) 4599
DOI: 10.1021/acs.jafc.5b00461 J. Agric. Food Chem. 2015, 63, 4597−4605
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Journal of Agricultural and Food Chemistry ments were needed. The experimental data were simulated in order that the yields calculated from the mathematical model have the smallest deviation with the lutein yields obtained under different experimental conditions. The analysis of variance (ANOVA) was performed using origin 7.0, release 10 (OriginLab Corp., Northampton, MA, USA). Multiplerange tests were used to compare means of the estimated kinetic parameters. Evaluations were based on the p < 0.05 significance level. Scanning Electron Micrographs (SEM). To further reveal the mechanism of USCCE, the present study investigated the effects of various extraction methods on C. pyrenoidosa powder microstructure. C. pyrenoidosa powders obtained with different extraction methods (Soxhlet extraction, subcritical CO2 extraction, USCCE without pretreatment, USCCE with pretreatment) were observed by SEM. The control was dried in a vacuum oven at 45 °C for 12 h. The surface characteristics of dried cell were observed and recorded by using a Japanese Electronics Corp. JSM-6490LV scanning electron microscope. The samples were placed in a preparation chamber, and the morphology of the samples was magnified and digitally recorded.
cell can be better exposed to the solvent, thereby accelerating the release and extraction of the active ingredients and improving the yield of lutein. Effect of Temperature on Lutein Yield. The results are shown in Figure 2A, when the extraction pressure was 17 MPa and the optimum temperature 27 °C. When the temperature was 27 °C, the yield began to decrease. The effect of temperature on the extraction efficiency was mainly reflected in two aspects. On the one hand, when the temperature rises, the pressure of solute vapor increases, and the thermal motion of molecules becomes faster, the chance of colliding with each other increases, and the opportunity of subcritical fluid associating with lutein increases. On the other hand, an elevated temperature under a certain pressure will decrease the density of the CO2 and the ability to dissolve, resulting in the lutein yield reducing. This phenomenon has been reported elsewhere when some natural components were extracted by supercritical CO2.28,29 Lutein having the highest yield at 27 °C is a combined effect of these two aspects. Effect of Pressure on Lutein Yield. The results are shown in Figure 2B: with the extraction pressure increasing, lutein yield increases, and when the pressure exceeds 21 MPa, increasing the extraction pressure makes the yields decline, so the best extraction pressure is 21 MPa. The ability of subcritical CO2 to dissolve a substance is closely related to its density; changing the extraction pressure can make the subcritical CO2 density change, thereby increasing or reducing its ability to dissolve lutein. When the temperature remains constant, extraction pressure rising leads to an increase in density, so there will be two opposite effects: increasing the ability of CO2 to dissolve a substance, but reducing the diffusion of the substance.30 At relatively low temperature, the ability of CO2 to dissolve a substance is the dominant factor, so with pressure increasing, lutein yield increases; however, when the pressure exceeds 21 MPa, the diffusion of lutein was assumed to be inhibited, and this effect is greater than the positive effects brought about by the increased ability of CO2 to dissolve the substance, and therefore yields begin to decline. Effect of Flow Rate of CO2 on Lutein Yield. The CO2 flow rate also has some influence on the extraction capacity. It works mainly in two ways: when the fluid flow increases, CO2 goes through the material layer more quickly, the agitation and contact with the material are enhanced, and the mass transfer coefficient and contact area are increased accordingly and promote the solubility of the fluid, thereby increasing the extraction speed. On the contrary, excessive flow decreases the relative retention time of the fluid in the extractor, so the solute and fluid cannot work sufficiently and the yield is limited. In addition, excessive velocity will also lead to subcritical fluid
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RESULTS AND DISCUSSION Effect of Pretreatment on Lutein Yield. The parameters remained constant as the effects of the different factors on the Table 2. Variance Analysis between Factors Levels of Extraction Yield factor
dev sq
degrees of freedom (DOF)
A B C D error
15.033 361.143 73.787 184.526 15.03
2 2 2 2 2
F ratio (F)
critical value (α = 0.05)
24.023 4.908 12.275
19.000 19.000 19.000
significance *
“ultrasound enhanced subcritical CO2 extraction” of lutein were investigated. After enzymatic and soaking treatments of the C. pyrenoidosa, the yields of lutein were 85.05 and 69.02 mg/100 g C. pyrenoidosa, respectively. The better pretreatment method was enzymatic treatment; therefore, in subsequent experiments enzymatic treatment was adopted as the pretreatment method of C. pyrenoidosa. C. pyrenoidosa has a tough cellulose layer cell wall; it is not conducive for the solvent to penetrate into the cells to extract intracellular substances, so without any pretreatment, the extraction effect of lutein is poor. After soaking in high concentrations of polar solvents, its cells swelled, the structure loosened, and ingredients were released; the extraction became more conducive, so after soaking treatment the yield of lutein significantly increased. This effect has also been reported by the study conducted by Fang and others;27 they studied the supercritical CO2 extraction technology on the extraction of the colchicine roots. After enzymatic treatment, the cell wall of C. pyrenoidosa was degraded by cellulose, and the material in the
Table 3. Comparison between Different Extraction Technologies of Lutein extraction method
temperature (°C)
pressure (MPa)
ultrasound power (W)
time (h)
lutein yield (mg 100 g−1)
SE SWE SCE SCCE SCCE with pretreatment USCCE with pretreatment
43 150 50 27 27 27
0.1 5 25 21 21 21
0 0 0 0 0 1000
18 1/3 4 4 4 (+3 h pretreatment) 4 (+3 h pretreatment)
54.64 0 39.33 42.29 92.15 124.01
4600
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Figure 3. HPLC of extraction by different methods: (A) lutein standard; (B) SE; (C) SWE; (D) SCE; (E) SCCE; (F) SCCE with pretreatment; (G) USCCE with pretreatment.
separated in separation kettles I and II, respectively, but after the addition of ethanol, most of the extracts were brought into separation vessel II, resulting in lutein not being separated out. Experimental results are shown in Figure 2D: the yield of lutein increases as cosolvent increases rapidly in the range of 0−1.0 mL/g, but when the amount is >1.0 mL/g, the effect of cosolvent started to slow; considering the cost, the amount of cosolvent was chosen as 1.0 mL/g. Effect of Extraction Time on Lutein Yield. The results are shown in Figure 2E: with the extension of the extraction time, yield also has a corresponding increase, but the slope of the yield curve is gradually reduced and eventually levels off. A similar result was presented in research by Deenu et al.,11 in which when the temperature was fixed, the lutein yield increased until a certain amount of time (approximately 5 h) and then decreased. This could be explained as the chemical decomposition of the bioactive compound present in the extract may occur as the extraction time prolongs, resulting in a decrease in the extraction yield. Considering the technology and production cost, the preferred time for the extraction of lutein is 4 h. Effect of Ultrasound Power on Lutein Yield. The results are shown in Figure 2F: with the introduction of ultrasound,
carrying the solute into the circulation system before complete separation in the separation kettle, thus reducing the yield rate. Experimental results are shown in Figure 2C: with the increase of the fluid flow, lutein yield first increased and then decreased. The optimum fluid flow rate was 30 kg/h. Effect of Cosolvent on Lutein Yield. Adding an appropriate amount of suitable cosolvent can significantly improve the solubility of certain extracted components in subcritical fluid and the selectivity of subcritical fluid. CO2 is a nonpolar substance, whereas lutein is a polar material, its dipole moment being 1.863.31 High pressure can increase lutein’s solubility in subcritical carbon dioxide, but this effect is limited. Ethanol is the only organic reagent that is permitted for use in functional food and pharmaceutical processing.32 As a cosolvent, ethanol can increase the density of a subcritical fluid, and its penetration ability is strong in the matrix diffusion, so after the addition of a cosolvent, the yield of lutein greatly improved. However, the positive effect of cosolvent on the extraction of lutein is limited; besides increasing the subcritical fluid solubility, it would also weaken the selective ability of an extraction system, resulting in an increase of total extracts. This phenomenon has been reported in a study by Wu;23 before the addition of ethanol, lutein and other substances could be well 4601
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Figure 5. Model fitting curve of lutein extraction yield for USCCE and SCCE. The k value of USCCE was 0.6599, and the k value of SCCE was 0.5683.
with the findings of many other researchers on natural product extraction.11,14,33−35 In research conducted by Riera et al.,33 mass transfer enhancement in supercritical fluids extraction (SFE) by means of power ultrasound was calculated; at the end of the extraction time (8.5 h), the yield of the oil was significantly increased when the SFE was ultrasonically assisted. The improvement was about 20%. At the same time the process was accelerated by using ultrasound. Similar extraction yields were obtained in about 30% shorter time. Optimization of Extraction Conditions of Lutein. Experiments were performed to determine the importance order of the factors influencing ultrasound-enhanced subcritical CO2 extraction of lutein and calculate the optimum process parameters; according to the result of the orthogonal test, range analysis and variance analysis of factors are made. The orthogonal experiment results and the analysis results are shown in Tables 1 and 2, respectively. Table 1 shows that in the four factors, extraction pressure had the maximum effect; extraction temperature had the minimum effect. According to Table 1, the importance order of each factor on lutein yield was B > D > C > A, and the optimization of process parameters of USCCE was A2B2C3D3 in which extraction temperature was 27 °C, extraction pressure was 21 MPa, dosage of ethanol was 1.5 mL/g, and ultrasonic power was 1000 W. Experiments were performed under the optimal conditions to verify the credibility of the best level combination, and results showed that the yield of lutein, 124.01 mg/100 g C. pyrenoidosa, was even higher than the highest yield in the orthogonal experiment, 117.75 mg/100 g C. pyrenoidosa (Table 1), so the best conditions were determined as A2B2C3D3. As can be seen from the results of the analysis variance in Table 2, when α = 0.05, the effects of extraction pressure (factor B) on the yield of lutein in broken C. pyrenoidosa reached a significant level, whereas the effects of the ultrasonic power (factor D) and cosolvent dosage (factor C) were not significant. The effect of ultrasonic power, by contrast, is more significant than that of cosolvent dosage, and with the increase of ultrasonic power, lutein yield increases. The extraction temperature (factor A) has a smaller effect on extraction; in the horizontal range of the experiments, the significance of factor A was the least, considering the production cost, and compared
Figure 4. Absorption spectrum of different components: (A, top) lutein standard (retention time, 8.35); (B, middle) substance retains at 10.06 min; (C, bottom) substance retains at 10.93 min.
Table 4. Extraction Yield of Lutein from Chlorella pyrenoidosa by SCCE and USCCE yield (mglutein/100gChlorella)
yield (mglutein/100gChlorella)
time (h)
USCCE
SCCE
time (h)
USCCE
SCCE
0 0.5 1 1.5 2 2.5 3
0 50.23 62.46 80.1 89.98 102.06 113.01
0 31.12 46.91 56.6 64.29 74.36 83.85
3.5 4 4.5 5 5.5 6
117.21 124.01 125.62 126.73 127.44 127.77
90.36 92.15 95.37 96.45 97.01 97.39
lutein yield rises rapidly. Because of the increase in sound power, sound intensity and the effect of ultrasonic vibration increases, leading to accelerated movement of solute molecules and the enhancement of the mass transfer inside the material, thereby improving the yield of lutein. This result is consistent 4602
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Figure 6. Scanning electron micrographs of seed powder from treatments of (A) control, (B) Soxhlet extraction (SD), (C) subcritical CO2 extraction (SCCE), (D) USCCE without pretreatment, and (E) USCCE with pretreatment.
with other research, the temperature could be chosen as room temperature.11 Comparison of USCCE with Other Extraction Methods. It can be seen from Table 3, in terms of the extraction temperature, the temperature of USCCE and SCCE with or without pretreatment was room temperature, which was lower than the other three methods. In terms of the extraction pressure, the pressure of USCCE and SCCE is lower than that of SCE; therefore, the former two methods have a relatively low requirement for the equipment. In terms of extraction time, supercritical and subcritical CO2 extraction needed at most 7 h, compared with 18 h for Soxhlet extraction. We assume efficiency as extraction rate per unit time; the least efficiency of supercritical and subcritical CO2 extraction was 9.8, whereas the efficiency of Soxhlet was 3.04. Extraction time could be greatly reduced and still achieve higher efficiency. Compared with SCCE without pretreatment, the extraction yield of lutein after pretreatment is greatly improved, pretreatment being more conducive to the extraction of effective constituents in C. pyrenoidosa. Because of the introduction of ultrasound and pretreatment, USCCE with pretreatment had a much higher yield than the other four methods; yield had a 100−200% increase. Compared with the
SCCE with pretreatment, the yield also had a 34.5% increase. To sum up, the USCCE with pretreatment is the best to extract lutein; the yield of SCCE was slightly higher than the SCE; Soxhlet extraction yield is medium, but it was so timeconsuming that its efficiency is not high. The HPLC conditions achieved baseline separation of lutein from the total extract, the retention time of lutein was in the vicinity of 8.35 min, and in Figure 3A lutein typical strong shoulder peak can be seen at 445 and 474 nm. Except for the subcritical water extracts, the other five extracts have the other two substances detected at retention times of 10.06 and 10.93 min; the two substances were full wavelength scanned, and the results are shown in Figure 4B,C. The main absorption band profile of these two substances was in the 400−500 nm range and has a shoulder peak or even three absorption peaks of typical carotenoid,36 so these two substances were inferred as carotenoids, too, but their specific structure needs more analysis. Extraction Kinetics. The results of USCCE and SCCE are shown in Table 4. According to the experimental data, we used the nonlinear fitting method to get the E∞ and k values in Origin7.0 software. As is shown in Figure 5 and Table 4, the kinetic model and experimental data have a rather good fitting 4603
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*(J.-G.J.) E-mail: [email protected]. Phone: +86-2087113849. Fax: +86-20-87113843.
degree. The k value of USCCE was 0.6599, larger than that of SCCE 0.5683, suggesting that ultrasound can increase the diffusion coefficient of internal solute and material; E∞ increased significantly after using ultrasound, suggesting that ultrasound had an effect on the adsorption/desorption balance of solute between the interface of solid and liquid phases, and it made the balance tilt in the desorption direction. This conclusion is consistent with research conducted by Romdhane.30 Overall, E∞ and k increase at the same time, suggesting that ultrasound can enhance subcritical CO2 extraction. Microstructure of C. pyrenoidosa Powder. The images of C. pyrenoidosa treated by ultrasound with or without enzymatic pretreatment and conventional extraction are shown in Figure 6. The microstructure of the microalgal cell by SD (Figure 6B) had a lot of holes and interspaces because of a long time soaking (18 h). Part of the microalgal cell surface by SCCE (Figure 6C) floated up and had stepped lamella, which was because 4 h is not enough for CO2 to penetrate into the interior. The microstructure of the microalgal cell by USCCE (Figure 6D) had more interspaces and holes than the microalgal cell by SCCE. This was the same as the results of Li et al.37 and Zhao et al.,38 in which ultrasound treatment disrupted tissues and cell walls. Moreover, the physical disruption of C. pyrenoidosa cells by ultrasound, together with enzymatic pretreatment, was the most efficient method to destroy microalgal cell walls (Figure 6E). Enzymatic pretreatment has recently been shown to be another alternative method, which opens up cell walls through biodegradation and releases bioactive compounds from microalga.39 The cell walls were damaged by the application of ultrasound and enzymatic pretreatment, which resulted in the greater penetration of solvent into the sample matrix and increased the contact surface area between the solid and liquid phase, and, as a result, the solute quickly diffused from the solid phase to the solvent. Hence, USCCE with pretreatment is much more efficient and rapid for the extraction of the bioactive compound. Therefore, the sonication played an important role in breaking up the microalgal cell walls to enhance the extraction yield. The extraction conditions for USCCE with enzyme pretreatment of lutein were evaluated and optimized with regard to the extraction efficiency. Results indicate that lutein was successfully extracted using USCCE with enzyme pretreatment due to its high efficiency, especially compared to other extraction methods. Among the four significant factors that affect USCCE with enzyme pretreatment (extraction temperature, extraction pressure, cosolvent dosage, and ultrasonic power), extraction pressure has maximum effect and extraction temperature has minimum effect. The optimal parameters of USCCE with enzyme pretreatment are extraction temperature of 27 °C, extraction pressure of 21 MPa, ethanol dosage of 1.5 mL/g, and ultrasonic power of 1000 W. Experiments performed under the optimal conditions reached 124.01 mg/100 g C. pyrenoidosa. Kinetic models of USCCE and SCCE in C. pyrenoidosa were established; different E∞ and k values illustrate that ultrasound could strengthen the subcritical CO2 extraction, and the model can also be a significant guide to the study of the amplification of the extraction process.
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Funding
This project was supported by the National Natural Foundation of China (Grant 21406074), Guangdong Province Science and Technology plan project (2013B020311006), and Guangdong Provincial Bureau of Ocean and Fishery Science and Technology to promote a special (A201301B04). Notes
The authors declare no competing financial interest.
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Corresponding Authors
*(T.-Q.Q.) E-mail: [email protected]. Phone: +86-2087113849. Fax: +86-20-87113843. 4604
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