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Ultrasound assisted extraction of β-carotene from Spirulina platensis
Ultrasonics Sonochemistry 20 (2013) 271–276
Contents lists available at SciVerse ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Ultrasound assisted extraction of b-carotene from Spirulina platensis Soumen Dey, Virendra K. Rathod ⇑ Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
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Article history: Received 7 January 2012 Received in revised form 18 May 2012 Accepted 20 May 2012 Available online 29 May 2012 Keywords: UAE b-carotene Spirulina platensis n-heptane Electrical acoustic intensity Yield
a b s t r a c t This paper illustrates the Ultrasound Assisted Extraction (UAE) of b-carotene from Spirulina platensis. Various parameters such as extraction time, solvent type, biomass to solvent ratio, temperature, electrical acoustic intensity, length of the probe tip dipped into the solvent, duty cycle and pre treatment effect were explored for the extraction of b-carotene. From economic point of view, the optimal conditions for the extraction of b-carotene from Spirulina were 1.5 g Spirulina (2 min pre soaked in methanol) in 50 ml n-heptane at 30 °C temperature, 167 W/cm2 electrical acoustic intensity and 61.5% duty cycle for 8 min with probe tip length of 0.5 cm dipped into the extracting solvent from the surface. The maximum extraction achieved under the above mentioned optimum parameters was 47.10%. The pre-treatment time showed a promising effect on the yield as pre-treating the biomass with methanol for 2 min before ultrasonication showed 12 times increase in extraction yield of b-carotene. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Various microalgae such as Dunalliela, Chlorella, Chlamydomonas and Haematococcus were used as sources to extract oil, pigments, immunological diagnostics, nutritional supplements, anti-cancer drugs, polyunsaturated fatty acids and recombinant proteins [1]. Spirulina platensis (Spirulina), a cyanobacterium, is a single celled alga living in warm, alkaline fresh water and is a rich source of protein. It has been certified as Generally Regarded as Safe and hence can be used in food products [2]. It contains various valuable pigments such as phycocyanin, phycoerythrin, chlorophyll and also carotenoids. In Spirulina, b-carotene represents 67–79% of total carotenoids present which is equivalent to 53% more retinol equivalent than the amount present in carrot [3]. Despite above advantages, literature related to Spirulina and b-carotene are limited and most of the content and extraction studies related to b-carotene have been carried out on microalgae Dunalliela salina. Carotenoids are the family of isoprenoids having forty carbon atoms. These are the most important photosynthetic pigments coupled with protection of chlorophyll and thylakoid membrane from photo oxidative damage [4]. b-carotene is one of the hydrocarbon carotenoids having chromophore characteristic due to the presence of eleven conjugated double bonds in its structure. It is synthesized in plants, algae and some photosynthetic microorganisms as an important intermediate in the biosynthetic pathway of
⇑ Corresponding author. Tel.: +91 22 33612020; fax: +91 22 33611020. E-mail addresses: [email protected], [email protected] (V.K. Rathod). 1350-4177/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.05.010
various carotenoids such as zeaxanthin, from phytoene. Out of the large pool of carotenoids bearing provitamin A activity, b-carotene holds the prime position [5]. It helps protect against certain chronic diseases such as cancer, diabetes [6], cardiovascular diseases [7] etc. Traditionally, extraction of various bioactive compounds from natural products has been performed in many industries using different solvents. The conventional extraction methods such as maceration and soxhlet extraction have many drawbacks like large amount of solvent utilization, long extraction time and lower extraction yield. Recently, investigators have focused on novel extraction techniques like ultrasound assisted extraction (UAE), microwave assisted extraction, supercritical fluid extraction and pressurized fluid extraction to overcome these drawbacks [8]. But amongst all these novel techniques, UAE is an inexpensive and simple substitute to traditional extraction techniques [9]. Applications of UAE for the extraction of lipids, proteins, flavonoids, carotenoids, hemicelluloses, triterpenoids and aromatic compounds are well reported in literature [10–14]. UAE uses acoustic cavitation for producing cavitation bubbles which implodes resulting into high shear forces [15,16]. This helps in disrupting the cell wall allowing the solvent to penetrate into the plant material [17] and increases the contact surface area between the solvent and compound of interest, resulting into increased mass transfer along with good mixing. Therefore, UAE provides increased extraction yield, increased rate of extraction, reduced extraction time and higher processing throughput along with the advantage of usage of reduced temperature and solvent volume [18] which is very useful for the extraction of heat labile compounds [19].
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Although UAE gives many advantages, there are no reports available on extraction of b-carotene from Spirulina using this technique. This paper aims to study the effect of various parameters such as electrical acoustic intensity, extraction time, biomass to solvent ratio, solvent type, tip height, temperature, duty cycle and pre soaking effect which affects UAE. Hence, the main objective of this work is to optimize the various above-mentioned parameters for the extraction of b-carotene from Spirulina using UAE. 2. Materials and methods Analytical grade Methanol and n-heptane were purchased from SD Fine Chemicals Ltd, Mumbai, India. b-carotene standard type-II was procured from Sigma Aldrich, USA. Spray dried S. platensis of 60 mesh particle size was acquired from Parry Nutraceuticals, India and stored at 2-4 °C in the nitrogen environment. 0.22 l PTFE microfiltration membrane assembly was purchased from AllPure, USA. 2.1. Calculation of electrical acoustic intensity The electrical acoustic intensity (I) dissipated from the probe microtip was calculated according to the following formula:
IðW=cm2 Þ ¼
P
pr2
Where r is the radius of the probe microtip (cm), and P is the input power (W). In the present study, the input power levels were randomly adjusted to 50, 90, 117, 131, 145 and 165 W. The corresponding ultrasonic intensities were 64, 115, 149, 167, 185 and 210 W/cm2 respectively. 2.2. Calculation of extraction yield The extraction yield of b-Carotene was calculated according to the following formula:
Yieldðmg=gÞ ¼
weight of b-carotene extracted ðmgÞ weight of dried sample ðgÞ
probe was fitted with PZT transducer with tip diameter of 1 cm. The schematic representation of the experimental setup has been shown in Fig. 1. The general protocol used for the Ultrasound Assisted Extraction was as follows: In this study, effects of different factors of ultrasound treatment on the extraction yield of b-carotene were tested. 0.5 g of the spray-dried Spirulina biomass (250 micron particle size) was mixed with minimal amount of methanol for 2 min and then transferred into a specially designed round bottomed 100 ml glass vessel having a narrow neck. The main drawback of using flat bottomed vessel was that there were silent regions at the corners where plant material accumulates and no longer accessible to UAE. Thus to avoid such silent regions, a round bottomed vessel was used. Then 50 ml of n-heptane was poured into that extraction vessel. The ultrasound was introduced using the ultrasonic probe for 5 min operating in 50% duty cycle (5 s ON and 5 s OFF). 0.5 cm length of the ultrasonic probe tip was dipped into the solvent from its surface, for passing ultrasound. An electrical acoustic intensity of 167 W/cm2 was used for the extraction. Samples were withdrawn after 5 min intervals and then filtered using 0.22 lm PTFE microfiltration membranes to get clear liquid extract. The extract was then saponified using 20% (v/v) alc. KOH. Carotenoids in natural environments are predominantly esterified by fatty acids. In order to simplify the separation, the sample was saponified to remove the fatty acids and liberate the parent carotenoids. Chlorophylls were also degraded. When chlorophylls were treated with alcoholic KOH, carboxylic acid was produced which forms salts that were soluble in water. After saponification, the saponified extract was washed four times with distilled water. Then the water washed extract was further used for analysis using UV–Vis Double Beam Spectrophotometer 6.84 (Chemito, Mumbai, India) and HPLC. During the extraction process, the disruptor was held in a thermostat-controlled water bath at 30 °C. For the pre treatment of the biomass, 0.5 g of spray dried spirulina biomass was mixed with minimal amount of methanol, just enough to wet the biomass. The wetted biomass was kept for 2 min. Pre treating the biomass with methanol helps in loosening of the cell wall structure and ultimately helps in UAE. In order to study influence of solvent, two more solvents i.e. diethyl ether and hexane were selected. For selecting the best
2.3. Calculation of % extraction yield The % extraction yield of b-Carotene was calculated according to the following formula:
%Extraction yield ¼
extraction yield of b-carotene ðmg Þ g Þ maximum b-carotene content ðmg g
100
2.4. Calculation of duty cycle The pulse duration and pulse interval refer to ‘‘ON’’ time and ‘‘OFF’’ time of the sonochemical reactor. The total time of a pulse duration period plus a pulse interval period is the cycle time. A duty cycle (expressed as %) is the proportion of the pulse duration period to the cycle time.
Duty cycle ð%Þ ¼
\ON"time in sec 100 ½\ON"time in sec þ \OFF"time in sec
2.5. Experimental design The sonochemical reactor set up used in the present work consisted of an ultrasonic probe (Dakshin India Ltd., Mumbai) operating at a frequency of 20 kHz with rated power of 200 W. Ultrasonic
Fig. 1. Schematic representation of the experimental setup.
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has also been carried out in a small batch extractor at room temperature keeping all the parameters same. It has been observed that the yield obtained after 8 min of ultrasound was almost doubled (1.15 mg/g) when compared with the conventional extraction yield (0.56 mg/g) for 24 h indicating positive role of ultrasound for the intensification of extraction process.
solvent out of the three, 50 ml of n-heptane in the above mentioned method was replaced with 50 ml of diethyl ether and 50 ml of hexane, in two separate runs, keeping all other above mentioned parameters same. The above mentioned conditions were kept constant for all the treatments, unless otherwise mentioned. Single factor experimental design was used to quantify the effect of different factors such as solvent type, solid to solvent ratio, temperature, time, electrical acoustic intensity, tip length dipped into the solvent, duty cycle and pre-treatment time on the extraction yield of b-carotene. All experiments were performed three times and the average values have been reported with standard deviation.
3.2. Selection of solvent The extraction of b-carotene was performed using different solvents like hexane, n-heptane and diethyl ether. Fig. 3 shows that diethyl ether gave slightly higher extraction yield followed by n-heptane and hexane. Since, the diethyl ether has higher vapor pressure, it was expected that the extraction yield would have been obtained lower as compared to other two solvents. The probable reason for the higher yield in case of diethyl ether is due to its higher polarity and lower viscosity. The polar solvents help in increasing the permeability of the cell wall of the microalgae and low viscosity increases the diffusion of solvent as well as at low viscosity, acoustic cavitation takes place very easily. However, diethyl ether is not selected as the solvent for UAE due to its low boiling point of 34 °C. With a slight heat up during sonication the solvent will start vaporizing. Also the fumes generated are harmful and on storage leads to peroxides formation which on heating increases the risk of explosion. Out of hexane and heptane, the former showed relatively lower extraction yield as compared to the later. So, n-heptane proved to be a better solvent and also its having solubility of 0.0003 (% w/w) in water. It is also more hydrophobic than hexane, hence more selective for b-carotene. Hence, heptane was selected for the present study.
2.6. Analytical method UV–Vis Double Beam Spectrophotometer 6.84 was used to analyze the extracts from the above method. The concentration of the b-carotene extracted was measured at 450 nm. Quantifications of the sample were done using the calibration curve prepared from the standard b-carotene of different known concentrations. The concentration obtained was expressed in lg/ml, which was finally converted to mg/g. 3. Results and discussion 3.1. Effect of extraction time Fig. 2 shows the change in extraction yield of b-carotene from Spirulina powder at different extraction times. It has been observed that the extraction yield increases exponentially till few minutes (4 min), later increases gradually (8 min) and then becomes constant. The bubbles formed due to cavitation collapses giving a large amount of energy to surrounding which causes disruption of cell wall. The initial sharp increase in the rate of extraction was due to the large b-carotene concentration gradient between the extracting solvent and the cell and also due to easier extraction of constituents from outer part. With an increase in the extraction time, the concentration gradient decreased; as the mass transfer was increased with continuous exposure to ultrasound as well as the extraction became difficult due to interior location of cells. The continuous increase in release of the product of interest due to extraction, the solvent became saturated with the product. So, after that there was negligible mass transfer and extraction. Balachandran et al. [20] reported similar trend in their research. In order to compare the influence of the cavitation, the extraction
3.3. Effect of biomass to solvent ratio Biomass to solvent ratio is one of the most important parameters to be optimized in an extraction process. Thus, the extraction yield was measured at different biomass to solvent ratios ranging from 0.01 to 0.05 g/ml and results are reported in Fig. 4. It was observed that biomass to solvent ratio of 0.03 g/ml gave maximum extraction yield and the trend showed a gradual increase from 0.01 g/ml to 0.03 g/ml and then remained almost constant with further increase in ratio. The reason for the increase in extraction yield with biomass to solvent ratio was that initially in the case of 0.01 g/ml, the solvent volume was high and the concentration of compound to be extracted was low. When the biomass concentration was increased, the solvent slowly became saturated
1.4 1.2
Yield (mg/g)
1.0 0.8 0.6 0.4 0.2 0.0 0
2
4
6
8
10
12
14
16
Time (minutes) Fig. 2. Effect of extraction time on extraction of b-carotene.
18
20
22
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resulting into increased mass transfer of intracellular products into the solvent. At low temperature, the thermal effect was negligible and hence the extraction yield was less. As the temperature was increased to 30 °C, both the effects were equally dominating and the combination effect resulted into relatively higher extraction yield. As the temperature was further increased to 40 °C, the cavitation effect starts decreasing. Increasing temperature had a negative effect on cavitation because the cavitation intensity decreases with increase in temperature [23–25]. So, at 40 °C only thermal effect was pronounced and hence resulted in lesser extraction yield. Further increasing the temperature does not add into increase in the extraction yield.
1.4
Yield (mg/g)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 Heptane
Hexane Solvent type
Diethyl ether
Fig. 3. Effect of solvent type on extraction of b-carotene.
3.5. Effect of electrical acoustic intensity 1.4
Yield (mg/g)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
0.01
0.02 0.03 0.04 Biomass to solvent ratio (g/ml)
0.05
0.06
Fig. 4. Effect of biomass to solvent ratio on extraction of b-carotene.
resulting into non-complete interaction of the solvent with the biomass [21]. Same reason holds true for the reason of similar extraction yield at 0.04 and 0.05 g/ml biomass to solvent ratio. The phenomenon of lower extraction yield at lower biomass/solvent ratio was also observed by Vongsangnak et al. [22], who found that a larger solvent volume did not lead to a higher saponin yield from cultured cells of Panax notoginseng by microwave extraction. 3.4. Effect of temperature Effect of temperature on extraction yield was studied by varying the temperature from 10 °C to 50 °C with the interval of 10 °C. Since b-carotene is heat sensitive, higher extraction temperatures have not been studied. It was observed that with an increase in temperature from 10 °C to 30 °C, the extraction yield also showed increasing trend (Fig. 5). In UAE, two phenomenons play important role, namely cavitation effect and thermal effect. Cavitation effect works by imploding cavitation bubbles or cavities and thermal effect works by swelling and loosening the cell structure
In order to see the effect of electrical acoustic intensity on extraction yield of b-carotene, the ultrasonicator was operated at 64, 115, 149, 167, 185 and 210 W/cm2. It was observed that the extraction yield increased till 167 W/cm2 and then decreased at 185 W/cm2 but increased at 210 W/cm2, as shown in Fig. 6. With increase in electrical acoustic intensity more energy was getting transferred for cavitation phenomenon to occur and the cavities formed imploded energetically [26]. This resulted into the increase in the extraction yield from 64 to 167 W/cm2. With further increase in electrical acoustic intensity, more bubbles were formed which hampers the propagation of shock waves [24]. Also, the bubbles may coalesce to form bigger ones and implode weakly. Hence, the extraction yield decreased from 167 to 185 W/cm2. The increase in the extraction from 185 to 210 W/cm2 could be due to the thermal effect as the heat generated at this electrical acoustic intensity was very high.
3.6. Effect of tip length dipped in the solution The tip of the probe was dipped into the extracting solvent from the surface at different heights (0.5, 1, 1.5 and 2 cm) to study the effect of the tip length on the extraction yield of b-carotene. Fig. 7 reveals that more the tip dipped into the solvent (from the surface), lesser was the extraction yield. This may be due to improper distribution of the cavitation bubbles. As the tip was inserted more into the solution, the cavities generated directly hits the wall of the vessel. This resulted into damping of the energy generated by the cavities and hence the extraction yield of b-carotene decreases. When the tip of the probe was dipped upto 0.5 cm, the bubbles were properly scattered into the solvent and the shock wave or the energy released due to imploding bubbles were correctly dispersed in the solvent resulting into higher extraction yield.
1.4 1.4
1.2 1.2 1.0 Yield (mg/g)
Yield (mg/g)
1.0 0.8 0.6 0.4 0.2
0.8 0.6 0.4 0.2
0.0
0.0
0
10
20 30 40 Temperature (°C)
50
Fig. 5. Effect of temperature on extraction of b-carotene.
60
0
20
40
60 80 100 120 140 160 180 Electrical acoustic intensity (W/cm2 )
200
Fig. 6. Effect of electrical acoustic intensity on extraction of b-carotene.
220
275
1.4
1.4
1.2
1.2
1.0
1.0
Yield (mg/g)
Yield (mg/g)
S. Dey, V.K. Rathod / Ultrasonics Sonochemistry 20 (2013) 271–276
0.8 0.6 0.4
0.8 0.6 0.4 0.2
0.2
0.0
0.0 0
0.5
1
1.5
2
0
Tip length dipped into the solvent (cm) Fig. 7. Effect of tip length dipped into the solvent on extraction of b-carotene.
2
4 6 Pre treatment time (minutes)
8
10
Fig. 9. Effect of pre treatment time on extraction of b-carotene.
4. Conclusion 3.7. Effect of duty cycle The effect of duty cycle on the extraction yield of b-carotene was studied at 0%, 28.6%, 50%, 61.5%, 70.6%, 80% and 100% duty cycles. Fig. 8 has clearly shown that the extraction yield increased with an increase in the duty cycle from 0% to 61.5% and then became almost steady till 100% duty cycle. Ultrasonicator can be operated both in pulse mode and continuous mode. Operating in continuous mode creates problems such as erosion of the tip and also it is energy inefficient as compared to pulsed mode [27]. So, for such reasons pulse mode was selected. Pan et al. [27] also reported that operating in both pulsed and continuous mode at a particular intensity gave similar yield. Hence, it is confirmed from this study that proper ON–OFF timing must be determined of the ultrasonicator for better extraction, as unnecessary increase in the ON timing would lead to excessive heating and unnecessary electrical consumption. 3.8. Effect of pre-treatment time/ soaking time The effect of pre-treatment time of the dried biomass on the extraction was studied. The dried biomass was treated with a known volume of methanol for 0, 2, 4, 6 and 10 min. It was observed that pre-treatment played a very important role in increasing the extraction yield significantly. The extraction increased from 3.2% to 39% with 2 min of methanol pre-treatment (Fig. 9). Methanol being polar in nature helps in increasing the permeability of the microalgal cell wall which makes easy for ultrasound to break the cells. This study would be helpful in saving large amount of solvent used for the extraction. It was also observed that with further increase in pre-treatment time from 2 to 10 min, there was negligible change in b-carotene extraction.
1.4 1.2
Yield (mg/g)
1.0 0.8 0.6 0.4 0.2 0.0 0
20
40 60 Duty cycle (%)
80
Fig. 8. Effect of duty cycle on extraction of b-carotene.
100
In this study, various parameters affecting the UAE of b-carotene from Spirulina were investigated. As confirmed by the results, selection of proper extracting solvent was very important. Heptane was selected, though diethyl ether gave higher extraction. With an increase in biomass to solvent ratio, extraction yield of b-carotene showed a unique trend, giving highest extraction at 0.03 g/ml biomass to solvent ratio. As b-carotene is heat sensitive and also due to economical point of view, a temperature of 30 °C was selected as optimal. The rate of b-carotene extraction was very high initially till 4 min and then slowed down reaching saturation at 8 min with the highest of 47% extraction. Results from the electrical acoustic intensity study were quite interesting as the trend showed an increase of extraction from 64 to 167 W/cm2 and then decrease till 185 W/cm2 and again increased in 210 W/cm2 at tip length of 0.5 cm dipped into the solvent. Proper ON–OFF timing should be optimized for an extraction process as after 61.5% duty cycle the extraction was almost constant. The pre soaking time also had a significant effect on the extraction of b-carotene as 2 min of soaking resulted into 12 times increase in extraction. Hence, the valuable information gained from this study should enlighten further studies in developing and optimizing the UAE process for industries to make it a simple and profitable venture. References [1] T.L. Walker, S. Purton, D.K. Becker, C. Collet, Microalgae as bioreactors, Plant Cell Rep. 24 (2005) 629–641. [2] C.C. Moraes, J.F. De Medeiros Burkert, S.J. Kalil, C-Phycocyanin extraction process for large-scale use, J. Food Biochem. 34 (2010) 133–148. [3] A. Jassby, Spirulina: A Model for Microalgae as Human Food, in: C.A. Lumb, J.R. Waaland (Eds.), Algae and Human Affairs, Cambridge University Press, Cambridge, 1988, pp. 149–179. [4] J.C. Bauerfeind, G.B. Brubacher, H.M. Kliäui, W.L. Marusich, Use of Carotenoids, in: O. Isler (Ed.), Carotenoids, Birkhaüser Verlag, Basel, 1971, pp. 743–770. [5] J. Szpylka, J.W. DeVries, Determination of b-carotene in supplements and raw materials by reversed-phase high pressure liquid chromatography: Collaborative study, J. AOAC Int. 88 (2005) 1279–1291. [6] E.S. Ford, J.C. Will, B.A. Bowman, K.M. Narayan, Diabetes mellitus and serum carotenoids: findings from the third national health and nutrition examination survey, Am. J. Epidemiol. 149 (1999) 168–176. [7] C.O. Perera, G.M. Yen, Functional properties of carotenoids in human health, Int. J. Food Prop. 10 (2007) 201–230. [8] L. Wang, C.L. Weller, Recent advances in extraction of neutraceuticals from plants, Trends Food Sci. Technol. 17 (2006) 300–312. [9] W. Jing, S. Baoguo, C. Yanping, T. Yuan, L. Xuehong, Optimization of ultrasound-assisted extraction of phenolic compounds from wheat bran, Food Chem. 106 (2008) 804–810. [10] H.Z. Li, L. Pordesimo, J. Weiss, High intensity ultrasound-assisted extraction of oil from soya beans, Food Res. Int. 37 (2007) 731–738. [11] F. Chen, Y.Z. Sun, G.H. Zhao, X.J. Liao, X.S. Hu, J.H. Wu, Z.F. Wang, Optimization of ultrasound-assisted extraction of anthrocyanins in red raspberries and identification of anthrocyanins in extract using high-performance liquid chromatography–mass spectrometry, Ultrason. Sonochem. 14 (2007) 767–778. [12] X.H. Yue, W. Prinyawiwatkul, J.M. King, Improving extraction of lutein from egg yolk using an ultrasound-assisted solvent method, J. Food Sci. 71 (2006) 239–241.
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Contents lists available at SciVerse ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Ultrasound assisted extraction of b-carotene from Spirulina platensis Soumen Dey, Virendra K. Rathod ⇑ Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history: Received 7 January 2012 Received in revised form 18 May 2012 Accepted 20 May 2012 Available online 29 May 2012 Keywords: UAE b-carotene Spirulina platensis n-heptane Electrical acoustic intensity Yield
a b s t r a c t This paper illustrates the Ultrasound Assisted Extraction (UAE) of b-carotene from Spirulina platensis. Various parameters such as extraction time, solvent type, biomass to solvent ratio, temperature, electrical acoustic intensity, length of the probe tip dipped into the solvent, duty cycle and pre treatment effect were explored for the extraction of b-carotene. From economic point of view, the optimal conditions for the extraction of b-carotene from Spirulina were 1.5 g Spirulina (2 min pre soaked in methanol) in 50 ml n-heptane at 30 °C temperature, 167 W/cm2 electrical acoustic intensity and 61.5% duty cycle for 8 min with probe tip length of 0.5 cm dipped into the extracting solvent from the surface. The maximum extraction achieved under the above mentioned optimum parameters was 47.10%. The pre-treatment time showed a promising effect on the yield as pre-treating the biomass with methanol for 2 min before ultrasonication showed 12 times increase in extraction yield of b-carotene. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Various microalgae such as Dunalliela, Chlorella, Chlamydomonas and Haematococcus were used as sources to extract oil, pigments, immunological diagnostics, nutritional supplements, anti-cancer drugs, polyunsaturated fatty acids and recombinant proteins [1]. Spirulina platensis (Spirulina), a cyanobacterium, is a single celled alga living in warm, alkaline fresh water and is a rich source of protein. It has been certified as Generally Regarded as Safe and hence can be used in food products [2]. It contains various valuable pigments such as phycocyanin, phycoerythrin, chlorophyll and also carotenoids. In Spirulina, b-carotene represents 67–79% of total carotenoids present which is equivalent to 53% more retinol equivalent than the amount present in carrot [3]. Despite above advantages, literature related to Spirulina and b-carotene are limited and most of the content and extraction studies related to b-carotene have been carried out on microalgae Dunalliela salina. Carotenoids are the family of isoprenoids having forty carbon atoms. These are the most important photosynthetic pigments coupled with protection of chlorophyll and thylakoid membrane from photo oxidative damage [4]. b-carotene is one of the hydrocarbon carotenoids having chromophore characteristic due to the presence of eleven conjugated double bonds in its structure. It is synthesized in plants, algae and some photosynthetic microorganisms as an important intermediate in the biosynthetic pathway of
⇑ Corresponding author. Tel.: +91 22 33612020; fax: +91 22 33611020. E-mail addresses: [email protected], [email protected] (V.K. Rathod). 1350-4177/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.05.010
various carotenoids such as zeaxanthin, from phytoene. Out of the large pool of carotenoids bearing provitamin A activity, b-carotene holds the prime position [5]. It helps protect against certain chronic diseases such as cancer, diabetes [6], cardiovascular diseases [7] etc. Traditionally, extraction of various bioactive compounds from natural products has been performed in many industries using different solvents. The conventional extraction methods such as maceration and soxhlet extraction have many drawbacks like large amount of solvent utilization, long extraction time and lower extraction yield. Recently, investigators have focused on novel extraction techniques like ultrasound assisted extraction (UAE), microwave assisted extraction, supercritical fluid extraction and pressurized fluid extraction to overcome these drawbacks [8]. But amongst all these novel techniques, UAE is an inexpensive and simple substitute to traditional extraction techniques [9]. Applications of UAE for the extraction of lipids, proteins, flavonoids, carotenoids, hemicelluloses, triterpenoids and aromatic compounds are well reported in literature [10–14]. UAE uses acoustic cavitation for producing cavitation bubbles which implodes resulting into high shear forces [15,16]. This helps in disrupting the cell wall allowing the solvent to penetrate into the plant material [17] and increases the contact surface area between the solvent and compound of interest, resulting into increased mass transfer along with good mixing. Therefore, UAE provides increased extraction yield, increased rate of extraction, reduced extraction time and higher processing throughput along with the advantage of usage of reduced temperature and solvent volume [18] which is very useful for the extraction of heat labile compounds [19].
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Although UAE gives many advantages, there are no reports available on extraction of b-carotene from Spirulina using this technique. This paper aims to study the effect of various parameters such as electrical acoustic intensity, extraction time, biomass to solvent ratio, solvent type, tip height, temperature, duty cycle and pre soaking effect which affects UAE. Hence, the main objective of this work is to optimize the various above-mentioned parameters for the extraction of b-carotene from Spirulina using UAE. 2. Materials and methods Analytical grade Methanol and n-heptane were purchased from SD Fine Chemicals Ltd, Mumbai, India. b-carotene standard type-II was procured from Sigma Aldrich, USA. Spray dried S. platensis of 60 mesh particle size was acquired from Parry Nutraceuticals, India and stored at 2-4 °C in the nitrogen environment. 0.22 l PTFE microfiltration membrane assembly was purchased from AllPure, USA. 2.1. Calculation of electrical acoustic intensity The electrical acoustic intensity (I) dissipated from the probe microtip was calculated according to the following formula:
IðW=cm2 Þ ¼
P
pr2
Where r is the radius of the probe microtip (cm), and P is the input power (W). In the present study, the input power levels were randomly adjusted to 50, 90, 117, 131, 145 and 165 W. The corresponding ultrasonic intensities were 64, 115, 149, 167, 185 and 210 W/cm2 respectively. 2.2. Calculation of extraction yield The extraction yield of b-Carotene was calculated according to the following formula:
Yieldðmg=gÞ ¼
weight of b-carotene extracted ðmgÞ weight of dried sample ðgÞ
probe was fitted with PZT transducer with tip diameter of 1 cm. The schematic representation of the experimental setup has been shown in Fig. 1. The general protocol used for the Ultrasound Assisted Extraction was as follows: In this study, effects of different factors of ultrasound treatment on the extraction yield of b-carotene were tested. 0.5 g of the spray-dried Spirulina biomass (250 micron particle size) was mixed with minimal amount of methanol for 2 min and then transferred into a specially designed round bottomed 100 ml glass vessel having a narrow neck. The main drawback of using flat bottomed vessel was that there were silent regions at the corners where plant material accumulates and no longer accessible to UAE. Thus to avoid such silent regions, a round bottomed vessel was used. Then 50 ml of n-heptane was poured into that extraction vessel. The ultrasound was introduced using the ultrasonic probe for 5 min operating in 50% duty cycle (5 s ON and 5 s OFF). 0.5 cm length of the ultrasonic probe tip was dipped into the solvent from its surface, for passing ultrasound. An electrical acoustic intensity of 167 W/cm2 was used for the extraction. Samples were withdrawn after 5 min intervals and then filtered using 0.22 lm PTFE microfiltration membranes to get clear liquid extract. The extract was then saponified using 20% (v/v) alc. KOH. Carotenoids in natural environments are predominantly esterified by fatty acids. In order to simplify the separation, the sample was saponified to remove the fatty acids and liberate the parent carotenoids. Chlorophylls were also degraded. When chlorophylls were treated with alcoholic KOH, carboxylic acid was produced which forms salts that were soluble in water. After saponification, the saponified extract was washed four times with distilled water. Then the water washed extract was further used for analysis using UV–Vis Double Beam Spectrophotometer 6.84 (Chemito, Mumbai, India) and HPLC. During the extraction process, the disruptor was held in a thermostat-controlled water bath at 30 °C. For the pre treatment of the biomass, 0.5 g of spray dried spirulina biomass was mixed with minimal amount of methanol, just enough to wet the biomass. The wetted biomass was kept for 2 min. Pre treating the biomass with methanol helps in loosening of the cell wall structure and ultimately helps in UAE. In order to study influence of solvent, two more solvents i.e. diethyl ether and hexane were selected. For selecting the best
2.3. Calculation of % extraction yield The % extraction yield of b-Carotene was calculated according to the following formula:
%Extraction yield ¼
extraction yield of b-carotene ðmg Þ g Þ maximum b-carotene content ðmg g
100
2.4. Calculation of duty cycle The pulse duration and pulse interval refer to ‘‘ON’’ time and ‘‘OFF’’ time of the sonochemical reactor. The total time of a pulse duration period plus a pulse interval period is the cycle time. A duty cycle (expressed as %) is the proportion of the pulse duration period to the cycle time.
Duty cycle ð%Þ ¼
\ON"time in sec 100 ½\ON"time in sec þ \OFF"time in sec
2.5. Experimental design The sonochemical reactor set up used in the present work consisted of an ultrasonic probe (Dakshin India Ltd., Mumbai) operating at a frequency of 20 kHz with rated power of 200 W. Ultrasonic
Fig. 1. Schematic representation of the experimental setup.
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has also been carried out in a small batch extractor at room temperature keeping all the parameters same. It has been observed that the yield obtained after 8 min of ultrasound was almost doubled (1.15 mg/g) when compared with the conventional extraction yield (0.56 mg/g) for 24 h indicating positive role of ultrasound for the intensification of extraction process.
solvent out of the three, 50 ml of n-heptane in the above mentioned method was replaced with 50 ml of diethyl ether and 50 ml of hexane, in two separate runs, keeping all other above mentioned parameters same. The above mentioned conditions were kept constant for all the treatments, unless otherwise mentioned. Single factor experimental design was used to quantify the effect of different factors such as solvent type, solid to solvent ratio, temperature, time, electrical acoustic intensity, tip length dipped into the solvent, duty cycle and pre-treatment time on the extraction yield of b-carotene. All experiments were performed three times and the average values have been reported with standard deviation.
3.2. Selection of solvent The extraction of b-carotene was performed using different solvents like hexane, n-heptane and diethyl ether. Fig. 3 shows that diethyl ether gave slightly higher extraction yield followed by n-heptane and hexane. Since, the diethyl ether has higher vapor pressure, it was expected that the extraction yield would have been obtained lower as compared to other two solvents. The probable reason for the higher yield in case of diethyl ether is due to its higher polarity and lower viscosity. The polar solvents help in increasing the permeability of the cell wall of the microalgae and low viscosity increases the diffusion of solvent as well as at low viscosity, acoustic cavitation takes place very easily. However, diethyl ether is not selected as the solvent for UAE due to its low boiling point of 34 °C. With a slight heat up during sonication the solvent will start vaporizing. Also the fumes generated are harmful and on storage leads to peroxides formation which on heating increases the risk of explosion. Out of hexane and heptane, the former showed relatively lower extraction yield as compared to the later. So, n-heptane proved to be a better solvent and also its having solubility of 0.0003 (% w/w) in water. It is also more hydrophobic than hexane, hence more selective for b-carotene. Hence, heptane was selected for the present study.
2.6. Analytical method UV–Vis Double Beam Spectrophotometer 6.84 was used to analyze the extracts from the above method. The concentration of the b-carotene extracted was measured at 450 nm. Quantifications of the sample were done using the calibration curve prepared from the standard b-carotene of different known concentrations. The concentration obtained was expressed in lg/ml, which was finally converted to mg/g. 3. Results and discussion 3.1. Effect of extraction time Fig. 2 shows the change in extraction yield of b-carotene from Spirulina powder at different extraction times. It has been observed that the extraction yield increases exponentially till few minutes (4 min), later increases gradually (8 min) and then becomes constant. The bubbles formed due to cavitation collapses giving a large amount of energy to surrounding which causes disruption of cell wall. The initial sharp increase in the rate of extraction was due to the large b-carotene concentration gradient between the extracting solvent and the cell and also due to easier extraction of constituents from outer part. With an increase in the extraction time, the concentration gradient decreased; as the mass transfer was increased with continuous exposure to ultrasound as well as the extraction became difficult due to interior location of cells. The continuous increase in release of the product of interest due to extraction, the solvent became saturated with the product. So, after that there was negligible mass transfer and extraction. Balachandran et al. [20] reported similar trend in their research. In order to compare the influence of the cavitation, the extraction
3.3. Effect of biomass to solvent ratio Biomass to solvent ratio is one of the most important parameters to be optimized in an extraction process. Thus, the extraction yield was measured at different biomass to solvent ratios ranging from 0.01 to 0.05 g/ml and results are reported in Fig. 4. It was observed that biomass to solvent ratio of 0.03 g/ml gave maximum extraction yield and the trend showed a gradual increase from 0.01 g/ml to 0.03 g/ml and then remained almost constant with further increase in ratio. The reason for the increase in extraction yield with biomass to solvent ratio was that initially in the case of 0.01 g/ml, the solvent volume was high and the concentration of compound to be extracted was low. When the biomass concentration was increased, the solvent slowly became saturated
1.4 1.2
Yield (mg/g)
1.0 0.8 0.6 0.4 0.2 0.0 0
2
4
6
8
10
12
14
16
Time (minutes) Fig. 2. Effect of extraction time on extraction of b-carotene.
18
20
22
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resulting into increased mass transfer of intracellular products into the solvent. At low temperature, the thermal effect was negligible and hence the extraction yield was less. As the temperature was increased to 30 °C, both the effects were equally dominating and the combination effect resulted into relatively higher extraction yield. As the temperature was further increased to 40 °C, the cavitation effect starts decreasing. Increasing temperature had a negative effect on cavitation because the cavitation intensity decreases with increase in temperature [23–25]. So, at 40 °C only thermal effect was pronounced and hence resulted in lesser extraction yield. Further increasing the temperature does not add into increase in the extraction yield.
1.4
Yield (mg/g)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 Heptane
Hexane Solvent type
Diethyl ether
Fig. 3. Effect of solvent type on extraction of b-carotene.
3.5. Effect of electrical acoustic intensity 1.4
Yield (mg/g)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
0.01
0.02 0.03 0.04 Biomass to solvent ratio (g/ml)
0.05
0.06
Fig. 4. Effect of biomass to solvent ratio on extraction of b-carotene.
resulting into non-complete interaction of the solvent with the biomass [21]. Same reason holds true for the reason of similar extraction yield at 0.04 and 0.05 g/ml biomass to solvent ratio. The phenomenon of lower extraction yield at lower biomass/solvent ratio was also observed by Vongsangnak et al. [22], who found that a larger solvent volume did not lead to a higher saponin yield from cultured cells of Panax notoginseng by microwave extraction. 3.4. Effect of temperature Effect of temperature on extraction yield was studied by varying the temperature from 10 °C to 50 °C with the interval of 10 °C. Since b-carotene is heat sensitive, higher extraction temperatures have not been studied. It was observed that with an increase in temperature from 10 °C to 30 °C, the extraction yield also showed increasing trend (Fig. 5). In UAE, two phenomenons play important role, namely cavitation effect and thermal effect. Cavitation effect works by imploding cavitation bubbles or cavities and thermal effect works by swelling and loosening the cell structure
In order to see the effect of electrical acoustic intensity on extraction yield of b-carotene, the ultrasonicator was operated at 64, 115, 149, 167, 185 and 210 W/cm2. It was observed that the extraction yield increased till 167 W/cm2 and then decreased at 185 W/cm2 but increased at 210 W/cm2, as shown in Fig. 6. With increase in electrical acoustic intensity more energy was getting transferred for cavitation phenomenon to occur and the cavities formed imploded energetically [26]. This resulted into the increase in the extraction yield from 64 to 167 W/cm2. With further increase in electrical acoustic intensity, more bubbles were formed which hampers the propagation of shock waves [24]. Also, the bubbles may coalesce to form bigger ones and implode weakly. Hence, the extraction yield decreased from 167 to 185 W/cm2. The increase in the extraction from 185 to 210 W/cm2 could be due to the thermal effect as the heat generated at this electrical acoustic intensity was very high.
3.6. Effect of tip length dipped in the solution The tip of the probe was dipped into the extracting solvent from the surface at different heights (0.5, 1, 1.5 and 2 cm) to study the effect of the tip length on the extraction yield of b-carotene. Fig. 7 reveals that more the tip dipped into the solvent (from the surface), lesser was the extraction yield. This may be due to improper distribution of the cavitation bubbles. As the tip was inserted more into the solution, the cavities generated directly hits the wall of the vessel. This resulted into damping of the energy generated by the cavities and hence the extraction yield of b-carotene decreases. When the tip of the probe was dipped upto 0.5 cm, the bubbles were properly scattered into the solvent and the shock wave or the energy released due to imploding bubbles were correctly dispersed in the solvent resulting into higher extraction yield.
1.4 1.4
1.2 1.2 1.0 Yield (mg/g)
Yield (mg/g)
1.0 0.8 0.6 0.4 0.2
0.8 0.6 0.4 0.2
0.0
0.0
0
10
20 30 40 Temperature (°C)
50
Fig. 5. Effect of temperature on extraction of b-carotene.
60
0
20
40
60 80 100 120 140 160 180 Electrical acoustic intensity (W/cm2 )
200
Fig. 6. Effect of electrical acoustic intensity on extraction of b-carotene.
220
275
1.4
1.4
1.2
1.2
1.0
1.0
Yield (mg/g)
Yield (mg/g)
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0.8 0.6 0.4
0.8 0.6 0.4 0.2
0.2
0.0
0.0 0
0.5
1
1.5
2
0
Tip length dipped into the solvent (cm) Fig. 7. Effect of tip length dipped into the solvent on extraction of b-carotene.
2
4 6 Pre treatment time (minutes)
8
10
Fig. 9. Effect of pre treatment time on extraction of b-carotene.
4. Conclusion 3.7. Effect of duty cycle The effect of duty cycle on the extraction yield of b-carotene was studied at 0%, 28.6%, 50%, 61.5%, 70.6%, 80% and 100% duty cycles. Fig. 8 has clearly shown that the extraction yield increased with an increase in the duty cycle from 0% to 61.5% and then became almost steady till 100% duty cycle. Ultrasonicator can be operated both in pulse mode and continuous mode. Operating in continuous mode creates problems such as erosion of the tip and also it is energy inefficient as compared to pulsed mode [27]. So, for such reasons pulse mode was selected. Pan et al. [27] also reported that operating in both pulsed and continuous mode at a particular intensity gave similar yield. Hence, it is confirmed from this study that proper ON–OFF timing must be determined of the ultrasonicator for better extraction, as unnecessary increase in the ON timing would lead to excessive heating and unnecessary electrical consumption. 3.8. Effect of pre-treatment time/ soaking time The effect of pre-treatment time of the dried biomass on the extraction was studied. The dried biomass was treated with a known volume of methanol for 0, 2, 4, 6 and 10 min. It was observed that pre-treatment played a very important role in increasing the extraction yield significantly. The extraction increased from 3.2% to 39% with 2 min of methanol pre-treatment (Fig. 9). Methanol being polar in nature helps in increasing the permeability of the microalgal cell wall which makes easy for ultrasound to break the cells. This study would be helpful in saving large amount of solvent used for the extraction. It was also observed that with further increase in pre-treatment time from 2 to 10 min, there was negligible change in b-carotene extraction.
1.4 1.2
Yield (mg/g)
1.0 0.8 0.6 0.4 0.2 0.0 0
20
40 60 Duty cycle (%)
80
Fig. 8. Effect of duty cycle on extraction of b-carotene.
100
In this study, various parameters affecting the UAE of b-carotene from Spirulina were investigated. As confirmed by the results, selection of proper extracting solvent was very important. Heptane was selected, though diethyl ether gave higher extraction. With an increase in biomass to solvent ratio, extraction yield of b-carotene showed a unique trend, giving highest extraction at 0.03 g/ml biomass to solvent ratio. As b-carotene is heat sensitive and also due to economical point of view, a temperature of 30 °C was selected as optimal. The rate of b-carotene extraction was very high initially till 4 min and then slowed down reaching saturation at 8 min with the highest of 47% extraction. Results from the electrical acoustic intensity study were quite interesting as the trend showed an increase of extraction from 64 to 167 W/cm2 and then decrease till 185 W/cm2 and again increased in 210 W/cm2 at tip length of 0.5 cm dipped into the solvent. Proper ON–OFF timing should be optimized for an extraction process as after 61.5% duty cycle the extraction was almost constant. The pre soaking time also had a significant effect on the extraction of b-carotene as 2 min of soaking resulted into 12 times increase in extraction. Hence, the valuable information gained from this study should enlighten further studies in developing and optimizing the UAE process for industries to make it a simple and profitable venture. References [1] T.L. Walker, S. Purton, D.K. Becker, C. Collet, Microalgae as bioreactors, Plant Cell Rep. 24 (2005) 629–641. [2] C.C. Moraes, J.F. De Medeiros Burkert, S.J. Kalil, C-Phycocyanin extraction process for large-scale use, J. Food Biochem. 34 (2010) 133–148. [3] A. Jassby, Spirulina: A Model for Microalgae as Human Food, in: C.A. Lumb, J.R. Waaland (Eds.), Algae and Human Affairs, Cambridge University Press, Cambridge, 1988, pp. 149–179. [4] J.C. Bauerfeind, G.B. Brubacher, H.M. Kliäui, W.L. Marusich, Use of Carotenoids, in: O. Isler (Ed.), Carotenoids, Birkhaüser Verlag, Basel, 1971, pp. 743–770. [5] J. Szpylka, J.W. DeVries, Determination of b-carotene in supplements and raw materials by reversed-phase high pressure liquid chromatography: Collaborative study, J. AOAC Int. 88 (2005) 1279–1291. [6] E.S. Ford, J.C. Will, B.A. Bowman, K.M. Narayan, Diabetes mellitus and serum carotenoids: findings from the third national health and nutrition examination survey, Am. J. Epidemiol. 149 (1999) 168–176. [7] C.O. Perera, G.M. Yen, Functional properties of carotenoids in human health, Int. J. Food Prop. 10 (2007) 201–230. [8] L. Wang, C.L. Weller, Recent advances in extraction of neutraceuticals from plants, Trends Food Sci. Technol. 17 (2006) 300–312. [9] W. Jing, S. Baoguo, C. Yanping, T. Yuan, L. Xuehong, Optimization of ultrasound-assisted extraction of phenolic compounds from wheat bran, Food Chem. 106 (2008) 804–810. [10] H.Z. Li, L. Pordesimo, J. Weiss, High intensity ultrasound-assisted extraction of oil from soya beans, Food Res. Int. 37 (2007) 731–738. [11] F. Chen, Y.Z. Sun, G.H. Zhao, X.J. Liao, X.S. Hu, J.H. Wu, Z.F. Wang, Optimization of ultrasound-assisted extraction of anthrocyanins in red raspberries and identification of anthrocyanins in extract using high-performance liquid chromatography–mass spectrometry, Ultrason. Sonochem. 14 (2007) 767–778. [12] X.H. Yue, W. Prinyawiwatkul, J.M. King, Improving extraction of lutein from egg yolk using an ultrasound-assisted solvent method, J. Food Sci. 71 (2006) 239–241.
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