Ultrasonic Extraction of Active Compounds from Saffron
R. Kadkhodaee, A. Hemmati-Kakhki
Department of Food Technology
Khorasan Research Centre for Technology Development
Mashad
Iran
Keywords: crocins, picrocrocine, pulsed sonication, safranal, ultrasound
Abstract
The active compounds of saffron were extracted using high power ultrasound at a constant frequency of 30 kHz. The effect of acoustic intensity, time and mode of sonication on the extraction yield of three major constituents of saffron was investigated at 20 °C. The efficiency of the process was compared with that of cold water extraction method proposed by ISO. The results showed that ultrasound largely improved the extraction rate and incredibly reduced the process time. The extraction yield increased with the increase of time and amplitude of sonication. It was also found that the use of pulsed ultrasound with short pulse intervals was more efficient than continuous sonication.
INTRODUCTION
Saffron is the dried stigmas of the flowers of Crocus sativa Linnaeus, which is cultivated in many countries including Iran, Spain, Greece, India, and china; with the former producing greater than 90 % of the world’s total annual production (Sampathu et al., 1984; Negbie, 1999). It is the most expensive spice in the world increasingly gaining interest among the customers for the delicate flavour, bitter taste and attractive yellow colour it impacts on food. The main constituents responsible for these characteristics are crocins, picrocrocin and safranal. Crocins are water soluble derivatives of crocetin, glucosyl esters of 8,8’-diapocarotene-8,8’-dioic acid, representing the yellow pigments of saffron (Sampathu et al., 1984). Picrocrocin, 4-(β-D-glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde, is the glucoside responsible for the bitter taste of saffron from which safranal is derived (Zarghami and Heinz, 1971). Safranal, 2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, is a monoterpene considered to be the major component constituting saffron volatile components (Zarghami and Heinz, 1971; Tarantilis et al., 1994).
The quality of saffron is determined by its coloring strength, aroma and taste. These quality attributes are highly dependent on soil, climate, rainfall, time of harvest, and post harvest operations of saffron. Numerous methods have been proposed for quality determination of saffron involving preparation of saffron extract followed by measurement the quantity of crocins, picrocrocins, and safranal (Basker and Negbi, 1985; Sujata et al., 1992; Orfanou and Tsimidou, 1996; ISO, 2003; Kanakis et al., 2004). Preparation of extract has a crucial impact on the accuracy of the results. It has been reported that the type of solvent, time and method of extraction not only considerably affect the diffusion rate of the components across the cell wall but also their stability (Himeno and Sano, 1987; Sujata et al.,1992; Tarantilis et al.,1994; Orfanou and Tsimidou, 1996). Crocins have been shown to undergo degradation during prolonged extraction time in aqueous media of high water activity. It has also been demonstrated that the use of alcohol or water-alcohol results in higher extraction rate compared to water (Orfanou and Tsimidou, 1996). Accordingly, the methods of extraction are being steadily revised and modified techniques proposed.
The use of high power ultrasound in the extraction of functional and bioactive components of plant materials has been reported by many researchers (Salisova et al., 1997; Vinatoru et al., 1997; Melecchi et al., 2002; Bruni et al., 2002; Albu et al., 2004; Schinor et al., 2004; Wang and Weller, 2006). It has been shown that ultrasound largely improves the extraction rate by disrupting plant cells and hence increasing the diffusion of the cell contents across the cell wall. The beneficial effects of sound waves on extraction are attributed to the formation and asymmetrical collapse of microcavities in the vicinity of cell walls leading to the generation of microjets rupturing the cells. It is also thought that the pulsation of bubbles causes acoustic streaming which improves mass transfer rate by preventing the solvent layer surrounding the plant tissue from getting saturated and hence enhancement of convection.
The research reported here is aimed at the ultrasound-assisted extraction of active principles from saffron. Observations and comparisons will be made with reference to the methodology proposed by the International Standards Organization (ISO 3632-2:2003).
MATERIALS AND METHODS
Commercial samples of saffron stigmas were purchased from local market. The samples were ground with a pestle and mortar and passed through a 0.5 mm mesh. The prepared sample was stored in a hermetic bag at 4 °C temperature until use.
Preparation of Extracts
1. Cold Water Extraction Using Ultrasound. 0.25 g of sample was extracted with 20 ml of distilled water in a flat-bottomed glass tube using direct sonication, Dr. Hielscher ultrasonic processor, Germany (Model UP 50H), at a frequency of 30 kHz for 1, 3, 5 and 10 min. A titanium sonotrode of 3 mm in diameter was used in all experiments with the tip placed 1.5 cm below the surface of liquid. The temperature was kept constant at 20 °C throughout sonication by immersing the glass tube in a refrigerated water bath. Continuous and pulsed sonication at 0.2 and 0.5 duty cycles and 20, 60 and 100 % of the available amplitude were used. The above sonication times were corrected with respect to the duty cycle used when working on pulse mode. After sonication the sample was immediately transferred into a 500 ml volumetric flask and made to the volume with distilled water. 20 ml of this suspension was then diluted to 200 ml followed by filtration through cellulose acetate membrane (pore size: 0.45 µm). The absorbance readings of the filtrate were measured on a UV-Vis recording spectrophotometer, Shimadzu, Japan (Model UV-160A) at 257, 330 and 440 nm representing for picrocrocine, safranal, and crocins, respectively. Distilled water was used as blank. The results were expressed as:
where A is the absorbance at λmax, m the mass of saffron sample (g), and H is the mass fraction of moisture and volatile content of the sample. H was determined to be 6.5 % for the samples used in this study.
2. Cold Water Extraction without Ultrasound. 0.25 g of sample was extracted with 500 ml of distilled water for 1 hr according to ISO 3632-2: 2003. The suspension was stirred using a magnetic stirrer during extraction and the temperature kept constant at 20 °C by immersing the flask in a refrigerated water bath. A 20 ml aliquot of this solution was transferred to a 200 ml volumetric flask, diluted to the mark with distilled water and then filtered as explained above. Absorbance readings were taken at the same wavelengths given in section 1.
3. Statistical Analysis. Triplicate samples were used. A completely randomized factorial design was performed to determine the significance of difference between means by calculating Fisher static (F) at a 5 % confidence interval. Comparison among means of the two extraction methods was carried out by Duncan’s Multiple Range Test. Microsoft Excel and SAS 8.2 (SAS Institute In., Cary, USA) were used for data analysis.
RESULTS AND DISCUSSION
The results clearly indicated that increasing sonication time increased the yield, with up to 15 % enhancement for safranal towards the end of extraction period. The rate of extraction was high during the first 5 minutes and then decreased thereafter. An explanation for this is that there is a large concentration gradient for the solutes in the plant cells and extraction solvent at the initial stage of extraction resulting in higher extraction rates. Additionally, the constituents located in the surface layers of the particles are more readily accessible than those in the deeper regions which hardly diffuse out and hence slow down the rate of extraction.
Comparison of the results obtained in the presence of ultrasound with those of ISO procedure revealed that continuous sonication resulted in greater values at all times and amplitudes tested, whereas and gained higher values only at longer sonication times or higher amplitudes (Table 1). Pulsed sonication led to various extraction rates clearly indicating that using low duty cycle did not have positive effect on extraction yield, although sonication at the maximum amplitude for 10 min resulted in values comparable to those of cold water extraction (Table 2). On the other hand, as the data given in Table 3 show, sonication at high burst cycle beneficially impacted the process of extraction causing larger amounts of the plant constituents to diffuse out and hence values much greater than those of continuous sonication. This is depicted in Figures 1a, b and c illustrating changes in values in the course of extraction at the amplitude of 100 %. These observations indicate that a pulse is the more efficient, the shorter the interval between pulses. In a pulsed ultrasound field the bubbles volume concentration in the path of the acoustic wave is lower than that of a continuous field which improves the acoustic transparency of the cavitation field. This is due to the fact that in the pulsed field the time between two pulses acts as a rest time during which the small bubbles and the unstable cavities generated by bubble collapses, depending on their sizes, dissolve away or float out of the cavitation zone and the initial conditions of the liquid are restored (Atchley et al., 1988; Johri et al., 1988; Francescutto et al., 1999). This reduces the bubble-bubble interactions and the probability of losing the spherical shape of the bubble at the early stage of collapse. Therefore, the bubble collapses are more efficient and hence higher amount of heat and pressure is generated in the core of bubble upon implosion. Another reason for the improvement of sonochemical reactions in a pulsed field can be the disintegration of the stable agglomerations of cavitation bubbles or prevention of their formation. The bubbles inside the cluster are shielded from the ultrasound field by the bubbles at the outside part of the cluster and act strongly on each other. Both theses factors diminish the efficiency of the bubble collapse. The pulse modulation of the ultrasound prevents the clustering and this can favor the clarification of the cavitation zone and thus maximization of the process yield as reported by some authors (Flynn and Curch, 1988a, b; Wan et al., 1996).
The effect of acoustic amplitude on extraction of crocins, safranal and picrocrocine is shown in Figures 2-4. As can be seen increasing amplitude increases, with up to 11, 13, and 12 % enhancement for picrocrocine, safranal, and crocins, respectively, when pulsed sonication at 100 % amplitude and 0.5 duty cycle was used. The increased yield suggests that either additional cells have been ruptured or extraction from the inner parts of the matrix has been accelerated. These can be attributed to the cavitational effects, which caused the intensification of mass transfer and thus closed interaction between the solvent and the plant tissues. It is demonstrated that at increasing amplitudes, cavitational bubble collapse is more violent since the resonant bubble size is proportional to the amplitude of ultrasounic wave (Suslick et al., 1987; Suslick and Price, 1999, Li et al., 2004). Bubble collapse in the vicinity of plant membranes may cause strong shear forces to be exerted that can cause microfractures to be formed in plant tissues (Vinatoru et al., 1997; Vinatoru, 2001). Additionally, increasing amplitude may cause microcavities to be formed inside the cells that could contribute to disruption of the walls from the inside (Vinatoru et al., 1997).
CONCLUSIONS
These results obtained in this study propose ultrasound as a potent alternative for efficient extraction of active components from saffron. In comparison to cold water extraction ultrasound improves the efficiency and reduces the processing time. The rate of extraction was shown to be a function of time, amplitude and mode of sonication. The increase of time and amplitude had a positive effect on the extraction yield. Pulsed sonication at short intervals was found to be more efficient than continuous sonication. Although these findings demonstrate that the use of ultrasound produces greater extraction yields and thus can be considered in the future revision of the ISO specification standard, further experiments are yet required before it is introduced for industrial applications.
ACKNOWLEGEMENTS
Financial support for this project from Khorasan Science and technologyPark is gratefully acknowledged. The authors also thank Ms. Sara Sobhani and Ms. Zahra Adabi for their great assistance in the experimentation and analysis.
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Table 1. Comparison of values obtained using continuous sonication and ISO procedure
Amplitude (%) / Sonication time (min) / / /20
60
100
ISO / 1
3
5
10
1
3
5
10
1
3
5
10 / 84.2±0.8
85.3±1.1
87.4±2.6
88.1±4.6
85.3±1.1
87.7±1.9
88.6±1.0
90.1±1.7
87.4±0.6
89.0±1.5
89.3±2.1
91.8±0.7
89.0±1.6 / 35.9±0.8
37.1±0.6
37.4±0.1
39.0±1.2
37.0±0.5
37.2±0.1
37.9±0.8
39.4±0.6
37.0±0.5
37.8±0.4
38.6±3.4
39.4±0.6
38.6±1.1 / 233.3±0.7
234.3±3.3
235.9±2.7
237.5±3.0
233.9±4.3
236.6±3.3
237.0±2.3
238.3±3.9
234.0±3.3
236.6±3.1
237.6±1.1
239.3±3.3
226.9±2.6
Table 2. Comparison of values obtained using pulsed sonication at 0.2 cycle and ISO procedure
Amplitude (%) / Sonication time (min) / / /20
60
100
ISO / 1
3
5
10
1
3
5
10
1
3
5
10 / 80.7±0.8
85.0±0.4
85.5±1.1
86.4±1.9
82.9±3.8
86.3±1.2
86.8±1.2
89.3±1.2
84.7±1.6
86.8±2.4
87.6±0.5
90.0±1.9
89.0±1.6 / 33.1±0.7
35.4±0.05
36.4±0.7
36.5±0.8
34.4±2.3
36.2±0.3
37.0±0.3
38.8±1.6
35.1±1.3
36.5±1.8
37.2±0.6
38.9±0.7
38.6±1.1 / 194.3±3.9
210.0±0.5
215.1±2.5
220.2±6.6
204.0±16
210.4±0.9
216.3±4.8
222.6±0.8
204.8±5.4
212.0±2.9
218.5±0.5
226.9±1.7
226.9±2.6
Table 3. Comparison of values obtained using pulsed sonication at 0.5 cycle and ISO procedure
Amplitude (%) / Sonication time (min) / / /20
60
100
ISO / 1
3
5
10
1
3
5
10
1
3
5
10 / 88.5±1.8
89.6±0.4
91.8±0.7
95.0±1.5
89.2±1.3
90.0±0.5
95.2±2.1
95.3±1.6
89.6±1.4
90.5±0.05
95.4±2.6
96.1±1.8
89.0±1.6 / 33.2±0.9
33.7±0.05
39.1±1.1
39.1±1.2
37.7±0.5
38.7±0.3
39.70.2
40.5±1.2
38.7±0.8
39.3±1.1
40.5±1.5
41.1±1.0
38.6±1.1 / 224.2±7.9
228.5±1.5
232.0±2.2
242.8±4.8
226.2±5.6
233.8±2.0
239.5±1.4
243.3±1.6
227.9±6.2
234.4±0.3
245.4±3.6
246.1±5.4
226.9±2.6
Fig. 1.Changes in as a function of time during sonication at 100 % amplitude. (a) crocins, (b) safranal, and (c) picrocrocine. ♦, continuous sonication; ▲, pulsed sonication at 0.2 cycle; ■, pulsed sonication at 0.5 cycle.
Fig. 2. Influence of acoustic amplitude on the extraction yield of crocins. (a) continuous sonication, (b), pulsed sonication at 0.2 cycle, (c) pulsed sonication at 0.5 cycle. ■, 1min; ▲, 3 min; ♦, 5 min; ×, 10 min.
Fig. 3. Influence of acoustic amplitude on the extraction yield of safranal. (a) continuous sonication, (b), pulsed sonication at 0.2 cycle, (c) pulsed sonication at 0.5 cycle. ■, 1min; ▲, 3 min; ♦, 5 min; ×, 10 min.
Fig. 4. Effect of acoustic amplitude on the extraction yield of picrocrocine. (a) continuous sonication, (b), pulsed sonication at 0.2 cycle, (c) pulsed sonication at 0.5 cycle. ■, 1min; ▲, 3 min; ♦, 5 min; ×, 10 min.