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Use of Microwaves to Extract Chlorogenic Acids from Green Coffee Beans
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Use of Microwaves to Extract Chlorogenic Acids from Green Coffee Beans Lingamallu Jagan Mohan Rao, Kulathooran Ramalakshmi Plantation Products, Spices and Flavour Technology Department, Central Food Technological Research Institute, Mysore, India
• T he yields of MAE under optimum conditions were higher than those from the conventional solvent extraction at 5 min and 50°C and the extracts showed radical-scavenging activity of > 75%, even at a concentration of 25 ppm. • The MAE process can thus be predicted and controlled for industrial application.
CHAPTER POINTS • M icrowave-assisted extraction (MAE) showed obvious advantages in terms of short duration and high efficiency in the recovery of chlorogenic acid from raw plant materials in comparison with conventional heat-reflux extraction. • The mechanism of the enhanced extraction by microwave assistance was studied by observing cell destruction of plant material after MAE treatment by scanning electron microscopy. The enhanced extraction was related partly to a greater extent of cell rupture of the plant materials, and this was observed by scanning electron microscopy. The plant materials were significantly destroyed due to the cell rupture during MAE treatment. • In general, extraction is the first step in the preparation of pharmaceutical formulations from raw plant material and significantly affects the cost of the whole manufacture process. • MAE has been considered as a potential alternative to conventional solvent extraction for the isolation of phenolic compounds from plants. • Under optimum conditions of time (5 min), temperature (50°C), and wattage (800 W), the maximum quantities of chlorogenic acids and caffeine could be extracted with water as solvent. • The extracts contained chlorogenic acids and caffeine in the ranges of 31–62% and 22–40%, respectively.
Processing and Impact on Active Components in Food http://dx.doi.org/10.1016/B978-0-12-404699-3.00070-6
INTRODUCTION Electromagnetic Radiation Electromagnetic radiation is a form of energy that is transmitted through a space at an enormous velocity. It is said to have a dual nature, exhibiting both wave and particle characteristics. As the name implies, electromagnetic radiation is an alternating electrical and associated magnetic force field in space. Thus, an electromagnetic wave has an electric component and a magnetic component. Both the components oscillate in planes perpendicular to the direction of the propagation of the radiation. The entire range over which electromagnetic radiation exists is known as electromagnetic spectrum. The electromagnetic spectrum consists of gamma, X-ray, ultraviolet, visible, infrared, microwave, radiofrequency, and ultrasound radiations (Ali and Hardiman, 2005; Susanne, 2012). Infrared Radiation Infrared radiation (IR) is a part of electromagnetic spectrum with a wavelength in the range of 0.78 μm to 1000 μm. Application of infrared heating has recently been employed in certain food-processing
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70. MICROWAVES TO EXTRACT CHLOROGENIC ACIDS FROM COFFEE
applications, because of its superiority in terms of costs and the product quality (color, organoleptic and nutritional value) as compared with conventional heating. Infrared heating offers many advantages over conventional heating under similar processing temperatures. Both the situations involve simultaneous heat and mass transfer, and in the case of conventional heating both are in opposite directions. These are in the same (from within to outside) direction in case of IR, which results in a higher rate of heat transfer to the material (Robyn et al., 2000). Microwave Radiation Microwave radiation (MW; wavelength in the range of 1 mm to 1 m) lies between IR and radiofrequency (RF) and is known as dielectric radiation. Material exposed to MW is heated due to dipolar rotation and ionic polarization. Hence, material containing a polar substance such as water is heated faster with MW. The typical domestic MW oven has a frequency of 2450 MHz. The applications of MW in food processing are blanching, drying, pasteurization, baking, etc. Compared to standard microwave technology the need to use higher frequencies such as 24,150 MHz for industrial applications has to be carefully verified with respect to special physical/ engineering advantages. MICROWAVE-ASSISTED EXTRACTION
MAE is a process that uses microwave energy along with solvent to extract target compounds from various matrices. The volumetric heating, localized temperature, and pressure can cause selective migration of t arget compounds from the material to the surroundings at a more rapid rate and with similar or better recoveries compared with conventional extractions. MAE was used for extraction of selected components from a wide variety of sample matrices and has been used as a promising alternative sample preparation technique for a number of applications (Zhou and Liu, 2006; Gao et al., 2006; Zhang and Xu, 2007; Rostagno et al., 2007). MAE can considerably reduce both extraction time and solvent consumption compared to conventional methods. Radiofrequency Waves RF waves are also being used for thermal processing food materials. In a radio frequency heating system, the RF generator creates an alternating electric field between two electrodes. The material is placed between the electrodes where the alternating energy causes polar molecules in the material to continuously reorient them to face opposite poles in much the same way that bar magnets behave in an alternating magnetic field. The friction resulting from molecular movement causes the material to rapidly heat throughout its entire mass. For those applications that require uniform
heating and precise temperature control, RF offers many advantages over conventional drying methods, namely uniform heating through the entire thickness, 2- to 20-times faster than conventional drying methods, energy efficient, uniform moisture profiling, low maintenance, and eco-friendly process. The permitted frequencies for industrial applications are 13, 27, and 40 MHz. A few reports on the existence of a non-thermal effect of RF energy on microorganisms are available. Freshness and nutrients are retained in foods when RF energy inactivates microorganisms non-thermally (Ali and Hardiman, 2005). Generally foodstuffs are complex mixtures of different large biochemical molecules (simple sugars, amino acids, vitamins etc), biochemical polymers (complex sugars, proteins, lipids etc.), inorganic salts and water. The strongest absorption is often localizable within the so-called group frequency, which is generated by the vibrations of these molecular aggregates (or molecular structural groups). The wavelength region of interest in discussing the interaction with foodstuffs can be restricted to the infrared range 0.78–15 μm even for high-temperature radiators, the spectral flux, [(W/m2)/μm] ascribed to wavelengths larger than 15 μm represents a small fraction of total radiation power.
HOW THE COMPOSITION ALTERED Chlorogenic Acids Chlorogenic acid (5-O-caffeoyl-quinic acid), an ester of caffeic acid with quinic acid, has received considerable attention for its wide distribution and potential biological effects (Clifford, 1999). It is one of the most abundant polyphenols in the human diet with coffee, fruits, and vegetables as its major sources. It is also an important bioactive compound and rich in selected raw materials used in traditional Chinese medicine, such as flowers and buds of Lonicera japonica Thumb, and the leaves of Eucommia ulmodies, both which have been used for the treatment of exopathogenic wind-heat or epidemic febrile disease at the early stage, carbuncles, furuncles, and swellings for centuries. The chlorogenic acids (CGA) are an important group of compounds in green coffee beans. Although 71 different species of CGA have now been identified (Rohit and Jaganmohanrao, 2013) the vast majority of the compounds belong to three classes: caffeoylquinic acids (CQA; 3CQA, 4CQA, and 5CQA), di-caffeoylquinic acids (diCQA; 3,4diCQA, 3,5diCQA, and 4,5diCQA) and feruloylquinic acids (FQA) as shown in Figure 70.1. Other quinic acids are CiQA, cinnamoyl quinic acids; GQA, galloyl quinic acids; iFQA, isoferuloyl quinic acids; oCoQA, o-coumaroyl quinic acids; SQA, sinapoyl quinic
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FIGURE 70.1 Chlorogenic acid isomers.
acids, which are known either naturally or synthetically. Although CGA (primarily as 5-caffeoylquinic acid) are widely distributed in plant materials, their content in green coffee is among the highest found in plants, ranging from 4 to 14% (Trugo and Macrae, 1984; Farah et al., 2008). The levels of CGA in green coffee beans have been reported to vary from approximately 7.88 to 14.4% dry matter for Coffea canephora (robusta) and approximately 3.4 to 4.8% for Coffea arabica (Ky et al., 2001). The analytical methods for CGA estimation include chromatographic and spectroscopic techniques. Chromatographic techniques reported are thin-layer chromatography, gas chromatography, and liquid chromatography (Bicchi et al., 1995; Rehwald et al., 1994). Spectral techniques include spectrophotometry, IR spectrometry, 1H NMR spectroscopy and chemiluminescence. Microwave Extraction In general, CGA are extracted from dried and powdered plant materials using solvent/aqueous extraction.
However, this technique is very time consuming as well as requiring high energy. This drawback prevents the application of the reported method. Development of a new technique for separation and purification of CGA is essential. Growing advancements in the isolation/separation techniques in recent years have made the purification and quantification of CGA easy, sensitive, and specific, with marked improvements in yield in much less time compared to traditional methods. Generally, absorption of the microwave energy increases with the dielectric constant of the molecule, resulting in power dissipation inside the solvent and plant materials and then generating more effective molecular movement and heating. As a polar solvent, water can efficiently absorb microwave energy and leads to efficient heating. MAE was used for extraction of interested components from a wide variety of sample matrices and has been used as a promising alternative sample preparation technique for a number of applications. Compared to conventional methods, MAE can considerably reduce both extraction time and solvent consumption.
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Chlorogenic Acids from Flower Buds of Lonicera japonica Thunb An efficient MAE technique has been developed to recover CGA from flower buds of Lonicera japonica, using a mixture of water and ethanol (Gao et al., 2003; Dan et al., 2003). Ethanol (50%) as MAE solvent gave the best yield of CGA in 5 min at a temperature of 60°C. For MAE, the choice of extraction solvent takes into account not only its solubility for target component but also its ability to absorb microwave energy. The yield of CGA increased with the increase of material : solvent ratio (amount of plant material : volume of extraction solvent) up to 1 : 10 (w/v) and further increase has no effect on the yield (Criado et al., 2004). The yield of CGA increased with time up to 30 min at 40°C whereas the same yield of CGA is obtained within 5 min at 60°C under microwave extraction and further increase in temperature has no effect on the yield. The yield of CGA reached 6.14% at 60°C within 5 min, which was significantly higher than that at 40°C. There was no obvious difference on yield of CGA between 60°C and 80°C; therefore, it was reported that the MAE temperature of 60°C was suitable for CGA extraction from flower buds. A conventional heat-reflux extraction of CGA from raw plant materials was carried out at 60°C. The yield of CGA reached 5.19% at an optimal ethanol concentration of 50% as extraction solvent within 5 min; further increase in the yield is slow and reached a maximum of 6% within 30 min. In comparison to the heat-flux extraction, the MAE showed obvious advantages in terms of short duration and high efficiency to extract CGA. This is mainly due to the fact that microwave energy is delivered efficiently to materials through molecular interaction with the electromagnetic field and offers a rapid transfer of energy to the extraction solvent and raw plant materials. Control and extracted samples of raw materials were subjected to Scanning Electron Microscopy. The changes in the raw material during heat-reflux extraction were not considerably different from the untreated sample, and only few slight ruptures happened on its surface. The surface of the MAE sample was greatly destroyed because that microwave irradiation accelerated cell rupture by sudden temperature rise and internal pressure increase inside the cells of plant sample. During the rupture process, a rapid exudation of the chemical substance within the cells into the surrounding solvents took place. Chlorogenic Acids from Coffee Coffee, an aromatic, non-alcoholic brew is known for its stimulating and refreshing taste. Two species are of significant economic importance namely, Arabica (Coffea arabica) and Robusta (Coffea canephora) (Varnam and Sutherland, 1994). In recent years, due to the increasing
interest in finding physiologically functional foodstuffs, the relationship between coffee and health has been extensively investigated (George et al., 2008). Coffee is reported to exhibit a number of bioactivities, such as antioxidant (Ramalakshmi et al., 2008, 2009, 2011), anticarcinogenic, and antimutagenic activity (Giovannucci, 1998). Coffee is known to show a protective effect on cancer and other cardiovascular diseases due to its antioxidant activity. It also protects low-density-lipoproteins from oxidation. This protective effect is the result of the action of several polyphenolic constituents. The physical and chemical properties of individual phenolics, strongly affect their antioxidant activities. In addition, these molecules can have a synergistic or antagonistic effect when present in complex mixtures. Indeed, the polyphenol composition of the beverage varies in species of coffee and tea (Richelle, 2001). In the case of coffee, robusta exhibits a higher antioxidant activity than arabica, which could be due to the higher amount of CGA. Following light roasting, the antioxidant activities of both coffees decreased markedly but further roasting produced the heterocyclic compounds having the antioxidant activity, and as these polyphenolic rich beverages are often consumed with milk, the effect of milk on different beverages (coffee, cocoa, black tea) were evaluated and coffee was found to have high antioxidant activity. An understanding of the protective role of dietary antioxidants in vivo requires a better characterization of the polyphenol composition of the antioxidant matrix as well as quantitative data on their absorption, their tissue distribution, their metabolism, and their biological actions. Indeed, after consumption, polyphenols have to cross the intestinal wall but must also resist further catabolism. The metabolism of polyphenols involves two important organs—the liver, where biotransformation enzymes convert them or their metabolites into conjugated forms such as glucuronides or sulphates, and the colon where microorganisms degrade unabsorbed ones. At present, only a little information is available on the absorption of the vast diversity of polyphenols present in these beverages (Richelle, 2001). So the antioxidant activity of coffee has to be considered while estimating the daily ingested dose of polyphenols. Thus, the beneficial effects of coffee may be attributed in part to polyphenols and caffeine serving as antioxidants. These positive properties of coffee on human health are attributed to the presence of phenolic compounds such as CGA with strong antioxidant and radical-scavenging activities present in green coffee. Earlier reports (Clifford and Wilson, 1985; Debabrata et al., 2011) suggest that the content of CGA in C. canephora varies from 7 to 10% (mfb), whereas C. arabica varies from 5 to 7.5% (mfb). In addition to having different total CGA levels, the relative amounts of CQA, diCQA, and FQA have also been shown to vary in mature C. canephora and C. arabica grain. For example Ky et al. (2001) found
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that CQA, diCQA and FQA represent roughly 67, 20, and 13%, respectively, of the total CGA content of C. canephora variety versus 80, 15, and 5%, respectively, of the total CGA content of C. arabica variety. In addition to being found in coffee, these compounds are also found at significant levels in plant foods such as apples, pears, tomato, potato, and eggplant (Niggeweg et al., 2004). Total CGA content of green and roasted beans of different varieties is provided in Table 70.1. Extraction of Chlorogenic Acids using Microwaves MAE showed obvious advantages in terms of short duration and high efficiency to recover CGA from raw plant materials in comparison with conventional heatreflux extraction. The mechanism of the enhanced extraction by microwave assistance was studied by observing cell destruction of plant material after MAE treatment by scanning electron microscopy. The enhanced extraction was related partly to a greater extent of cell rupture of the plant materials, and this was observed by TABLE 70.1 Chlorogenic Acid Content in Green and Roasted Coffee Varieties Coffee Variety
Bean Variety
Chlorogenic Acid (mg/g)
Coffea arabica
Dry green coffee beans
68–70
Medium roasted
26–30
Dark roasted
22–25
Very dark roasted
7–9
Coffea canephora Dry green coffee beans
85–88
scanning electron microscopy. The plant materials were significantly destroyed due to the cell rupture during MAE treatment. In this context, conditions for microwave-assisted extraction of green coffee beans were optimized and the quality of the extracts was monitored through determination of the contents of CGA, caffeine, and total polyphenols as well as evaluating the radical scavenging activity. A comparative analysis between MAE and conventional heat reflux methods was also made. EXTRACTION METHODOLOGY
Robusta cherry green coffee beans (Figure 70.2) were ground and sieved (< 720 μm) using Hammer mill and then defatted. Defatted coffee powder was extracted with different solvents such as water, methanol, and ethanol using Microwave lab station [(Model: STARTS configuration with control terminal 260, Milestone, Italy); built-in focused IR sensor and magnetic stirrer; Magnetron: SN: 133613; Frequency 50 Hz] in a closed system under different sets of conditions of temperature and wattage for specific periods of time. The slurry obtained was filtered to get a clear extract that was used for quantitative analysis. The quantitative analyses of extracts for percentage of yield, caffeine, CGA, and total polyphenols (TPP) contents, and radical scavenging activity (RSA) of MAE-treated defatted ground robusta cherry coffee were presented. The caffeine, CGA, RSA, and TPP content were analyzed spectrophotometrically at λmax275, 325, 517, and 760 nm, respectively. The percentage yield of analyte under different conditions/ parameters of MAE, i.e., time, microwave wattage, and temperature, are presented in the Table 70.2.
Medium roasted
35–37
Dark roasted
20–22
YIELD OF MAE EXTRACTS
Very dark roasted
6–7
The yields are in the range of 15–20% with water as solvent of extraction and found to be higher than alcohol FIGURE 70.2 Robusta coffee berries (Coffea robusta).
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TABLE 70.2 Microwave-Assisted Extraction of Green Coffee using Aqueous Medium Variable Parameter
Constant Parameter
Variables
Caffeine (%)
Chlorogenic acids (%)
Yield (%)
Time
Temperature (50°C) Wattage (800 W)
2 min
3.75 ± 0.07
6.05 ± 0.07
17.15 ± 0.07
5 min
4.60 ± 0.14
6.75 ± 0.07
17.50 ± 0.14
10 min
4.40 ± 0.14
6.35 ± 0.07
17.05 ± 0.07
400 W
3.70 ± 0.14
4.70 ± 0.14
15.40 ± 0.14
600 W
4.25 ± 0.07
5.80 ± 0.14
17.75 ± 0.07
800 W
7.25 ± 0.07
8.40 ± 0.28
18.10 ± 0.14
30°C
6.68 ± 0.07
6.65 ± 0.07
17.60 ± 0.07
50°C
7.25 ± 0.07
8.10 ± 0.04
18.05 ± 0.07
70°C
4.75 ± 0.07
6.75 ± 0.07
17.60 ± 0.14
90°C
4.65 ± 0.07
6.35 ± 0.07
16.75 ± 0.07
Wattage
Temperature
Time (5 min) Temperature (50°C)
Time (5 min) Wattage (800 W)
Rohit et al. (2012).
extracts (8.5–9.5%), indicating better extraction efficiency in water than in organic solvents. The reason could be that the dielectric constant of water is higher than hat of the alcohol, which helps in absorption of microwaves. It was found that the yield was highest (18%) with water as solvent for extraction under MAE conditions of 5 min, 800 W, and 50°C compared to other conditions. Chen and Spiro (1995) reported that the yield of extracts from rosemary leaves improved with an increase in microwave power and the induction stage appeared. However, Chemat et al. (2005) confirmed that there is not much difference in the extraction yield when the microwave power was increased from 50 to 150 W during the extraction of caraway seeds. The difference may be due to the nature of the plant material and the components to be extracted. CHLOROGENIC ACIDS IN EXTRACT
The percentage yields of CGA in the aqueous extracts obtained at different MAE parameters/conditions are presented in Table 70.2. The yield of CGA is in the range of 4.7–8.1% in aqueous extractions, whereas it is in the range of 4.9–5.6% in alcohols. The yield of CGA increases up to 5 min. The yield of CGA increases with increased wattage up to 800 W; experiments could not be carried out beyond 800 W, due to limitations with the equipment. Extraction yield of CGA is increased along with temperature up to 50°C, and decreased at higher temperatures, which could be due to degradation at higher temperatures. The highest yield of CGA (8.4 ± 0.28%) was obtained at MAE conditions of 5 min, 800 W and 50°C and water as solvent of extraction. CAFFEINE IN EXTRACT
The caffeine content in the aqueous extracts obtained at MAE times of 2, 5, and 10 min were 3.75 ± 0.07%, 4.6 ± 0.14%,
and 4.4 ± 0.14%, respectively. The yield of caffeine did not increase after 5 min. With MAE wattage of 400, 600, and 800 W, the yields in aqueous extracts were 3.7 ± 0.14%, 4.25 ± 0.07%, and 7.25 ± 0.07%, respectively. This indicates that the extraction of caffeine increases with increase in wattage. Experiments could not be carried out beyond 800 W, due to limitations with the equipment. At MAE temperature of 30, 50, 70, and 90°C, the yields obtained were 6.35 ± 0.07%, 7.25 ± 0.07%, 4.75 ± 0.07, and 4.65 ± 0.07%, respectively. Extraction yield of caffeine is increased along with temperature up to 50°C, and decreased at higher temperatures; this may be due to the degradation of caffeine at higher temperatures. When ethanol and methanol were used as extraction solvents, the yield obtained in the extracts were 3.05 ± 0.07% and 3.75 ± 0.07% respectively at MAE conditions of 5 min, 800 W and 50°C and lower than water as solvent. The highest yield (7.25%) of caffeine obtained MAE conditions of 5 min, 800 W, and 50°C in aqueous extraction. The comparative analysis of percentage extraction yields of caffeine at different MAE parameters is shown in Table 70.2. Pan et al. (2003) conducted experiments of MAE for polyphenols and caffeine at the microwave power of 700 W from green tea leaves. It is reported that MAE for 4 min provided maximum extraction of caffeine and polyphenols from tea leaves. TPP IN EXTRACT
The TPP content in coffee extract is measured in terms of gallic acid equivalents. During the estimation, Folin–Ciocalteu Reagent (FCR) was added to the extract. The FCR reacts with polyphenol compounds in the alkaline medium (sodium carbonate) and forms complexes. The various polyphenols that are present in the coffee are caffeic acid, protocatecuic acid, gallic acid, quinic acid, pyrogallol, etc., apart from CGA.
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The total amounts of polyphenol content in extracts under different MAE conditions using different solvents are presented in Table 70.3. TPP content was observed in the range of 2.6–5.5 mg/g gallic acid equivalents in aqueous extractions, where as it is in the range of 0 .95–1.5 mg/g gallic acid equivalents in alcohol extracts. TPP increased with time up to 5 min. TPP content is decreased along with increase in wattage as well as temperature. This may be due to degradation of other phenolic compounds at higher wattages as well as at higher temperatures. Also, the TPP content was found to be higher in methanol extracts compared to ethanol extracts indicating more solubility TABLE 70.3 Microwave-Assisted Extraction of Green Coffee using Different Solvents Solvent
Extraction Conditions
Caffeine (%)
Ethanol
Time (5 min) 3.05 ± 0.07 Temperature Methanol (50°C) 3.44 ± 0.07 Wattage Water 7.25 ± 0.07 (800 W)
Chlorogenic Acids (%) Yield (%) 4.95 ± 0.07
8.95 ± 0.07
5.60 ± 0.14
9.05 ± 0.07
8.40 ± 0.28
18.10 ± 0.14
Rohit et al. (2012).
of polyphenols in methanol than ethanol. The greater polyphenols content will help to increase the antioxidant potential of coffee extracts. RADICAL SCAVENGING ACTIVITY OF COFFEE EXTRACTS
RSA in coffee extract was measured in terms of ercentage inhibition of DPPH free radicals. RSA of difp ferent extracts under different MAE conditions using different solvents are presented in the Table 70.4. RSA in extracts were found to be in the range of 75–81% even at 25 ppm concentration. The RSA in water extracts was higher compared to ethanol (38%) and methanol (45%) at 25 ppm concentration. It was also found to be higher in methanol extracts compared to ethanol extracts indicating better antioxidant activity in methanol extracts. The greater polyphenols content will help to increase the antioxidant potential of coffee extracts. On the basis of results obtained from different sets of experiments conducted using MAE, it was observed that the following sets of conditions give the optimum extraction of CGA and caffeine from defatted grounded robusta cherry coffee samples. MAE conditions, to provide higher quantities of CGA and caffeine were as follows: Time: 5 min; microwave wattage: 800 W;
TABLE 70.4 TPP and Radical Scavenging Activity (RSA) of Extracts using Microwave-Assisted Extraction RSA (%) Exp set
Variable Parameter
A
FOR AQUEOUS EXTRACT
I
Time*
II
III
Wattage**
Temperature***
Variables
TPP (mg/g)
25 ppm
50 ppm
2 min
2.95 ± 0.07
77.15 ± 0.07
79.50 ± 0.14
5 min
3.45 ± 0.07
78.55 ± 0.21
82.10 ± 0.14
10 min
3.10 ± 0.14
77.80 ± 0.14
82.20 ± 0.14
400 W
3.50 ± 0.14
78.35 ± 0.21
81.55 ± 0.21
600 W
2.60 ± 0.14
78.70 ± 0.14
81.95 ± 0.07
800 W
2.20 ± 0.14
81.35 ± 0.21
83.10 ± 0
30°C
4.20 ± 0.14
78.50 ± 0
81.20 ± 0.14
50°C
3.85 ± 0.07
79.60 ± 0.14
82.35 ± 0.21
70°C
3.60 ± 0.14
77.60 ± 0.14
80.10 ± 0.40
90°C
2.85 ± 0.07
75.10 ± 0.14
78.75 ± 0.07
EXTRACT +
B
FOR SOLVENT
IV
Ethanol
0.95 ± 0.07
38.5 ± 0.14
56.4 ± 0.14
V
Methanol
1.50 ± 0.14
45.65 ± 0.07
61.7 ± 0.14
#As
gallic acid equivalents. *Temperature, 50°C; wattage, 800 W. **Temperature, 50°C; time, 5 min ***Wattage, 800 W; time, 5 min; +Temperature, 50°C; wattage, 800 W; time, 5 min. Rohit et al. (2012).
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TABLE 70.5 Comparative Analyses between Conventional Heat-Reflux Extraction and MAE %Yield Method of Extraction
Parameters
Caffeine
Chlorogenic acids
Conventional heat reflux method of extraction
Time (5 min) Temperature (50°C) Sample : solvent (1 : 4)
3.05 ± 0.21
3.95 ± 0.21
MicrowaveAssisted Extraction (MAE)
Time (5 min) Temperature (50°C) Wattage (800 W) Sample : solvent (1 : 4)
7.25 ± 0.07
8.4 ± 0.28
Rohit et al. (2012).
and Temperature: 50°C. The extraction efficiency in MAE was shown to be higher in water than in organic solvents, namely ethanol and methanol. This may due to the different dielectric constants of the respective solvents. COMPARATIVE ANALYSIS OF MAE AND CONVENTIONAL EXTRACTS
The comparative analysis of extraction efficiency between conventional heat-reflux and MAE at optimal extraction conditions is presented in Table 70.5. It is evident that MAE has shown significantly higher and better extraction yield than conventional heat reflux for both CGA and caffeine at optimal extraction conditions.
References Ali, Z., Hardiman, C., 2005. Electromagnetic radiation and human health: A review of sources and effects. High Freq. Electron. 16–26 July. Bicchi, C.P., Binello, A.E., Pellegrino, G.M., Vanni, A.C., 1995. Characterization of Green and Roasted Coffees through the Chlorogenic Acid Fraction by HPLC-UV and Principal Component Analysis. J. Agri. Food Chem. 14, 1549. Chemat, S., Amar, H.A., Lagha, A., Esveld, D.C., 2005. Microwave-assisted extraction kinetics of terpenes from caraway seeds. Chem. Eng. Proc. 44, 1320–1326. Chen, S.S., Spiro, M., 1995. Kinetics of microwave extraction of rosemary leaves in hexane, ethanol and a hexane + ethanol mixture. Flavour Fragrance J. 10, 101–112. Clifford, M.N., 1999. Chlorogenic acids and other cinnamates—nature, occurrence and dietary burden. J. Sci. Food Agri. 79, 362–372. Clifford, M.N., Wilson, K.C., 1985. Coffee—Botany, Biochemistry and Production of Bean and Beverages. Croom Helm, London. Criado, M.R., Torre, S.P., Pereiro, I.R., Torrijos, R.C., 2004. Optimization of a microwave-assisted derivatization-extraction procedure for the determination of chloro phenols in ash samples. J. Chromatogr. A 1024, 155–163. Dan, L., Zhao, G.L., Liu, J.J., 2003. Study on extraction process of chlorogenic acid from Flos lonicerae. Nat. Prod. Res. Drug Dev. 15, 124–126. Debabrata Patra, K., Ramalakshmi, L., Jagan Mohan Rao. Recent trends in coffee in Recent trends in soft beverages, 2011. Woodhead Publishing Limited, New Delhi, http://www.woodheadpublishing. com/en/book.
Farah, A., Perrone, D., Donangelo, C.M., de Paulis, T., Martin, P.R., 2008. Comprehensive analysis of major and minor chlorogenic acids and lactones in economically relevant Brazilian coffee cultivars. Food Chem. 106, 859–867. Gao, J.R., Hu, Sun, J.S., 2003. Study on extraction process of chlorogenic acid from Flos lonicerae. J. China Agri. Univ. 8, 5–8. Gao, M., Song, B.Z., Liu, C.Z., 2006. Dynamic microwave-assisted extraction of flavonoids from Saussurea medusa Maxim cultured cells. Biochem. Eng. J. 32, 79–83. George, S.E., Ramalakshmi, K., Jagan Mohan Rao, L., 2008. A perception on health benefits of coffee. Crit. Rev. Food Sci. Nutr. 48, 264–286. Giovannucci, E., 1998. Meta-analysis of coffee-consumption and risk of colorectal cancer. Am. J. Epidemiol. 147, 1043–1052. Ky, C.L., Louarn, J., Dussert, S., Guyot, B., Hamon, S., Noirot, M., 2001. Caffeine, trigonelline, chlorogenic acids and sucrose diversity in wild Coffea arabica L. and C-canephora P. accessions. Food Chem. 75, 223–230. Niggeweg, R., Michael, A.J., Martin, C., 2004. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22, 746–754. Pan, X., Niu, G., Liu, H., 2003. Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chem. Eng. Process. 42, 129–133. Ramalakshmi, K., Jagan Mohan Rao, L., Yuko Takano-Ishikawa, Masao Goto, 2009. Bioactivities of low-grade green coffee and spent coffee in different in vitro model systems. Food Chem. 115, 79–85. Ramalakshmi, K., Rahath Kubra, Jagan Mohan Rao, L., 2008. Antioxidant potential of low grade coffee beans. Food Res. Int. 41, 96–103. Ramalakshmi, K., Hithamani, G., Asha, K.R., Jagan Mohan Rao, L., 2011. Separation and characterization of chlorogenic acid rich conserves from low grade coffee beans and their radical scavenging potential. Int. J. Food Sci. Technol. 46, 109–115. Rehwald, A., Meier, M., Stiche, O., 1994. Qualitative and quantitative reversed-phase high-performance liquid chromatography of flavonoids in Passiflora incarnate L. Pharma. Acta Helvetiae 69, 153–158. Richelle, M., 2001. Effect of various antioxidants in vegetable oils. J. Agri. Food Chem. 49 3438–3344. Robyn, A.L., David, R.M., Jocelyn, A.L., Peter, W.F., 2000. Biological effects of electromagnetic fields—Mechanisms for the effects of pulsed microwave radiation on protein conformation. J. Theortical Biol. 206, 291–298. Rohit, U., Jaganmohanrao, L., 2013. An outlook on chlorogenic acids— occurrence, chemistry, technology, and biological activities. Crit. Rev. Food Sci. Nutr. DOI: 10.1080/10408398.2011.576319. Rohit, U., Ramalakshmi, K., Jaganmohanrao, L., 2012. Microwaveassisted extraction of chlorogenic acids from green coffee beans, Food Chem. 130, 184–188. Rostagno, M.A., Palma, M., Barroso, C.G., 2007. Microwave assisted extraction of soyisoflavones. Anal. Chim. Acta 588, 274–282. Susanne, N.M.H., 2012. Students’ Conceptions about ‘Radiation’: Results from an Explorative Interview Study of 9th Grade Students. J. Sci. Educ. Technol. 21, 826–834. Trugo, L.C., Macrae, R., 1984. A study of the effect of roasting on the chlorogenic acid composition of coffee using HPLC. Food Chem. 15, 219–227. Varnam, A.H., Sutherland, J.P., 1994. Beverages: Technology, chemistry and microbiology. Boundary Row, London: Chapman and Hall. pp. 191–255. Zhang, W.B., Xu, S.Y., 2007. Microwave-assisted extraction of secoisolariciresinol diglucoside from flaxseed hull. J. Sci. Food Agri. 87, 1455–1462. Zhou, H.Y., Liu, C.Z., 2006. Rapid determination of solanesol in tobacco by high performance liquid chromatography with evaporative light scattering detection following microwave-assisted extraction. J. Chromatogr. B 835, 119–122.
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Use of Microwaves to Extract Chlorogenic Acids from Green Coffee Beans Lingamallu Jagan Mohan Rao, Kulathooran Ramalakshmi Plantation Products, Spices and Flavour Technology Department, Central Food Technological Research Institute, Mysore, India
• T he yields of MAE under optimum conditions were higher than those from the conventional solvent extraction at 5 min and 50°C and the extracts showed radical-scavenging activity of > 75%, even at a concentration of 25 ppm. • The MAE process can thus be predicted and controlled for industrial application.
CHAPTER POINTS • M icrowave-assisted extraction (MAE) showed obvious advantages in terms of short duration and high efficiency in the recovery of chlorogenic acid from raw plant materials in comparison with conventional heat-reflux extraction. • The mechanism of the enhanced extraction by microwave assistance was studied by observing cell destruction of plant material after MAE treatment by scanning electron microscopy. The enhanced extraction was related partly to a greater extent of cell rupture of the plant materials, and this was observed by scanning electron microscopy. The plant materials were significantly destroyed due to the cell rupture during MAE treatment. • In general, extraction is the first step in the preparation of pharmaceutical formulations from raw plant material and significantly affects the cost of the whole manufacture process. • MAE has been considered as a potential alternative to conventional solvent extraction for the isolation of phenolic compounds from plants. • Under optimum conditions of time (5 min), temperature (50°C), and wattage (800 W), the maximum quantities of chlorogenic acids and caffeine could be extracted with water as solvent. • The extracts contained chlorogenic acids and caffeine in the ranges of 31–62% and 22–40%, respectively.
Processing and Impact on Active Components in Food http://dx.doi.org/10.1016/B978-0-12-404699-3.00070-6
INTRODUCTION Electromagnetic Radiation Electromagnetic radiation is a form of energy that is transmitted through a space at an enormous velocity. It is said to have a dual nature, exhibiting both wave and particle characteristics. As the name implies, electromagnetic radiation is an alternating electrical and associated magnetic force field in space. Thus, an electromagnetic wave has an electric component and a magnetic component. Both the components oscillate in planes perpendicular to the direction of the propagation of the radiation. The entire range over which electromagnetic radiation exists is known as electromagnetic spectrum. The electromagnetic spectrum consists of gamma, X-ray, ultraviolet, visible, infrared, microwave, radiofrequency, and ultrasound radiations (Ali and Hardiman, 2005; Susanne, 2012). Infrared Radiation Infrared radiation (IR) is a part of electromagnetic spectrum with a wavelength in the range of 0.78 μm to 1000 μm. Application of infrared heating has recently been employed in certain food-processing
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applications, because of its superiority in terms of costs and the product quality (color, organoleptic and nutritional value) as compared with conventional heating. Infrared heating offers many advantages over conventional heating under similar processing temperatures. Both the situations involve simultaneous heat and mass transfer, and in the case of conventional heating both are in opposite directions. These are in the same (from within to outside) direction in case of IR, which results in a higher rate of heat transfer to the material (Robyn et al., 2000). Microwave Radiation Microwave radiation (MW; wavelength in the range of 1 mm to 1 m) lies between IR and radiofrequency (RF) and is known as dielectric radiation. Material exposed to MW is heated due to dipolar rotation and ionic polarization. Hence, material containing a polar substance such as water is heated faster with MW. The typical domestic MW oven has a frequency of 2450 MHz. The applications of MW in food processing are blanching, drying, pasteurization, baking, etc. Compared to standard microwave technology the need to use higher frequencies such as 24,150 MHz for industrial applications has to be carefully verified with respect to special physical/ engineering advantages. MICROWAVE-ASSISTED EXTRACTION
MAE is a process that uses microwave energy along with solvent to extract target compounds from various matrices. The volumetric heating, localized temperature, and pressure can cause selective migration of t arget compounds from the material to the surroundings at a more rapid rate and with similar or better recoveries compared with conventional extractions. MAE was used for extraction of selected components from a wide variety of sample matrices and has been used as a promising alternative sample preparation technique for a number of applications (Zhou and Liu, 2006; Gao et al., 2006; Zhang and Xu, 2007; Rostagno et al., 2007). MAE can considerably reduce both extraction time and solvent consumption compared to conventional methods. Radiofrequency Waves RF waves are also being used for thermal processing food materials. In a radio frequency heating system, the RF generator creates an alternating electric field between two electrodes. The material is placed between the electrodes where the alternating energy causes polar molecules in the material to continuously reorient them to face opposite poles in much the same way that bar magnets behave in an alternating magnetic field. The friction resulting from molecular movement causes the material to rapidly heat throughout its entire mass. For those applications that require uniform
heating and precise temperature control, RF offers many advantages over conventional drying methods, namely uniform heating through the entire thickness, 2- to 20-times faster than conventional drying methods, energy efficient, uniform moisture profiling, low maintenance, and eco-friendly process. The permitted frequencies for industrial applications are 13, 27, and 40 MHz. A few reports on the existence of a non-thermal effect of RF energy on microorganisms are available. Freshness and nutrients are retained in foods when RF energy inactivates microorganisms non-thermally (Ali and Hardiman, 2005). Generally foodstuffs are complex mixtures of different large biochemical molecules (simple sugars, amino acids, vitamins etc), biochemical polymers (complex sugars, proteins, lipids etc.), inorganic salts and water. The strongest absorption is often localizable within the so-called group frequency, which is generated by the vibrations of these molecular aggregates (or molecular structural groups). The wavelength region of interest in discussing the interaction with foodstuffs can be restricted to the infrared range 0.78–15 μm even for high-temperature radiators, the spectral flux, [(W/m2)/μm] ascribed to wavelengths larger than 15 μm represents a small fraction of total radiation power.
HOW THE COMPOSITION ALTERED Chlorogenic Acids Chlorogenic acid (5-O-caffeoyl-quinic acid), an ester of caffeic acid with quinic acid, has received considerable attention for its wide distribution and potential biological effects (Clifford, 1999). It is one of the most abundant polyphenols in the human diet with coffee, fruits, and vegetables as its major sources. It is also an important bioactive compound and rich in selected raw materials used in traditional Chinese medicine, such as flowers and buds of Lonicera japonica Thumb, and the leaves of Eucommia ulmodies, both which have been used for the treatment of exopathogenic wind-heat or epidemic febrile disease at the early stage, carbuncles, furuncles, and swellings for centuries. The chlorogenic acids (CGA) are an important group of compounds in green coffee beans. Although 71 different species of CGA have now been identified (Rohit and Jaganmohanrao, 2013) the vast majority of the compounds belong to three classes: caffeoylquinic acids (CQA; 3CQA, 4CQA, and 5CQA), di-caffeoylquinic acids (diCQA; 3,4diCQA, 3,5diCQA, and 4,5diCQA) and feruloylquinic acids (FQA) as shown in Figure 70.1. Other quinic acids are CiQA, cinnamoyl quinic acids; GQA, galloyl quinic acids; iFQA, isoferuloyl quinic acids; oCoQA, o-coumaroyl quinic acids; SQA, sinapoyl quinic
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FIGURE 70.1 Chlorogenic acid isomers.
acids, which are known either naturally or synthetically. Although CGA (primarily as 5-caffeoylquinic acid) are widely distributed in plant materials, their content in green coffee is among the highest found in plants, ranging from 4 to 14% (Trugo and Macrae, 1984; Farah et al., 2008). The levels of CGA in green coffee beans have been reported to vary from approximately 7.88 to 14.4% dry matter for Coffea canephora (robusta) and approximately 3.4 to 4.8% for Coffea arabica (Ky et al., 2001). The analytical methods for CGA estimation include chromatographic and spectroscopic techniques. Chromatographic techniques reported are thin-layer chromatography, gas chromatography, and liquid chromatography (Bicchi et al., 1995; Rehwald et al., 1994). Spectral techniques include spectrophotometry, IR spectrometry, 1H NMR spectroscopy and chemiluminescence. Microwave Extraction In general, CGA are extracted from dried and powdered plant materials using solvent/aqueous extraction.
However, this technique is very time consuming as well as requiring high energy. This drawback prevents the application of the reported method. Development of a new technique for separation and purification of CGA is essential. Growing advancements in the isolation/separation techniques in recent years have made the purification and quantification of CGA easy, sensitive, and specific, with marked improvements in yield in much less time compared to traditional methods. Generally, absorption of the microwave energy increases with the dielectric constant of the molecule, resulting in power dissipation inside the solvent and plant materials and then generating more effective molecular movement and heating. As a polar solvent, water can efficiently absorb microwave energy and leads to efficient heating. MAE was used for extraction of interested components from a wide variety of sample matrices and has been used as a promising alternative sample preparation technique for a number of applications. Compared to conventional methods, MAE can considerably reduce both extraction time and solvent consumption.
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Chlorogenic Acids from Flower Buds of Lonicera japonica Thunb An efficient MAE technique has been developed to recover CGA from flower buds of Lonicera japonica, using a mixture of water and ethanol (Gao et al., 2003; Dan et al., 2003). Ethanol (50%) as MAE solvent gave the best yield of CGA in 5 min at a temperature of 60°C. For MAE, the choice of extraction solvent takes into account not only its solubility for target component but also its ability to absorb microwave energy. The yield of CGA increased with the increase of material : solvent ratio (amount of plant material : volume of extraction solvent) up to 1 : 10 (w/v) and further increase has no effect on the yield (Criado et al., 2004). The yield of CGA increased with time up to 30 min at 40°C whereas the same yield of CGA is obtained within 5 min at 60°C under microwave extraction and further increase in temperature has no effect on the yield. The yield of CGA reached 6.14% at 60°C within 5 min, which was significantly higher than that at 40°C. There was no obvious difference on yield of CGA between 60°C and 80°C; therefore, it was reported that the MAE temperature of 60°C was suitable for CGA extraction from flower buds. A conventional heat-reflux extraction of CGA from raw plant materials was carried out at 60°C. The yield of CGA reached 5.19% at an optimal ethanol concentration of 50% as extraction solvent within 5 min; further increase in the yield is slow and reached a maximum of 6% within 30 min. In comparison to the heat-flux extraction, the MAE showed obvious advantages in terms of short duration and high efficiency to extract CGA. This is mainly due to the fact that microwave energy is delivered efficiently to materials through molecular interaction with the electromagnetic field and offers a rapid transfer of energy to the extraction solvent and raw plant materials. Control and extracted samples of raw materials were subjected to Scanning Electron Microscopy. The changes in the raw material during heat-reflux extraction were not considerably different from the untreated sample, and only few slight ruptures happened on its surface. The surface of the MAE sample was greatly destroyed because that microwave irradiation accelerated cell rupture by sudden temperature rise and internal pressure increase inside the cells of plant sample. During the rupture process, a rapid exudation of the chemical substance within the cells into the surrounding solvents took place. Chlorogenic Acids from Coffee Coffee, an aromatic, non-alcoholic brew is known for its stimulating and refreshing taste. Two species are of significant economic importance namely, Arabica (Coffea arabica) and Robusta (Coffea canephora) (Varnam and Sutherland, 1994). In recent years, due to the increasing
interest in finding physiologically functional foodstuffs, the relationship between coffee and health has been extensively investigated (George et al., 2008). Coffee is reported to exhibit a number of bioactivities, such as antioxidant (Ramalakshmi et al., 2008, 2009, 2011), anticarcinogenic, and antimutagenic activity (Giovannucci, 1998). Coffee is known to show a protective effect on cancer and other cardiovascular diseases due to its antioxidant activity. It also protects low-density-lipoproteins from oxidation. This protective effect is the result of the action of several polyphenolic constituents. The physical and chemical properties of individual phenolics, strongly affect their antioxidant activities. In addition, these molecules can have a synergistic or antagonistic effect when present in complex mixtures. Indeed, the polyphenol composition of the beverage varies in species of coffee and tea (Richelle, 2001). In the case of coffee, robusta exhibits a higher antioxidant activity than arabica, which could be due to the higher amount of CGA. Following light roasting, the antioxidant activities of both coffees decreased markedly but further roasting produced the heterocyclic compounds having the antioxidant activity, and as these polyphenolic rich beverages are often consumed with milk, the effect of milk on different beverages (coffee, cocoa, black tea) were evaluated and coffee was found to have high antioxidant activity. An understanding of the protective role of dietary antioxidants in vivo requires a better characterization of the polyphenol composition of the antioxidant matrix as well as quantitative data on their absorption, their tissue distribution, their metabolism, and their biological actions. Indeed, after consumption, polyphenols have to cross the intestinal wall but must also resist further catabolism. The metabolism of polyphenols involves two important organs—the liver, where biotransformation enzymes convert them or their metabolites into conjugated forms such as glucuronides or sulphates, and the colon where microorganisms degrade unabsorbed ones. At present, only a little information is available on the absorption of the vast diversity of polyphenols present in these beverages (Richelle, 2001). So the antioxidant activity of coffee has to be considered while estimating the daily ingested dose of polyphenols. Thus, the beneficial effects of coffee may be attributed in part to polyphenols and caffeine serving as antioxidants. These positive properties of coffee on human health are attributed to the presence of phenolic compounds such as CGA with strong antioxidant and radical-scavenging activities present in green coffee. Earlier reports (Clifford and Wilson, 1985; Debabrata et al., 2011) suggest that the content of CGA in C. canephora varies from 7 to 10% (mfb), whereas C. arabica varies from 5 to 7.5% (mfb). In addition to having different total CGA levels, the relative amounts of CQA, diCQA, and FQA have also been shown to vary in mature C. canephora and C. arabica grain. For example Ky et al. (2001) found
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that CQA, diCQA and FQA represent roughly 67, 20, and 13%, respectively, of the total CGA content of C. canephora variety versus 80, 15, and 5%, respectively, of the total CGA content of C. arabica variety. In addition to being found in coffee, these compounds are also found at significant levels in plant foods such as apples, pears, tomato, potato, and eggplant (Niggeweg et al., 2004). Total CGA content of green and roasted beans of different varieties is provided in Table 70.1. Extraction of Chlorogenic Acids using Microwaves MAE showed obvious advantages in terms of short duration and high efficiency to recover CGA from raw plant materials in comparison with conventional heatreflux extraction. The mechanism of the enhanced extraction by microwave assistance was studied by observing cell destruction of plant material after MAE treatment by scanning electron microscopy. The enhanced extraction was related partly to a greater extent of cell rupture of the plant materials, and this was observed by TABLE 70.1 Chlorogenic Acid Content in Green and Roasted Coffee Varieties Coffee Variety
Bean Variety
Chlorogenic Acid (mg/g)
Coffea arabica
Dry green coffee beans
68–70
Medium roasted
26–30
Dark roasted
22–25
Very dark roasted
7–9
Coffea canephora Dry green coffee beans
85–88
scanning electron microscopy. The plant materials were significantly destroyed due to the cell rupture during MAE treatment. In this context, conditions for microwave-assisted extraction of green coffee beans were optimized and the quality of the extracts was monitored through determination of the contents of CGA, caffeine, and total polyphenols as well as evaluating the radical scavenging activity. A comparative analysis between MAE and conventional heat reflux methods was also made. EXTRACTION METHODOLOGY
Robusta cherry green coffee beans (Figure 70.2) were ground and sieved (< 720 μm) using Hammer mill and then defatted. Defatted coffee powder was extracted with different solvents such as water, methanol, and ethanol using Microwave lab station [(Model: STARTS configuration with control terminal 260, Milestone, Italy); built-in focused IR sensor and magnetic stirrer; Magnetron: SN: 133613; Frequency 50 Hz] in a closed system under different sets of conditions of temperature and wattage for specific periods of time. The slurry obtained was filtered to get a clear extract that was used for quantitative analysis. The quantitative analyses of extracts for percentage of yield, caffeine, CGA, and total polyphenols (TPP) contents, and radical scavenging activity (RSA) of MAE-treated defatted ground robusta cherry coffee were presented. The caffeine, CGA, RSA, and TPP content were analyzed spectrophotometrically at λmax275, 325, 517, and 760 nm, respectively. The percentage yield of analyte under different conditions/ parameters of MAE, i.e., time, microwave wattage, and temperature, are presented in the Table 70.2.
Medium roasted
35–37
Dark roasted
20–22
YIELD OF MAE EXTRACTS
Very dark roasted
6–7
The yields are in the range of 15–20% with water as solvent of extraction and found to be higher than alcohol FIGURE 70.2 Robusta coffee berries (Coffea robusta).
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TABLE 70.2 Microwave-Assisted Extraction of Green Coffee using Aqueous Medium Variable Parameter
Constant Parameter
Variables
Caffeine (%)
Chlorogenic acids (%)
Yield (%)
Time
Temperature (50°C) Wattage (800 W)
2 min
3.75 ± 0.07
6.05 ± 0.07
17.15 ± 0.07
5 min
4.60 ± 0.14
6.75 ± 0.07
17.50 ± 0.14
10 min
4.40 ± 0.14
6.35 ± 0.07
17.05 ± 0.07
400 W
3.70 ± 0.14
4.70 ± 0.14
15.40 ± 0.14
600 W
4.25 ± 0.07
5.80 ± 0.14
17.75 ± 0.07
800 W
7.25 ± 0.07
8.40 ± 0.28
18.10 ± 0.14
30°C
6.68 ± 0.07
6.65 ± 0.07
17.60 ± 0.07
50°C
7.25 ± 0.07
8.10 ± 0.04
18.05 ± 0.07
70°C
4.75 ± 0.07
6.75 ± 0.07
17.60 ± 0.14
90°C
4.65 ± 0.07
6.35 ± 0.07
16.75 ± 0.07
Wattage
Temperature
Time (5 min) Temperature (50°C)
Time (5 min) Wattage (800 W)
Rohit et al. (2012).
extracts (8.5–9.5%), indicating better extraction efficiency in water than in organic solvents. The reason could be that the dielectric constant of water is higher than hat of the alcohol, which helps in absorption of microwaves. It was found that the yield was highest (18%) with water as solvent for extraction under MAE conditions of 5 min, 800 W, and 50°C compared to other conditions. Chen and Spiro (1995) reported that the yield of extracts from rosemary leaves improved with an increase in microwave power and the induction stage appeared. However, Chemat et al. (2005) confirmed that there is not much difference in the extraction yield when the microwave power was increased from 50 to 150 W during the extraction of caraway seeds. The difference may be due to the nature of the plant material and the components to be extracted. CHLOROGENIC ACIDS IN EXTRACT
The percentage yields of CGA in the aqueous extracts obtained at different MAE parameters/conditions are presented in Table 70.2. The yield of CGA is in the range of 4.7–8.1% in aqueous extractions, whereas it is in the range of 4.9–5.6% in alcohols. The yield of CGA increases up to 5 min. The yield of CGA increases with increased wattage up to 800 W; experiments could not be carried out beyond 800 W, due to limitations with the equipment. Extraction yield of CGA is increased along with temperature up to 50°C, and decreased at higher temperatures, which could be due to degradation at higher temperatures. The highest yield of CGA (8.4 ± 0.28%) was obtained at MAE conditions of 5 min, 800 W and 50°C and water as solvent of extraction. CAFFEINE IN EXTRACT
The caffeine content in the aqueous extracts obtained at MAE times of 2, 5, and 10 min were 3.75 ± 0.07%, 4.6 ± 0.14%,
and 4.4 ± 0.14%, respectively. The yield of caffeine did not increase after 5 min. With MAE wattage of 400, 600, and 800 W, the yields in aqueous extracts were 3.7 ± 0.14%, 4.25 ± 0.07%, and 7.25 ± 0.07%, respectively. This indicates that the extraction of caffeine increases with increase in wattage. Experiments could not be carried out beyond 800 W, due to limitations with the equipment. At MAE temperature of 30, 50, 70, and 90°C, the yields obtained were 6.35 ± 0.07%, 7.25 ± 0.07%, 4.75 ± 0.07, and 4.65 ± 0.07%, respectively. Extraction yield of caffeine is increased along with temperature up to 50°C, and decreased at higher temperatures; this may be due to the degradation of caffeine at higher temperatures. When ethanol and methanol were used as extraction solvents, the yield obtained in the extracts were 3.05 ± 0.07% and 3.75 ± 0.07% respectively at MAE conditions of 5 min, 800 W and 50°C and lower than water as solvent. The highest yield (7.25%) of caffeine obtained MAE conditions of 5 min, 800 W, and 50°C in aqueous extraction. The comparative analysis of percentage extraction yields of caffeine at different MAE parameters is shown in Table 70.2. Pan et al. (2003) conducted experiments of MAE for polyphenols and caffeine at the microwave power of 700 W from green tea leaves. It is reported that MAE for 4 min provided maximum extraction of caffeine and polyphenols from tea leaves. TPP IN EXTRACT
The TPP content in coffee extract is measured in terms of gallic acid equivalents. During the estimation, Folin–Ciocalteu Reagent (FCR) was added to the extract. The FCR reacts with polyphenol compounds in the alkaline medium (sodium carbonate) and forms complexes. The various polyphenols that are present in the coffee are caffeic acid, protocatecuic acid, gallic acid, quinic acid, pyrogallol, etc., apart from CGA.
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The total amounts of polyphenol content in extracts under different MAE conditions using different solvents are presented in Table 70.3. TPP content was observed in the range of 2.6–5.5 mg/g gallic acid equivalents in aqueous extractions, where as it is in the range of 0 .95–1.5 mg/g gallic acid equivalents in alcohol extracts. TPP increased with time up to 5 min. TPP content is decreased along with increase in wattage as well as temperature. This may be due to degradation of other phenolic compounds at higher wattages as well as at higher temperatures. Also, the TPP content was found to be higher in methanol extracts compared to ethanol extracts indicating more solubility TABLE 70.3 Microwave-Assisted Extraction of Green Coffee using Different Solvents Solvent
Extraction Conditions
Caffeine (%)
Ethanol
Time (5 min) 3.05 ± 0.07 Temperature Methanol (50°C) 3.44 ± 0.07 Wattage Water 7.25 ± 0.07 (800 W)
Chlorogenic Acids (%) Yield (%) 4.95 ± 0.07
8.95 ± 0.07
5.60 ± 0.14
9.05 ± 0.07
8.40 ± 0.28
18.10 ± 0.14
Rohit et al. (2012).
of polyphenols in methanol than ethanol. The greater polyphenols content will help to increase the antioxidant potential of coffee extracts. RADICAL SCAVENGING ACTIVITY OF COFFEE EXTRACTS
RSA in coffee extract was measured in terms of ercentage inhibition of DPPH free radicals. RSA of difp ferent extracts under different MAE conditions using different solvents are presented in the Table 70.4. RSA in extracts were found to be in the range of 75–81% even at 25 ppm concentration. The RSA in water extracts was higher compared to ethanol (38%) and methanol (45%) at 25 ppm concentration. It was also found to be higher in methanol extracts compared to ethanol extracts indicating better antioxidant activity in methanol extracts. The greater polyphenols content will help to increase the antioxidant potential of coffee extracts. On the basis of results obtained from different sets of experiments conducted using MAE, it was observed that the following sets of conditions give the optimum extraction of CGA and caffeine from defatted grounded robusta cherry coffee samples. MAE conditions, to provide higher quantities of CGA and caffeine were as follows: Time: 5 min; microwave wattage: 800 W;
TABLE 70.4 TPP and Radical Scavenging Activity (RSA) of Extracts using Microwave-Assisted Extraction RSA (%) Exp set
Variable Parameter
A
FOR AQUEOUS EXTRACT
I
Time*
II
III
Wattage**
Temperature***
Variables
TPP (mg/g)
25 ppm
50 ppm
2 min
2.95 ± 0.07
77.15 ± 0.07
79.50 ± 0.14
5 min
3.45 ± 0.07
78.55 ± 0.21
82.10 ± 0.14
10 min
3.10 ± 0.14
77.80 ± 0.14
82.20 ± 0.14
400 W
3.50 ± 0.14
78.35 ± 0.21
81.55 ± 0.21
600 W
2.60 ± 0.14
78.70 ± 0.14
81.95 ± 0.07
800 W
2.20 ± 0.14
81.35 ± 0.21
83.10 ± 0
30°C
4.20 ± 0.14
78.50 ± 0
81.20 ± 0.14
50°C
3.85 ± 0.07
79.60 ± 0.14
82.35 ± 0.21
70°C
3.60 ± 0.14
77.60 ± 0.14
80.10 ± 0.40
90°C
2.85 ± 0.07
75.10 ± 0.14
78.75 ± 0.07
EXTRACT +
B
FOR SOLVENT
IV
Ethanol
0.95 ± 0.07
38.5 ± 0.14
56.4 ± 0.14
V
Methanol
1.50 ± 0.14
45.65 ± 0.07
61.7 ± 0.14
#As
gallic acid equivalents. *Temperature, 50°C; wattage, 800 W. **Temperature, 50°C; time, 5 min ***Wattage, 800 W; time, 5 min; +Temperature, 50°C; wattage, 800 W; time, 5 min. Rohit et al. (2012).
8. BEVERAGES
590
70. MICROWAVES TO EXTRACT CHLOROGENIC ACIDS FROM COFFEE
TABLE 70.5 Comparative Analyses between Conventional Heat-Reflux Extraction and MAE %Yield Method of Extraction
Parameters
Caffeine
Chlorogenic acids
Conventional heat reflux method of extraction
Time (5 min) Temperature (50°C) Sample : solvent (1 : 4)
3.05 ± 0.21
3.95 ± 0.21
MicrowaveAssisted Extraction (MAE)
Time (5 min) Temperature (50°C) Wattage (800 W) Sample : solvent (1 : 4)
7.25 ± 0.07
8.4 ± 0.28
Rohit et al. (2012).
and Temperature: 50°C. The extraction efficiency in MAE was shown to be higher in water than in organic solvents, namely ethanol and methanol. This may due to the different dielectric constants of the respective solvents. COMPARATIVE ANALYSIS OF MAE AND CONVENTIONAL EXTRACTS
The comparative analysis of extraction efficiency between conventional heat-reflux and MAE at optimal extraction conditions is presented in Table 70.5. It is evident that MAE has shown significantly higher and better extraction yield than conventional heat reflux for both CGA and caffeine at optimal extraction conditions.
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