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Process integration for purification and concentration of red cabbage (Brassica oleracea L.) anthocyanins
Separation and Purification Technology 141 (2015) 10–16
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Process integration for purification and concentration of red cabbage (Brassica oleracea L.) anthocyanins Chandrasekhar Jampani, K.S.M.S. Raghavarao ⇑ Department of Food Engineering, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 25 March 2014 Received in revised form 8 November 2014 Accepted 15 November 2014 Available online 29 November 2014 Keywords: Anthocyanins Process integration Aqueous two phase extraction Osmotic membrane distillation Forward osmosis
a b s t r a c t Multistage aqueous two phase extraction was carried out using polyethylene glycol (PEG) 4000/magnesium sulfate (14.8/10.3%, w/w) system. The phase forming polymer (PEG 4000) was successfully separated from anthocyanins employing organic–aqueous extraction. Different processes employed for the purification and concentration of anthocyanins were compared with one another. The highest concentration of anthocyanins (3123.45 mg/L and 43 °Brix) was obtained in case of integration of aqueous two phase extraction with forward osmosis. Non-enzymatic browning index (0.11) and degradation constant (0.21) were found to be lowest in case of the integrated process involving aqueous two phase extraction followed by forward osmosis when compared to other processes. Color density was found to increase from 0.6 to 14.56 and stability of anthocyanins (with respect to pH and temperature) was found to be more in case of integrated process (aqueous two phase extraction followed by forward osmosis). Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Color is an imperative component of food or any edible stuff as it can enhance the appearance [1]. Food colorants are being used in beverages, desserts, jams, jellies, sauces, cosmetics, toothpaste, etc. In addition medicines including tablets, capsules and syrups are colored with food colorants. Numerous studies have demonstrated the effect of artificial colorants in food, which include the inhibition of the immune system and allergic reactions [2]. In addition, the use of non-permitted colors or overindulgence of permitted synthetic colors may also cause thyroid tumors, asthma, abdominal pain, nausea, liver and kidney damage and cancer. In view of this, interest in natural food colorants has increased considerably, mainly because of the apparent lack of toxicity and ecofriendliness [3]. Natural colors such as betalains from beet root, carotenoids from carrots and anthocyanins from grapes, and red cabbage. are some of the examples. Anthocyanins are glycosylated polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium (flavylium) salts accounting for the colors in several fruits, vegetables and flowers [4]. The hydrophilic nature of anthocyanins facilitates their incorporation in food systems. A number of properties have been reported such as antioxidant [5], antiulcer [6], anticancer, antitumor and anti-mutagenic [7], antidiabetes [8,9], antibacterial activity [10], ocular[11] and prevention of cardiovas⇑ Corresponding author. Tel.: +91 821 2513910; fax: +91 821 2517233. E-mail address: [email protected] (K.S.M.S. Raghavarao). http://dx.doi.org/10.1016/j.seppur.2014.11.024 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.
cular diseases [12]. Several factors such as concentration of free sugars, oxygen, pH [13], temperature, light [14] and concentration of colorant itself [15] influence the stability of anthocyanins. Removal of free sugars from the extract of anthocyanins is very much desirable otherwise leads to Maillard reaction [16,17]. Several chromatographic techniques are used for the purification of anthocyanins [18–22]. However, in case of methods such as column chromatography and electrophoresis scale up becomes uneconomical unless the product is of high value. Aqueous two-phase extraction (ATPE), a liquid–liquid extraction (LLE) strategy, has been recognized as a potential technique because of its multiple advantages including: biological compatibility, low interfacial tension, high capacity, scale up easiness and scope for continuous operation [23–25]. ATPE has been successfully employed for the preliminary purification of anthocyanins [26–28]. The increase in concentration of anthocyanins improves their stability through self-association [3]. Concentration of natural color extracts by conventional method such as evaporation results in low quality product due to loss of hue and chroma [29,30]. Membrane processes such as microfiltration, ultrafiltration and reverse osmosis were being employed for clarification and concentration of natural color extracts [31–33]. There are many limitations of these membrane processes such as requirement of high pressure, limit to maximum attainable concentration and concentration polarization [29,30]. On the other hand, the non-pressure driven membrane processes such as osmotic membrane
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11
Fig. 1. Overall flow chart for the separation and concentration of anthocyanins using different processes.
distillation (OMD) and forward osmosis (FO) are the promising technologies (as they are osmotic gradient driven) for the concentration of thermally sensitive liquid foods [29,30,34–37]. These athermal membrane processes, operated at ambient temperature, practically eliminate the thermal damage to the product thereby retaining the color. Objective of the present work is to integrate ATPE with membrane processes such as OMD and FO for concentration of red cabbage anthocyanins. Further, comparison of these integrated processes with an integrated membrane process (involving ultrafiltration followed by FO) and conventional method (thermal evaporation).
a very thin semi-permeable non-porous active skin layer of cellulose triacetate and porous support layer. The membrane was embedded in a nylon mesh for an increased strength. The active layer effectively excludes the passage of solutes with a molecular weight cut-off of 100 Da. 2.3. Methods The overall process flow diagram for purification and concentration of anthocyanins using different processes is shown in Fig. 1.
2.2. Membranes
2.3.1. Extraction of anthocyanins Red cabbage (Brassica oleracea L.) was procured from local supermarket. Leaves were cut into small pieces and extraction was carried out by maintaining the ratio of solid (cut leaves) to extraction media (eco-solvent–water) at 1:2 (w/v) while providing thorough contact in a mixing unit (Singer FP-450, India). The extract of anthocyanins obtained was filtered (using a muslin cloth) to remove the coarse particles. The filtrate obtained was centrifuged (Eltek-TC 4100D, Elektrocrafts, India) at 8000 rpm for 15 min to remove the fine suspended particles. Ascorbic acid (0.1%, w/v) was added to arrest the activity of polyphenol oxidase which otherwise causes enzymatic browning. The crude extract of anthocyanins thus obtained was stored at 4–6 °C and required quantities were taken for different experiments.
Hydrophobic polypropylene (PP) membranes (Accurel, Enka, Germany) of pore size 0.2 and 0.05 lm were used for osmotic membrane distillation. Hydrophilic membrane (Osmotek, Inc., Corvallis, OR, USA) used for forward osmosis is asymmetric having
2.3.2. Aqueous two phase extraction PEG 4000 (14.8% w/w) and magnesium sulfate (10.3% w/w) were added to the crude extract of anthocyanins (74.9% w/w) making the total weight of the system 100% on w/w basis. The contents
2. Materials and methods 2.1. Chemicals Calcium chloride dihydrate (CaCl22H2O) and sodium chloride (NaCl) were obtained from Ranbaxy chemicals, Mumbai, India. Polyethylene glycol 4000 Da was procured from Sisco research laboratories, Mumbai, India. Double distilled water was used for all the experiments. All the experiments were carried out in triplicate and average values are reported. All the other chemicals used were of analytical grade.
2.3.3. Osmotic membrane distillation and Forward osmosis A polyester mesh (0.25 mm, on osmotic agent side), membrane (area – 0.012 m2) and a viton gasket (3.0 mm, on the feed side) were supported between two stainless steel (SS 316) frames of a flat module. Feed (anthocyanins solution) and osmotic solutions were circulated on either side of the membrane in co-current mode using peristaltic pumps (Model 72-315-230, Barnant Company, USA). The ratio of feed to osmotic solution was maintained as 1:3 for all the experiments. The transmembrane flux was calculated by measuring the increase in weight of the osmotic solution at regular intervals of 1 h. Calcium chloride and sodium chloride solutions were used as osmotic solutions in OMD and FO, respectively. All the experiments were carried out at room temperature (27 ± 1 °C). 2.3.4. Thermal evaporation Crude extract of anthocyanins was concentrated by conventional process of thermal evaporation in agitated falling film vacuum evaporator (Model No. 04-012, Chemetron corporation, USA) and used as control. 2.3.5. Integrated membrane process Ultrafiltration (Serial No. P5PN8322003, Millipore, USA) was employed for clarification of the crude extract of anthocyanins using a membrane cartridge (PLCTK 30, Millipore, USA) of 30 kDa cut-off. The permeate obtained in this process was subjected to forward osmosis in order to concentrate the anthocyanins. 2.3.6. Freeze drying The concentrated solutions of anthocyanins obtained after different integrated processes (shown in Fig. 1) were subjected to freeze drying (Lyodryer LT5B, Lyophilisation systems INC., USA) to obtain the pigment in powder form. 2.3.7. Stability studies In order to examine the effect of pH on the stability of anthocyanins, buffer solutions of different pH (1, 3 and 5) were prepared. Pigment, obtained after freeze drying, was dissolved (10 mg) in these buffer solutions (5 mL) in separate volumetric flasks and incubated at room temperature (27 ± 1 °C) for 7 days. The total amount of anthocyanins in each solution was determined at regular intervals of 24 h using pH differential method [38]. In order to study the effect of temperature on the stability of anthocyanins, diluted samples were incubated in a shaking water bath (BANI 115, Jayadeep Engineers, India) for 5 h at 30, 40, 60, 80 and 100 °C. Then the absorbance was determined at 530 nm. The anthocyanins (ACN, % w/w) remained after time ‘t’ was calculated using the following equation.
ACN ð%Þ ¼
Ct 100 C0
ð1Þ
where C0 is the initial amount of anthocyanins (mg) and Ct is the amount of anthocyanins (mg) at time t (day in case of pH stability studies and hr in case of temperature stability studies).
30
2.5
25
2
20
1.5
15
1
10
0.5
Brix (0)
3
5
0
0 0
2
4
6
8
10
12
14
Time (hr) Fig. 2. Variation of transmembrane flux and total soluble solids during osmotic membrane distillation.
2.4. Analytical procedures 2.4.1. Concentration of anthocyanins Anthocyanin concentration of the samples was determined according to the pH differential method [38]. This method is based on the anthocyanins structural transformation (reversible) that occurs with a change in pH. Aliquots of the anthocyanin solution were diluted with buffers of pH 1.0 (0.025 M potassium chloride) and 4.5 (0.4 M sodium acetate), respectively. These samples were thoroughly vortexed (Spinix MC-01, Tarsons products Pvt. Ltd, India) and then placed in the dark for 30 min to equilibrate. The absorbance of each solution was measured using a spectrophotometer (UV-1700, Shimadzu, Japan) at a wavelength of maximum absorption (kvis-max – 530 nm) and at haze detection absorption (700 nm) against a blank (distilled water). Concentration of anthocyanins was calculated using the following equation [39]
Anthocyanins concentration ðmg=LÞ ¼
A MW DF 1000 eL
ð2Þ
where A = [(A530 A700)pH1.0 – (A530 - A700)pH4.5]; MW, the molecular weight of anthocyanins (cyanidin-3-glycoside – 449.2 g/mol); DF, the dilution factor; e, the extinction coefficient (26,900 L/cm mol) and L is the path length (1 cm).
10
50
9
45
8
40
7
35
6
30
5
25
4
20
3
15
2
10
1
5
0
Brix ( 0 )
were mixed thoroughly using a magnetic stirrer for an hour to equilibrate, and the mixture was allowed for phase separation. After clear separation, the top and bottom phases were collected, volumes noted and were subjected to analysis of anthocyanins as well as sugars. Multistage ATPE was carried out in order to remove maximum possible sugars from PEG-rich (top) phase followed by organic–aqueous extraction employing chloroform for the removal of the phase forming polymer (PEG) from this phase.
Transmembrane flux (L/(m2h)
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Transmembrane flux (L/(m2h)
12
0 0
2
4
6
8
10
12
14
Time (hr) Fig. 3. Variation of transmembrane flux and total soluble solids during forward osmosis (after PEG removal).
C. Jampani, K.S.M.S. Raghavarao / Separation and Purification Technology 141 (2015) 10–16
2.4.2. Indices for pigment degradation, polymeric color and browning Indices for anthocyanin degradation of an aqueous extract can be calculated from a few absorbance readings of the sample that has been treated with sodium bisulfite (20% w/v sodium bisulfate). The absorbance at 420 nm of the bisulfite treated sample serves as an index for browning. Color density is defined as the sum of absorbances at the kvis-max and 420 nm. The ratio between monomeric and total anthocyanins is used to determine degradation index [39]. Color density of the control sample (treated with water) was calculated as follows:
Color density ¼ ½ðA420 nm A700 nm Þ þ ðA530 A700 nm Þ DF Degradation constant (Kd) of the extract was determined considering first-order degradation kinetics as per the following equation [40]
Ct C0
ln
¼ K d t
ð3Þ
where C0 is the initial concentration of anthocyanins (mg/L) and Ct is the concentration of anthocyanins (mg/L) at a specified time t. The degradation studies were carried out by measuring the anthocyanin content each day, up to 15 days, at room temperature (27 ± 1 °C). 2.4.3. Determination of pH, soluble solids and total sugars The pH of the extract of anthocyanins was measured using a pH meter (APX 175, Control Dynamics, India). Total soluble solid content expressed as brix was measured using refractometer (ERMA, Japan). The Dubois method [41] was used for the estimation of total sugars present in the extract. The sugars have absorption maxima at 480 nm. Dextrose was used as a standard for the determination of sugars. 2.4.4. Statistical analysis Significant differences between means were determined by t test (paired two samples for mean) using Microsoft Excel. Significance of differences was defined at p < 0.05. 3. Results and discussion 3.1. Multistage aqueous two phase extraction In our earlier work ATPE is carried out employing different salts namely potassium phosphate, sodium citrate, ammonium sulfate,
10
40 35
8 30 7 25
6 5
20
4
Brix (0 )
Transmembrane flux (L/m2h)
9
15
3 10 2 5
1 0
0 0
2
4
6
8
10
12
14
Time (hr) Fig. 4. Variation of transmembrane flux and total soluble solids (after ultrafiltration) during forward osmosis.
13
magnesium sulfate and sodium sulfate along with PEG and the results were presented elsewhere [42]. In case of potassium phosphate (pH-7) and sodium citrate (pH-8) degradation of anthocyanins was observed. Anthocyanins showed higher stability in case of (NH4)2SO4, MgSO4 and Na2SO4 because pH of these systems was around 5 (where anthocyanins are stable). In case of magnesium sulfate, the highest yield of anthocyanins (96.2%) in the PEG-rich (top) phase was observed with good stripping of sugars (58.64%) into the salt-rich (bottom) phase. Hence, MgSo4 was selected in the present study. Process parameters such as molecular weight of phase forming polymer, tie line length and phase volume ratio were standardized in order to maximize the differential partitioning of anthocyanins and sugars into opposite phases during ATPE. PEG 4000/magnesium sulfate (14.8%/10.3% w/w) was found to be the most suitable system [42]. Tie line length of 32.61% and volume ratio of 0.73 have resulted in maximum partitioning of anthocyanins to the PEG-rich phase (yield – 98.19%) and sugars to the salt-rich phase (yield – 73.16%). In order to explore the possibility of achieving further increase in purity of anthocyanins (removal of sugars), multistage ATPE was carried out as presented in the flow chart shown in Fig. 1. At the end of the fourth stage, it was found that the yield of anthocyanins and sugars in the PEG-rich (top) phase were 91.35% and 3.56%, respectively (data is not shown). In order to use the anthocyanins for intended applications, the PEG has to be removed from the top phase. PEG was removed successfully by an organic–aqueous extraction employing a volume ratio of 1.0, which extracted PEG to the organic (chloroform) phase (bottom phase) while the anthocyanins remained in the aqueous phase (top phase). Nitrogen gas was passed through the aqueous (top) phase containing only anthocyanins to remove residual chloroform, if any, present in it. In order to confirm the complete removal of polymer, phenol solution was added to the anthocyanins solution and no precipitation was observed, indicating the absence of PEG.
3.2. Membrane processes The increase in concentration of anthocyanins improves their stability through self-association [17,43]. In order to concentrate the purified anthocyanins obtained after multistage ATPE, membrane processes such as osmotic membrane distillation and forward osmosis were employed and the results are discussed in the following sections.
3.2.1. Osmotic membrane distillation Osmotic membrane distillation (OMD) was carried out using CaCl22H2O (12 M) as the osmotic agent (due to its high osmotic activity) at room temperature (27 ± 1 °C), for the concentration of purified anthocyanins (obtained after removal of the PEG). The flow rate of the feed and osmotic agent was maintained at 100 mL/min. In order to concentrate, around 700 mL of anthocyanin solution obtained after organic–aqueous extraction (concentration of red cabbage anthocyanins – 508.05 mg/L, soluble solids – 10 °Brix) was subjected to OMD for 12 h. The transmembrane flux and concentration of anthocyanins in terms of soluble solids were measured at regular intervals of 1 h during the process and the results are presented in Fig. 2. It can be observed from the figure that the transmembrane flux decreased with an increase in process time while the brix increased, as expected. The decrease in flux can be attributed to the decrease in vapor pressure difference (the driving force) and also to the concentration polarization (caused due to the increase in concentration of solute particles near the membrane surface). The concentration of anthocyanins was observed to increase from 508.05 mg/L (10 °Brix) to 945.34 mg/L (26 °Brix).
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Table 1 Physicochemical characteristics of red cabbage anthocyanins concentrated by different processes. Physicochemical characteristic
Crude extract
ATPE + OMD
a
Anthocyanin concentration (mg/L) Brix (°) Degradation constant (day1) Non-enzymatic browning index (-) Color density (-) Polymeric color (%)
b
221.22 ± 2.0 4 ± 0.2a 1.17 ± 0.12a 0.78 ± 0.02a 0.67 ± 0.03a 0.46 ± 0.04a
945.32 ± 6.0 26 ± 1.0b 0.30 ± 0.1b 0.17 ± 0.01b 8.9 ± 1.8b 8.16 ± 0.33b
ATPE + FO
UF + FO c
3123.45 ± 9.1 43 ± 1.2c 0.21 ± 0.03c 0.11 ± 0.02c 14.56 ± 1.2c 12.56 ± 0.8c
Thermal evaporation d
1952.19 ± 8.1 33 ± 1.0d 0.51 ± 0.02d 0.30 ± 0.01d 7.89 ± 1.0d 7.89 ± 0.43d
1412.56 ± 11.8e 21 ± 0.4e 3.12 ± 0.5e 0.89 ± 0.08e 3.11 ± 0.08e 6.13 ± 0.23e
ATPE – aqueous two phase extraction, OMD – osmotic membrane distillation. FO – forward osmosis, UF – ultrafiltration. Means of the same row followed by different letters differ significantly (p < 0.05).
Amount anthocyanins remained (%)
(a) 120
pH 1
100 80
Crude ATPE+OMD
60
ATPE+FO UF+FO
40
Thermal evaporaon
20 0 0
1
2
3
4
5
6
7
8
Days
feed and osmotic agent was maintained at 100 mL/min. In order to concentrate, around 700 mL of pigment solution (concentration of red cabbage anthocyanins 508.05 mg/L; soluble solids 10 °Brix), obtained after organic–aqueous extraction, was subjected to forward osmosis for 12 h. Transmembrane flux and concentration of anthocyanins in terms of soluble solids were obtained at regular intervals of 1 h during the process and the results are presented in Fig. 3. It can be observed from the figure that the transmembrane flux decreased with an increase in process time while the brix increased, as expected. The decrease in flux can be attributed to the decrease in osmotic pressure difference (the driving force) and also to the concentration polarization (caused due to the increase in concentration of solute particles near the membrane surface). The concentration of anthocyanins was observed to increase from 508.05 mg/L (10 °Brix) to 3123.45 mg/L (43 °Brix).
(b) 120 Amount of anthocyanins (%)
pH 3 100 80 Crude ATPE+OMD
60
ATPE+FO 40
UF+FO Thermal evaporaon
20 0 0
2
4
6
8
Days pH 5
(c) 120
Crude
Amount of anthocyanins (%)
ATPE+OMD 100
ATPE+FO UF+FO
80
3.2.3. Integrated membrane process An integrated membrane process involving ultrafiltration followed by forward osmosis was employed for the concentration of crude extract of anthocyanins. Ultrafiltration (Tangential flow filtration) was employed for the clarification of crude anthocyanins extract. It was observed that the concentration of the extract (permeate) remained almost the same (4 °Brix) after ultrafiltration, however, with much improved clarity (visual observation) than that of the crude extract. This can be attributed to the removal of tannins, pectins and the suspended solids, which generally cause haziness. The clarified color extract was subjected to forward osmosis to concentrate the anthocyanins and the results are presented in Fig. 4. It can be observed that the concentration of anthocyanins increased from 240.32 mg/L (4 °Brix) to 1989.98 mg/L (33 °Brix) while the flux decreased from 9.0 to 4.62 L/m2 h. The decrease in flux can be attributed to the decrease in osmotic pressure difference and the concentration polarization (caused due to the increase in concentration of solute particles near the membrane surface).
Thermal evaporaon
3.3. Comparison of different integrated processes
60 40 20 0 0
2
4
6
8
Days Fig. 5. Effect of pH on stability of anthocyanins.
3.2.2. Forward osmosis Forward osmosis was carried out using NaCl (6 M) as the osmotic agent at room temperature (27 ± 1 °C), for the concentration of purified anthocyanins (after removal of PEG). The flow rate of the
The anthocyanin concentrates produced by different integrated processes were compared with one another and also with the conventional process (thermal evaporation) by evaluating the physiochemical characteristics of anthocyanin solution and the results are presented in Table 1. It can be observed from the table that the highest concentration (3123.45 mg/L) and brix (43 °Brix) of anthocyanins were obtained in case of integrated process involving ATPE followed by FO. Evaluation of the colorant properties such as non-enzymatic browning index and degradation constant confirmed that the stability of anthocyanins to be high in case of integrated processes involving ATPE followed by membrane process (OMD/FO). Non-enzymatic browning index was found to be 0.11, Color density was found to increase from 0.6 to 14.56, and polymeric color was found to be highest (13.22) in case of integrated
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C. Jampani, K.S.M.S. Raghavarao / Separation and Purification Technology 141 (2015) 10–16 Table 2 Effect of temperature on stability of anthocyanins. Temperature (°C)
30 40 60 80
Amount of anthocyanins (%, w/w) remained after 5 h of incubation Crude extract
ATPE + OMD
ATPE + FO
UF + FO
Thermal evaporation
65 ± 2a 51 ± 1a 45 ± 2a 40 ± 1a
97 ± 2b 93 ± 2b 89 ± 3b 82 ± 2b
99 ± 1b 93 ± 2b 90 ± 1b 85 ± 3b
83 ± 3c 74 ± 2c 64 ± 4c 49 ± 2c
75 ± 3d 49 ± 3d 43 ± 1d 23 ± 3d
ATPE – aqueous two phase extraction, OMD – osmotic membrane distillation. FO – forward osmosis, UF – ultrafiltration. Means of the same row followed by different letters differ significantly (p < 0.05).
method involving ATPE followed by FO. The transmembrane flux was also observed to be high in case FO compared to OMD (can be seen from Figs. 2 and 3). Hence, it can be concluded that integrated process involving ATPE followed by FO can be a potential alternative for the purification and concentration of anthocyanins.
Acknowledgements Authors thank the Director, CSIR-CFTRI, Mysore, for the infrastructural facilities at the Institute. Chandrasekhar Jampani gratefully acknowledges CSIR, Government of India for the research fellowship.
3.4. Stability aspects of anthocyanins
References
In order to investigate the effect of pH on the stability of anthocyanins, 10 mg of pigment obtained by each process was dissolved in buffers of different pH (1, 3 and 5). The amount of anthocyanins (%, w/w) was calculated at regular interval of 24 h by measuring the absorbance at 530 nm and the results are presented in Fig. 5. It can be observed from the figure that the degradation of anthocyanins was found to increase with an increase in pH from 1 to 5. However, the decrease in concentration of anthocyanins (ACN, % w/w) was found to be less in case of anthocyanins obtained by integrated processes such as ATPE + FO and ATPE + OMD when compared to anthocyanins obtained by other processes (UF + FO and thermal evaporation) and crude extract. This can be attributed to the presence of free sugars along with anthocyanins in case of thermal evaporation, crude extract and integrated membrane process (involving UF followed by FO). In order to examine the effect of temperature on the stability of anthocyanins, 10 mg of pigment was dissolved in a buffer of pH 1 and this solution was incubated in a water bath at temperatures of 30, 40, 60 and 80 °C, respectively. The amount of anthocyanins (ACN, % w/w) was calculated by measuring the absorbance at 530 nm after 5 h and the results are presented in Table 2. It can be observed from the table that the concentration of anthocyanins decreased from 100% to 23% with an increase in temperature from 30 to 80 °C. However, the degradation of anthocyanins is less in case of anthocyanins obtained by integrated processes, namely, ATPE + FO and ATPE + OMD when compared to anthocyanins obtained by other processes (UF + FO and thermal evaporation). This can be attributed to the stability associated with co-pigmentation of anthocyanins at high concentrations.
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4. Conclusions Different methods were employed for the purification and concentration of anthocyanins from red cabbage. An integrated process involving ATPE followed by FO was found to be the most suitable process for the purification and concentration of anthocyanins (which increased from 508.05 mg/L (10 °Brix) to 3123.45 mg/L (43 °Brix)) when compared to other processes. Evaluation of the final product with respect to degradation constant, non-enzymatic browning index and color density indicated an increase in the stability of anthocyanins obtained by the integrated process involving ATPE followed by FO (which has potential to be scaled-up into a large scale process).
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[24] M.R. Kula, K.H. Kroner, H. Hustedt, Purification of enzymes by liquid–liquid extraction, Adv. Biochem. Eng. 24 (1982) 73–118. [25] K.S.M.S. Raghavarao, T. Ranganathan, N. Srinivas, R.S. Barhate, Aqueous two phase extraction—an environmentally benign technique, Clean Technol. Environ. Policy 5 (2003) 136–141. [26] Y. Wu, W. Zhang, Y. Wang, J. Han, Y. Liu, Y. Hu, L. Ni, Extraction and preliminary purification of anthocyanins from grape juice in aqueous twophase system, Sep. Purif. Technol. 124 (2014) 170–178. [27] X. Liu, T. Mu, H. Sun, M. Zhang, Jingwang Chen, Optimisation of aqueous twophase extraction of anthocyanins from purple sweet potatoes by response surface methodology, Food Chem. 141 (3) (2013) 3034–3041. [28] Y. Liu, J. Han, Y. Wang, Y. Lu, G. Zhang, C. Sheng, Y. Yan, Selective separation of flavones and sugars from honeysuckle by alcohol/salt aqueous two-phase system and optimization of extraction process, Sep. Purif. Technol. 118 (2013) 776–783. [29] Voltaire Sant’ Anna, Ligia Damasceno Ferreira Marczak, Isabel Cristina Tessaro, Membrane concentration of liquid foods by forward osmosis: Process and quality view, J. Food Eng. 111 (2012) 483–489. [30] Susmit Ilame, Satyavir Singh, Application of membrane separation in fruit and vegetable juice processing: a review, Crit. Rev. Food Sci. Nut., doi: http:// dx.doi.org/10.1080/10408398.2012.679979. [31] B. Girard, L.R. Fukomoto, Membrane processing of fruit juices, and beverages: a review, Crit. Rev. Food Sci. Nutr. 40 (2000) 91–101. [32] L.E. Rodriguez-Sanoa, M.M. Giusti, W.D. Robert, R.E. Wrolstad, Development and process optimization of red radish concentration extract as potential natural red colorant, J. Food Process. Preserv. 25 (2001) 165–182. [33] H. Nawaz, J. Shi, G.S. Mittal, Y. Kakudac, Extraction of polyphenols from grape seeds and concentration by ultrafiltration, Sep. Purif. Technol. 48 (2006) 176– 181.
[34] A. Cassano, A. Figoli, A. Tagarelli, G. Sindona, E. Drioli, Integrated membrane process for the production of highly nutritional kiwifruit juice, Desalination 189 (1) (2006) 21–30. [35] B.R. Babu, N.K. Rastogi, K.S.M.S. Raghavarao, Concentration and temperature polarization effects during osmotic membrane distillation, J. Membr. Sci. 322 (2008) 146–153. [36] J. Ren, J.R. McCutcheon, A new commercial thin film composite membrane for forward osmosis, Desalination, doi: http://dx.doi.org/10.1016/ j.desal.2013.11.026. [37] M. Xie, W.E. Price, L.D. Nghiem, Rejection of pharmaceutically active compounds by forward osmosis: role of solution pH and membrane orientation, Sep. Purif. Technol. 104 (2013) 107–114. [38] T. Flueki, F.J. Francis, Quantitative methods for anthocyanins 2. Determination of total anthocyanin and degradation index for cranberry juice, J. Food Sci. 33 (1986) 78–83. [39] M.M. Giusti, R.E. Wrolstad, Characterization and measurement of anthocyanins by UV–Visible spectroscopy, Curr. Protoc. Food Analyt. Chem. (2001) (F1.2.1–F1.2). [40] A. Ibarz, S. Garza, J. Pagan, Non-enzymatic browning of selected fruit juices affected by D-galacturonic acid, Int. J. Food Sci. Technol. 43 (2008) 908. [41] M. Dubois, K.A. Gilles, K. Hamilton, P.A. Rebers, Smith, Colorimetric method for the determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [42] J. Candrasekhar, Adsorption and liquid–liquid extraction for the downstream processing of Anthocyanins, Ph.D. Thesis, University of Mysore, India, 2014. [43] L.T. Dao, G.R. Takeoka, R.H. Edwards, J.D.J. Berrios, Improved method for the stabilization of anthocyanidins, J. Agri. Food Chem. 46 (1998) 3564–3569.
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Process integration for purification and concentration of red cabbage (Brassica oleracea L.) anthocyanins Chandrasekhar Jampani, K.S.M.S. Raghavarao ⇑ Department of Food Engineering, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 25 March 2014 Received in revised form 8 November 2014 Accepted 15 November 2014 Available online 29 November 2014 Keywords: Anthocyanins Process integration Aqueous two phase extraction Osmotic membrane distillation Forward osmosis
a b s t r a c t Multistage aqueous two phase extraction was carried out using polyethylene glycol (PEG) 4000/magnesium sulfate (14.8/10.3%, w/w) system. The phase forming polymer (PEG 4000) was successfully separated from anthocyanins employing organic–aqueous extraction. Different processes employed for the purification and concentration of anthocyanins were compared with one another. The highest concentration of anthocyanins (3123.45 mg/L and 43 °Brix) was obtained in case of integration of aqueous two phase extraction with forward osmosis. Non-enzymatic browning index (0.11) and degradation constant (0.21) were found to be lowest in case of the integrated process involving aqueous two phase extraction followed by forward osmosis when compared to other processes. Color density was found to increase from 0.6 to 14.56 and stability of anthocyanins (with respect to pH and temperature) was found to be more in case of integrated process (aqueous two phase extraction followed by forward osmosis). Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Color is an imperative component of food or any edible stuff as it can enhance the appearance [1]. Food colorants are being used in beverages, desserts, jams, jellies, sauces, cosmetics, toothpaste, etc. In addition medicines including tablets, capsules and syrups are colored with food colorants. Numerous studies have demonstrated the effect of artificial colorants in food, which include the inhibition of the immune system and allergic reactions [2]. In addition, the use of non-permitted colors or overindulgence of permitted synthetic colors may also cause thyroid tumors, asthma, abdominal pain, nausea, liver and kidney damage and cancer. In view of this, interest in natural food colorants has increased considerably, mainly because of the apparent lack of toxicity and ecofriendliness [3]. Natural colors such as betalains from beet root, carotenoids from carrots and anthocyanins from grapes, and red cabbage. are some of the examples. Anthocyanins are glycosylated polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium (flavylium) salts accounting for the colors in several fruits, vegetables and flowers [4]. The hydrophilic nature of anthocyanins facilitates their incorporation in food systems. A number of properties have been reported such as antioxidant [5], antiulcer [6], anticancer, antitumor and anti-mutagenic [7], antidiabetes [8,9], antibacterial activity [10], ocular[11] and prevention of cardiovas⇑ Corresponding author. Tel.: +91 821 2513910; fax: +91 821 2517233. E-mail address: [email protected] (K.S.M.S. Raghavarao). http://dx.doi.org/10.1016/j.seppur.2014.11.024 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.
cular diseases [12]. Several factors such as concentration of free sugars, oxygen, pH [13], temperature, light [14] and concentration of colorant itself [15] influence the stability of anthocyanins. Removal of free sugars from the extract of anthocyanins is very much desirable otherwise leads to Maillard reaction [16,17]. Several chromatographic techniques are used for the purification of anthocyanins [18–22]. However, in case of methods such as column chromatography and electrophoresis scale up becomes uneconomical unless the product is of high value. Aqueous two-phase extraction (ATPE), a liquid–liquid extraction (LLE) strategy, has been recognized as a potential technique because of its multiple advantages including: biological compatibility, low interfacial tension, high capacity, scale up easiness and scope for continuous operation [23–25]. ATPE has been successfully employed for the preliminary purification of anthocyanins [26–28]. The increase in concentration of anthocyanins improves their stability through self-association [3]. Concentration of natural color extracts by conventional method such as evaporation results in low quality product due to loss of hue and chroma [29,30]. Membrane processes such as microfiltration, ultrafiltration and reverse osmosis were being employed for clarification and concentration of natural color extracts [31–33]. There are many limitations of these membrane processes such as requirement of high pressure, limit to maximum attainable concentration and concentration polarization [29,30]. On the other hand, the non-pressure driven membrane processes such as osmotic membrane
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11
Fig. 1. Overall flow chart for the separation and concentration of anthocyanins using different processes.
distillation (OMD) and forward osmosis (FO) are the promising technologies (as they are osmotic gradient driven) for the concentration of thermally sensitive liquid foods [29,30,34–37]. These athermal membrane processes, operated at ambient temperature, practically eliminate the thermal damage to the product thereby retaining the color. Objective of the present work is to integrate ATPE with membrane processes such as OMD and FO for concentration of red cabbage anthocyanins. Further, comparison of these integrated processes with an integrated membrane process (involving ultrafiltration followed by FO) and conventional method (thermal evaporation).
a very thin semi-permeable non-porous active skin layer of cellulose triacetate and porous support layer. The membrane was embedded in a nylon mesh for an increased strength. The active layer effectively excludes the passage of solutes with a molecular weight cut-off of 100 Da. 2.3. Methods The overall process flow diagram for purification and concentration of anthocyanins using different processes is shown in Fig. 1.
2.2. Membranes
2.3.1. Extraction of anthocyanins Red cabbage (Brassica oleracea L.) was procured from local supermarket. Leaves were cut into small pieces and extraction was carried out by maintaining the ratio of solid (cut leaves) to extraction media (eco-solvent–water) at 1:2 (w/v) while providing thorough contact in a mixing unit (Singer FP-450, India). The extract of anthocyanins obtained was filtered (using a muslin cloth) to remove the coarse particles. The filtrate obtained was centrifuged (Eltek-TC 4100D, Elektrocrafts, India) at 8000 rpm for 15 min to remove the fine suspended particles. Ascorbic acid (0.1%, w/v) was added to arrest the activity of polyphenol oxidase which otherwise causes enzymatic browning. The crude extract of anthocyanins thus obtained was stored at 4–6 °C and required quantities were taken for different experiments.
Hydrophobic polypropylene (PP) membranes (Accurel, Enka, Germany) of pore size 0.2 and 0.05 lm were used for osmotic membrane distillation. Hydrophilic membrane (Osmotek, Inc., Corvallis, OR, USA) used for forward osmosis is asymmetric having
2.3.2. Aqueous two phase extraction PEG 4000 (14.8% w/w) and magnesium sulfate (10.3% w/w) were added to the crude extract of anthocyanins (74.9% w/w) making the total weight of the system 100% on w/w basis. The contents
2. Materials and methods 2.1. Chemicals Calcium chloride dihydrate (CaCl22H2O) and sodium chloride (NaCl) were obtained from Ranbaxy chemicals, Mumbai, India. Polyethylene glycol 4000 Da was procured from Sisco research laboratories, Mumbai, India. Double distilled water was used for all the experiments. All the experiments were carried out in triplicate and average values are reported. All the other chemicals used were of analytical grade.
2.3.3. Osmotic membrane distillation and Forward osmosis A polyester mesh (0.25 mm, on osmotic agent side), membrane (area – 0.012 m2) and a viton gasket (3.0 mm, on the feed side) were supported between two stainless steel (SS 316) frames of a flat module. Feed (anthocyanins solution) and osmotic solutions were circulated on either side of the membrane in co-current mode using peristaltic pumps (Model 72-315-230, Barnant Company, USA). The ratio of feed to osmotic solution was maintained as 1:3 for all the experiments. The transmembrane flux was calculated by measuring the increase in weight of the osmotic solution at regular intervals of 1 h. Calcium chloride and sodium chloride solutions were used as osmotic solutions in OMD and FO, respectively. All the experiments were carried out at room temperature (27 ± 1 °C). 2.3.4. Thermal evaporation Crude extract of anthocyanins was concentrated by conventional process of thermal evaporation in agitated falling film vacuum evaporator (Model No. 04-012, Chemetron corporation, USA) and used as control. 2.3.5. Integrated membrane process Ultrafiltration (Serial No. P5PN8322003, Millipore, USA) was employed for clarification of the crude extract of anthocyanins using a membrane cartridge (PLCTK 30, Millipore, USA) of 30 kDa cut-off. The permeate obtained in this process was subjected to forward osmosis in order to concentrate the anthocyanins. 2.3.6. Freeze drying The concentrated solutions of anthocyanins obtained after different integrated processes (shown in Fig. 1) were subjected to freeze drying (Lyodryer LT5B, Lyophilisation systems INC., USA) to obtain the pigment in powder form. 2.3.7. Stability studies In order to examine the effect of pH on the stability of anthocyanins, buffer solutions of different pH (1, 3 and 5) were prepared. Pigment, obtained after freeze drying, was dissolved (10 mg) in these buffer solutions (5 mL) in separate volumetric flasks and incubated at room temperature (27 ± 1 °C) for 7 days. The total amount of anthocyanins in each solution was determined at regular intervals of 24 h using pH differential method [38]. In order to study the effect of temperature on the stability of anthocyanins, diluted samples were incubated in a shaking water bath (BANI 115, Jayadeep Engineers, India) for 5 h at 30, 40, 60, 80 and 100 °C. Then the absorbance was determined at 530 nm. The anthocyanins (ACN, % w/w) remained after time ‘t’ was calculated using the following equation.
ACN ð%Þ ¼
Ct 100 C0
ð1Þ
where C0 is the initial amount of anthocyanins (mg) and Ct is the amount of anthocyanins (mg) at time t (day in case of pH stability studies and hr in case of temperature stability studies).
30
2.5
25
2
20
1.5
15
1
10
0.5
Brix (0)
3
5
0
0 0
2
4
6
8
10
12
14
Time (hr) Fig. 2. Variation of transmembrane flux and total soluble solids during osmotic membrane distillation.
2.4. Analytical procedures 2.4.1. Concentration of anthocyanins Anthocyanin concentration of the samples was determined according to the pH differential method [38]. This method is based on the anthocyanins structural transformation (reversible) that occurs with a change in pH. Aliquots of the anthocyanin solution were diluted with buffers of pH 1.0 (0.025 M potassium chloride) and 4.5 (0.4 M sodium acetate), respectively. These samples were thoroughly vortexed (Spinix MC-01, Tarsons products Pvt. Ltd, India) and then placed in the dark for 30 min to equilibrate. The absorbance of each solution was measured using a spectrophotometer (UV-1700, Shimadzu, Japan) at a wavelength of maximum absorption (kvis-max – 530 nm) and at haze detection absorption (700 nm) against a blank (distilled water). Concentration of anthocyanins was calculated using the following equation [39]
Anthocyanins concentration ðmg=LÞ ¼
A MW DF 1000 eL
ð2Þ
where A = [(A530 A700)pH1.0 – (A530 - A700)pH4.5]; MW, the molecular weight of anthocyanins (cyanidin-3-glycoside – 449.2 g/mol); DF, the dilution factor; e, the extinction coefficient (26,900 L/cm mol) and L is the path length (1 cm).
10
50
9
45
8
40
7
35
6
30
5
25
4
20
3
15
2
10
1
5
0
Brix ( 0 )
were mixed thoroughly using a magnetic stirrer for an hour to equilibrate, and the mixture was allowed for phase separation. After clear separation, the top and bottom phases were collected, volumes noted and were subjected to analysis of anthocyanins as well as sugars. Multistage ATPE was carried out in order to remove maximum possible sugars from PEG-rich (top) phase followed by organic–aqueous extraction employing chloroform for the removal of the phase forming polymer (PEG) from this phase.
Transmembrane flux (L/(m2h)
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Transmembrane flux (L/(m2h)
12
0 0
2
4
6
8
10
12
14
Time (hr) Fig. 3. Variation of transmembrane flux and total soluble solids during forward osmosis (after PEG removal).
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2.4.2. Indices for pigment degradation, polymeric color and browning Indices for anthocyanin degradation of an aqueous extract can be calculated from a few absorbance readings of the sample that has been treated with sodium bisulfite (20% w/v sodium bisulfate). The absorbance at 420 nm of the bisulfite treated sample serves as an index for browning. Color density is defined as the sum of absorbances at the kvis-max and 420 nm. The ratio between monomeric and total anthocyanins is used to determine degradation index [39]. Color density of the control sample (treated with water) was calculated as follows:
Color density ¼ ½ðA420 nm A700 nm Þ þ ðA530 A700 nm Þ DF Degradation constant (Kd) of the extract was determined considering first-order degradation kinetics as per the following equation [40]
Ct C0
ln
¼ K d t
ð3Þ
where C0 is the initial concentration of anthocyanins (mg/L) and Ct is the concentration of anthocyanins (mg/L) at a specified time t. The degradation studies were carried out by measuring the anthocyanin content each day, up to 15 days, at room temperature (27 ± 1 °C). 2.4.3. Determination of pH, soluble solids and total sugars The pH of the extract of anthocyanins was measured using a pH meter (APX 175, Control Dynamics, India). Total soluble solid content expressed as brix was measured using refractometer (ERMA, Japan). The Dubois method [41] was used for the estimation of total sugars present in the extract. The sugars have absorption maxima at 480 nm. Dextrose was used as a standard for the determination of sugars. 2.4.4. Statistical analysis Significant differences between means were determined by t test (paired two samples for mean) using Microsoft Excel. Significance of differences was defined at p < 0.05. 3. Results and discussion 3.1. Multistage aqueous two phase extraction In our earlier work ATPE is carried out employing different salts namely potassium phosphate, sodium citrate, ammonium sulfate,
10
40 35
8 30 7 25
6 5
20
4
Brix (0 )
Transmembrane flux (L/m2h)
9
15
3 10 2 5
1 0
0 0
2
4
6
8
10
12
14
Time (hr) Fig. 4. Variation of transmembrane flux and total soluble solids (after ultrafiltration) during forward osmosis.
13
magnesium sulfate and sodium sulfate along with PEG and the results were presented elsewhere [42]. In case of potassium phosphate (pH-7) and sodium citrate (pH-8) degradation of anthocyanins was observed. Anthocyanins showed higher stability in case of (NH4)2SO4, MgSO4 and Na2SO4 because pH of these systems was around 5 (where anthocyanins are stable). In case of magnesium sulfate, the highest yield of anthocyanins (96.2%) in the PEG-rich (top) phase was observed with good stripping of sugars (58.64%) into the salt-rich (bottom) phase. Hence, MgSo4 was selected in the present study. Process parameters such as molecular weight of phase forming polymer, tie line length and phase volume ratio were standardized in order to maximize the differential partitioning of anthocyanins and sugars into opposite phases during ATPE. PEG 4000/magnesium sulfate (14.8%/10.3% w/w) was found to be the most suitable system [42]. Tie line length of 32.61% and volume ratio of 0.73 have resulted in maximum partitioning of anthocyanins to the PEG-rich phase (yield – 98.19%) and sugars to the salt-rich phase (yield – 73.16%). In order to explore the possibility of achieving further increase in purity of anthocyanins (removal of sugars), multistage ATPE was carried out as presented in the flow chart shown in Fig. 1. At the end of the fourth stage, it was found that the yield of anthocyanins and sugars in the PEG-rich (top) phase were 91.35% and 3.56%, respectively (data is not shown). In order to use the anthocyanins for intended applications, the PEG has to be removed from the top phase. PEG was removed successfully by an organic–aqueous extraction employing a volume ratio of 1.0, which extracted PEG to the organic (chloroform) phase (bottom phase) while the anthocyanins remained in the aqueous phase (top phase). Nitrogen gas was passed through the aqueous (top) phase containing only anthocyanins to remove residual chloroform, if any, present in it. In order to confirm the complete removal of polymer, phenol solution was added to the anthocyanins solution and no precipitation was observed, indicating the absence of PEG.
3.2. Membrane processes The increase in concentration of anthocyanins improves their stability through self-association [17,43]. In order to concentrate the purified anthocyanins obtained after multistage ATPE, membrane processes such as osmotic membrane distillation and forward osmosis were employed and the results are discussed in the following sections.
3.2.1. Osmotic membrane distillation Osmotic membrane distillation (OMD) was carried out using CaCl22H2O (12 M) as the osmotic agent (due to its high osmotic activity) at room temperature (27 ± 1 °C), for the concentration of purified anthocyanins (obtained after removal of the PEG). The flow rate of the feed and osmotic agent was maintained at 100 mL/min. In order to concentrate, around 700 mL of anthocyanin solution obtained after organic–aqueous extraction (concentration of red cabbage anthocyanins – 508.05 mg/L, soluble solids – 10 °Brix) was subjected to OMD for 12 h. The transmembrane flux and concentration of anthocyanins in terms of soluble solids were measured at regular intervals of 1 h during the process and the results are presented in Fig. 2. It can be observed from the figure that the transmembrane flux decreased with an increase in process time while the brix increased, as expected. The decrease in flux can be attributed to the decrease in vapor pressure difference (the driving force) and also to the concentration polarization (caused due to the increase in concentration of solute particles near the membrane surface). The concentration of anthocyanins was observed to increase from 508.05 mg/L (10 °Brix) to 945.34 mg/L (26 °Brix).
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Table 1 Physicochemical characteristics of red cabbage anthocyanins concentrated by different processes. Physicochemical characteristic
Crude extract
ATPE + OMD
a
Anthocyanin concentration (mg/L) Brix (°) Degradation constant (day1) Non-enzymatic browning index (-) Color density (-) Polymeric color (%)
b
221.22 ± 2.0 4 ± 0.2a 1.17 ± 0.12a 0.78 ± 0.02a 0.67 ± 0.03a 0.46 ± 0.04a
945.32 ± 6.0 26 ± 1.0b 0.30 ± 0.1b 0.17 ± 0.01b 8.9 ± 1.8b 8.16 ± 0.33b
ATPE + FO
UF + FO c
3123.45 ± 9.1 43 ± 1.2c 0.21 ± 0.03c 0.11 ± 0.02c 14.56 ± 1.2c 12.56 ± 0.8c
Thermal evaporation d
1952.19 ± 8.1 33 ± 1.0d 0.51 ± 0.02d 0.30 ± 0.01d 7.89 ± 1.0d 7.89 ± 0.43d
1412.56 ± 11.8e 21 ± 0.4e 3.12 ± 0.5e 0.89 ± 0.08e 3.11 ± 0.08e 6.13 ± 0.23e
ATPE – aqueous two phase extraction, OMD – osmotic membrane distillation. FO – forward osmosis, UF – ultrafiltration. Means of the same row followed by different letters differ significantly (p < 0.05).
Amount anthocyanins remained (%)
(a) 120
pH 1
100 80
Crude ATPE+OMD
60
ATPE+FO UF+FO
40
Thermal evaporaon
20 0 0
1
2
3
4
5
6
7
8
Days
feed and osmotic agent was maintained at 100 mL/min. In order to concentrate, around 700 mL of pigment solution (concentration of red cabbage anthocyanins 508.05 mg/L; soluble solids 10 °Brix), obtained after organic–aqueous extraction, was subjected to forward osmosis for 12 h. Transmembrane flux and concentration of anthocyanins in terms of soluble solids were obtained at regular intervals of 1 h during the process and the results are presented in Fig. 3. It can be observed from the figure that the transmembrane flux decreased with an increase in process time while the brix increased, as expected. The decrease in flux can be attributed to the decrease in osmotic pressure difference (the driving force) and also to the concentration polarization (caused due to the increase in concentration of solute particles near the membrane surface). The concentration of anthocyanins was observed to increase from 508.05 mg/L (10 °Brix) to 3123.45 mg/L (43 °Brix).
(b) 120 Amount of anthocyanins (%)
pH 3 100 80 Crude ATPE+OMD
60
ATPE+FO 40
UF+FO Thermal evaporaon
20 0 0
2
4
6
8
Days pH 5
(c) 120
Crude
Amount of anthocyanins (%)
ATPE+OMD 100
ATPE+FO UF+FO
80
3.2.3. Integrated membrane process An integrated membrane process involving ultrafiltration followed by forward osmosis was employed for the concentration of crude extract of anthocyanins. Ultrafiltration (Tangential flow filtration) was employed for the clarification of crude anthocyanins extract. It was observed that the concentration of the extract (permeate) remained almost the same (4 °Brix) after ultrafiltration, however, with much improved clarity (visual observation) than that of the crude extract. This can be attributed to the removal of tannins, pectins and the suspended solids, which generally cause haziness. The clarified color extract was subjected to forward osmosis to concentrate the anthocyanins and the results are presented in Fig. 4. It can be observed that the concentration of anthocyanins increased from 240.32 mg/L (4 °Brix) to 1989.98 mg/L (33 °Brix) while the flux decreased from 9.0 to 4.62 L/m2 h. The decrease in flux can be attributed to the decrease in osmotic pressure difference and the concentration polarization (caused due to the increase in concentration of solute particles near the membrane surface).
Thermal evaporaon
3.3. Comparison of different integrated processes
60 40 20 0 0
2
4
6
8
Days Fig. 5. Effect of pH on stability of anthocyanins.
3.2.2. Forward osmosis Forward osmosis was carried out using NaCl (6 M) as the osmotic agent at room temperature (27 ± 1 °C), for the concentration of purified anthocyanins (after removal of PEG). The flow rate of the
The anthocyanin concentrates produced by different integrated processes were compared with one another and also with the conventional process (thermal evaporation) by evaluating the physiochemical characteristics of anthocyanin solution and the results are presented in Table 1. It can be observed from the table that the highest concentration (3123.45 mg/L) and brix (43 °Brix) of anthocyanins were obtained in case of integrated process involving ATPE followed by FO. Evaluation of the colorant properties such as non-enzymatic browning index and degradation constant confirmed that the stability of anthocyanins to be high in case of integrated processes involving ATPE followed by membrane process (OMD/FO). Non-enzymatic browning index was found to be 0.11, Color density was found to increase from 0.6 to 14.56, and polymeric color was found to be highest (13.22) in case of integrated
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C. Jampani, K.S.M.S. Raghavarao / Separation and Purification Technology 141 (2015) 10–16 Table 2 Effect of temperature on stability of anthocyanins. Temperature (°C)
30 40 60 80
Amount of anthocyanins (%, w/w) remained after 5 h of incubation Crude extract
ATPE + OMD
ATPE + FO
UF + FO
Thermal evaporation
65 ± 2a 51 ± 1a 45 ± 2a 40 ± 1a
97 ± 2b 93 ± 2b 89 ± 3b 82 ± 2b
99 ± 1b 93 ± 2b 90 ± 1b 85 ± 3b
83 ± 3c 74 ± 2c 64 ± 4c 49 ± 2c
75 ± 3d 49 ± 3d 43 ± 1d 23 ± 3d
ATPE – aqueous two phase extraction, OMD – osmotic membrane distillation. FO – forward osmosis, UF – ultrafiltration. Means of the same row followed by different letters differ significantly (p < 0.05).
method involving ATPE followed by FO. The transmembrane flux was also observed to be high in case FO compared to OMD (can be seen from Figs. 2 and 3). Hence, it can be concluded that integrated process involving ATPE followed by FO can be a potential alternative for the purification and concentration of anthocyanins.
Acknowledgements Authors thank the Director, CSIR-CFTRI, Mysore, for the infrastructural facilities at the Institute. Chandrasekhar Jampani gratefully acknowledges CSIR, Government of India for the research fellowship.
3.4. Stability aspects of anthocyanins
References
In order to investigate the effect of pH on the stability of anthocyanins, 10 mg of pigment obtained by each process was dissolved in buffers of different pH (1, 3 and 5). The amount of anthocyanins (%, w/w) was calculated at regular interval of 24 h by measuring the absorbance at 530 nm and the results are presented in Fig. 5. It can be observed from the figure that the degradation of anthocyanins was found to increase with an increase in pH from 1 to 5. However, the decrease in concentration of anthocyanins (ACN, % w/w) was found to be less in case of anthocyanins obtained by integrated processes such as ATPE + FO and ATPE + OMD when compared to anthocyanins obtained by other processes (UF + FO and thermal evaporation) and crude extract. This can be attributed to the presence of free sugars along with anthocyanins in case of thermal evaporation, crude extract and integrated membrane process (involving UF followed by FO). In order to examine the effect of temperature on the stability of anthocyanins, 10 mg of pigment was dissolved in a buffer of pH 1 and this solution was incubated in a water bath at temperatures of 30, 40, 60 and 80 °C, respectively. The amount of anthocyanins (ACN, % w/w) was calculated by measuring the absorbance at 530 nm after 5 h and the results are presented in Table 2. It can be observed from the table that the concentration of anthocyanins decreased from 100% to 23% with an increase in temperature from 30 to 80 °C. However, the degradation of anthocyanins is less in case of anthocyanins obtained by integrated processes, namely, ATPE + FO and ATPE + OMD when compared to anthocyanins obtained by other processes (UF + FO and thermal evaporation). This can be attributed to the stability associated with co-pigmentation of anthocyanins at high concentrations.
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4. Conclusions Different methods were employed for the purification and concentration of anthocyanins from red cabbage. An integrated process involving ATPE followed by FO was found to be the most suitable process for the purification and concentration of anthocyanins (which increased from 508.05 mg/L (10 °Brix) to 3123.45 mg/L (43 °Brix)) when compared to other processes. Evaluation of the final product with respect to degradation constant, non-enzymatic browning index and color density indicated an increase in the stability of anthocyanins obtained by the integrated process involving ATPE followed by FO (which has potential to be scaled-up into a large scale process).
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