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Comparison of different ultrasound assisted extraction techniques for pectin from tomato processing waste
Journal Pre-proofs Comparison of different ultrasound assisted extraction techniques for pectin from tomato processing waste Animesh Singh Sengar, Ashish Rawson, Manimekalai Muthiah, Suresh Kumar Kalakandan PII: DOI: Reference:
S1350-4177(19)30860-0 https://doi.org/10.1016/j.ultsonch.2019.104812 ULTSON 104812
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
3 June 2019 24 September 2019 27 September 2019
Please cite this article as: A.S. Sengar, A. Rawson, M. Muthiah, S. Kumar Kalakandan, Comparison of different ultrasound assisted extraction techniques for pectin from tomato processing waste, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.104812
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1
Comparison of different ultrasound assisted extraction techniques for pectin from
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tomato processing waste Animesh Singh Sengar, Ashish Rawson*, Manimekalai Muthiah, Suresh Kumar Kalakandan Indian Institute of Food Processing Technology Thanjavur- 613 005, Tamil Nadu, India. Corresponding author: Dr Ashish Rawson, e-mail: [email protected], [email protected]
3 4
Abstract
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Concept of waste to wealth is a hot topic with research ongoing globally to reduce carbon
6
footprint. In an effort to follow up this cause present study focused on tomato industry waste
7
specifically the peel of tomatoes for extraction of pectin. Pectin extraction was performed
8
using five different extraction techniques (Ultrasound assisted extraction (UAE); microwave
9
assisted extraction (MAE); ohmic heating assisted extraction (OHAE); ultrasound assisted
10
microwave extraction (UAME) and ultrasound assisted ohmic heating extraction (UAOHE) at
11
different power levels to study its extraction and degradation kinetics and in turn to optimize
12
the extraction process. The extracted pectin yield ranged from 9.30% for OHAE to 25.42 %
13
for MAE. Also, there was very less difference in the yield of MAE and UAME extracted
14
pectin, but at the cost of major difference in degree of esterification 59.76±0.70 and
15
73.33±1.76 %, respectively. In addition, all the pectin extracted under optimized conditions
16
was having acceptable purity, [Galacturonic acid (GalA) content ranged from 675.8±11.31 to
17
913.3±20.50 g/kg of pectin]. FTIR analysis confirmed the presence of functional groups in
18
the finger print region of identification for polysaccharide in all the extracted pectin.
19
According to obtained results, UAME can be considered as better green extraction
20
technology in terms of extraction yield as well as in quality of pectin compared to the other
21
treatments used. Therefore, results suggest that UAME can be used as an efficient pectin
22
extraction method from tomato processing waste.
23 24 25
Keywords: Ultrasonication, Microwave, Ohmic heating, extraction, Pectin.
26
1. Introduction
27
Tomato (Solanum lycopersicum) is widely consumed crop in the world as raw and in
28
processed forms [1]. According to FAOSTAT (2017)[2], total world production of tomato
29
accounted to be 18,23,01,395 tonnes. Out of this significant volume is processed yearly to
30
create products like tomato juice, paste, puree, ketchup and sauce for human utilization,
31
generating huge amounts of wastes from the food processing sector. Products which remain
32
after pressing of tomato majorly includes 27% peel, 33% seed and furthermore, 40% part of
33
pomace [3]. Tomato waste are usually disposed and/or utilized for animal feed or as manure
34
[4]. The waste produced along these food processing industries are viewed as the rich well-
35
spring of functional components like pectin, lycopene, and essential oils and so forth.
36
Moreover, they are low-cost raw materials and hence been under research in current years. In
37
recent research, tomato byproducts were used as ingredients for newly developed extruded
38
product [5].
39
The primary plant cell of fruits and vegetables is made out of polysaccharide like pectin,
40
hemicellulose and cellulose. The word Pectin is derived from a Greek word "Pektikos" which
41
implies “Coagulant” and is one of the most interesting functional component due to its
42
solubility and polyanionic nature [6]. It is majorly found in the portion of primary cell wall,
43
middle lamella and also in secondary cell walls of plant. Conventionally, pectin is isolated
44
from various food waste produced in food processing industries including citrus peel (85%),
45
apple pomace (14%) and sugar beet (<1%) [7].
46
Pectin is a heteropolysaccharide which is mainly composed of polymer of -galacturonic
47
acid (GalA) (not less than 65% according to standards for food grade pectin defined under
48
EU guideline no. 231/2012) which is an isomer of D-glucuronic acid (D-GlcA). Pectin,
49
according to the International numbering system has been defined as a food additive (Code
50
no. 440) and has no recommended maximum level for addition (limited to use lowest
51
possible level to achieve the desired effect). In terms of the degree of methylation (DM), it
52
can be very well defined into two categories with their applications as high methoxyl pectin
53
(DM 50%) and low methoxyl pectin (DM < 50%) [8]. High-methoxyl pectin can form gel
54
when heated in sugar solution having solution concentration higher than 55Brix and pH
55
maintained lower than 3.5. Though, low-methoxyl pectin can also form gel in the of presence
56
calcium ions. Pectin with higher degree of methylation are favored for food products for
57
achieving the desired consistency and structure in jam, jellies, etc. Whereas, low-methoxyl
58
pectin are used as fat replacer in ice creams, heat reversible bakery glazing agent, emulsified
59
meat products, low calorie beverages and also in fruit preparation for yoghurt [9].
60
Traditionally, pectin is isolated from citrus fruit peel with techniques utilizing
61
acidified water which are not only time and energy consuming, but also give poor yield and
62
lead to the destruction of pectin structure. Though, novel techniques like MAE, UAE, OHAE
63
and combination of these were used and has been demonstrated to give better extraction
64
yields to overcome the limitations of conventional extraction method. Ultrasonication has
65
always proved to be a better method for extraction, modification and blanching, which
66
creates new opportunities to produce bioactive compounds [10-14].
67
In addition, pectin isolation highlights the multiple stage physicochemical process which are
68
influenced by parameters, for example, temperature, pH, extraction time, solvent, solid/liquid
69
ratio and number of extractions. As the extraction of pectin at industrial scale majorly relies
70
upon citrus fruit peels and apple pomace, hence newer potential sources for pectin extraction
71
are continuously being explored. Yang et al. [15] isolated pectin from potato pulp, similarly
72
various researchers [16-21] studied the pectin extraction from grapefruit. Likewise, de
73
Oliveira et al. [22] and Liew et al. [23] utilized passion fruit for isolating pectin. Also Yang et
74
al. [24] investigated that, sisal waste can be utilized for the extraction of pectin by
75
combination of enzymatic and ultrasonic method.
76
Despite the fact that, pectin is present in the majority of plant cells, however, the source used
77
for pectin extraction are mainly restricted to apple pomace and citrus fruit peel at commercial
78
scale. Owing to the fact that conventional extraction can lead to wastage of large amount of
79
solvents, energy, time and generation of carbon footprints. There has been an increased
80
demand as well as focus on green novel technologies for extraction of important metabolites.
81
Hence, the objective of this research were to establish new source and enhanced extraction
82
technique utilizing different novel technologies (UAE; MAE; OHAE; UAME and UAOHE)
83
for pectin extraction and furthermore to consider the impact of these techniques on the yield
84
of pectin and on its characteristic properties.
85
2. Materials and methods:
86
2.1. Materials:
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Fresh tomato (Solanum lycopersicum) were procured from local market, Thanjavur, India.
88
These were steam blanched (100oC for 15 min) and peel were removed manually. Tomato
89
peels were dried at 60C up to constant moisture level and were milled using electronic
90
pulverizer (Pulverisette 14, FRITSCH, Germany) to maintain the particle size 600 microns
91
by passing it through mesh sieve. Subsequently, the tomato peel powder (TPP) was vacuum
92
packed to avert moisture absorption.
93
2.2. Extraction and purification:
94
Parameters for extraction were studied in the initial trials as well those referred from
95
literature [19, 25] and the best proven ones were used for further extraction of pectin. The
96
details of which is discussed below.
97
2.2.1. Sample preparation for extraction:
98
Dried TPP was conditioned with the distilled water with solid-to-liquid ratio of 1:50 g/mL (to
99
avoid initial saturation of solvent during extraction process). Further pH of this solution was
100
maintained to 1.5 using 0.5 N HCl (extracting agent). Temperature of the solution was
101
monitored throughout extraction process [19, 25]. Figure 1 shows process flow chart for all
102
the extraction methods.
103
2.2.2. Ultrasound assisted extraction (UAE):
104
Ultrasound was applied using horn-type probe system (Sonic CV334, 220 V, 50/60 Hz). The
105
system with specification: power output 750 W, 20 KHz and having probe tip diameter 19
106
mm, with 12 mm immersed in solvent. Sonication was conducted at constant temperature of
107
60C on three power inputs 450 W, 600 W and 750 W. Treatment time was increased with
108
constant interval from 2, 4, 6, 8, 10, 12, 14 and 16 min with pulsation of 50% (30 s ON: 30 s
109
OFF).
110
2.2.3. Microwave extraction:
111
System used for extraction was household microwave system (900 W/ 2450 MHz) which
112
allows it to work at three different power 540, 720 and 900 W. Sample treatment time for
113
extraction was chosen with equal break of interims (0.667 min ie. 40 s) starting from 0.667,
114
1.334, 2, 2.667, 3.334, 4, 4.667 and 5.334 min. And the temperature was continuously
115
monitored during the study.
116
2.2.4. Ohmic heating assisted extraction (OHAE):
117
The extraction process in ohmic heating was performed using cuboidal chamber (80 80
118
30) mm3 made from acrylic sheet (5 mm thick). The stainless steel plates having thickness 1
119
mm and area 2291 mm2 were used as electrodes. To investigate the changes in yield with
120
increasing power level and treatment time, the extraction was performed at 40V, 50V and
121
60V for 1, 2, 3, 4, 5, 6, 7 and 8 min, respectively. And the temperature was continuously
122
monitored during the study.
123
2.2.5. Ultrasound assisted microwave extraction (UAME):
124
In this, the TPP was subjected to sonication as pretreatment without any pH change though,
125
the solid to liquid ratio was maintained as 1:50 g/ml similar to the previously studied work by
126
[25]. As pretreatment, sonication was carried out at power levels of 450, 600 and 750 W for
127
time durations of 2, 4, 6, 8, 10, 12, 14 and 16 min with pulsation of 50% (30 s ON: 30 s
128
OFF). Following this the pH of the sample was adjusted to 1.5 using 0.5 N HCl which was
129
subsequently subjected to microwave extraction for 4 min at power level 540 W, with
130
continuous monitoring of temperature.
131
2.2.6. Ultrasound assisted Ohmic heating extraction (UAOHE):
132
The sonication as pretreatment was performed as mentioned in the method (2.2.5.) followed
133
by ohmic heating for 5 min at 60 V. And the temperature was continuously monitored.
134
2.2.7. Purification of extracted pectin:
135
After extraction, pH of solution was adjusted to 3.5 using 0.5 N NaOH. Supernatant of the
136
solution was separated by centrifugation at 5000 rpm for 20 min (10C). To this clear
137
solution, three volumes of ethanol (96%) was added and kept undisturbed for 8-10h at room
138
temperature to precipitate the dissolved pectin. This was further separated by centrifugation
139
at 5000 rpm for 20 min (10C). The extracted pectin was purified by washing it three times
140
with ethanol (95%) and dried at 50oC up to constant weight [16]. This purified sample was
141
further used for characterization of pectin.
142
Pectin yield (%), was estimated as ratio of dried extracted pectin mass (Mp) obtained after
143
extraction to the initial mass of tomato peel powder (Mw) used for extraction [1].
144
𝑀𝑝
𝑃𝑒𝑐𝑡𝑖𝑛 𝑦𝑖𝑒𝑙𝑑 (%) = 𝑀 × 100 𝑤
(1)
145
2.3. Study of extraction kinetics:
146
Extraction of pectin from plant cell structure goes in two transformations during the process:
147
(i) conversion of in-soluble pectin (protopectin) as soluble pectin and subsequent diffusion of
148
this from plant cell to solvent (dissolution rate constant, K1), (ii) degradation of partially
149
dissolved pectin during the extraction process (degradation rate constant, K2) [19].
150
Maximum extractable pectin % (Y0) was obtained by analysis of extract obtained following
151
three repeated continuous extractions from the same sample. However, yields for all the
152
extraction process were recorded and study of extraction kinetics was performed using
153
MATLAB R2019a. First-order two step rate equation was fitted to understand the extraction
154
kinetics of pectin for all extraction techniques used [26].
155 𝑌𝑎
𝐾1
= (𝐾 ― 𝐾 ) × (exp ( ― 𝐾1𝑡) ― exp ( ― 𝐾2𝑡)) 𝑌0 2 1
156
(2)
157 158
where,
159
𝑌𝑎 = Yield of pectin (%)
160
𝑌0 = Maximum extractable pectin (%)
161
K1 = Dissolution rate constant (min-1)
162
K2 = Degradation rate constant (min-1)
163
t = actual extraction time (min)
164 165 166
2.4. Methods for evaluation of qualitative parameters: 1) Estimation of degree of esterification:
167
Titrimetric method as described by [24], was used for the determination of degree of
168
esterification (DE) and results were compared for all the extracted pectin.
169
2) Galacturonic acid content analysis:
170
Determination of GalA content of the extracted pectin was done by the spectrophotometric
171
method [27]. Results were compared for all the extracted pectin.
172
3) Pectin color determination:
173
The color of all extracted pectin was measured with calibrated Hunter Color Lab colorimeter
174
(Model: Color Quest XE, USA). These were expressed as L (Lightness: 0 as black, 100 as
175
white), a (-a for greenness, +a for redness) and b (-b for blueness, +b for yellowness) values.
176
4) FT-IR spectrum:
177
Fourier transform infrared spectroscopy (FT-IR) was used for characterization of purified
178
pectin structure. IR spectra was collected using FT-IR spectrophotometer, PerkinElmer, USA
179
[Spectrum two]. Spectra was used for structural analysis of pectin and curves were produced
180
at resolution of 4 cm-1 with 16 scans ranging from 400 to 4000 cm-1.
181
5) Scanning electron microscopy:
182
Morphological observations of the residue after extraction were obtained by Scanning
183
Electron Microscopy. Samples were mounted on SEM specimen stub along double-side tape
184
prior to coating. Microscopy was carried out with (Model: VEGA3 TESCAN, Czech
185
Republic) system at 10 kV accelerating voltage with 1.00 KX magnification.
186
6) Data analysis:
187
All experiments were done in three replicates and Minitab Express 2018 used for analysis of
188
variance.
189
3. Results and discussion:
190
3.1. Extraction kinetics:
191
Extraction process are governed by mass transfer principles and isolation of pectin is a
192
function of: (a) Extraction through direct interaction of cell matrix with the wave, leading to a
193
release of target compound in the cool solvent; (b) Heating of a solvent to its boiling point,
194
leading to dissolution of the target compound [28]. For better understanding of extraction
195
process and to determine the effect of parameters, kinetics of extraction was performed using
196
first order two step kinetic model. The two constants used to explain the extraction process
197
were, dissolution rate constant (K1) and degradation rate constant (K2) as given in equation 2.
198
However, the predicted extraction time was calculated using equation 3.
199
3.1.1 UAE:
200
For UAE, (Figure 2 (a)) a maximum yield of 15.21% was obtained at power of 600 W, time
201
8.61 min and temperature 60C. Furthermore, it was observed that, with prolonged extraction
202
time the yield decreased to 14.29% in 16 min for the same power. For extraction at 750 W,
203
the maximum yield attained was 11.75%, whereas at 450 W, the yield obtained was 13.00%
204
which suggests the inadequate power input for efficient extraction. This phenomenon of
205
lower yield at higher power can be explained by the fact that long exposer time and
206
increasing power input causes disintegration of dissolved pectin and creates simpler
207
monosaccharides [29]. Dissolution rate constant (K1) was observed to increase with power,
208
further it was observed that initial rate of increase was higher and found to be 1.1410-1 min-1
209
for 450 W and 1.6210-1 min-1 for 600 W. Though, on further increase in power to 750 W, a
210
low rate of increase in K1 (1.6410-1 min-1) was observed. Degradation rate constant (K2)
211
was also observed to be increasing, 0.79510-1, 0.79810-1 and 1.10710-1 min-1 with power
212
input 450, 600 and 750 W, respectively. This illustrates that higher energy treatment
213
conditions results in accelerated structural breakdown of dissolved pectin side chain. Also,
214
the decrease in extraction yield with increasing power was observed which may be due to
215
higher bubble volume concentration or probably due to formation of cavity bubbles around
216
the tip of probe which could screen and create reduction in the energy transmission into the
217
medium, this is also considered as saturation effect [19].
218
3.1.2 MAE:
219
MAE showed highest yield of 25.42% at 900 W, temperature 88.7C and time 3.34 min
220
among all the extraction process (Table 1). However, on further increase in extraction time
221
for 4, 4.67 and 5.34 min at same power, it caused decline in yield of pectin to 24.97, 24.63
222
and 24.51%, respectively (figure 2 (b)). In addition, maximum yield obtained during MAE at
223
720 and 540 W were 24.45 (88.1C, 4.0 min) and 20.83% (85.3C, 4.0 min), respectively.
224
Thus the efficient extraction time for MAE to achieve the maximum yield at power of 900 W
225
was about 3.34 min. The mechanism behind MAE was the absorption of microwave energy
226
by the system to initiate accelerated dissolution of pectin and on further increasing of
227
extraction time, it provokes the degradation of pectin due to its thermolabile nature. The
228
values for constant K1 and K2 were obtained from equation 2, to study the effect on yield
229
with increasing time and power these were obtained for 540 W, 720 W and 900 W which
230
were K1, 3.11110-1, 3.76510-1 and 5.17510-1 min-1; K2, 0.26510-1, 0.29110-1 and
231
0.312710-1 min-1, respectively. The increasing trend was obtained for dissolution and
232
degradation rate constant with elevating power. For both the constants K1 and K2, the
233
increase was gradual for power level 540 to 720 W, whereas at 900 W it increased
234
drastically; this phenomenon may be due to higher temperature accumulation at elevated
235
power. It must be underlined that, temperature in the system used for MAE increased with the
236
increase in power and it could not be controlled. As reported earlier, microwave causes
237
loosening of cell wall structure and causes cleaving of parenchymal cells [30]. Thus, skin
238
tissues are unfolded up by microwave radiation at a substantial rate which increases the
239
interaction between the solvent and the extracting material. It clearly illustrates that, higher
240
energy promotes the permeation of the pectin though the resultant temperature increase
241
causes degradation of pectin which is undesirable and unavoidable.
242
3.1.3 OHAE:
243
OHAE (Figure 2 (c)) exhibit maximum yield of 10.65% at 60 V after 5.0 min treatment time
244
and attaining temperature 81C. However, the maximum yield obtained at 40 V and 50 V
245
after about 7.0 min (50.2C) and 5.0 min (62.4C) of extraction were 9.30 and 9.60%,
246
respectively. This may be because of better cell permeabilization was attained at 60 V due to
247
higher electric field strength and raised temperature. The dissolution rate K1 were found to be
248
2.0410-1, 2.2410-1 and 2.3910-1 min-1 for 40 V, 50 V and 60 V, respectively. The result
249
shows gradual increase in dissolution rate (K1) with increasing power along, moreover it was
250
found to be directly proportional to extraction yield. During OHAE, degradation rate (K2)
251
increased from 1.4910-1, 1.81810-1 and 1.9410-1 min-1 for 40 V, 50 V and 60 V,
252
respectively. This may be due to gradual increase in the temperature and permeation with
253
treatment time at lower electric field, so that it eliminates the thermal degradation of
254
extracted pectin. It was reported in previous study that, permeation of intercellular material
255
through cell wall increases with increasing electric field strength and creates more effective
256
extraction even at lower temperature [31].
257
3.1.4 UAME:
258
In this case varying ultrasound (US) pretreatment was conducted, followed by microwave
259
treatment at selected power level of 540 W for 4 min. During extraction, maximum yield
260
following UAME was 18.00%
261
temperature was 85.1C (Figure 2 (d)). It was found to be less when compared to yield
262
obtained following MAE alone which was 20.83% at same power of 540 W. This could be
263
due to continuous diffusion of pectin from plant cell during US pretreatment, which further
264
led to hydrolysis of partially dissolved pectin, as it was freely available during microwave
265
heating.
266
However, pectin obtained following UAME with US pretreatment of 600 W for 10.0 min,
267
and 750 W for 8.0 min were 16.70 and 15.25% respectively, with the final temperature of the
268
samples at 87.7C and 86.3oC in the same order.
(US pretreatment: 450 W after 8.0 min), and final
269
Dissolution rate (K1) for UAME were found to be increasing with US pretreatment
270
power input (450 W, 600 W, 750 W) to 1.68710-1, 1.71710-1 and 1.73410-1 min-1.
271
Whereas, higher degradation rate (K2) was obtained for UAME (at US pretreatment of 450
272
W) compared to MAE alone which was 0.54410-1 min-1 and 0.265810-1 min-1,
273
respectively, at same microwave power of 540 W. This shows that, degradation in case of
274
UAME was more as increasing temperature with US pretreatment followed by microwave
275
treatment accelerates the hydrolysis of pectin. Though Bagherian, Ashtiani, Fouladitajar and
276
Mohtashamy [25] observed opposite phenomenon in which UAME proved to give better
277
extraction yield compared to MAE for pectin extraction from grapefruit.
278
3.1.5 UAOHE:
279
In this case varying US pretreatment was conducted, followed by Ohmic heating at selected
280
power level of 60 V for 5 min. The maximum yield of 14.60% was obtained following
281
UAOHE (US pretreatment at 450 W for 10.0 min; Figure 2 (e)). Whereas the temperature
282
increased to 68.9C following UAOHE treatment. Whereas, UAOHE extraction yield for US
283
pretreatment of 600 W, 8.0 min and 750 W, 6.0 min was 13.20% and 11.50%, respectively.
284
This decrease in the yield of pectin with increasing power input of ultrasound may be because
285
of increasing viscosity which can have diminishing ohmic heating effect or due to hydrolysis
286
of pectin with increasing temperature during ohmic heating. Moreover, higher yield was
287
obtained following UAOHE extraction 14.6% compared to OHAE 10.65% at 60 V. This may
288
be because ultrasound disintegrates the peel cuticle and create larger surface area for solute-
289
to-solvent interaction. US pretreatment followed by ohmic heating develops higher electric
290
field strength and increases the temperature which causes accelerated permeation of
291
intercellular materials.
292
The dissolution rate (K1) for UAOHE was 1.1510-1, 1.5610-1 and 1.5910-1 min-1,
293
which was directly proportional to the US pretreatment as it was gradually increased.
294
Whereas degradation rate (K2) were 1.02410-1, 1.0710-1 and 1.07710-1 min-1, which
295
shows an increase in degradation rate kinetics of pectin following UAOHE treatment with
296
increase in US pretreatment. Also decrease in extraction yield at US pretreatment power of
297
600 W and 750 W was observed compared to 450 W, this may be due to higher disintegration
298
of cell cuticle which causes increase in freely available pectin, which can degrade with
299
increasing power.
300
3.1.6 General discussion of extraction technologies
301
The above results illustrates that in UAE, cavitation produced during treatment can
302
directly rupture the cell wall structure and initiate the extraction [32]. Whereas in MAE and
303
OHAE, the solvent gets heated up to dissolve pectin, though the mode of heating varies,
304
which affects the extraction efficiency.
305
The results for all the extraction techniques showed increasing dissolution rate and
306
degradation rate constants with increasing power and temperature. One can notice major
307
difference in dissolution rate of MAE (540 W) and UAME (450 W-US) table 1, however, the
308
difference in their yield was not much. It’s because of longer extraction time in case of
309
UAME, as present research was focused on to study the effect of ultrasound pretreatment
310
over different extraction technologies. Yield in all the extraction process for particular power
311
level increased rapidly with initial increase in treatment time followed by gradual decline in
312
the extraction rate. This decline in extraction rate or increase in degradation rate was due to
313
thermal and high ultrasound intensity degradation of pectin, as observed in the previous
314
studies [19, 29, 33]. Furthermore, the extraction yield is dependent on the rate of dissolution
315
as well as rate of degradation which act opposite to each other in effective yield of extraction.
316
Higher dissolution rate may lead to high extraction yield; where as higher degradation rate
317
leads to lower yield, hence these two factors work simultaneously contrary to each other and
318
together determine the final yield.
319
Also results illustrates that, all the methods which were used for extraction gave the
320
maximum possible yield up to some extent of power level and on further power
321
input/increasing time, the yield was found to decrease. In case of MAE and OHAE this may
322
be because of increase in temperature and thermolabile nature of pectin, which causes
323
hydrolysis of dissolved pectin. Whereas for UAE, increasing power intensity (cavitation)
324
degraded the structure of pectin by rupturing the side chains and converting it into simpler
325
monosaccharides which are not precipitated with alcohol [29]. Lower viscosity and surface
326
tension could be the reason behind the initial increase in pectin extraction during UAE.
327
Similar results were also suggested in previous studies [19]. The results obtained during
328
study indicates that power, temperature and extraction time creates significant influence on
329
the kinetic of extraction.
330
3.2. Characterization of extracted pectin sample:
331
3.2.1 Degree of esterification (DE%):
332
All the extracted pectin from tomato peel waste belonged to high methoxyl pectin, as evident
333
by the degree of esterification (DE) which were higher than 50%, regardless to the method of
334
extraction (Table 2). DE for all the extracted pectin ranged from 59.76 - 76.00%, so these
335
could be used as gelling agent and for other food applications. Also, the lowest DE was
336
obtained for MAE extracted pectin (59.76 %), this may be because of harsh temperature
337
extraction, thus the pectin obtained following MAE would take longer time for gel setting.
338
Whereas UAME had a DE of 73.33 % which was much higher showing that the pectin
339
obtained has good gelling strength. Previous studies [25] reported higher DE values, thus the
340
reduction in DE for all the extracted pectin may be because extraction was performed at
341
lower pH (1.5). Since, the DE results depends up on the nature of extraction material and
342
conditions maintained during extraction. In previous study, Bagherian, Ashtiani, Fouladitajar
343
and Mohtashamy [25] reported the same trend of decrease in DE for MAE. They found
344
comparative decrease in DE was more in MAE to UAME, 79.35 and 82.61%, respectively,
345
for pectin extraction from grapefruit.
346
3.2.2 Galacturonic acid content (GalA):
347
As shown in Table 2, GalA content of all the extracted pectin was much higher than 650 g
348
GalA kg-1 Pectin, thus these polysaccharides extracted can be considered as pectin and can be
349
used as food additive. MAE and UAME extracted pectin were having higher and statistically
350
similar (p0.05) galacturonic acid content among the five-extraction methods used. This
351
indicated that MAE and UAME were able to separate pectin from tomato peel waste more
352
efficiently compared to all the other extraction processes and thus the galacturonic acid
353
content was highest [24]. Also, it supports the lowest yield obtained in case of OHAE since
354
the galacturonic acid content was least for the same. Similar trend of results were reported
355
for tomato extracted pectin in previous study [34].
356
3.2.3 Color of pectin:
357
Color would be important parameter for pectin as it plays major role for acceptance before
358
being used as food additives. Tomato extracted pectin color was predominantly due to the
359
lycopene (as its insoluble in water and ethanol) and other water-insoluble pigments (Table 2).
360
During the extraction process, disintegration of cell wall makes the water-soluble pigments
361
available to get dissolved in solvent. These pigment gets trapped in pectin during the
362
precipitation stage. The values of L*, a* and b* i.e., color measuring parameter indicates the
363
effect of extraction method on the color of the pectin. UAME extracted pectin was having the
364
highest lightness (L*) value and least lycopene color was observed this may be due to US
365
pretreatment which extracts the lycopene to the solvent and followed microwave heating
366
causes destruction of lycopene into fragments like acetone, glyoxal, laevulinic aldehyde and
367
methyl-heptenone [35]. Whereas, UAE and MAE extracted pectin was having higher values
368
for a* and b*, respectively. Compared to all the extraction methods studied the highest
369
lightness value was observed for UAME pectin and hence can be considered as better quality,
370
due to higher consumer acceptability (table 2).
371
3.2.4 Structural analysis:
372
FTIR spectra analysis of extracted pectin from tomato peel waste were done to endorse the
373
identity of extracted pectin and to show the effect of extraction method on structural
374
properties of pectin. It was observed that, spectra of all the extracted pectin exhibited similar
375
transmittance pattern in accordance with commercial pectin spectra (Figure 3). The spectra
376
wavelength range from 950 to 1200 cm-1 are predominantly known as finger print region of
377
carbohydrate as it identifies the presence of major functional groups of polysaccharides [36,
378
37]. The absorption bands observed from 1011 to 1220 cm-1 corresponds to C-O-C glycoside
379
ring bond stretching, O-H bending and stretching of C-O bond in COOH. One can see this
380
region is same for all the pectin, insignificant to method of extraction. The spectra obtained
381
from commercial pectin shows higher intensity peaks which may be due to higher
382
concentration, whereas all other extracted ones were having lower intensities. The region
383
from 890 to 1000 cm-1 represents the low and moderate intensity bands assigned to C-C
384
skeletal vibrations. Whereas, these were only present in commercial pectin and not in
385
extracted pectin. It could be due acidic pH maintained during all the extraction process. The
386
band from 625 to 640 cm-1 are related to lower frequency vibrations of pyranoid ring, i.e., C-
387
C deformation vibration of pectin ring skeletal [1]. Also, the UAE extracted pectin was
388
having the least variation intensity, compared to the pectin obtained from other extraction
389
method. It could be due to cleavage of bonds during the high intensity ultrasound treatment.
390
Thus, the data obtained from FTIR shows similar pattern to that of obtained from commercial
391
pectin and corresponds to presence of esterified form of pectin.
392
3.2.5 Scanning Electron Microscopic Imaging:
393
The images produced by Scanning Electron Microscopy (SEM) clearly shows the difference
394
between the mechanism of all the extraction treatments employed. Different morphological
395
changes in the raw material surface after extraction were seen. Figure 4. (A), (B) and (C)
396
images are of UAE, MAE and OHAE residues after extraction, respectively. These all showed
397
significantly different tissue morphology and surface modification. Ultrasound treated tomato
398
peel sample (A) was having porous and loosened structure (shown in 1 and 2) which may be
399
created due to rupture of cavitation bubble. Encircled portion 1 and 2 shows deep holes on
400
the surface which indicates the targeted force of microjet produced during rupture of bubble.
401
Whereas, microwave treated tomato peel sample showed uneven surface, could be due to
402
microwave targeted strikes during the treatment. The highlighted portion on (B) as 3 and 4,
403
illustrates the area of depression and hump creation during microwave treatment. Ohmic
404
heating treated sample was having the least effect compared to UAE and MAE, which clearly
405
explains the inefficient extraction of pectin from the sample. Encircled area in (C) 5 and 6,
406
the lines around the depression indicates the permeation of intercellular material during
407
extraction process. In both the combination treatments, (figure 4. (D) and (E)) the effect of
408
ultrasound as well as following extraction method are visible. The UAME created more
409
disintegrated structure as one can see in encircled portion of (D) 7 and 8, which could be due
410
to combined effect of UAE and MAE. Whereas, UAOHE residues (figure 4. (E)) seems to
411
have depressions (due to cavitation) on the surface (indicated as 9) and those lines (region 10)
412
around them may represent the permeation (due to electric field strength) of intercellular
413
matter. This could be explained as Ultrasound and microwave has more effect on the
414
microstructure and proved to give better yield than ohmic heated, so the resultant
415
combination effect of both caused more disintegration.
416
4. Conclusion:
417
This work was done to create a new approach towards the valorization of tomato processing
418
waste. Different eco-friendly extraction technologies were used and their kinetics of
419
extraction were studied. All the results illustrated that, lower energy ultrasound followed with
420
microwave extraction can give better results in terms of quantity as well as quality. And also,
421
pectin extracted from tomato peel can be used as food additive as it fulfills all the standard
422
requirements.
423 424
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530 531
B
C
E
D
F
532 Tomato processing waste (TPP) Drying of tomato peel at 50C up to constant weight
MAE
UAE
OHAE
UAOHE
UAME
Centrifuged to separate supernatant Three parts of ethanol (96%) was added and kept undisturbed for 8 h. Precipitate was separated and washed three times with ethanol (95%). Dried at 50C up to constant weight.
MAE
UAME
UAE
UAOHE
Figure 1: Process flow chart for all the extraction methods.
OHAE
533 534
Figure 2: Effect of different extraction method on the yield of pectin are plotted. (a) UAE, (b) MAE, (c) OHAE, (d) UAME and (e) UAOHE.
535
536 537 538
Figure 3: FTIR spectra of pectin obtained from different extraction methods.
A
B
1
3
2
4
C
D
7
5
8
6
E
F
10 9
Figure 4: SEM images of tomato peel residue (A) UAE (B) MAE (C) OHAE (D) UAME (E) UAOHE and (F) untreated TPP.
Table 1: Kinetic parameters for extraction of pectin from tomato peel using different extraction methods
Extraction method
Po wer leve l
Dissolu tion rate (K1) (10-1 min-1)
Degrada tion rate (K2) (10-1 min-1)
Extrac ted pectin (%)
Actu al extra ction time (min)
Tempera ture of extractio n (C)
R2
RMS E
UAE
45 0 W 60 0 W 75 0 W
1.14
0.795
13.0 0
10.0 0
60.0
0. 95
0.03 08
1.62
0.796
15.2 1
8.00
60.0
0. 96
0.03 14
1.64
1.107
11.7 5
6.00
60.0
0. 93
0.03 20
1.68
0.544
18.0 0
8.00
85.1
0. 99
0.01 72
1.71
0.724
16.7 0
10.0 0
87.7
0. 94
0.03 97
1.73
0.828
15.2 5
8.00
86.3
0. 94
0.03 69
1.15
1.024
14.6 0
10.0 0
68.9
0. 91
0.03 39
UAM E*
UAOH E*
US (45 0 W) M W (54 0 W) US (60 0 W) M W (54 0 W) US (75 0 W) M W (54 0 W) US (45 0 W) O H (60
V) US (60 0 W) O H (60 V) US (75 0 W) O H (60 V)
1.56
1.070
13.2 0
8.00
72.1
0. 87
0.05 36
1.59
1.077
11.5 0
6.00
77.3
0. 92
0.04 17
Other Technologies
MAE
OHAE
54 0 W 72 0 W 90 0 W 40 V 50 V 60 V
3.11
0.265
20.8 3
4.00
85.3
0. 99
0.02 63
3.76
0.291
24.4 5
4.00
88.1
0. 97
0.04 48
5.17
0.312
25.4 2
3.34
88.7
0. 98
0.03 90
2.04
1.498
9.30
7.00
50.2
2.24
1.818
9.60
5.00
62.4
2.39
1.943
10.6 5
5.00
81.0
0. 91 0. 91 0. 92
0.03 07 0.03 02 0.02 71
* Extraction temperature mentioned in these methods are of followed microwave and ohmic heating technique.
Table 2: GalA content, DE% and color of extracted pectin for all the extracted pectin. Extraction method
Extraction conditions Pow er
UAE
600 W
MAE
900 W
OHAE
60 V
Time (t) (min) 8.00
GalA1
DE2
Color L*
a*
b*
825.0±22. 63b
66.43±1. 45c
54.92±0. 36c
13.49±0. 04a
24.22±0. 56a
3.34
911.7±18. 24a
59.76±0. 70d
50.96±0. 49d
11.07±0. 22c
24.9±0.6 6a
5.00
675.8±11. 31d
74.33±0. 74b
58.65±0. 35b
5.49±0.1 6e
19.87±0. 18c
US US 913.3±20. 73.33±1. 67.22±0. (450 (8.00 50a 76b 00a W) ) MW MW (540 (4.00 W) ) UAOHE US US 779.3±12. 76.00±0. 47.6±0.4 (450 (10.0 58c 88a 9e W) 0) OH OH (60 (5 V) min) 1 All the values are in g kg-1 of pectin. 2 Values are in percentage. Values are mean ± standard deviation (n=3). Values with different letters in same column differ significantly.
8.58±1.8 7d
21.72±0. 04b
12.69±0. 05b
23.28±0. 40a
UAME
Graphical Abstract
Highlights 1) Valorization of tomato processing waste using innovative technologies 2) UAME gave highest dissolution rate and lower degradation rate compared to UAOHE and UAE 3) UAME gave the best quality pectin with high DE % , GalA content and least colour. 4) Highest extraction yield of pectin was obtained with MAE
S1350-4177(19)30860-0 https://doi.org/10.1016/j.ultsonch.2019.104812 ULTSON 104812
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
3 June 2019 24 September 2019 27 September 2019
Please cite this article as: A.S. Sengar, A. Rawson, M. Muthiah, S. Kumar Kalakandan, Comparison of different ultrasound assisted extraction techniques for pectin from tomato processing waste, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.104812
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1
Comparison of different ultrasound assisted extraction techniques for pectin from
2
tomato processing waste Animesh Singh Sengar, Ashish Rawson*, Manimekalai Muthiah, Suresh Kumar Kalakandan Indian Institute of Food Processing Technology Thanjavur- 613 005, Tamil Nadu, India. Corresponding author: Dr Ashish Rawson, e-mail: [email protected], [email protected]
3 4
Abstract
5
Concept of waste to wealth is a hot topic with research ongoing globally to reduce carbon
6
footprint. In an effort to follow up this cause present study focused on tomato industry waste
7
specifically the peel of tomatoes for extraction of pectin. Pectin extraction was performed
8
using five different extraction techniques (Ultrasound assisted extraction (UAE); microwave
9
assisted extraction (MAE); ohmic heating assisted extraction (OHAE); ultrasound assisted
10
microwave extraction (UAME) and ultrasound assisted ohmic heating extraction (UAOHE) at
11
different power levels to study its extraction and degradation kinetics and in turn to optimize
12
the extraction process. The extracted pectin yield ranged from 9.30% for OHAE to 25.42 %
13
for MAE. Also, there was very less difference in the yield of MAE and UAME extracted
14
pectin, but at the cost of major difference in degree of esterification 59.76±0.70 and
15
73.33±1.76 %, respectively. In addition, all the pectin extracted under optimized conditions
16
was having acceptable purity, [Galacturonic acid (GalA) content ranged from 675.8±11.31 to
17
913.3±20.50 g/kg of pectin]. FTIR analysis confirmed the presence of functional groups in
18
the finger print region of identification for polysaccharide in all the extracted pectin.
19
According to obtained results, UAME can be considered as better green extraction
20
technology in terms of extraction yield as well as in quality of pectin compared to the other
21
treatments used. Therefore, results suggest that UAME can be used as an efficient pectin
22
extraction method from tomato processing waste.
23 24 25
Keywords: Ultrasonication, Microwave, Ohmic heating, extraction, Pectin.
26
1. Introduction
27
Tomato (Solanum lycopersicum) is widely consumed crop in the world as raw and in
28
processed forms [1]. According to FAOSTAT (2017)[2], total world production of tomato
29
accounted to be 18,23,01,395 tonnes. Out of this significant volume is processed yearly to
30
create products like tomato juice, paste, puree, ketchup and sauce for human utilization,
31
generating huge amounts of wastes from the food processing sector. Products which remain
32
after pressing of tomato majorly includes 27% peel, 33% seed and furthermore, 40% part of
33
pomace [3]. Tomato waste are usually disposed and/or utilized for animal feed or as manure
34
[4]. The waste produced along these food processing industries are viewed as the rich well-
35
spring of functional components like pectin, lycopene, and essential oils and so forth.
36
Moreover, they are low-cost raw materials and hence been under research in current years. In
37
recent research, tomato byproducts were used as ingredients for newly developed extruded
38
product [5].
39
The primary plant cell of fruits and vegetables is made out of polysaccharide like pectin,
40
hemicellulose and cellulose. The word Pectin is derived from a Greek word "Pektikos" which
41
implies “Coagulant” and is one of the most interesting functional component due to its
42
solubility and polyanionic nature [6]. It is majorly found in the portion of primary cell wall,
43
middle lamella and also in secondary cell walls of plant. Conventionally, pectin is isolated
44
from various food waste produced in food processing industries including citrus peel (85%),
45
apple pomace (14%) and sugar beet (<1%) [7].
46
Pectin is a heteropolysaccharide which is mainly composed of polymer of -galacturonic
47
acid (GalA) (not less than 65% according to standards for food grade pectin defined under
48
EU guideline no. 231/2012) which is an isomer of D-glucuronic acid (D-GlcA). Pectin,
49
according to the International numbering system has been defined as a food additive (Code
50
no. 440) and has no recommended maximum level for addition (limited to use lowest
51
possible level to achieve the desired effect). In terms of the degree of methylation (DM), it
52
can be very well defined into two categories with their applications as high methoxyl pectin
53
(DM 50%) and low methoxyl pectin (DM < 50%) [8]. High-methoxyl pectin can form gel
54
when heated in sugar solution having solution concentration higher than 55Brix and pH
55
maintained lower than 3.5. Though, low-methoxyl pectin can also form gel in the of presence
56
calcium ions. Pectin with higher degree of methylation are favored for food products for
57
achieving the desired consistency and structure in jam, jellies, etc. Whereas, low-methoxyl
58
pectin are used as fat replacer in ice creams, heat reversible bakery glazing agent, emulsified
59
meat products, low calorie beverages and also in fruit preparation for yoghurt [9].
60
Traditionally, pectin is isolated from citrus fruit peel with techniques utilizing
61
acidified water which are not only time and energy consuming, but also give poor yield and
62
lead to the destruction of pectin structure. Though, novel techniques like MAE, UAE, OHAE
63
and combination of these were used and has been demonstrated to give better extraction
64
yields to overcome the limitations of conventional extraction method. Ultrasonication has
65
always proved to be a better method for extraction, modification and blanching, which
66
creates new opportunities to produce bioactive compounds [10-14].
67
In addition, pectin isolation highlights the multiple stage physicochemical process which are
68
influenced by parameters, for example, temperature, pH, extraction time, solvent, solid/liquid
69
ratio and number of extractions. As the extraction of pectin at industrial scale majorly relies
70
upon citrus fruit peels and apple pomace, hence newer potential sources for pectin extraction
71
are continuously being explored. Yang et al. [15] isolated pectin from potato pulp, similarly
72
various researchers [16-21] studied the pectin extraction from grapefruit. Likewise, de
73
Oliveira et al. [22] and Liew et al. [23] utilized passion fruit for isolating pectin. Also Yang et
74
al. [24] investigated that, sisal waste can be utilized for the extraction of pectin by
75
combination of enzymatic and ultrasonic method.
76
Despite the fact that, pectin is present in the majority of plant cells, however, the source used
77
for pectin extraction are mainly restricted to apple pomace and citrus fruit peel at commercial
78
scale. Owing to the fact that conventional extraction can lead to wastage of large amount of
79
solvents, energy, time and generation of carbon footprints. There has been an increased
80
demand as well as focus on green novel technologies for extraction of important metabolites.
81
Hence, the objective of this research were to establish new source and enhanced extraction
82
technique utilizing different novel technologies (UAE; MAE; OHAE; UAME and UAOHE)
83
for pectin extraction and furthermore to consider the impact of these techniques on the yield
84
of pectin and on its characteristic properties.
85
2. Materials and methods:
86
2.1. Materials:
87
Fresh tomato (Solanum lycopersicum) were procured from local market, Thanjavur, India.
88
These were steam blanched (100oC for 15 min) and peel were removed manually. Tomato
89
peels were dried at 60C up to constant moisture level and were milled using electronic
90
pulverizer (Pulverisette 14, FRITSCH, Germany) to maintain the particle size 600 microns
91
by passing it through mesh sieve. Subsequently, the tomato peel powder (TPP) was vacuum
92
packed to avert moisture absorption.
93
2.2. Extraction and purification:
94
Parameters for extraction were studied in the initial trials as well those referred from
95
literature [19, 25] and the best proven ones were used for further extraction of pectin. The
96
details of which is discussed below.
97
2.2.1. Sample preparation for extraction:
98
Dried TPP was conditioned with the distilled water with solid-to-liquid ratio of 1:50 g/mL (to
99
avoid initial saturation of solvent during extraction process). Further pH of this solution was
100
maintained to 1.5 using 0.5 N HCl (extracting agent). Temperature of the solution was
101
monitored throughout extraction process [19, 25]. Figure 1 shows process flow chart for all
102
the extraction methods.
103
2.2.2. Ultrasound assisted extraction (UAE):
104
Ultrasound was applied using horn-type probe system (Sonic CV334, 220 V, 50/60 Hz). The
105
system with specification: power output 750 W, 20 KHz and having probe tip diameter 19
106
mm, with 12 mm immersed in solvent. Sonication was conducted at constant temperature of
107
60C on three power inputs 450 W, 600 W and 750 W. Treatment time was increased with
108
constant interval from 2, 4, 6, 8, 10, 12, 14 and 16 min with pulsation of 50% (30 s ON: 30 s
109
OFF).
110
2.2.3. Microwave extraction:
111
System used for extraction was household microwave system (900 W/ 2450 MHz) which
112
allows it to work at three different power 540, 720 and 900 W. Sample treatment time for
113
extraction was chosen with equal break of interims (0.667 min ie. 40 s) starting from 0.667,
114
1.334, 2, 2.667, 3.334, 4, 4.667 and 5.334 min. And the temperature was continuously
115
monitored during the study.
116
2.2.4. Ohmic heating assisted extraction (OHAE):
117
The extraction process in ohmic heating was performed using cuboidal chamber (80 80
118
30) mm3 made from acrylic sheet (5 mm thick). The stainless steel plates having thickness 1
119
mm and area 2291 mm2 were used as electrodes. To investigate the changes in yield with
120
increasing power level and treatment time, the extraction was performed at 40V, 50V and
121
60V for 1, 2, 3, 4, 5, 6, 7 and 8 min, respectively. And the temperature was continuously
122
monitored during the study.
123
2.2.5. Ultrasound assisted microwave extraction (UAME):
124
In this, the TPP was subjected to sonication as pretreatment without any pH change though,
125
the solid to liquid ratio was maintained as 1:50 g/ml similar to the previously studied work by
126
[25]. As pretreatment, sonication was carried out at power levels of 450, 600 and 750 W for
127
time durations of 2, 4, 6, 8, 10, 12, 14 and 16 min with pulsation of 50% (30 s ON: 30 s
128
OFF). Following this the pH of the sample was adjusted to 1.5 using 0.5 N HCl which was
129
subsequently subjected to microwave extraction for 4 min at power level 540 W, with
130
continuous monitoring of temperature.
131
2.2.6. Ultrasound assisted Ohmic heating extraction (UAOHE):
132
The sonication as pretreatment was performed as mentioned in the method (2.2.5.) followed
133
by ohmic heating for 5 min at 60 V. And the temperature was continuously monitored.
134
2.2.7. Purification of extracted pectin:
135
After extraction, pH of solution was adjusted to 3.5 using 0.5 N NaOH. Supernatant of the
136
solution was separated by centrifugation at 5000 rpm for 20 min (10C). To this clear
137
solution, three volumes of ethanol (96%) was added and kept undisturbed for 8-10h at room
138
temperature to precipitate the dissolved pectin. This was further separated by centrifugation
139
at 5000 rpm for 20 min (10C). The extracted pectin was purified by washing it three times
140
with ethanol (95%) and dried at 50oC up to constant weight [16]. This purified sample was
141
further used for characterization of pectin.
142
Pectin yield (%), was estimated as ratio of dried extracted pectin mass (Mp) obtained after
143
extraction to the initial mass of tomato peel powder (Mw) used for extraction [1].
144
𝑀𝑝
𝑃𝑒𝑐𝑡𝑖𝑛 𝑦𝑖𝑒𝑙𝑑 (%) = 𝑀 × 100 𝑤
(1)
145
2.3. Study of extraction kinetics:
146
Extraction of pectin from plant cell structure goes in two transformations during the process:
147
(i) conversion of in-soluble pectin (protopectin) as soluble pectin and subsequent diffusion of
148
this from plant cell to solvent (dissolution rate constant, K1), (ii) degradation of partially
149
dissolved pectin during the extraction process (degradation rate constant, K2) [19].
150
Maximum extractable pectin % (Y0) was obtained by analysis of extract obtained following
151
three repeated continuous extractions from the same sample. However, yields for all the
152
extraction process were recorded and study of extraction kinetics was performed using
153
MATLAB R2019a. First-order two step rate equation was fitted to understand the extraction
154
kinetics of pectin for all extraction techniques used [26].
155 𝑌𝑎
𝐾1
= (𝐾 ― 𝐾 ) × (exp ( ― 𝐾1𝑡) ― exp ( ― 𝐾2𝑡)) 𝑌0 2 1
156
(2)
157 158
where,
159
𝑌𝑎 = Yield of pectin (%)
160
𝑌0 = Maximum extractable pectin (%)
161
K1 = Dissolution rate constant (min-1)
162
K2 = Degradation rate constant (min-1)
163
t = actual extraction time (min)
164 165 166
2.4. Methods for evaluation of qualitative parameters: 1) Estimation of degree of esterification:
167
Titrimetric method as described by [24], was used for the determination of degree of
168
esterification (DE) and results were compared for all the extracted pectin.
169
2) Galacturonic acid content analysis:
170
Determination of GalA content of the extracted pectin was done by the spectrophotometric
171
method [27]. Results were compared for all the extracted pectin.
172
3) Pectin color determination:
173
The color of all extracted pectin was measured with calibrated Hunter Color Lab colorimeter
174
(Model: Color Quest XE, USA). These were expressed as L (Lightness: 0 as black, 100 as
175
white), a (-a for greenness, +a for redness) and b (-b for blueness, +b for yellowness) values.
176
4) FT-IR spectrum:
177
Fourier transform infrared spectroscopy (FT-IR) was used for characterization of purified
178
pectin structure. IR spectra was collected using FT-IR spectrophotometer, PerkinElmer, USA
179
[Spectrum two]. Spectra was used for structural analysis of pectin and curves were produced
180
at resolution of 4 cm-1 with 16 scans ranging from 400 to 4000 cm-1.
181
5) Scanning electron microscopy:
182
Morphological observations of the residue after extraction were obtained by Scanning
183
Electron Microscopy. Samples were mounted on SEM specimen stub along double-side tape
184
prior to coating. Microscopy was carried out with (Model: VEGA3 TESCAN, Czech
185
Republic) system at 10 kV accelerating voltage with 1.00 KX magnification.
186
6) Data analysis:
187
All experiments were done in three replicates and Minitab Express 2018 used for analysis of
188
variance.
189
3. Results and discussion:
190
3.1. Extraction kinetics:
191
Extraction process are governed by mass transfer principles and isolation of pectin is a
192
function of: (a) Extraction through direct interaction of cell matrix with the wave, leading to a
193
release of target compound in the cool solvent; (b) Heating of a solvent to its boiling point,
194
leading to dissolution of the target compound [28]. For better understanding of extraction
195
process and to determine the effect of parameters, kinetics of extraction was performed using
196
first order two step kinetic model. The two constants used to explain the extraction process
197
were, dissolution rate constant (K1) and degradation rate constant (K2) as given in equation 2.
198
However, the predicted extraction time was calculated using equation 3.
199
3.1.1 UAE:
200
For UAE, (Figure 2 (a)) a maximum yield of 15.21% was obtained at power of 600 W, time
201
8.61 min and temperature 60C. Furthermore, it was observed that, with prolonged extraction
202
time the yield decreased to 14.29% in 16 min for the same power. For extraction at 750 W,
203
the maximum yield attained was 11.75%, whereas at 450 W, the yield obtained was 13.00%
204
which suggests the inadequate power input for efficient extraction. This phenomenon of
205
lower yield at higher power can be explained by the fact that long exposer time and
206
increasing power input causes disintegration of dissolved pectin and creates simpler
207
monosaccharides [29]. Dissolution rate constant (K1) was observed to increase with power,
208
further it was observed that initial rate of increase was higher and found to be 1.1410-1 min-1
209
for 450 W and 1.6210-1 min-1 for 600 W. Though, on further increase in power to 750 W, a
210
low rate of increase in K1 (1.6410-1 min-1) was observed. Degradation rate constant (K2)
211
was also observed to be increasing, 0.79510-1, 0.79810-1 and 1.10710-1 min-1 with power
212
input 450, 600 and 750 W, respectively. This illustrates that higher energy treatment
213
conditions results in accelerated structural breakdown of dissolved pectin side chain. Also,
214
the decrease in extraction yield with increasing power was observed which may be due to
215
higher bubble volume concentration or probably due to formation of cavity bubbles around
216
the tip of probe which could screen and create reduction in the energy transmission into the
217
medium, this is also considered as saturation effect [19].
218
3.1.2 MAE:
219
MAE showed highest yield of 25.42% at 900 W, temperature 88.7C and time 3.34 min
220
among all the extraction process (Table 1). However, on further increase in extraction time
221
for 4, 4.67 and 5.34 min at same power, it caused decline in yield of pectin to 24.97, 24.63
222
and 24.51%, respectively (figure 2 (b)). In addition, maximum yield obtained during MAE at
223
720 and 540 W were 24.45 (88.1C, 4.0 min) and 20.83% (85.3C, 4.0 min), respectively.
224
Thus the efficient extraction time for MAE to achieve the maximum yield at power of 900 W
225
was about 3.34 min. The mechanism behind MAE was the absorption of microwave energy
226
by the system to initiate accelerated dissolution of pectin and on further increasing of
227
extraction time, it provokes the degradation of pectin due to its thermolabile nature. The
228
values for constant K1 and K2 were obtained from equation 2, to study the effect on yield
229
with increasing time and power these were obtained for 540 W, 720 W and 900 W which
230
were K1, 3.11110-1, 3.76510-1 and 5.17510-1 min-1; K2, 0.26510-1, 0.29110-1 and
231
0.312710-1 min-1, respectively. The increasing trend was obtained for dissolution and
232
degradation rate constant with elevating power. For both the constants K1 and K2, the
233
increase was gradual for power level 540 to 720 W, whereas at 900 W it increased
234
drastically; this phenomenon may be due to higher temperature accumulation at elevated
235
power. It must be underlined that, temperature in the system used for MAE increased with the
236
increase in power and it could not be controlled. As reported earlier, microwave causes
237
loosening of cell wall structure and causes cleaving of parenchymal cells [30]. Thus, skin
238
tissues are unfolded up by microwave radiation at a substantial rate which increases the
239
interaction between the solvent and the extracting material. It clearly illustrates that, higher
240
energy promotes the permeation of the pectin though the resultant temperature increase
241
causes degradation of pectin which is undesirable and unavoidable.
242
3.1.3 OHAE:
243
OHAE (Figure 2 (c)) exhibit maximum yield of 10.65% at 60 V after 5.0 min treatment time
244
and attaining temperature 81C. However, the maximum yield obtained at 40 V and 50 V
245
after about 7.0 min (50.2C) and 5.0 min (62.4C) of extraction were 9.30 and 9.60%,
246
respectively. This may be because of better cell permeabilization was attained at 60 V due to
247
higher electric field strength and raised temperature. The dissolution rate K1 were found to be
248
2.0410-1, 2.2410-1 and 2.3910-1 min-1 for 40 V, 50 V and 60 V, respectively. The result
249
shows gradual increase in dissolution rate (K1) with increasing power along, moreover it was
250
found to be directly proportional to extraction yield. During OHAE, degradation rate (K2)
251
increased from 1.4910-1, 1.81810-1 and 1.9410-1 min-1 for 40 V, 50 V and 60 V,
252
respectively. This may be due to gradual increase in the temperature and permeation with
253
treatment time at lower electric field, so that it eliminates the thermal degradation of
254
extracted pectin. It was reported in previous study that, permeation of intercellular material
255
through cell wall increases with increasing electric field strength and creates more effective
256
extraction even at lower temperature [31].
257
3.1.4 UAME:
258
In this case varying ultrasound (US) pretreatment was conducted, followed by microwave
259
treatment at selected power level of 540 W for 4 min. During extraction, maximum yield
260
following UAME was 18.00%
261
temperature was 85.1C (Figure 2 (d)). It was found to be less when compared to yield
262
obtained following MAE alone which was 20.83% at same power of 540 W. This could be
263
due to continuous diffusion of pectin from plant cell during US pretreatment, which further
264
led to hydrolysis of partially dissolved pectin, as it was freely available during microwave
265
heating.
266
However, pectin obtained following UAME with US pretreatment of 600 W for 10.0 min,
267
and 750 W for 8.0 min were 16.70 and 15.25% respectively, with the final temperature of the
268
samples at 87.7C and 86.3oC in the same order.
(US pretreatment: 450 W after 8.0 min), and final
269
Dissolution rate (K1) for UAME were found to be increasing with US pretreatment
270
power input (450 W, 600 W, 750 W) to 1.68710-1, 1.71710-1 and 1.73410-1 min-1.
271
Whereas, higher degradation rate (K2) was obtained for UAME (at US pretreatment of 450
272
W) compared to MAE alone which was 0.54410-1 min-1 and 0.265810-1 min-1,
273
respectively, at same microwave power of 540 W. This shows that, degradation in case of
274
UAME was more as increasing temperature with US pretreatment followed by microwave
275
treatment accelerates the hydrolysis of pectin. Though Bagherian, Ashtiani, Fouladitajar and
276
Mohtashamy [25] observed opposite phenomenon in which UAME proved to give better
277
extraction yield compared to MAE for pectin extraction from grapefruit.
278
3.1.5 UAOHE:
279
In this case varying US pretreatment was conducted, followed by Ohmic heating at selected
280
power level of 60 V for 5 min. The maximum yield of 14.60% was obtained following
281
UAOHE (US pretreatment at 450 W for 10.0 min; Figure 2 (e)). Whereas the temperature
282
increased to 68.9C following UAOHE treatment. Whereas, UAOHE extraction yield for US
283
pretreatment of 600 W, 8.0 min and 750 W, 6.0 min was 13.20% and 11.50%, respectively.
284
This decrease in the yield of pectin with increasing power input of ultrasound may be because
285
of increasing viscosity which can have diminishing ohmic heating effect or due to hydrolysis
286
of pectin with increasing temperature during ohmic heating. Moreover, higher yield was
287
obtained following UAOHE extraction 14.6% compared to OHAE 10.65% at 60 V. This may
288
be because ultrasound disintegrates the peel cuticle and create larger surface area for solute-
289
to-solvent interaction. US pretreatment followed by ohmic heating develops higher electric
290
field strength and increases the temperature which causes accelerated permeation of
291
intercellular materials.
292
The dissolution rate (K1) for UAOHE was 1.1510-1, 1.5610-1 and 1.5910-1 min-1,
293
which was directly proportional to the US pretreatment as it was gradually increased.
294
Whereas degradation rate (K2) were 1.02410-1, 1.0710-1 and 1.07710-1 min-1, which
295
shows an increase in degradation rate kinetics of pectin following UAOHE treatment with
296
increase in US pretreatment. Also decrease in extraction yield at US pretreatment power of
297
600 W and 750 W was observed compared to 450 W, this may be due to higher disintegration
298
of cell cuticle which causes increase in freely available pectin, which can degrade with
299
increasing power.
300
3.1.6 General discussion of extraction technologies
301
The above results illustrates that in UAE, cavitation produced during treatment can
302
directly rupture the cell wall structure and initiate the extraction [32]. Whereas in MAE and
303
OHAE, the solvent gets heated up to dissolve pectin, though the mode of heating varies,
304
which affects the extraction efficiency.
305
The results for all the extraction techniques showed increasing dissolution rate and
306
degradation rate constants with increasing power and temperature. One can notice major
307
difference in dissolution rate of MAE (540 W) and UAME (450 W-US) table 1, however, the
308
difference in their yield was not much. It’s because of longer extraction time in case of
309
UAME, as present research was focused on to study the effect of ultrasound pretreatment
310
over different extraction technologies. Yield in all the extraction process for particular power
311
level increased rapidly with initial increase in treatment time followed by gradual decline in
312
the extraction rate. This decline in extraction rate or increase in degradation rate was due to
313
thermal and high ultrasound intensity degradation of pectin, as observed in the previous
314
studies [19, 29, 33]. Furthermore, the extraction yield is dependent on the rate of dissolution
315
as well as rate of degradation which act opposite to each other in effective yield of extraction.
316
Higher dissolution rate may lead to high extraction yield; where as higher degradation rate
317
leads to lower yield, hence these two factors work simultaneously contrary to each other and
318
together determine the final yield.
319
Also results illustrates that, all the methods which were used for extraction gave the
320
maximum possible yield up to some extent of power level and on further power
321
input/increasing time, the yield was found to decrease. In case of MAE and OHAE this may
322
be because of increase in temperature and thermolabile nature of pectin, which causes
323
hydrolysis of dissolved pectin. Whereas for UAE, increasing power intensity (cavitation)
324
degraded the structure of pectin by rupturing the side chains and converting it into simpler
325
monosaccharides which are not precipitated with alcohol [29]. Lower viscosity and surface
326
tension could be the reason behind the initial increase in pectin extraction during UAE.
327
Similar results were also suggested in previous studies [19]. The results obtained during
328
study indicates that power, temperature and extraction time creates significant influence on
329
the kinetic of extraction.
330
3.2. Characterization of extracted pectin sample:
331
3.2.1 Degree of esterification (DE%):
332
All the extracted pectin from tomato peel waste belonged to high methoxyl pectin, as evident
333
by the degree of esterification (DE) which were higher than 50%, regardless to the method of
334
extraction (Table 2). DE for all the extracted pectin ranged from 59.76 - 76.00%, so these
335
could be used as gelling agent and for other food applications. Also, the lowest DE was
336
obtained for MAE extracted pectin (59.76 %), this may be because of harsh temperature
337
extraction, thus the pectin obtained following MAE would take longer time for gel setting.
338
Whereas UAME had a DE of 73.33 % which was much higher showing that the pectin
339
obtained has good gelling strength. Previous studies [25] reported higher DE values, thus the
340
reduction in DE for all the extracted pectin may be because extraction was performed at
341
lower pH (1.5). Since, the DE results depends up on the nature of extraction material and
342
conditions maintained during extraction. In previous study, Bagherian, Ashtiani, Fouladitajar
343
and Mohtashamy [25] reported the same trend of decrease in DE for MAE. They found
344
comparative decrease in DE was more in MAE to UAME, 79.35 and 82.61%, respectively,
345
for pectin extraction from grapefruit.
346
3.2.2 Galacturonic acid content (GalA):
347
As shown in Table 2, GalA content of all the extracted pectin was much higher than 650 g
348
GalA kg-1 Pectin, thus these polysaccharides extracted can be considered as pectin and can be
349
used as food additive. MAE and UAME extracted pectin were having higher and statistically
350
similar (p0.05) galacturonic acid content among the five-extraction methods used. This
351
indicated that MAE and UAME were able to separate pectin from tomato peel waste more
352
efficiently compared to all the other extraction processes and thus the galacturonic acid
353
content was highest [24]. Also, it supports the lowest yield obtained in case of OHAE since
354
the galacturonic acid content was least for the same. Similar trend of results were reported
355
for tomato extracted pectin in previous study [34].
356
3.2.3 Color of pectin:
357
Color would be important parameter for pectin as it plays major role for acceptance before
358
being used as food additives. Tomato extracted pectin color was predominantly due to the
359
lycopene (as its insoluble in water and ethanol) and other water-insoluble pigments (Table 2).
360
During the extraction process, disintegration of cell wall makes the water-soluble pigments
361
available to get dissolved in solvent. These pigment gets trapped in pectin during the
362
precipitation stage. The values of L*, a* and b* i.e., color measuring parameter indicates the
363
effect of extraction method on the color of the pectin. UAME extracted pectin was having the
364
highest lightness (L*) value and least lycopene color was observed this may be due to US
365
pretreatment which extracts the lycopene to the solvent and followed microwave heating
366
causes destruction of lycopene into fragments like acetone, glyoxal, laevulinic aldehyde and
367
methyl-heptenone [35]. Whereas, UAE and MAE extracted pectin was having higher values
368
for a* and b*, respectively. Compared to all the extraction methods studied the highest
369
lightness value was observed for UAME pectin and hence can be considered as better quality,
370
due to higher consumer acceptability (table 2).
371
3.2.4 Structural analysis:
372
FTIR spectra analysis of extracted pectin from tomato peel waste were done to endorse the
373
identity of extracted pectin and to show the effect of extraction method on structural
374
properties of pectin. It was observed that, spectra of all the extracted pectin exhibited similar
375
transmittance pattern in accordance with commercial pectin spectra (Figure 3). The spectra
376
wavelength range from 950 to 1200 cm-1 are predominantly known as finger print region of
377
carbohydrate as it identifies the presence of major functional groups of polysaccharides [36,
378
37]. The absorption bands observed from 1011 to 1220 cm-1 corresponds to C-O-C glycoside
379
ring bond stretching, O-H bending and stretching of C-O bond in COOH. One can see this
380
region is same for all the pectin, insignificant to method of extraction. The spectra obtained
381
from commercial pectin shows higher intensity peaks which may be due to higher
382
concentration, whereas all other extracted ones were having lower intensities. The region
383
from 890 to 1000 cm-1 represents the low and moderate intensity bands assigned to C-C
384
skeletal vibrations. Whereas, these were only present in commercial pectin and not in
385
extracted pectin. It could be due acidic pH maintained during all the extraction process. The
386
band from 625 to 640 cm-1 are related to lower frequency vibrations of pyranoid ring, i.e., C-
387
C deformation vibration of pectin ring skeletal [1]. Also, the UAE extracted pectin was
388
having the least variation intensity, compared to the pectin obtained from other extraction
389
method. It could be due to cleavage of bonds during the high intensity ultrasound treatment.
390
Thus, the data obtained from FTIR shows similar pattern to that of obtained from commercial
391
pectin and corresponds to presence of esterified form of pectin.
392
3.2.5 Scanning Electron Microscopic Imaging:
393
The images produced by Scanning Electron Microscopy (SEM) clearly shows the difference
394
between the mechanism of all the extraction treatments employed. Different morphological
395
changes in the raw material surface after extraction were seen. Figure 4. (A), (B) and (C)
396
images are of UAE, MAE and OHAE residues after extraction, respectively. These all showed
397
significantly different tissue morphology and surface modification. Ultrasound treated tomato
398
peel sample (A) was having porous and loosened structure (shown in 1 and 2) which may be
399
created due to rupture of cavitation bubble. Encircled portion 1 and 2 shows deep holes on
400
the surface which indicates the targeted force of microjet produced during rupture of bubble.
401
Whereas, microwave treated tomato peel sample showed uneven surface, could be due to
402
microwave targeted strikes during the treatment. The highlighted portion on (B) as 3 and 4,
403
illustrates the area of depression and hump creation during microwave treatment. Ohmic
404
heating treated sample was having the least effect compared to UAE and MAE, which clearly
405
explains the inefficient extraction of pectin from the sample. Encircled area in (C) 5 and 6,
406
the lines around the depression indicates the permeation of intercellular material during
407
extraction process. In both the combination treatments, (figure 4. (D) and (E)) the effect of
408
ultrasound as well as following extraction method are visible. The UAME created more
409
disintegrated structure as one can see in encircled portion of (D) 7 and 8, which could be due
410
to combined effect of UAE and MAE. Whereas, UAOHE residues (figure 4. (E)) seems to
411
have depressions (due to cavitation) on the surface (indicated as 9) and those lines (region 10)
412
around them may represent the permeation (due to electric field strength) of intercellular
413
matter. This could be explained as Ultrasound and microwave has more effect on the
414
microstructure and proved to give better yield than ohmic heated, so the resultant
415
combination effect of both caused more disintegration.
416
4. Conclusion:
417
This work was done to create a new approach towards the valorization of tomato processing
418
waste. Different eco-friendly extraction technologies were used and their kinetics of
419
extraction were studied. All the results illustrated that, lower energy ultrasound followed with
420
microwave extraction can give better results in terms of quantity as well as quality. And also,
421
pectin extracted from tomato peel can be used as food additive as it fulfills all the standard
422
requirements.
423 424
References:
425
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530 531
B
C
E
D
F
532 Tomato processing waste (TPP) Drying of tomato peel at 50C up to constant weight
MAE
UAE
OHAE
UAOHE
UAME
Centrifuged to separate supernatant Three parts of ethanol (96%) was added and kept undisturbed for 8 h. Precipitate was separated and washed three times with ethanol (95%). Dried at 50C up to constant weight.
MAE
UAME
UAE
UAOHE
Figure 1: Process flow chart for all the extraction methods.
OHAE
533 534
Figure 2: Effect of different extraction method on the yield of pectin are plotted. (a) UAE, (b) MAE, (c) OHAE, (d) UAME and (e) UAOHE.
535
536 537 538
Figure 3: FTIR spectra of pectin obtained from different extraction methods.
A
B
1
3
2
4
C
D
7
5
8
6
E
F
10 9
Figure 4: SEM images of tomato peel residue (A) UAE (B) MAE (C) OHAE (D) UAME (E) UAOHE and (F) untreated TPP.
Table 1: Kinetic parameters for extraction of pectin from tomato peel using different extraction methods
Extraction method
Po wer leve l
Dissolu tion rate (K1) (10-1 min-1)
Degrada tion rate (K2) (10-1 min-1)
Extrac ted pectin (%)
Actu al extra ction time (min)
Tempera ture of extractio n (C)
R2
RMS E
UAE
45 0 W 60 0 W 75 0 W
1.14
0.795
13.0 0
10.0 0
60.0
0. 95
0.03 08
1.62
0.796
15.2 1
8.00
60.0
0. 96
0.03 14
1.64
1.107
11.7 5
6.00
60.0
0. 93
0.03 20
1.68
0.544
18.0 0
8.00
85.1
0. 99
0.01 72
1.71
0.724
16.7 0
10.0 0
87.7
0. 94
0.03 97
1.73
0.828
15.2 5
8.00
86.3
0. 94
0.03 69
1.15
1.024
14.6 0
10.0 0
68.9
0. 91
0.03 39
UAM E*
UAOH E*
US (45 0 W) M W (54 0 W) US (60 0 W) M W (54 0 W) US (75 0 W) M W (54 0 W) US (45 0 W) O H (60
V) US (60 0 W) O H (60 V) US (75 0 W) O H (60 V)
1.56
1.070
13.2 0
8.00
72.1
0. 87
0.05 36
1.59
1.077
11.5 0
6.00
77.3
0. 92
0.04 17
Other Technologies
MAE
OHAE
54 0 W 72 0 W 90 0 W 40 V 50 V 60 V
3.11
0.265
20.8 3
4.00
85.3
0. 99
0.02 63
3.76
0.291
24.4 5
4.00
88.1
0. 97
0.04 48
5.17
0.312
25.4 2
3.34
88.7
0. 98
0.03 90
2.04
1.498
9.30
7.00
50.2
2.24
1.818
9.60
5.00
62.4
2.39
1.943
10.6 5
5.00
81.0
0. 91 0. 91 0. 92
0.03 07 0.03 02 0.02 71
* Extraction temperature mentioned in these methods are of followed microwave and ohmic heating technique.
Table 2: GalA content, DE% and color of extracted pectin for all the extracted pectin. Extraction method
Extraction conditions Pow er
UAE
600 W
MAE
900 W
OHAE
60 V
Time (t) (min) 8.00
GalA1
DE2
Color L*
a*
b*
825.0±22. 63b
66.43±1. 45c
54.92±0. 36c
13.49±0. 04a
24.22±0. 56a
3.34
911.7±18. 24a
59.76±0. 70d
50.96±0. 49d
11.07±0. 22c
24.9±0.6 6a
5.00
675.8±11. 31d
74.33±0. 74b
58.65±0. 35b
5.49±0.1 6e
19.87±0. 18c
US US 913.3±20. 73.33±1. 67.22±0. (450 (8.00 50a 76b 00a W) ) MW MW (540 (4.00 W) ) UAOHE US US 779.3±12. 76.00±0. 47.6±0.4 (450 (10.0 58c 88a 9e W) 0) OH OH (60 (5 V) min) 1 All the values are in g kg-1 of pectin. 2 Values are in percentage. Values are mean ± standard deviation (n=3). Values with different letters in same column differ significantly.
8.58±1.8 7d
21.72±0. 04b
12.69±0. 05b
23.28±0. 40a
UAME
Graphical Abstract
Highlights 1) Valorization of tomato processing waste using innovative technologies 2) UAME gave highest dissolution rate and lower degradation rate compared to UAOHE and UAE 3) UAME gave the best quality pectin with high DE % , GalA content and least colour. 4) Highest extraction yield of pectin was obtained with MAE