Ultrasonics - Sonochemistry 42 (2018) 805–813

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Ultrasonics - Sonochemistry
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Ultrasound assisted enzymatic degumming of crude soybean oil T

⁎
Nishant S. More, Parag R. Gogate
Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India

A R T I C L E I N F O A B S T R A C T

Keywords: The present work deals with ultrasound assisted enzymatic degumming (UAED) of crude soybean oil quantifying
Enzymatic degumming the extent of degumming (EOD), cavitational yield and synergistic index (f) for the combination approaches. The
Ultrasound effect of different operating parameters such as enzyme loading, pH, presence of water, temperature and ul-
Crude soybean oil trasonic power on the EOD has been investigated. Ultrasound combined with enzyme at loading of 2.0 ml/L
Phospholipase
resulted in EOD as 92.2% under ambient conditions. Addition of water (5%) in combination with ultrasound and
Phospholipids
enzyme at 2.0 ml/L loading and pH of 5 resulted in maximum EOD (98.4%) in 120 min of treatment. The extent
of phospholipid separation was also observed to be dependent on the power dissipation and maximum phos-
pholipids separation was obtained at 100 W. Scale-up studies were performed at 500 ml and 1 L operating vo-
lume under optimized conditions of 2.0 ml/L as the enzyme loading, pH of 5, 5% water addition and ultrasonic
power of 100 W where 93.63% and 91.15% phospholipid separation respectively was obtained. The effects of
ultrasonic treatment were also quantified in terms of the acid value reduction and oxidative stability for the
processed oil. It was demonstrated that suitable reduction in acid value (final value less than 1) and oxidative
stability (TOTOX less than 4) is effectively obtained using UAED. Overall the approach of UAED was established
to show much higher efficacy for soybean oil processing as compared to only ultrasound or only enzymatic
treatment.

1. Introduction phospholipids can be separated by intense oxidation essentially aimed

India is the second largest soybean oil producer in Asia and accounts
for 3.95 percent soybean oil production all over the world. Soybean oil
consumption rate is also increasing consistently due to number of ap-
plications in domestic and commercial sectors. Soybean oil is widely
used in food, pharmaceutical, and cosmetic industries as well as for
biodiesel production. Soybean oil is extracted from soya seeds using the
solvent extraction method and further purified using different sequen-
tial refining operations consisting of degumming, neutralization,
bleaching, dewaxing, decolourization and deodorization. Among all
these steps, degumming is an important step aimed at separating major
quantum of phospholipids and mucilaginous gums from crude soybean
oil. Conventional degumming approaches are generally classified into
two categories as chemical and physical processes. Chemical degum-
ming approach includes use of acid and base whereas physical de-
gumming approach includes mainly the membrane degumming.
Crude soybean oil contains phospholipids which may further clas-
sified into hydratable (lecithin) and non-hydratable phospholipids
(calcium and magnesium salt as well as phosphatidic acid). Hydratable
matter can be easily separated by simply water addition which is also
the most common industrially applied method. Non-hydratable
to convert the non-hydratable matter into hydratable so that it can be
easily separated from the crude oil in the next processing step. Acid
degumming is a commonly applied approach based on addition of
substantial amount of phosphoric acid, citric acid or oxalic acid to
oxidize the non-hydratable phospholipids and further separate the same
using water addition. Within course of reaction, reverse miscella can be
produced from association of monovalent ions which majorly affects
the efficacy of degumming process. Hence, it is preferred to perform
alkali degumming with the addition of sodium hydroxide as compared
to acid degumming which helps in separating the phospholipids from
crude oil more effectively. Addition of sodium hydroxide may however
reduce the efficacy of the main degumming purpose by causing adverse
reaction of saponification and hence the loading of sodium hydroxide
used need to be critically decided. A review of existing literature on
acid/alkali based degumming (mostly considered as the conventional
routes for processing) and subsequent applications has been presented
to establish the importance of present work.
Albert and Martin [1] investigated total degumming approach based
on acid-base treatment of crude soybean oil and reported that the re-
sidual phosphorous concentration can be reduced to below 5 ppm.
McDonnell et al. [2] performed experiments to drive diesel engine with

⁎
Corresponding author.
E-mail address: pr.gogate@ictmumbai.edu.in (P.R. Gogate).

https://doi.org/10.1016/j.ultsonch.2017.12.031
Received 18 October 2017; Received in revised form 14 December 2017; Accepted 15 December 2017
Available online 16 December 2017
1350-4177/ © 2017 Elsevier B.V. All rights reserved.
N.S. More, P.R. Gogate
the blended fuel of diesel and degummed oil using hot water and re-
ported better fuel efficiency as compared to the diesel fuel, though the
actual value was dependent on the blending oil proportions. Tasan et al.
[3] evaluated the effects of solvent extraction method and storage
condition on EOD and reported that the degumming treatment resulted
in better quality and stability characteristics of oil along with drastic
separation of the phosphorous and metal content. Use of alkali based
refining approach was also reported to reduce the acid value from 2.82
to 0.52 in addition to the main objective of reducing the phospholipids
concentration by removing free fatty acids and intermediates formed
during the refining process [4,5]. Analysis of the literature revealed that
though EOD was significant, the diverse effects of addition of acidic and
basic reagents for achievement of desired degumming yield also caused
undesirable product changes in fatty acid composition and also resulted
in the generation of waste material such as soaps, esters, gums etc.
Membrane degumming was proposed as an effective alternative to
the chemical oxidation based routes. Membrane based degumming was
investigated by Lin and Rhee [6] and it was reported that maximum
phospholipid separation was obtained at 26.81 L/m2 h flux, 40 °C
temperature and 300 psi pressure. Alicieo et al. [7,8] evaluated the
effect of temperature and pressure on the degree of separation using
ceramic membrane and hollow fibre membrane and reported 99% and
73% phosphorous removal from crude soybean oil for the ceramic and
hollow fibre membranes respectively at optimum conditions of 50 °C
and 6.0 bar. Cross flow microfiltration degumming approach was also
reported to give desired characteristics for crude sunflower oil based on
the use of 0.5 μm and 0.2 μm alumina membrane [9]. Koris and Marki
[10,11] performed pre-ultrafiltration followed by ceramic membrane
based separation at 2 bar feed pressure which was reported to yield
97% phospholipid separation. Paula et al. [12] investigated degumming
for oil/hexane miscella by the application of multi-channel ceramic
membrane at 2.9–3.9 tangential velocity and 1–2 bar transmembrane
pressure and 99.7% phospholipid separation was reported. From the
analysis of overviewed membrane degumming approaches, it has been
observed that crude oil with high phospholipid concentration can also
cause constraints in the operation such as lower oil handling capacity,
higher processing time, requirement of additional reagent etc. Apart
from this, membrane based approaches commonly offered fouling,
chocking and clogging problems which can again extend the processing
time, cost and maintenance.
An alternative to membrane degumming or the chemical oxidation
based degumming is the enzymatic degumming approach. Clausen [13]
investigated use of phospholipase A1 for degumming of mediated ve-
getable oils and reported that complete degumming was obtained at
50 °C and pH of 5 within 2 h treatment. Jiang et al. [14,15] studied
degumming of different vegetable oils using phospholipase type-B and
reported that the phosphorous content could be reduced to below 5 mg/
kg within 5 h at 40 °C. Yu et al. [16,17] compared degumming approach
based on free enzyme and immobilized enzyme supported with mag-
netic nanoparticles and reported significant reduction in phospholipid
concentration within 7 h and 50 °C under optimized conditions. Sam-
paio et al. [18] studied the degumming of crude soybean oil and ra-
peseed oil using phospholipase A1 and reported that degumming effi-
ciency depends on composition of the crude oil though for most of the
cases, residual oil with less than 10 ppm phospholipid concentration
could be obtained. Though the processing using enzymes gives better
results for EOD, it was also established from the analysis that higher
treatment times are required and hence it is imperative to use some
process intensification approach. From the analysis of literature for
various conventional degumming approaches, it was also established
that each approach has its own limitations and disadvantages which
provides scope for research. It is important to consider all the opera-
tional constraints and override the demerits of processing approaches to
develop a new protocol that can give maximum degumming yield,
minimum maintenance and better handling capacity for oil processing.
The present work aims at investigating the efficacy of cavitation in
806
Ultrasonics - Sonochemistry 42 (2018) 805–813
combination with enzyme as an improved approach for degumming of
crude oil.
Cavitation based approaches have been reported to intensify che-
mical processing including the oxidation reactions and the separations
[19]. One of the major effects of cavitation is the release of significant
magnitude of energy and generation of turbulence [20] locally which
can reduce the mass transfer effects playing a major limiting role in
enzymatic reactions. Cavitation assisted enzymatic approaches have
also been reported to give effective results for various processes in
terms of better productivity and reduced processing time [21,22],
though limited studies can be seen for enhancing the effectiveness of
degumming.
Ultrasound assisted enzymatic degumming (UAED) approach was
studied by Jiang et al. [23] depicting the understanding into the effect
of pH, temperature, water addition, power intensity and treatment
time. It was reported that less than 10 ppm residual phospholipid was
obtained at optimum condition of pH 5, 50 °C and 2.5% of water ad-
dition in 7 h treatment time. In our earlier work, ultrasonic assisted
degumming protocol for treatment of crude soybean oil was established
[28] with intensification studies using various oxidizing agents like
hydrogen peroxide and ozone gas. It was reported that complete
phospholipid separation can be obtained at laboratory scale using ul-
trasound with intensification benefits in terms of reduced reaction time
and processing cost. Apart from these studies, no other studies were
observed for intensified degumming of soybean oil based on the use of
ultrasound/enzyme and hence the importance of the current work is
established. The present work deals with the application of ultrasound
in combination with enzyme for intensification of the degumming of
crude soybean oil. The effect of various parameters like ultrasonic
power, temperature, pH, enzyme loading and water addition on the
EOD has also been investigated. The progress of degumming was
monitored by measuring the change in total phosphorous content. The
acid value, peroxide value and p-anisidine value were also character-
ized to check the possible positive effects of ultrasonic treatment.
2. Materials and methods
2.1. Materials
Crude soybean oil was obtained from Maharashtra Oil Extraction
Private Limited, Dhule, Maharashtra (India) and carefully stored in
dark place without exposure to light. Phospholipase A (Lecitase ultra)
was procured from Brenntag Ingredients (India) Private Limited,
Gurgaon (India). Analytical and lab grade chemicals like sodium mo-
lybdate, hydrazine sulphate, potassium hydroxide, sulphuric acid, po-
tassium dihydrogen orthophosphate, hydrochloric acid, zinc oxide, 2-2-
4 trimethylpentane, glacial acetic acid, p-anisidine, potassium iodide,
sodium thiosulphate, sodium dodecyl sulphate and potassium dichro-
mate were procured from SD Fine Chemicals Pvt. Ltd., Mumbai (India).
All the chemicals were received from authentic chemicals supplier in
Mumbai and used as received from the supplier.
2.2. Experimental set-up
Ultrasonic horn obtained from M/S Dakshin, Mumbai (India) and
operated over power dissipation range of 20–120 W and fixed fre-
quency of 20 kHz has been used in the study. A schematic representa-
tion of the experimental set-up based on ultrasonic horn is shown in
Fig. 1. Due to probability of enzyme denaturation, duty cycle was kept
constant at 50% (5-s ON and 5-s OFF) throughout the experimental
work so as to give a buffer for enzyme. The processing temperature was
controlled using water bath and the reactor was provided with mag-
netic stirrer to achieve uniform mixing. For only enzymatic treatment,
same setup was used without the immersion of ultrasonic horn into the
reactor.
N.S. More, P.R. Gogate
2.3. Experimental methodology
UAED experiments were performed mostly in 250 ml glass reactor
and also some using 500-ml and 1-L glass reactors to establish the scale-
up aspects. Determination of phospholipid concentration, acid value,
peroxide value and p-anisidine value of the received crude oil sample
were initially performed to establish the initial conditions. Analysis was
done using AOCS methods as discussed in details later.
Initially, effect of enzyme loading was investigated by using dif-
ferent loadings of 0.5, 1.0, 1.5, 2.0, 3.0, 5.0 ml/L at ambient tempera-
ture controlled with water bath. The optimized enzyme loading value
was then used to study the effect of pH by performing experiments at
varying pH as 6.5, 6.0, 5.5, 5.0, 4.0 and 2.0. 0.1 N sulphuric acid was
used to adjust the pH of crude oil sample. Effect of addition of water
was studied at 1, 2, 3, 4 and 5% loading using double distilled water as
the additive. Effect of temperature on UAED was studied using different
temperatures as 40 °C, 50 °C, 60 °C, 70 °C and 80 °C controlled using the
water bath. At fixed frequency of ultrasound as 20 kHz, effect of power
dissipation was studied using 20 W, 40 W, 60 W, 80 W and 100 W as the
ultrasonic power under the previously optimized conditions. Samples
were collected at every 10 min of interval and the reaction in the
sample was stopped by heating the sample for 5 min at 80 °C.
Subsequently, clear oil was obtained after centrifugation step in which
precipitated phospholipids get separated. Reproducibility of the results
was checked by repeating each experiment three times and reported
data is the average value. The experimental errors were observed to be
in the range of 1–2%. For further analysis of the oil, samples were
stored in a refrigerator for avoiding the direct exposure to light.
2.4. Analytical methods
2.4.1. Phospholipid analysis
Phospholipid concentration was measured in terms of total phos-
phorous content using the sodium molybdate dye formation method
(AOCS Ca 12-15) based on the analysis using UV spectrophotometer
(Thermofisher 2600) at 650 nm [6,24]. Phospholipid percentage was
calculated from multiplication of total phosphorous content by 30 as
the conversion factor as per the details mentioned in earlier work [25].
2.4.2. Acid value
Determination of free fatty acids was performed using the neu-
tralization of acids by the alcoholic potassium hydroxide based on
AOCS Ca 5a-40 method [26]. Acid value results were obtained in the
form of mg of KOH required to neutralize one gram of oil.
807
Ultrasonics - Sonochemistry 42 (2018) 805–813
Fig. 1. Schematic representation of experimental set up for
enzymatic degumming using ultrasonic horn.
2.4.3. Peroxide value
Peroxide value was determined using the acetic acid-isooctane
method (AOCS Cd 8b-90) in terms of milliequivalents of peroxide per
kilogram of test sample [27]. The method is commonly used in oil
processing industries.
2.4.4. P-anisidine value
The determination of p-anisidine value was performed using the
glacial acetic acid based on the p-anisidine dye formation method
(AOCS Cd 18-90) involving the quantification using UV spectro-
photometer at 350 nm [28]. This method determines the oxidative
stability of processed oil.
3. Results and discussion
3.1. Effect of enzyme loading on extent of degumming using ultrasound
Effect of enzyme loading on EOD using the ultrasonic horn was
studied at different loadings of 0.5, 1.0, 1.5, 2.0, 3.0, 5.0 ml/L at 34 °C
of temperature and 6.75 of pH. The obtained results for the final EOD
have been given in Table 1 whereas the graphical representation of the
trends in variation in the EOD with time at different loadings is de-
picted in Fig. 2. It can be seen from the Fig. 2 that the EOD after
120 min increased from 86.2% at 0.5 ml/L of enzyme loading to
91.52% at 2.0 ml/L enzyme loading. Significant changes in EOD were
not observed for a further increase in the enzyme loading beyond
2.0 ml/L. The actual EOD observed were 93.45% and 94.14% at loading
of 3.0 ml/L and 5.0 ml/L respectively. With the addition of phospholi-
pase A at higher loading beyond 2.0 ml/L, it can be said that the con-
trolling mechanism no longer remains as the limited availability of
enzyme and hence only marginal changes are obtained. Considering
these results, 2.0 ml/L enzyme loading was considered as an optimum
value and used in further experiments related to understanding the
effect of pH. The comparative studies of EOD using conventional me-
chanical stirring based approach (82.04%) revealed better yield for the
approach of combination of enzyme with ultrasound. From an earlier
study [25] where the use of only ultrasound for degumming was in-
vestigated at 120 W power dissipation, it was again established that
combination of ultrasound with enzyme was a superior approach as
individual operation of ultrasound resulted in 81.12% as the extent of
degumming. The enhancement in degumming rate in the presence of
ultrasound was attributed due to facilitated dispersion of phospholipase
A in the oil medium due to the turbulence generated by ultrasound [28]
and also the presence of enzymes provides catalytic action to drive the
EOD to a higher extent.
N.S. More, P.R. Gogate Ultrasonics - Sonochemistry 42 (2018) 805–813

Table 1 3.2. Effect of pH for combined approach of ultrasound and enzyme

Comparison of different approaches in terms of EOD.
Effect of pH on ultrasonic degumming in the presence of enzyme
Approaches Parameter Reaction time EOD (%)
range was studied at pH of 6.5, 6.0 5.5, 5.0, 4.5, 4.0 and 2.0. The obtained
results for the effect of pH on the EOD have been given in Fig. 3. It was
Combined US and Enzyme at 0.5 ml/L 120 min 86.20 observed that EOD increased marginally for a pH change from 6.5 to
different loading 1.0 ml/L 120 min 90.73
5.5. At pH below 5.5, significant changes in EOD were observed over
1.5 ml/L 120 min 91.52
2.0 ml/L 120 min 92.23 the range of 92.24 to 95.21% with maximum EOD at pH of 2. It is also
3.0 ml/L 120 min 93.45 important to note that the relative increase in EOD was lower below pH
5.0 ml/L 120 min 94.14 of 5, which was established as optimum. Qu et al. [17] also reported
2.0 ml/L 120 min 82.47 that maximum phospholipids removal was obtained at pH of 5 and
Without US
60 °C temperature using phospholipase A2. Jiang et al. [14] studied
Combined US and Enzyme (2.0 ml/L 6.5 120 min 91.32 enzymatic degumming using phospholipase B extracted from Pseudo-
loading) at different pH 6.0 120 min 91.48
monas fluorescence BIT-18 and reported that phospholipid retention was
5.5 120 min 92.17
5.0 120 min 93.24 below 5 ppm at optimum pH of 5.2 within 6 h. The pH of oil sample was
4.5 120 min 93.72 varied by using 0.1 N H2SO4 solution which can cause detrimental ef-
4.0 120 min 94.84 fects on final quality of oil and hence significant acidic conditions
2.0 120 min 95.21 should be avoided. The effect of pH is typically based on enhancement
Combined US/Enzyme (2.0 ml/L as 1% 120 min 92.37 in ionization rate and availability of more active sites on enzyme sur-
loading/5 as pH, ambient 2% 120 min 93.97 face [29]. The pH favourably influences the interaction between oil
temperature as 34 °C) at 3% 120 min 95.44
miscella and enzyme giving increased performance in terms of de-
different Water addition 4% 120 min 96.72
5% 120 min 98.38 gumming rate. Sadeghi [30] studied purification of soybean oil using
phospholipase A1 and reported that repulsive conditions occurred
Combined US/Enzyme (2.0 ml/L as 40 °C 120 min 96.62
loading/5 as pH, Water 50 °C 120 min 96.78 within intermolecular bonding of non-hydratable phospholipids at pH
addition at 5%) at different 60 °C 120 min 96.85 of 4.8 which effectively converted the non-hydratable phospholipids
Temperature 70 °C 120 min 96.88 into the water soluble ones. Based on the obtained results in the present
80 °C 120 min 96.94 work and presented analysis in terms of effect of pH, less pH adjustment
Combined US/Enzyme (2.0 ml/L as 20 W 120 min 85.49 was considered as optimized protocol and pH of 5 was used for further
loading/5 as pH, Water 40 W 120 min 89.58 experimental investigations as substantial increase in EOD with rela-
addition at 5%) at different 60 W 120 min 96.71
tively less addition of H2SO4 was observed at pH of 5.
Power 80 W 120 min 98.41
100 W 120 min 98.38

Scale-up studies 2.0 ml/L

500 ml Enzyme
3.3. Effect of addition of water
1000 ml loading 120 min 93.63
5.0 pH 120 min 91.15 Effect of addition of water for enzymatic degumming in combina-
5% water tion with ultrasound was investigated at 1, 2, 3, 4 and 5% loadings.
addition
Addition of water in the presence of ultrasound generates enhanced
34 °C
temperature hydroxyl radicals, which can contribute to the enhanced EOD. Also the
100 W US intensity of cavitation is expected to higher as compared to only oil due
power to suitable changes in the physicochemical properties of the liquid
medium. Presence of water is also likely to enhance the activity of
US-Ultrasound.
enzyme. The obtained results for the EOD at different water content
have been shown in Fig. 4. It has been observed that an increase in

100 Fig. 2. Effect of enzyme loading on EOD.

90

80
EXTENT OF DEGUMMING (%)

70

60
0.5 ml/L

50 1.0 ml/L

1.5 ml/L
40
2.0 ml/L
30 3.0 ml/L

5.0 ml/L
20
Without US
10
0 20 40 60 80 100 120
TIME (MIN)

808
N.S. More, P.R. Gogate Ultrasonics - Sonochemistry 42 (2018) 805–813

100 Fig. 3. Effect of pH on EOD.

90
EXTENT OF DEGUMMING (%)

80

70 pH 6.5
pH 6.0
60
pH 5.5
50 pH 5.0
pH 4.5
40
pH 4.0
30 pH 2.0

20
0 20 40 60 80 100 120
TIME (MIN)

water loading from 1% to 2% resulted in an increase in EOD from 92%
to 94%. Further increase in water loading from 3 to 5%, resulted in an
increase in EOD from 95% to 98%. It can be said that non-hydratable
phospholipids can be more easily separated from crude oil by addition
of water as a source of hydroxyl radicals, which helps in conversion to
hydratable form. Application of ultrasound for degumming of crude
soybean oil in the presence of water produced higher number of hy-
droxyl radicals and higher intensity cavitation due to the presence of
aqueous phase which is the major deciding factor for enhancement in
EOD. Presence of water also suitably affects the activity of the enzyme
also contributing to the enhanced EOD. Enzymatic degumming studies
performed by Jiang et al. [23] reported that maximum phospholipids
separation (final value below 10 ppm) was obtained with 2.5% of water
addition. Also, Dijkstra and Martin demonstrated that total degumming
approach resulted in phospholipid concentration below 5 ppm at 10%
of water loading. Enzymatic degumming studies performed by Jiang
et al. [15] on soybean oil, rapeseed oil, rice bran oil and camellia oil
also established that reduction to final value below 15 ppm was ob-
tained only with water addition at optimized loading. Li et al. [31]
established that the use of immobilized phospholipase A1 with 100 mg/
kg enzyme loading, 60 °C temperature and 2% water addition reduced
phospholipid concentration to below 10 ppm within 180 min of treat-
ment time. Comparison of the obtained results with literature clearly
established the existence of optimum water loading specific to oil being
processed and enzyme establishing the importance of the present work.
5% loading gave the best results in this work and hence it was used for
subsequent experiments related to effect of temperature.
3.4. Effect of temperature on ultrasonic degumming
Effect of temperature (using different values as 40 °C, 50 °C, 60 °C,
70 °C and 80 °C in addition to ambient conditions with temperature as
34 °C) on UAED approach was studied at the optimized 2.0 ml/L of
phospholipase A loading, pH of 5 and 5% as water loading and the
obtained results have been depicted in Fig. 5. It was observed that an
increase in temperature from 40 °C to 80 °C resulted in only slight
change in EOD from 96.62% to 96.94% and this range of EOD was
marginally lower as compared to EOD obtained at ambient temperature
of 34 °C (actual value of 98.38%). Jiang et al. [23] performed enzymatic
degumming studies at different temperatures and reported that max-
imum phospholipid retention (final value below 10 ppm) was observed
at 50 °C. Also, other studies of enzymatic degumming established
maximum EOD at 40 °C [23,30,32]. It is important to note that a spe-
cific enzyme has its own characteristics in terms of optimum catalytic
activity at a specific temperature [30] and perhaps, enzyme could get
denatured or deactivated at higher operating temperature. Based on the
obtained results and presented analysis, it was established that the most
suitable temperature for the highest enzyme activity was the ambient
temperature (34 °C) where maximum EOD as 98.38% was obtained.

100 Fig. 4. Effect of addition of water on EOD.

90

80
EXTENT OF DEGUMMING (%)

70

60 Water 1%

Water 2%
50
Water 3%
40 Water 4%

Water 5%
30

20
0 20 40 60 80 100 120
TIME(MIN)

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N.S. More, P.R. Gogate Ultrasonics - Sonochemistry 42 (2018) 805–813

100 Fig. 5. Effect of temperature on EOD.

90

80
EXTENT OF DEGUMMING (%)

70

60
40C

50 50C

60C
40
70C
30 80C

20
0 20 40 60 80 100 120
TIME(MIN)

3.5. Effect of ultrasonic power on EOD of power dissipation used in the study also affects the trends.

Effect of ultrasonic power on EOD was studied using different ul-

3.6. Scale-up studies
trasonic power as 20 W, 40 W, 60 W, 80 W and 100 W with other con-
stant conditions as 2.0 ml/L of enzyme loading, pH of 5, 5% as the
Scale-up studies for degumming of crude oil were performed using
water content and ambient temperature. Graphical representation of
500-ml and 1-L glass reactors with the optimized parameters as 2.0 ml/
obtained results has been depicted in Fig. 6. It was observed that
L of enzyme loading, pH of 5, 5% water addition and 100 W of ultra-
85–89% phospholipids removal was obtained using over power dis-
sonic power at ambient temperature and the obtained results have been
sipation over the range of 20–40 W. Rapid increase in EOD (96.71%)
depicted in Fig. 7. It was observed that EOD as 93.63% was obtained for
was observed at 60 W power dissipation and further increase in power
500 ml operating volume and 91.15% for 1 L processing within
from 60 to 100 W also resulted in a further increase in the EOD with
120 min. It can be seen that an increase in operating volume resulted in
98.38% phospholipids removal from crude soybean oil obtained at
gradual decrease in phospholipid removal. The demonstrated ultrasonic
100 W. Enhancement in phospholipids removal observed due to an in-
efficacy with optimized values at laboratory scale operation was re-
crease in power dissipation can be explained on the basis of higher
duced at larger operating volume mostly based on uneven distribution
cavitational activity due to production of higher number of bubbles
of cavitational activity when the same ultrasonic horn was applied in
eventually releasing higher energy driving enhanced degumming. Jiang
large scale operation with lower power density (< 50 W/L). Lower
et al. [23] also studied the effect of power intensity on UAED approach
power density means that the cavitational intensity would be lower also
and reported that maximum phospholipid removal with the final value
giving reduced intensification. It has been confirmed at laboratory scale
below 10 ppm was obtained at 0.09 W/cm2 intensity within 7 h treat-
of operation that cavitation based degumming approach could be a
ment. More and Gogate [25] also reported that ultrasonic degumming
better alternative method in comparison with conventional degumming
at 100 W power resulted in maximum EOD as 99% in the presence of
due to the offered advantages in processing. Also, it is certainly better
100 PPM of H2O2. It is important to understand that generally an in-
as compared to only enzymatic degumming considering the possible
crease in the ultrasonic power till an optimum is favourable though in
intensification benefits including enhanced EOD and lower requirement
the current work no such trend was observed confirming that the range
of enzymes for a similar change. It is also important to understand that

100 Fig. 6. Effect of ultrasonic power on EOD.

90

80
EXTENT OF DEGUMMING (%)

70

20 W
60
40 W
50 60 W
80 W
40
100 W
30

20
0 20 40 60 80 100 120
TIME(MIN)

810
N.S. More, P.R. Gogate Ultrasonics - Sonochemistry 42 (2018) 805–813

100 Fig. 7. Scale-up studies for enzymatic degumming at two

higher capacities of 500 ml and 1000 ml.

90
EXTENT OF DEGUMMING (%)

80

70

60

500 ml Batch
50
1000 ml
Batch
40

30
0 20 40 60 80 100 120
TIME (MIN)

UAED approach may not be scaled up to pilot scale using ultrasonic
horn but alternative configurations such as ultrasonic bath or ultrasonic
flow cell can be effective due to higher energy transfer efficiencies.
3.7. Supportive characterization data
Simultaneous effects of UAED approach on the acid value, peroxide
value, p-anisidine value, TOTOX factor and free fatty acid content in the
crude oil were also investigated to evaluate the quality of processed oil
and the obtained results have been given in Table 2. All the char-
acterization methods were as per the AOCS standard protocols re-
commended for oil quality assessment. From Table 2, it was observed
that acid value of treated oil decreased from 1.75 to below 1 in all the
approaches. Also, oxidative stability in terms of peroxide value and p-
anisidine value was observed to be below 1 after treatment. TOTOX is
the overall oxidative stability factor in correlation with both the per-
oxide value and p-anisidine value. As per the assessment protocol AOCS
Cg 3-91, TOTOX value less than 4 would be considered as a better
stability of oil. It was observed that almost all the approaches resulted
in TOTOX value less than 2. Thus the obtained results confirmed the
better quality of the processed oil based on the supportive character-
ization data. With this, it can be said that UAED approach resulted in
the desired degumming yield with simultaneous beneficial effects in
terms of reduction in acid value and better oxidative stability. It is
important to understand that the conventional degumming approaches
require more unit operations and processes to achieve these results.
Therefore, it can be said that improved UAED approach could be eco-
nomically advantageous in achieving the desired quality attributes as
compared to conventional degumming approaches.
3.8. Comparison of cavitational yield and synergistic index for different
approaches
Energy efficiency in terms of cavitational yield (mg of phospholipid
removal per joule of energy supplied) and synergistic index (f) was
calculated for different degumming approaches as per the methods
reported in literature [33–36] and the obtained data has been shown is
Table 3. The sample calculations have been given in Appendix A and B.
It was observed that higher cavitational yield was obtained for ap-
proach of ultrasound combined with enzyme (4.52 × 10−6 mg/J with
1.66 as the synergistic index) in comparison with only ultrasonic
treatment (3.61 × 10−6 mg/J) and conventional mechanical stirring
(2.23 × 10−6 mg/J). Combination of ultrasound and enzyme at opti-
mized pH and combined ultrasound and enzyme with optimum water
addition resulted in cavitational yield of 2.52 × 10−6 mg/J and
5.66 × 10−6 mg/J respectively. Also, maximum synergistic index as
2.34 was observed for the UAED with water addition. Combination of
ultrasound and enzyme with 5% water addition at 100 W power re-
sulted in the highest cavitational yield (5.66 × 10−6 mg/J) and max-
imum EOD (98.38%) as compared to the other degumming approaches.
For scale-up studies, 2.09 × 10−5 mg/L as the cavitational yield was
obtained for 500 ml operating volume under optimized conditions
whereas 1.87 × 10−5 mg/J as the cavitational yield was obtained for
1 L batch volume. The synergistic index and cavitational yield were
negatively affected by an increase in operating volume attributed to the
lower operating power density and un-uniform distribution of the ca-
vitational activity.
4. Conclusions
UAED approach was investigated for removal of phospholipids from
crude soybean oil. Different studies were performed to understand the
effect of enzyme loading, pH, water addition, temperature and ultra-
sonic power. Optimized conditions were established as 2.0 ml/L of
enzyme loading, pH of 5, 5% as the water loading and 100 W of ul-
trasonic power at ambient operating temperature (34 °C). The max-
imum EOD as 98.38% was obtained with 5.66 × 10−6 mg/J of cavita-
tional yield and 2.34 as the synergistic factor under these optimized
conditions. Scale-up studies for 500 ml and 1 L resulted in satisfactory
EOD yield as 93.63% and 91.15% respectively, though it was lesser as

Table 2
Results for acid value, peroxide value and p-anisidine value characterization methods.

Combined Crude oil Conventional US and US and enzyme at US and enzyme with US and enzyme at ambient US and enzyme at
Approach (Stirring) Enzyme optimum pH optimum Water addition Temperature optimum Power

Acid value 1.75 1.28 0.89 1.01 0.72 0.96 0.74

P-Anisidine Value 2.28 0.82 0.705 0.798 0.745 0.79 0.703
Peroxide Value 3.07 0.32 0.28 0.23 0.21 0.21 0.25
TOTOX 8.42 1.46 1.265 1.258 1.165 1.21 1.20

811
N.S. More, P.R. Gogate Ultrasonics - Sonochemistry 42 (2018) 805–813

Table 3
Comparison of EOD, synergistic index and cavitational yield for different approaches.

Scheme EOD (%) Synergistic index (F) Cavitational Yield (Including power required for heating and stirring) (mg/J)

Only Ultrasonic (US) Horn 81.12 – 3.61 × 10−6

Combined US Horn and Enzyme 92.23 1.66 2.52 × 10−6
Combined US Horn and enzyme at optimum pH 93.24 – 2.52 × 10−6
Combined US Horn and enzyme at optimum water 98.38 2.34 5.66 × 10−6
Combined US Horn and enzyme at optimum temperature 96.62 – 2.62 × 10−6
Combined US Horn and enzyme at optimum US power 98.38 1.67 2.52 × 10−6
Scale-up (500 ml) 93.63 1.85 1.09 × 10−5
Scale-up (1 L) 91.15 2.17 1.47 × 10−5

compared to laboratory scale confirming that different reactor config-
uration and optimized conditions of power may be required at large
scale. It was also established that the better quality of processed oil in
terms of acid value (less than 1) and oxidative stability was obtained for
all the approaches. Overall, UAED has been established to be an effi-
cient approach in comparison with only ultrasound or enzymatic pro-
cessing with demonstrated synergistic results. The possibility of scale
up can be based on the use of continuous ultrasonic flow cell and also
ultrasonic bath based on indirect irradiation as ultrasonic horn has
relatively poor scale up propositions.
Acknowledgement
Authors would like to acknowledge University Grants Commission -
Basic Scientific Research (UGC-BSR) scheme, Govt. of India, New Delhi,
India for providing funding to NSM and Maharashtra Oil Extraction
Limited, Dhule for supply of raw material.

Appendix A. Cavitational yield

A.1. Ultrasonic horn

Reaction time: 120 min

Operating volume: 250 ml
US power dissipation: 120 W
Power for centrifuge: 350 W
Power for magnetic Stirrer: 300 W
Total Power dissipation: 770 W
Net reduction in phospholipids= (EOD × Initial phospholipid concentration)/100
= (81.12 × 98.75)/100 = 80.106 mg/L

Power Density= (Total power dissipation × Treatment time in seconds)/Batch volume in L

= (770 × 120 × 60)/0.25 = 22176000 J/L

Cavitational Yield= Net reduction in phospholipids/Power Density

= 80.1/18480000 = 3.61 × 10−1 mg/J

Appendix B. Synergistic index (f)

B.1. Combination of ultrasonic (US) horn and enzyme (loading of 2.0 ml/L)

Rate constant for only ultrasonic horn (kUS) = 0.0005 min−1

Rate constant for only Enzyme = 0.0003 min−1
Rate constant for combined scheme i.e. US and Enzyme (kUS/Enzyme) = 0.005 min−1

Synergistic Index (f) for combined approach of US and Enzyme

= kUS /Enzyme/ kUS + kEnzyme
= 0.005/(0.0005 + 0.0025)
= 1.66

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