Veterinary World, EISSN: 2231-0916
Available at www.veterinaryworld.org/Vol.14/July-2021/08.pdf
RESEARCH ARTICLE
Open Access
A comparative study on the effects of boiling and ultrasonication on
radical scavenging activity, casein particle size, and whiteness of milk
Tanmay Hazra1
, Rohit Sindhav2
, Ch. V. K. Sudheendra3, Mitul Bumbadiya1, Radhika Govani4 and Vimal Ramani3
1. Department of Dairy Chemistry, College of Dairy Science, Kamdhenu University, Amreli, Gujarat, India; 2. Department
of Dairy Technology, College of Dairy Science, Kamdhenu University, Amreli, Gujarat, India; 3. Department of Dairy
Microbiology, College of Dairy Science, Kamdhenu University, Amreli, Gujarat, India; 4. Department of Dairy Business
Management, College of Dairy Science, Kamdhenu University, Amreli, Gujarat, India.
Corresponding author: Tanmay Hazra, e-mail: tanmayhazra08@gmail.com
Co-authors: RS: rohitsindhav7@gmail.com, ChVKS: sudheendracvk@yahoo.co.in, MB: mitulbumbadiya@gmail.com,
RG: govaniradhika@yahoo.com, VR: vimalmramani@gmail.com
Received: 15-03-2021, Accepted: 02-06-2021, Published online: 09-07-2021
doi: www.doi.org/10.14202/vetworld.2021.1784-1787 How to cite this article: Hazra T, Sindhav R, Sudheendra CVK,
Bumbadiya M, Govani R, Ramani V (2021) A comparative study on the effects of boiling and ultrasonication on radical
scavenging activity, casein particle size, and whiteness of milk, Veterinary World, 14(7): 1784-1787.
Abstract
Background and Aim: Different processing treatments affect the functional properties of milk. This study aimed to evaluate
the effects of boiling and ultrasonication on radical scavenging activity, micellar casein particle size, and the whiteness of
milk.
Materials and Methods: Milk was subjected to boiling and ultrasonication treatments. Then, the micellar casein size and
the whiteness of the milk were evaluated using L-Value- intensity of whiteness and the radical scavenging capacity of the
milk was evaluated using 1,1-diphenyl-2-picrylhydrazyl method.
Results: The radical scavenging activity of the milk was found to be reduced during the different processing treatments,
but this decrease was non-significant for the ultrasonication treatment (p>0.05). However, a significant reduction in radical
scavenging activity (p<0.05) was observed for the boiled milk. Micellar casein size reduction was observed after both
treatments, and boiling had a significant effect (p<0.05) on the micellar casein particle size. We found that the whiteness
of skim milk was significantly decreased after boiling treatment, but was not significantly decreased after ultrasonication.
Ultrasonication had a non-significant effect on the whiteness of ultrasonicated milk which was observed.
Conclusion: Ultrasonicated milk had a very non-significant effect on the antioxidant activity (radical scavenging activity)
of milk, whereas the effect of boiling was significant. Ultrasonication treatment increases the shelf-life of milk while
retaining its bioactive properties.
Keywords: antioxidant, radical scavenging activity, skim milk, ultrasonication.
Introduction
Milk is not only a stable source of high-quality
protein, lipids, and carbohydrates but is also a prominent source of various bioactive substances that
are essential for human growth and immunity [1,2].
Worldwide, cow, buffalo, goat, and sheep milk are
widely consumed and used for culinary purposes.
Water is the prominent substance in milk, making
up 80-85% of the total volume. Due to its high water
quantity and the presence of high-quality nutrients,
milk is an ideal medium for complex microbial ecosystems; however, these microbes adversely affect the
quality of milk during storage and processing [3,4].
Due to the highly perishable nature of milk, maintaining milk without changes to its nutritional, safety,
Copyright: Hazra, et al. Open Access. This article is distributed under
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(http://creativecommons.org/licenses/by/4.0/),
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article, unless otherwise stated.
Veterinary World, EISSN: 2231-0916
or functional properties is a challenging task. Heat
treatment (e.g. boiling, pasteurization, and thermization) is widely used to enhance the keeping quality of
milk [4,5]; however, heat treatments directly affect the
functional and nutritional qualities of milk, especially
milk proteins [6].
Milk proteins (casein and whey) play essential
roles in the processing and functional properties of a
variety of dairy products. The antioxidant and radical scavenging activity of milk has been well documented [7], and milk proteins play a significant role
in the antioxidant activity of milk [7,8]. In this current
era, consumers are aware of the nutritional profiles of
foods; therefore, adverse changes in milk due to heat
processing are undesirable. To address these concerns,
milk processors are now seeking novel, non-thermal
processing methods, such as ultrasonication treatment.
During ultrasonication treatment, acoustic
cavitation is produced, and this treatment has been
successfully applied to process milk and dairy products [9]. The effect of ultrasonication on the reduction
of bacterial load has been demonstrated in the previous study [10]. However, Lin et al. [11] observed morphological changes in casein during ultrasonication.
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The effect of ultrasonication on milk antioxidant
activities has not been fully investigated; therefore, in
this study, we aimed to ascertain the effects of boiling
and ultrasonication on the physicochemical and antioxidant properties of milk.
Materials and Methods
Ethical approval
Ethical approval was not necessary for this study
as milk was procured from farmers.
Study period and location
This study was conducted in the month of
September 2020 at College of Dairy Science, Amreli,
Gujarat
Procurement of milk
Cow milk was procured from local farmers in the
Amreli district and was skimmed by centrifugation at
3500 rpm for 45 min at 4°C.
Compositional analysis
The proximate composition of the cow skim milk
(CSM) samples was analyzed using the MilkoScan™
Mars instrument (Foss analytics- Hilleroed, Denmark).
Processing of milk
CSM samples were boiled at 100°C for 15 min.
For ultrasonication treatment, milk samples were sonicated at 20 kHz at 150 W for 10 min (JY92-IIN). The
samples were equilibrated at room temperature (25°C)
for 20 min before measuring the parameters.
Measurement of particle size of CSM
Casein micelle sizes were determined using a
Zetasizer- Malvern Pan analytical- Delhi, India. The
viscosity and refraction index of water were 0.8872
cP and 1.330, respectively. For each sample, the light
scattering measurements were carried out at 25°C.
Measurement of milk whiteness
The degree of change in whiteness was measured
using a Hunter colorimeter (Color Flex EZ colorimeter) and represented as an L* value (representing
lightness). The instrument was standardized with a
standard reference (scale 1-100). Higher score identified higher whiteness intensity.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of milk
The free radical scavenging activities of the
milk samples were measured using the DPPH method
described by Mann et al. [12]. This assay is generally
performed to determine the antioxidant activity in biological systems.
Statistical analysis
Data were presented as the mean±standard error
of the mean. Analysis of variance was used to perform
statistical comparisons between all the treatments,
where p<0.05 was considered statistically significant. Graphs were prepared using Microsoft Excel
version 2010.
Veterinary World, EISSN: 2231-0916
Results
Overall composition
The overall composition of the milk is shown
in Table-1. Solid not fat made up 8.78±0.21% of the
milk, lactose accounted for 4.28±0.06% of the overall
composition, and protein made up 4.1±0.07% of the
overall composition.
Effect of processing on micellar casein size
The effect of processing on the particle size of
micellar casein in the milk is presented in Figure-1.
The average size of micellar casein in the raw cow milk
was found to be 158.6±2.20 nm, whereas the average
size was 152.8±1.34 nm in the ultrasonicated milk and
133.12±0.14 nm in the boiled milk, a significant reduction (p<0.05) was observed for boiled milk only.
Effect of processing on the whiteness of CSM
The degree of change in whiteness was measured
using a Hunter colorimeter, and the results are presented in Figure-2. The average whiteness (L value)
Table-1: The overall composition of cow skim milk.
Constituents
Value
SNF
Lactose
Protein
8.78±0.21
4.28±0.06
4.1±0.07
SNF=Solid not fat
170
160
158.6
152.8
150
140
133.12
130
120
110
100
Average Micellar casein size (nm)
Untreated cow milk
Ultra sonicated cow milk
Boiled cow milk
Figure-1: The average size of micellar casein (nm) of cow
milk.
85
81.16
80.16
80
76.23
75
70
65
60
55
50
L-Value
Untreated cow milk
Ultra sonicated cow milk
Boiled cow milk
Figure-2: The average L-value (whiteness) of cow milk.
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of raw cow milk was found to be 81.16±0.56. The
effect of ultrasonication on the whiteness of the milk
was found to be non-significant (p>0.05), with an L
value of 80.16±0.12 nm. However, boiling treatment
significantly (p<0.05) reduced the milk whiteness
(76.23±0.10) by almost 7%.
Effects of processing on radical (DPPH) scavenging
activity of milk
The effects of processing on radical (DPPH) scavenging activity in milk are presented in Figure-3. The
radical scavenging activity of cow milk, boiled cow
milk, and ultrasonicated cow milk was 61.36±1.45%,
48.12±2.36%, and 59.75±0.86%, respectively. It
was observed that, for both processing treatments,
the antioxidant activities of milk in terms of radical
(DPPH) scavenging activity were reduced, but in the
case of boiling, the reduction was significant (21.50%,
p<0.05), whereas the reduction was not significant
(2.60%, p>0.05) in the case of ultrasonication.
Discussion
Milk is a complex biochemical fluid and an
ideal medium for the growth of various microorganisms. Therefore, maintaining the quality of milk is a
challenging task. In addition to the traditional heat
treatments, dairy producers are now considering
alternative non-thermal processing methods, such as
ultrasonication treatment, to enhance shelf-life while
retaining the nutritional and biofunctional properties
of milk [5].
Casein plays an essential role in terms of both
processing and the functional properties of milk [2,7].
Heat treatment modifies the structure of micellar
casein: Dephosphorylation and precipitation of calcium can be observed during treatment at high temperatures. In this study, it was observed that micellar
casein particles were reduced in size during heating, in
line with the earlier observation of Yang et al. [13] who
also reported that the average size of micellar caseins
was reduced after heat treatment. Liu et al. [14] also
reported that, during heating, shrinkage of micellar casein occurred and the sizes of casein particles
were reduced. Ultrasonication creates mechanical
65
61.36
Conclusion
59.76
60
55
48.12
50
45
40
35
30
DPPH (radical scavenging activity)
Untreated cow milk
Ultra Sonicated cow milk
Boiled cow milk
Figure-3: The average DPPH (radical scavenging activity)
of cow milk.
Veterinary World, EISSN: 2231-0916
wavelengths that disrupt micellar casein; therefore,
micellar casein size is usually reduced during this
process [11]. Indeed, the results of the current study
showed that micellar casein particle size was reduced
during ultrasonication treatment. Yang et al. [15]
reported that the secondary structure of proteins in
skim milk changes during ultrasonication treatment
and that the size of casein particles is reduced. The
size reduction of micellar casein due to ultrasonication might be due to disruption of casein or changes
in its structure. However, our findings suggest that
the effect of heat on the disruption of micellar casein
particles was more intense than that of ultrasonication
treatment.
The color of milk is usually white, and this whiteness is due to the reflection of light by fat globules and
micellar casein. Beta-carotene also affects the color of
milk [16] and creates a yellowish hue. We observed a
significant reduction in the whiteness of milk during
boiling treatment. Degradation and structural modification of casein occurs during heat treatment and may
account for the observed reduction in L value for the
boiled milk in this study. We also observed a non-significant effect on the whiteness of the milk after ultrasonication treatment. However, the previous studies
have reported no observed effect on milk color during
ultrasonication treatment [17].
The DPPH assay is used to determine antioxidant
or free radical scavenging activity in biosystems [12].
Milk is an ample source of antioxidant compounds
that often change during mechanical or thermal treatment [1]. In our study, it was observed that both heat
treatment and ultrasonication treatment reduced the
radical scavenging activities of milk. Several previous studies have reported that heat treatment reduced
the radical scavenging activities of milk [18]. Casein
has an effective role against lipid peroxidation and in
radical scavenging activity [7,8,19]. Micellar casein
is denatured during heating; therefore, the radical
scavenging activity of heat-treated milk is reduced
compared to non-treated milk. In the case of ultrasonication treatment, the denaturation of micellar casein
was not as severe; therefore, the radical scavenging
activity was higher compared to heat-treated milk.
The antioxidant activity in milk changes during
boiling and ultrasonication treatment, and boiling
treatment confers a greater reduction in antioxidant
activity compared to ultrasonication. Higher micellar casein degradation was observed in boiled milk
than in ultrasonicated milk. In view of the esthetic
quality of milk, higher whiteness was observed in
ultrasonicated milk samples as compared to boiled
milk samples. Our results reveal that the application
of ultrasonication treatment provides superior results
over boiling treatment in terms of the esthetic and
bioactive qualities of milk, as well as increasing the
shelf-life.
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Authors’ Contributions
TH: Conceptualized, performed the study, and
drafted the manuscript. RS and MB: Execution of
the project and drafted the manuscript. VR: Planning
of study and drafted the manuscript. ChVKS: Edited
the manuscript. RG: Data analysis and edited the
manuscript. All authors read and approved the final
manuscript.
Acknowledgments
The authors are thankful to the Kamdhenu
University, Gujarat, India, for providing necessary funds from the departmental grant (project
no. 16.10.3.37) and facilities for the study.
Competing Interests
8.
9.
10.
11.
12.
The authors declare that they have no competing
interests.
Publisher’s Note
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to jurisdictional claims in published institutional
affiliation.
14.
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