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The aqueous extract of scraped coconut kernel is known as coconut milk. Coconut milk preparations are also commercially available in the form of desiccated powders or liquids. While these various coconut milk preparations are heavily used in cooking in the Asian countries as a major source of dietary fat, limited studies have been conducted on their chemical and nutritional composition. In this study, we have determined the chemical composition and nutritional effects of both domestic preparations of coconut milk and the commercially available counterparts. The results indicate that the phenolic compounds of all coconut milk preparations provide protection against oxidative damage on lipids and inhibit oxidative damage of both proteins and DNA. The lipid profiles are not significantly affected by the consumption of the three coconut milk preparations despite their different fat contents.
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Research Article
Antioxidant and Nutritional Properties of Domestic and
Commercial Coconut Milk Preparations
Asiri N. Karunasiri,
1
Mahendra Gunawardane,
2
Chathuri M. Senanayake,
1
Nimanthi Jayathilaka ,
1
and Kapila N. Seneviratne
1
1
Department of Chemistry, Faculty of Science, University of Kelaniya, Kelaniya, Sri Lanka
2
Department of Microbiology, Faculty of Science, University of Kelaniya, Kelaniya, Sri Lanka
Correspondence should be addressed to Nimanthi Jayathilaka; njayathi@kln.ac.lk and Kapila N. Seneviratne; kapilas@kln.ac.lk
Received 13 November 2019; Revised 5 July 2020; Accepted 8 July 2020; Published 3 August 2020
Academic Editor: Amarat (Amy) Simonne
Copyright © 2020 Asiri N. Karunasiri et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
The aqueous extract of scraped coconut kernel is known as coconut milk. Coconut milk preparations are also commercially
available in the form of desiccated powders or liquids. While these various coconut milk preparations are heavily used in
cooking in the Asian countries as a major source of dietary fat, limited studies have been conducted on their chemical and
nutritional composition. In this study, we have determined the chemical composition and nutritional eects of both domestic
preparations of coconut milk and the commercially available counterparts. The results indicate that the phenolic compounds of
all coconut milk preparations provide protection against oxidative damage on lipids and inhibit oxidative damage of both
proteins and DNA. The lipid proles are not signicantly aected by the consumption of the three coconut milk preparations
despite their dierent fat contents.
1. Introduction
Coconut milk is the aqueous extract of the solid endosperm
(kernel) of coconut. In addition to the use for coconut oil
extraction by wet process, coconut milk is directly used as a
liquid medium in culinary applications to prepare dishes
including meat and vegetable dishes. Domestic coconut milk
is prepared by adding water to the scraped coconut kernel
and mixing in a blender followed by ltering coconut milk
through a strainer. Coconut milk is also available in the
market in powder form and in liquid form. Some nutritional
constituents and antioxidant properties of coconut milk have
been reported. Coconut milk is an emulsion containing
mainly lipid carbohydrates and proteins. It also contains
several minor compounds including phenolic substances
[1]. Antioxidant properties evaluated by ferric reducing
power (FRAP) assay and 1,1-diphenyl-2-picrylhydrazyl
(DPPH) assay indicate that coconut milk displays higher
antioxidant properties than cows milk [2]. Antioxidant
properties of the methanolic extracts of the coconut kernel
have also been tested with DPPH and 2,2-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) diammonium (ABTS)
assays, as a function of maturity, and the antioxidant activi-
ties increased up to 190 days from the date of pollination
and then decreased or remain unchanged [3]. Studies also
indicate that the serum LDL levels decreased while the
HDL levels increased in healthy subjects on a diet containing
coconut milk [4].
Although few studies have reported the chemical and
nutritional information of coconut milk, the chemical and
nutritional properties of dierent coconut milk preparations
have not been compared, and detailed studies on the protec-
tive eect of coconut milk antioxidants on oxidative stress-
induced macromolecular damage have not been reported to
the best of our knowledge. Harmful features of oxidative
stress arise when oxidative forces exceed the antioxidant
defense systems in biological systems. Oxidative stress is well
known to be closely related to cancer, atherosclerosis, hyper-
tension and diabetes mellitus. Phenolic antioxidants are well
known to confer protection against oxidative damage in
Hindawi
International Journal of Food Science
Volume 2020, Article ID 3489605, 9 pages
https://doi.org/10.1155/2020/3489605
biological systems. In addition to interacting with reactive
oxygen species (ROS) and neutralizing them, polyphenols
inhibit the enzymes that are involved in the production of
inammation mediators, preventing inammation and carci-
nogenesis induced by inammation [5]. Lipids, proteins, and
DNA are modied due to interactions with ROS, and the
changes in these macromolecules can result in loss of physi-
ological functioning which is also associated with the aging of
post mitotic cells. Thus, such changes to macromolecules can
be used as biomarkers of oxidative stress. Among lipid oxida-
tion products, thiobarbituric acid reactive substances
(TBARS) have been used as a marker of oxidative stress in
obstructive sleep apnea [6]. Among other biomacromole-
cules, DNA is also damaged by oxidative stress. mt-DNA,
which is more sensitive to oxidative stress damage, may alter
mitochondrial gene expression in many human cancer cells
[7]. Protein carbonyls that are formed due to oxidation of
proteins are also stable biomarkers of oxidative stress.
Though coconut milk is used for cooking in many coun-
tries on a daily basis as a major source of dietary fat, the
nutritional information of coconut milk has not suciently
been investigated. The present study was conducted to com-
pare basic chemical composition and the nutritional impact
of domestic coconut milk, powdered coconut milk, and
liquid coconut milk. The protective eect of the phenolic
extracts of the three coconut milk preparations on the oxida-
tive stress-induced macromolecular damage was evaluated
using Saccharomyces cerevisiae (yeast) as a biological model.
The nutritional eect of consumption of coconut milk based
on the lipid proles was evaluated using Wistar rats.
2. Materials and Methods
2.1. Sampling. Mature coconuts (12-14 months) collected
from Cocos nucifera L., typica (tall type) coconut trees,
characteristics of which have been reported, were used to pre-
pare domestic coconut milk. This coconut cultivar was con-
rmed based on morphological characteristics as previously
reported [8, 9]. For the analysis of commercial coconut milk
samples, powdered coconut milk and liquid coconut milk
with no added substances available in the market were used.
2.2. Preparation of Liquid Samples of Coconut Milk. To pre-
pare domestic coconut milk, scraped coconut kernel (100 g)
was added to distilled water (100 mL) and mixed using a
kitchen blender (3 min). The resultant slurry was squeezed
through cheesecloth to separate the milk portion, and
the resultant liquid was used as domestic coconut milk
(DCM). Commercially available coconut milk powder was
dissolved in distilled water according to manufacturers
instructions and the resultant liquid was used as powdered
coconut milk (PCM). Commercially available liquid coco-
nut milk was directly used as liquid coconut milk (LCM)
in the study based on the manufacturersinstructions.
The densities of DCM, PCM, and LCM were not signi-
cantly dierent (1:0±0:0 g/mL).
2.3. Preparation of Aqueous and Methanolic Extracts of
Coconut Milk. DCM, PCM, and LCM samples were frozen,
and the lipids were removed by centrifugation (1080 g,
10 min) to prepare aqueous extracts. To prepare the metha-
nolic extract of DCM, aqueous extracts of DCM (1 mL) were
mixed with methanol (1 mL) and chloroform (1 mL) and the
mixture was vortexed (30 Hz, 1 min). Then, the sample was
centrifuged (380 g, 10 min), and the top methanol/water layer
was separated. The same procedure was used to prepare
methanolic extracts of PCM and LCM.
2.4. Determination of Total Phenolic Content and
Antioxidant Activity. Each aqueous extract of DCM, PCM,
or LCM (15 μL) was diluted by adding deionized water
(135 μL). Then, Folin-Ciocalteu reagent (6 μL) and 25%
Na
2
CO
3
(15 μL) were added to it. The mixture was diluted
with deionized water (129 μL) and incubated for 1 hour at
room temperature. The absorbance of the sample was
measured at 765 nm using a UV-visible spectrophotometer
(Multiskan GO, Thermo Scientic, Finland) against a control
sample with no added coconut milk aqueous extract. Gallic
acid was used as the standard for the preparation of
calibration curves. DPPH radical scavenging activity and
ferric reducing power of the aqueous extracts of DCM,
PCM, and LCM were evaluated as reported by Seneviratne
and Kotuwegedara [10].
2.5. Total Sugar and Protein Content. Total sugar content of
coconut milk was determined by the method of Ting [11].
The standard curve was prepared by using glucose with
dierent concentrations. Protein content was determined
by Coomassie dye-binding assay (Bradford assay). Standard
series of protein samples with varying concentrations
(0.15 mg/mL to 2 mg/mL) were prepared by using Bovine
Serum Albumin (BSA). The negative controls were distilled
water and methanol-water (50% v/v) for the aqueous extract
of coconut milk and methanolic extract of coconut milk,
respectively, with all other reagents.
2.6. Total Fat Content. The fat extraction method was
adopted from a reported procedure used for the fat extraction
from coconut water [12]. Coconut milk (DCM, PCM, or
LCM) (10.00 mL) was mixed with hexane (10.00 mL) in a
separatory funnel and shaken thoroughly, and the top hexane
layer was collected. The extraction was continued with two
fresh 10.0 mL portions of hexane. Then, hexane was
removed, and the weight of the fat fraction was recorded.
2.7. Fatty Acid Composition. Methylation of fatty acids and
the sample preparation for GC analysis was conducted
according to a reported procedure [10]. A gas chromato-
graph (Shimadzu GC-2010 Plus, Japan) equipped with a
capillary column which was RtxR-WAX (crossbond with
PEG 30 m × 0:32 mm, i.d. 0.25 μm) and a ame ionization
detector (FID) were used for the analysis. The carrier gas
was helium with the ow rate of 0.5 mL/min, and the analyses
were performed on split mode (split ratio 100 : 1). Sample
(1 μL) was injected to the GC system. The temperatures of
the injector and detector were 230
°
C and 250
°
C, respectively.
A column temperature program of 130
°
C (3 min), 130
°
Cto
210
°
Cat45
°
C/min, and 210
°
C (12 min) was used.
2 International Journal of Food Science
2.8. Identication of Phenolic Compounds. Phenolic com-
pounds in the methanolic extracts of DCM, PCM, and
LCM were identied and quantied by a previously reported
method [13]. Briey, prepared phenolic extract (20 μL) was
injected to the HPLC system. HPLC experiments were
performed using an Agilent 1100-Innity liquid chromato-
graphic system (Agilent Technologies, Waldbronn, Germany)
equipped with an Agilent 1200 diode array detector and a
ZORBAX ECLIPSE Plus C18 column (Agilent Technologies,
USA) (4:6mm×100m3:5μmparticle size) maintained
at room temperature. Methanol (A) and 1×10
3mol dm3
H
2
SO
4
in deionized water (B) were used as the mobile phase.
The ow rate was 0.5 mL min
-1
, and the total running time
was 90min. The elution gradient began with 5% A and 95%
B. From 0 to 15min, A was increased to 10%, and this compo-
sition (10% A and 90% B) was continued up to 30min. Then,
A was increased to 20% from 30 to 40min, to 30% from 40 to
50 min, to 40% from 50 to 60 min, to 50% from 60 to 70 min,
and to 60% from 70 to 80 min, and this composition (60% A
and 30% B) was continued for 10min. Phenolic compounds
were detected at 280 nm and identied by comparison of the
retention times and UV spectra of authentic standards.
2.9. Inhibition of Macromolecular Damage by Coconut
Milk Antioxidants
2.9.1. Yeast Strain and Cultivation. Saccharomyces cerevisiae
(DMS 1333; ATCC 9763) was obtained from DSMZGer-
man Collection of Microorganisms and Cell Cultures, and
the culture was stored and maintained in a glycerol stock
(15%) at -80
°
C. The yeast cultures for the experiments were
grown in YPDA (yeast extract 10 g L
-1
, peptone 20 g L
-1
,
dextrose 20 g L
-1
, and agar 15 g L
-1
) and incubated at room
temperature. Broth cultures in YPD (yeast extract 10 g L
-1
,
peptone 20 g L
-1
, and dextrose 20 g L
-1
) were incubated at
room temperature at 250 rpm. Overnight cultures at room
temperature at 250 rpm inoculated from the glycerol stocks
were used to set up fresh cultures in the log phase for the anti-
oxidant activity assays according to a previously published
method with some modications [14]. The fresh cultures
were obtained by 1 : 40 dilution of the overnight culture into
sterile YPD medium and incubation at room temperature at
250 rpm for 3-4 hours until the cultures reach an OD of 0.5 at
620 nm.
2.9.2. Treatment of Yeast Cell Suspensions with Antioxidants.
Yeast cell suspensions were treated with phenolic extracts of
coconut milk solutions based on a reported method [15].
Methanolic extracts were used to make the stock antioxidant
solutions. For example, methanolic extracts of DCM were
evaporated, the residue was dissolved in distilled water to
obtain the stock phenolic extract (20.0 mg mL
-1
), and the
phenolic extract was sterilized by ltering through a
0.45 μm sterile lter. The procedure was repeated for metha-
nolic extracts of PCM and LCM. For every experiment, yeast
cultures at 0.5 OD maintained in YPD broth were treated
overnight with the abovementioned stock phenolic extract
at a 1 : 40 dilution in 1 mL of YPD broth culture to obtain
0.5 mg mL
-1
nal concentration. The control sample was
treated with the same volume of distilled water instead of
the phenolic extract. Sterile conditions were maintained
throughout all the steps of the experiments.
2.9.3. Oxidative Stress Induction of Yeast. YPD broth cultures
of yeast were used to induce the oxidative stress by H
2
O
2
after treating with phenolic extract at 0.5 mg mL
-1
[14]. The
broth cultures were centrifuged (100 g, 3 min) to harvest
yeast cell pellets, and the supernatants were removed along
with the antioxidants. Then, 1x PBS (1 mL) was added to
resuspend the pellets to wash residual antioxidants. Samples
were centrifuged (100 g, 3 min) again and supernatants were
removed. Then, the cell pellets were resuspended in 1x PBS
(1 mL), and 15 mM FeCl
2
was added to reach 150 μM FeCl
2
.
The mixture was incubated for 30 min at room temperature
at 250 rpm followed by induction of oxidative stress with
2mM H
2
O
2
(this concentration was deemed to be the maxi-
mum H
2
O
2
concentration that does not signicantly aect
the viability of the cells). The samples were incubated for 1
hour at room temperature at 250 rpm. The control sample
contained the same composition except water in the place
of H
2
O
2
.
2.9.4. Yeast Survival Assay. The oxidative stress-induced
macromolecular damage was assessed under the conditions
that do not signicantly aect the yeast cell viability accord-
ing to a previously reported method [14]. The yeast cells were
diluted 1 : 4000 with 1x PBS, and 25 μL was plated on YPDA
plates followed by incubation for 48 hours at room tempera-
ture to obtain viable cell colony counts. The percentage
survival of yeast was calculated based on the number of
colony-forming units (CFUs) according to the following
formula.
The percentage survival of yeast = CFU of sample × 100%
CFU control :
ð1Þ
2.9.5. Inhibition of Protein Carbonylation. Inhibition of pro-
tein carbonyl formation by coconut milk antioxidants was
assessed according to a reported method [16]. Oxidative
stress-induced yeast cells (1 mL) were harvested by centrifu-
gation (380 g, 10 min). The resultant pellet was resuspended
in 1x PBS (1 mL) to remove H
2
O
2
and stop oxidative damage
and centrifuged (380 g, 10 min) to harves t the cell pellet. The
cells were lysed in 100 μL 1x lysis buer (1% Tween 20, 0.1%
SDS, and 50 mM Tris HCl, pH 7) with vortexing (40 Hz, 10
seconds). TCA (30% w/v, 250 μL) was added to the mixture
to precipitate the protein and vortexed (40 Hz, 10 seconds).
Then, the mixture was centrifuged (300 g, 3 min) and protein
pellets were separated. 2,4-DNPH (10 mM in 2 M HCL,
250 μL) was added to the pellets, mixed, and incubated at
room temperature for 60 min in the dark. Then, TCA (20%,
250 μL) was added and centrifuged (11000 g, 3 min). The
pellet was separated, washed three times with ethanol : ethyl
acetate mixture (1 : 1 v/v) and incubated for 10 min. Then,
the mixture was centrifuged (11000 g, 3 min) and the pellet
was separated. Guanidine solutio n (6 M, 300 μL) was added
to the pellet to reconstitute the precipitated proteins and
3International Journal of Food Science
centrifuged (11000 g, 3 min). The absorbance of the superna-
tant was measured at 370 nm by using a UV-visible spectro-
photometer. The protein carbonyl content was calculated
using the following formula.
Protein carbonyl nmol/mL
ðÞ
=AsAc
X×300
200 ,ð2Þ
Xis the extinction coecient for DNPH (0.011 μM
-1
), As
is the absorbance of the sample, and Acis the absorbance of
the blank (guanidine solution).
2.9.6. Inhibition of Mitochondrial DNA (mt-DNA) Damage.
mt-DNA damage was assessed according to a previously
reported method with modications [17]. Yeast colonies
grown on YPDA plates after induction of oxidative stress
were copied onto the YPGA (yeast extract 1%, Bacto-
Peptone 2%, glycerol 2%, ethanol 2%, and agar 2%) plates
and incubated for 48 hours at room temperature. The per-
centage of survival based on the CFU in YPGA was used to
assess the percentage of cells with mt-DNA damage accord-
ing to following equation.
mtDNA damaged cells %
ðÞ
=CFU in YPDA CFU in YPGA
CFU in YPDA

100:
ð3Þ
2.9.7. Inhibition of Lipid Peroxidation. Thiobarbituric acid
reactive substances (TBARS) were measured using linoleic
acid as an external source of polyunsaturated fatty acids
according to a previously published method with some mod-
ications [14, 18]. Linoleic acid was homogenized in Tween
20 and H
2
O mixture (1 : 2000 v/v, respectively) at room tem-
perature for 5 min at 250 rpm. The homogenate was added to
the overnight cultures of antioxidant-treated yeast cells at an
end concentration of 0.03 mg/mL and incubated for 4 hrs at
room temperature at 250 rpm prior to the induction of oxida-
tive stress. The control sample contained the linoleic acid
homogenate, FeCl
2
, and H
2
O instead of H
2
O
2
. The cells were
harvested by centrifugation (380 g, 10 min) and washed with
1x PBS (1 mL). Resultant pellets were lysed as stated above,
and 1 mL thiobarbituric acid (TBA) (15% TCA, 0.8% TBA
in 0.25 N HCl) was added and mixed well. BHT (0.2%,
8μL) was added, and samples were heated for 15 min at
95
°
C. The mixture was allowed to cool and centrifuged at
380 g for 20 min. The absorbance of the supernatant was
measured at 532 nm by Multiskan GO spectrophotometer
(Thermo Scientic). The standard curve was prepared by
using malondialdehyde.
2.10. Animal Studies
2.10.1. Feeding Rats. Ethical approval for the animal study
using a rat model was obtained from the Ethics Review
Committee of the Faculty of Medical Sciences, University of
Sri Jayewardenepura (Application number: 25/16). Feeding
experiments were conducted as reported [19]. Briey,
seven-week-old male Wistar rats (weighing 240-300 g) were
selected from the Medical Research Institute, Colombo, Sri
Lanka. The animals were housed in cages in a room main-
tained at 25 ± 1°Cwith a 12 h light and dark cycle. Prior to
the commencement of the experiment, rats were acclimatized
to the basal diet for 6 days. Then, the rats were randomly
assigned into experimental groups (7 rats/group).
The control group was fed with the basal diet. The sec-
ond, third, and fourth groups of rats were fed with the test
diets containing 12 mL each of DCM, PCM, and LCM,
respectively, per kg of basal diet. The study was continued
for 150 days. Free access to water and diet was provided
throughout the experimental period. Rats were monitored
daily. Body weight and feed intake were measured weekly.
2.10.2. Analysis of Serum Lipid Proles. The rats were fasted
for 10-12 h prior to drawing blood on day 0, 30 days, 90 days,
120 days, and 150 days after feeding the experimental diets.
Blood (500 μL) was drawn from the tail vein and collected
into plain tubes and centrifuged to harvest serum. Serum
total cholesterol (TC), serum high-density lipoprotein
(HDL) cholesterol, and serum triglyceride (TG) levels were
determined using the test kit provided by G Cell (Beijing
Strong Biotechnologies, Inc., China). The method given in
the test kit was followed without any modications. Serum
low-density lipoprotein (LDL) cholesterol was determined
using the Friedewald equation (LDL = TC HDL ðTG/5Þ).
2.10.3. Dissection and Harvesting Tissues from Animals. At
the end of the feeding experiments, the animals were sub-
jected to barbiturate euthanasia. Body weight was measured
before dissecting the animals. The liver and heart were
harvested, and the liver size, liver weight, and heart weight
were recorded. Pericardium thickness was measured using a
Vernier caliper. Average weight gain during the study period
was calculated according to the following formula.
Average body weight gain = average final body weight
average initial body weight:
ð4Þ
2.11. Statistical Analysis. All experiments were run in tripli-
cate, and biological replicates were carried out unless
otherwise indicated. A two-sample t-test or one-way ANOVA
was carried out for the determination of signicant dierences
(p0:05) between the means. Data were analyzed using
Minitab (Version 17 for Windows).
3. Results and Discussion
3.1. Basic Nutritional Composition and Antioxidant Activity.
Basic nutrient composition in the aqueous DCM, PCM, and
LCM given in Table 1 indicates that there is no signicant
dierence in total phenol content, total sugar contents, and
power reduction of DCM, PCM, and LCM while the protein
content in LCM was signicantly lower compared to that in
DCM and PCM (p<0:05;n=27). In contrast to the power
reduction, DPPH radical scavenging activity showed signi-
cantly lower values in PCM and LCM compared to DCM,
despite the fact that no signicant dierences were observed
among the corresponding phenolic antioxidant contents of
4 International Journal of Food Science
the aqueous extracts. Such discrepancies could be due to
some dierences of the other nutrient contents as reported
[20]. For example, proteins have been reported to display
radical scavenging activity as determined by the DPPH assay
due to the antioxidant activity of sulfur-containing amino
acids such as methionines and cysteines [21]. Therefore,
methanolic extracts of DCM, PCM, and LCM were also pre-
pared by removing proteins using organic extraction with
chloroform to assess the DPPH radical scavenging activity.
Complete removal of proteins by organic extraction was con-
rmed by the Bradford assay. There were no signicant dif-
ferences among the percentage DPPH radical scavenging
activities of the methanolic extracts from the three dierent
coconut milk preparations after protein removal (DCM:
34:6±0:2, PCM: 32:2±10:7, and LCM: 31:2±12:9). There-
fore, proteins may have contributed to the signicant dier-
ences in antioxidant activity of the aqueous extracts of
coconut milk preparations as assessed by DPPH assay. In
addition, total fat contents of the three coconut milk samples
were signicantly dierent (p<0:05). Basic nutritional
parameters for DCM, PCM, and LCM are compared with
reported values for cows milk [2224], goats milk [23, 25,
26], and soy milk [24, 27, 28] in Table 1. However, basic
nutritional composition drastically changes with the diet,
environmental conditions, season, and breed in cows milk
and goats milk.
The composition of major fatty acids present in DCM,
PCM, and LCM is given in Table 2. The variation of fatty acid
composition in DCM, PCM, and LCM is well within the
reported range of the fatty acid composition of coconut fat
[29]. Table 2 also compares the composition of major fatty
acids in DCM, PCM, and LCM with the compositions of
those fatty acids in cows milk [30], goats milk [31], and
soy milk [32]. Except for palmitic acid, major fatty acids in
cows milk, goats milk, and soy milk are longer chain fatty
acids.
3.2. Identication of Phenolic Compounds. The phenolic
compounds in coconut milk originate from the brown coco-
nut testa and white coconut kernel. These phenolic sub-
stances are thermally stable at cooking temperatures [33].
Therefore, antioxidant properties of the phenolic substances
present in coconut milk may be retained during cooking.
Figure 1 shows the HPLC chromatogram of the phenolic
extract of DCM. Phenolic compounds present in PCM and
LCM are also similar according to the HPLC chromatograms
of PCM and LCM. HPLC chromatograms indicate that there
are seven phenolic compounds present in coconut milk.
Comparison of the quantities of phenolic substances present
in DCM, PCM, and LCM indicates that the phenolic compo-
sitions for most phenolic compounds are similar in the three
coconut milk types.
3.3. Antioxidant Activity under Stressed Conditions. Antioxi-
dant potential of plant extracts is usually evaluated by chem-
ical methods such as DPPH radical scavenging activity and
reducing power assays. However, antioxidant activities eval-
uated in chemical models may not always correlate in biolog-
ical systems [34]. Therefore, it is also important to use
biological models to investigate antioxidant activities of phe-
nolic extracts to get a clear idea about the true antioxidant
potential and to further conrm the results obtained in
chemical systems. Yeast cells were used in the present study
as a biological model to test the antioxidant potential of the
phenolic compounds of DCM, PCM, and LCM. Yeast cell
suspensions were pretreated with phenolic antioxidants of
DCM, PCM, and LCM followed by the induction of oxidative
stress using H
2
O
2
after removing the antioxidants. The oxi-
dative stress was induced at a concentration at which the cell
viability is above 80% to assess the eect of the antioxidants
on oxidative stress-induced macromolecular damage
(Figure 2). The antioxidant-treated cells have a signicantly
higher (p<0:05;n=4) percentage of viable cells under
induced stress conditions compared to untreated control
cells suggesting that coconut milk antioxidants provide pro-
tection against H
2
O
2
-induced cell death. However, there is
no signicant dierence among survival rates of yeast in the
samples treated with dierent coconut milk antioxidant
extracts followed by H
2
O
2
-induced oxidative stress.
To evaluate the eect of coconut milk antioxidants on the
inhibition of lipid peroxidation in yeast cells, levels of TBARS
were analyzed after subjecting yeast cells to oxidative stress.
Polyunsaturated fatty acids undergo oxidation to produce
TBARS. Yeast does not normally produce polyunsaturated
fatty acids though it can utilize exogenously provided polyun-
saturated fatty acids [14]. Antioxidants play an important role
in vivo in protecting cells from oxidative damage to polyunsat-
urated fatty acids. Therefore, eect of antioxidants on the
inhibition of TBARS formation in yeast cells can be evaluated
by providing polyunsaturated fatty acids to the medium.
Similarly, eect of antioxidants on the inhibition of pro-
tein carbonyl formation due to oxidative damage in yeast
Table 1: Basic nutrient composition of DCM, PCM, and LCM and reported values for cows milk, goats milk, and soy milk.
DCM PCM LCM Cows milk Goats milk Soya milk
Total phenol content (mg/L) 8:21 ± 0:13
a
8:23 ± 0:14
a
8:23 ± 0:12
a
13.31 [22] 87.9 [25] 61.4 [27]
Protein (%) 5:83 ± 0:48
a
4:53 ± 0:23
b
4:13 ± 0:19
b
2.82 [23] 3.48 [23] 4.50 [28]
Fat (%) 3:11 ± 0:16
b
1:83 ± 0:70
c
3:99 ± 0:40
a
3.42 [23] 5.23 [23] 4.30 [28]
Carbohydrates (%) 2:21 ± 0:01
a
(total sugars)
1:94 ± 0:26
a
(total sugars)
2:10 ± 0:59
a
(total sugars)
4.47 [23]
(lactose)
4.11 [23]
(lactose)
10.00 [28]
(total carbohydrates)
DPPH (%) 54:92 ± 3:32
a
44:08 ± 1:17
b
43:32 ± 0:06
b
8.70 [24] 56.55 [26] 33.51 [24]
Reducing power (%) 234:5±2:8
a
231:7±12:1
a
228:4±9:1
a
∗∗ ∗
Letters a, b and c were used to compare statistical signicance (p0:05) in the same row (n=27). Data not available under comparable conditions and units.
5International Journal of Food Science
cells was evaluated. Table 3 shows that both TBARS and pro-
tein carbonyl levels in yeast lysates from cultures pretreated
with the phenolic extracts of DCM, PCM, and LCM are sig-
nicantly lower (p<0:05;n=4) compared to the TBARS
and protein carbonyl levels of H
2
O
2
-stressed yeast lysates
without pretreatment with phenolic antioxidants while
phenolic extracts of both DCM- and PCM-treated samples
maintained lower protein carbonyl and TBARS levels
compared to yeast lysates treated with the phenolic extracts
of LCM.
Reactive oxygen species such as hydrogen peroxide
causes lesions in DNA [35]. mt-DNA shows a high suscepti-
bility towards oxidative damage. Therefore, DNA damage in
yeast cells can be assessed based on the fact that growth of
yeast cells on YPG (glycerol-containing) medium requires
mitochondrial respiration, while growth on YPD (glucose-
containing) medium is possible without it. As such, the
DNA damage assay in the present study is based on the
assumption that yeast cells with damaged mt-DNA are
unable to utilize glycerol as the source of carbon and energy
[17]. The mt-DNA damage assay indicates that phenolic
antioxidants have a statistically signicant (p<0:05;n=4)
protective eect against oxidative stress-induced DNA
damage compared to stressed yeast cells that were not
pretreated with antioxidants as indicated by the percentage
of respiratory-decient cells (Table 3).
3.4. Eect of Dierent Coconut Milk Preparations on Serum
Lipid Proles. Some studies indicate that saturated fat
increases the serum levels of cholesterol. However, a recent
report indicates that medium chain fatty acids decrease
serum cholesterol levels via reduction of bile absorption
[36]. As indicated in Table 2, major fatty acids of coconut
milk are medium chain fatty acids. Table 4 shows the eect
of feeding Wistar rats with DCM-, PCM-, and LCM-
containing diets on serum total cholesterol, LDL, and HDL
levels. There is no statistically signicant increase of total
cholesterol, LDL, and HDL levels over 150 days of feeding
DCM, PCM, and LCM compared to the control group
(n=7). Serum triglyceride levels increased signicantly in
rats fed with control or coconut milk fed rats over long-
term feeding of the diets (p<0:05). However, there is no
signicant dierence in TG levels in rats fed with diets
containing DCM, PCM, and LCM compared to control rats
during the study period. There is a higher saturated fat con-
tent in the DCM-, PCM-, and LCM-containing diets com-
pared to the control diet. However, increased fat content of
the test diets compared to the control diet has not altered
lipid proles signicantly. This observation may be due to
the presence of medium chain saturated fats in coconut milk.
At the conclusion of the dietary intervention, weight gain,
heart and liver size, heart and liver weight, and pericardium
thickness were measured to assess the impact of the test diets
on the physical parameters that have been associated with the
risk of developing cardiovascular disease due to deposition of
fat. There is no signicant dierence observed in nal body
weight, weight gain, liver size, liver weight, heart weight,
and pericardium thickness in rats in the studied groups.
Table 2: Fatty acid composition of DCM, PCM, and LCM and reported values for cows milk, goats milk, and soy milk.
Fatty acid composition (%) DCM PCM LCM Cows milk [30] Goats milk [31] Soya milk [32]
Caprylic acid (C8) 7:45 ± 0:12
b
6:64 ± 0:51
c
8:34 ± 0:05
a
2.7
Capric acid (C10) 6:84 ± 0:05
a
6:70 ± 0:04
a
6:93 ± 0:08
a
0.03 10.0
Lauric acid (C12) 55:52 ± 0:02
a
51:47 ± 0:80
b
55:01 ± 0:02
a
1.65 5.7
Myristic acid (C14) 22:08 ± 0:07
b
24:34 ± 0:73
a
21:66 ± 0:02
b
0.52 11.7 0.07
Palmitic acid (C16) 8:01 ± 0:07
b
10:84 ± 0:54
a
8:05 ± 0:14
b
34.02 26.3 10.7
Letters a, b, and c were used to compare statistical signicance (p0:05) in the same row (n=4).
mAU
70
60
50
40
30
20
10
0
020
1
2
3
5
4
6
7
40 60 80 min
–10
Figure 1: HPLC chromatogram of the phenolic extracts of DCM (1)
gallic acid, (2) chlorogenic acid, (3) parahydroxybenzoic acid, (4)
caeic acid, (5) vanillic acid, (6) syringic acid, and (7) ferulic acid.
100
Cell viability (%)
80
60
40
20
0
H2O2–+ –+ –+ –+
DCM – + +
PCM – + +
LCM – + +
Figure 2: Protective eect of phenolic extracts of DCM, PCM, and
LCM on the oxidative stress-induced damage of yeast cells given as
percentage viability (n=4).
6 International Journal of Food Science
4. Conclusions
Phenolic substances of coconut milk may protect macromol-
ecules such as lipids, proteins, and DNA against oxidative
damage in living systems. A considerable dierence in the
basic nutrient composition of DCM, PCM, and LCM could
be observed only for fat content. However, serum lipid pro-
les, body weight, average weight gain, liver size, liver weight,
heart weight, and pericardium thickness are not aected by
the dierences in fat contents of DCM, PCM, and LCM com-
pared to a control diet without coconut milk, suggesting that
consumption of coconut milk may not aect these health
parameters. However, more studies with larger quantities of
coconut milk in the diet are necessary to decide the healthy
limit of coconut milk in the diet.
Data Availability
The data used to support the ndings of this study are
included within the article and in the supplementary infor-
mation les provided with the paper.
Disclosure
Chathuri M. Senanayakes present address: Department of
Biosystems Technology, Faculty of Technology, University
of Sri Jayewardenepura, Sri Lanka.
Conflicts of Interest
Authors declare that they have no conict of interest.
Table 3: Levels of protein carbonyls, TBARS in yeast cell lysates, and respiratory-decient cells treated with dierent coconut milk
antioxidants.
Unstressed Stressed
ControlDCM PCM LCM
Protein carbonyl (nmol/mL) 9:1±0:3
c
15:2±0:3
a
14:2±0:1
b
14:0±0:1
b
14:8±0:1
a
TBARS (μg/mL) 0:00 ± 0:00
d
0:05 ± 0:00
a
0:02 ± 0:00
b
0:02 ± 0:0
b
0:02 ± 0:00
b
Respiratory-decient cells (%) 0:0±0:0
c
10:0±2:8
a
5:0±4:2
b
3:0±1:4
b
7:0±1:4
a
Letters a, b, and c were used to compare statistical signicance (p0:05) in the same row (n=4). Stress induced without prior exposure to antioxidants.
Table 4: Serum lipid proles of rats fed with diets containing DCM, PCM, and LCM.
Lipid parameter Day Value (mg/dL)
Control DCM PCM LCM
Total cholesterol
068:83 ± 3:14
aq
71:16 ± 4:03
aq
70:48 ± 5:59
aq
72:30 ± 5:63
aq
30 71:17 ± 6:59
bq
78:42 ± 5:41
ap
71:73 ± 3:83
bq
79:03 ± 6:43
aq
90 75:36 ± 3:04
ap
81:03 ± 9:60
ap
76:86 ± 9:30
ap
83:12 ± 9:37
ap
120 75:12 ± 2:44
ap
79:25 ± 5:34
ap
77:75 ± 4:53
ap
80:32 ± 5:65
ap
150 77:42 ± 6:97
bp
79:25 ± 4:36
bp
80:32 ± 5:21
bp
81:00 ± 3:46
ap
HDL cholesterol
027:81 ± 4:64
ap
30:14 ± 7:48
ap
28:51 ± 3:72
ap
30:60 ± 3:51
ap
30 25:05 ± 4:27
ap
27:86 ± 5:66
ap
25:79 ± 5:21
ap
28:97 ± 1:63
ap
90 27:08 ± 7:51
ap
27:08 ± 3:39
ap
29:66 ± 6:98
ap
30:84 ± 7:08
ap
120 27:08 ± 4:01
ap
27:75 ± 8:10
ap
28:43 ± 3:16
ap
29:98 ± 3:38
ap
150 25:42 ± 5:46
ap
31:59 ± 5:09
ap
26:73 ± 5:87
ap
30:81 ± 3:79
ap
LDL cholesterol
017:00 ± 5:17
ap
16:98 ± 6:76
ap
16:14 ± 3:69
ap
15:58 ± 4:76
ap
30 22:24 ± 8:36
ap
20:74 ± 6:79
ap
16:25 ± 6:24
ap
16:57 ± 7:94
ap
90 21:59 ± 8:19
ap
24:89 ± 7:23
ap
20:56 ± 7:41
ap
23:29 ± 9:98
ap
120 17:34 ± 4:60
ap
20:65 ± 7:50
ap
20:15 ± 5:09
ap
20:54 ± 6:34
ap
150 20:24 ± 6:41
ap
17:61 ± 5:57
ap
21:27 ± 8:31
ap
20:19 ± 3:07
ap
Triglycerides
0120:06 ± 8:05
aq
120:20 ± 3:64
aq
125:90 ± 12:49
aq
128:79 ± 15:60
aq
30 119:39 ± 12:72
aq
149:12 ± 13:93
bp
148:49 ± 15:63
bp
167:46 ± 20:11
bp
90 137:85 ± 14:93
aq
142:76 ± 14:39
ap
130:73 ± 8:27
aq
145:10 ± 10:73
ap
120 162:38 ± 14:88
ap
150:38 ± 17:92
ap
145:86 ± 14:95
ap
149:98 ± 18:05
ap
150 149:46 ± 11:61
ap
160:71 ± 14:82
ap
159:28 ± 17:79
ap
150:78 ± 12:37
ap
Letters a, b, and c were used to compare statistical signicance (p0:05) in the same row. Letters p and q were used to compare statistical signicance (p0:05)
in the same column within one lipid parameter. Each data point represents the mean ± standard deviation of seven replicates.
7International Journal of Food Science
Acknowledgments
The work was funded by the National Science Foundation,
Sri Lanka (RG/2015/AG/03) and University of Kelaniya
Research Grant RP/03/02/06/03/2018.
Supplementary Materials
Supplementary Figure 1, Supplementary Table 1, and Sup-
plementary Table 2. Supplementary Figure 1: gives the HPLC
chromatograms of the phenolic substances of PCM and
LCM. Supplementary Table 1: gives the quantities of pheno-
lic compounds of DCM, PCM, and LCM. Supplementary
Table 2: gives the body, liver, and heart characteristics of rats
fed with dierent types of coconut milk diets. (Supplementary
Materials)
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... Coconut milk is a milky fluid obtained by manual or mechanical extraction of fresh coconut (Cocos nucifera L) kernel with or without addition of water. As a coconut-producing country, coconut milk plays a vital role in the Sri Lankan diet [8]. It is valued mainly for its characteristic nutty flavor and for its nutritional content. ...
Experiment Findings
Spices have been a major influence on Sri Lankan cuisine since times immemorial. Spices are identified as one of the most distinctive ingredients for their indigenous flavor, aroma, and medicinal properties. In this study, coconut milk-based spicy ice cream was developed in compliance with the Sri Lankan standards to introduce a new perception of flavor using spices to the ice cream industry. Although coconut ice cream is commercially available in the local market, spicy flavored coconut ice cream is not yet available. Cinnamon (Cinnamomum verum), ginger (Zingiber officinale), and white pepper (Piper nigrum) are the spices used in the preparation of the ice cream as they are freely available and used as complementary spices in Sri Lanka. Physicochemical characteristics and sensory attributes of coconut milk-based spicy ice cream were compared with the existing normal coconut ice cream. In preparation of the ice cream, the same ice cream manufacturing process was followed with some modifications. Three different formulas (0.010%, 0.018%, and 0.025%) were developed by changing the percentage of spices added. The 0.018% spice-added sample was selected as the most acceptable ice cream with desired sensory attributes. pH (6:33 ± 0:01), titratable acidity (0:33 ± 0:05%), moisture (61:86 ± 0:33%), ash (0:41 ± 0:25%), total solids (38:02 ± 0:14%), overrun (66:76 ± 1:44%), protein (4:18 ± 0:16%), and fat content (11:66 ± 0:60%) were evaluated as physicochemical properties. Total phenolic content of the ice cream was expressed as 0:093 ± 0:002 mg gallic acid equivalents (GAE) per gram of sample in dry weight (mg/g). DPPH radical scavenging activity was 60:39 ± 0:02 mg ascorbic acid equivalents per gram of sample in dry weight (mg/g), and total antioxidant capacity was expressed as 0:36 ± 0:04 mmol ascorbic acid equivalent (AAE)/g of dry weight. Physicochemical properties of spicy coconut ice cream were more or less similar to that of normal coconut ice cream and in compliance with the Sri Lankan standards. Coconut milk-based spicy ice cream could be introduced to the market as a potential marketable nondairy product with spicy flavor, aroma, and smooth texture.
... Coconut milk is a milky fluid obtained by manual or mechanical extraction of fresh coconut (Cocos nucifera L) kernel with or without addition of water. As a coconut-producing country, coconut milk plays a vital role in the Sri Lankan diet [8]. It is valued mainly for its characteristic nutty flavor and for its nutritional content. ...
Article
Full-text available
Spices have been a major influence on Sri Lankan cuisine since times immemorial. Spices are identified as one of the most distinctive ingredients for their indigenous flavor, aroma, and medicinal properties. In this study, coconut milk-based spicy ice cream was developed in compliance with the Sri Lankan standards to introduce a new perception of flavor using spices to the ice cream industry. Although coconut ice cream is commercially available in the local market, spicy flavored coconut ice cream is not yet available. Cinnamon (Cinnamomum verum), ginger (Zingiber officinale), and white pepper (Piper nigrum) are the spices used in the preparation of the ice cream as they are freely available and used as complementary spices in Sri Lanka. Physicochemical characteristics and sensory attributes of coconut milk-based spicy ice cream were compared with the existing normal coconut ice cream. In preparation of the ice cream, the same ice cream manufacturing process was followed with some modifications. Three different formulas (0.010%, 0.018%, and 0.025%) were developed by changing the percentage of spices added. The 0.018% spice-added sample was selected as the most acceptable ice cream with desired sensory attributes. pH (), titratable acidity (), moisture (), ash (), total solids (), overrun (), protein (), and fat content () were evaluated as physicochemical properties. Total phenolic content of the ice cream was expressed as mg gallic acid equivalents (GAE) per gram of sample in dry weight (mg/g). DPPH radical scavenging activity was mg ascorbic acid equivalents per gram of sample in dry weight (mg/g), and total antioxidant capacity was expressed as mmol ascorbic acid equivalent (AAE)/g of dry weight. Physicochemical properties of spicy coconut ice cream were more or less similar to that of normal coconut ice cream and in compliance with the Sri Lankan standards. Coconut milk-based spicy ice cream could be introduced to the market as a potential marketable nondairy product with spicy flavor, aroma, and smooth texture. 1. Introduction Ice cream is a delicious, wholesome, and nutritious frozen dairy dessert, which is widely consumed globally, and it is very popular among people of all ages because of its taste and cool sensation property [1]. The main ingredients of ice cream are milk, cream, sweeteners, natural flavorings, and other optional ingredients such as eggs, nuts, fruits, chocolate, and candy. There are various types of ice creams available in the market including a wide range of flavors, colors, textures, and ingredients. Both the flavorings and the sweeteners are imparting sweet sensation to the ice cream. Sri Lanka is an Asian country which uses different flavorings in preparing meals, specially spices. Thus, Sri Lankans are more familiar with the flavor profiles of various spices. Spices also can be used as a flavoring for sweet products like ice creams [2]. Therefore, the main objective of this study was to formulate an ice cream with a new sensation which imparts both sweet and spicy tastes. Spices are the aromatic parts of tropical plants, dried seed, fruit, root, or a bark, traditionally used for flavoring, coloring, or preserving meals. Spices are consequential not only as foods but also as medicines. Cinnamon, ginger, and white pepper are complementary spices very well known among people due to their pleasant flavor, aroma, and medicinal value. Therefore, these three spices were selected to make the spicy ice cream. Cinnamon (Cinnamomum verum) is a spice obtained from the inner bark of several trees from the genus Cinnamomum that is used in both sweet and savory foods. Moreover, cinnamon is a powerful spice that has been used medicinally around the world for thousands of years [3]. Cinnamon bark contains several special compounds which are responsible for its many health-promoting properties, including cinnamaldehyde, cinnamic acid, and cinnamate. It has been revealed that special phenolic compounds, flavonoids, and antioxidants which are isolated from cinnamon are rich in antioxidant, antidiabetic, antimicrobial, immunity-boosting, and potential cancer and heart disease-protecting abilities [4]. Ginger (Zingiber officinale) is an underground stem or rhizome used as a flavoring and medicine in Asian and Arabic herbal traditions. Adding ginger as a flavoring makes dishes more delicious. Ginger is used as an ingredient for preparation of tea and production of some sweets and beverages [5]. It imparts many health benefits due to its antioxidants, antimicrobial activity, anti-inflammatory properties, and content of therapeutic compounds like gingerol, shogaol, paradol, and zingerone [6]. White pepper (Piper nigrum) is also one of the most popular culinary spices in the world. White pepper spice has had the black outer shell of the peppercorn removed, giving it a smooth mellow flavor. White pepper itself also contains immunity-boosting properties and anticancer, energy-boosting, anti-inflammatory, and antioxidant properties due to the compounds of capsaicin and piperine [7]. Though conventional ice cream is made by using dairy milk, over the years, nondairy milk has been more common, such as soy, almond, and coconut milk. Coconut milk ice cream may be an excellent alternative to those who suffer from lactose intolerance and benefit others as well. Coconut milk is a milky fluid obtained by manual or mechanical extraction of fresh coconut (Cocos nucifera L) kernel with or without addition of water. As a coconut-producing country, coconut milk plays a vital role in the Sri Lankan diet [8]. It is valued mainly for its characteristic nutty flavor and for its nutritional content. Coconut milk contains 56.3% moisture, 33.4% fat, 4.1% protein, 1.2% minerals, and 5.0% carbohydrates [9]. Development and quality evaluation of coconut milk-based soft ice cream has been reported [10]. Moreover, a study has been reported on physicochemical and sensory properties of ice cream formulated with virgin coconut oil [11]. However, the formulation of a spicy coconut ice cream and evaluation of its physicochemical and sensory attributes are yet to be investigated. 2. Materials and Methods 2.1. Preparation of Spices 2.1.1. Preparation of Cinnamon Powder Cinnamon quills (7 g) were purchased from a supermarket and washed using hot water (45°C) and dried at room temperature. Then, dried cinnamon quills were finely ground to obtain a powder with the help of a grinder (Philips HL1606, 500 W). 2.1.2. Preparation of White Pepper Powder Fresh white peppercorns (4 g) were purchased from a supermarket and cleaned using hot water (45°C) and dried at room temperature. Dried peppercorns were finely ground using a grinder (Philips HL1606, 500 W) to obtain the white pepper powder. 2.1.3. Preparation of Ginger Extract (Water Extraction) Fresh ginger rhizomes (15 g) were purchased from a supermarket. A mixture of 200 g of fresh, peeled, and cleaned ginger roots and 100 mL of filtered hot water (45°C) was blended using a medium speed level for 2 minutes using a blender (Wipro ANGEL WAM-L-55). Then, the blended mixture was filtered using a muslin cloth. 2.2. Ice Cream Production Mature fresh coconuts were split and scraped with the use of a stainless steel electric coconut scraper (Walvia, India). Milk was extracted by blending the scraped coconut with water (). 360 g sugar, 10 g ice cream stabilizer Cremodan (Cuisine tech), and 14 g gelatin (Motha Confectionery) as a thickening agent were mixed together and added to the coconut milk (1.4 mL) [9]. The mixture was heated to 40°C, and spices were added separately. Heating was continued to 90°C, and salt (8 g) was added. The mixture was pasteurized at 90°C for 20 minutes and cooled to room temperature. The mixture was filtered and homogenized. Then, it was kept for around 4 hours for aging. The mixture was fed into the instant ice cream-making machine (Softy line; capacity: 20 L/h). Ice cream was filled into a clean container (3 L) and stored in a freezer at -18°C prior to analysis. 2.3. Physicochemical Analysis of Spicy Coconut Ice Cream 2.3.1. pH of Ice Cream The pH of the melted ice cream sample was measured by using a pH meter (Starter 3000) [12]. 2.3.2. Titratable Acidity of Ice Cream Titratable acidity of the melted ice cream sample was calculated as the percentage of lauric acid was determined by titration with 0.1 N sodium hydroxide [13]. 2.3.3. Moisture Content of Ice Cream Moisture content of the spicy coconut ice cream and normal coconut ice cream (control) was measured according to the oven-dried method 925.10 as described in AOAC [14]. The empty moisture cans and lids were dried in the oven (Mammoth UF55, +20°C to +300°C) at 105°C for 3 hours and transferred to the desiccator to cool. The weight of cans with lids was taken. About 5 g each of spicy ice cream and control sample was weighed to the dishes, and samples were spread uniformly. The cans with samples were placed in the oven overnight. After drying, the dishes with partially covered lids were transferred to the desiccator to cool. Finally, the cans and samples were reweighed. Calculation [14]: where is the weight (g) of the sample before drying and is the weight (g) of the sample after drying. 2.3.4. Ash Content of Ice Cream Ash content of spicy coconut ice cream and control samples was determined using the dry ash method 925.10 as described in AOAC [14]. The crucibles and lids were placed in the muffle furnace (Hobersal Rex C 700) at 550°C for 5 hours to ensure that impurities on the surface of the crucible were burned off. The crucibles were cooled in a desiccator. Then, the clean and dry crucibles and lids were weighed. 5 g each of dried spicy ice cream and control samples was weighed into crucibles and placed in the furnace without covering with the lids. The sample was incinerated at 550°C for 5 hours. The lids were placed after completed heating to prevent loss of fluffy ash. After cooling down in the desiccator, ash with crucibles and lids was weighed. Calculation [14]: where is the weight (g) of ash and is the weight (g) of the dried sample. 2.3.5. Total Solids of Ice Cream (Gravimetric Method) Both spicy ice cream and control samples were subjected to the following testing. Samples were transferred to a beaker and gradually warmed in a water bath (35-40°C). Then, samples were cooled to room temperature. Dishes and lids were heated in an oven at 102°C for 1 hour. The lids were placed on the dishes and immediately transferred to the desiccator to cool. Then, weights were taken. About 5 mL of spicy ice cream and control samples was poured into the dishes separately. Lids were placed, and weights were taken. The dish was placed in boiling water bath without a lid (bottom of the dish, directly heated by steam). Heating was continued till most of the water is removed. The dish was removed from the water bath and placed in the oven at 102°C for 2 hours alongside lids. Thereafter, lids were placed and kept in a desiccator to cool. The weights were taken, and dishes were heated again with lids alongside in an oven for 1 hour. Then, lids were placed and transferred immediately to the desiccator, and weights were recorded [14]. Calculation [14]: where is the mass in g of the dish, is the mass in g of , and is the mass in g of . 2.3.6. Melt Down of Ice Cream Melt down of spicy coconut ice cream and control samples was determined according to method 941.08 as described in AOAC [14]. Melt down of ice cream samples was evaluated using a 50 g ice cream block (height: 2 cm, diameter: 6 cm) which was placed on a metric test sieve that was supported by a previously weighed beaker. The mass of melted ice cream collected in the beaker was recorded at 5-minute time intervals for a 60-minute duration. The mass of melted ice cream (g) was plotted against the time (min) [14]. 2.3.7. Overrun of Ice Cream Overrun of the ice cream was determined by using about 20 mL of ice cream mix and frozen ice cream. Calculation [14]: 2.3.8. Crude Protein Content of Ice Cream Crude protein content of the spicy coconut ice cream and control was determined by the Kjeldahl method: 920.87 as described in AOAC [14]. Approximately 0.5-1.0 g of dried ice cream sample was placed in a dry and cleaned digestion flask. 25 mL of concentrated sulfuric acid and Kjeldahl tablet were added to the digestion flask. Digestion was run for 3 hours using a mini Kjeldahl unit (Block Digestion Unit Model: K-424). After, the digestion flask was allowed to cool and conducted distillation using an automated distillation unit (BUCHI, USA). Finally, the sample was titrated with 0.25 N HCl solution, and crude protein content was determined (). 0.25 N HCl was standardized by titrating 0.25 N sodium carbonate solution. 2.3.9. Crude Fat Content of Ice Cream Fat content of the spicy coconut ice cream and control was determined by the Soxtherm method as described in AOAC [14]. Oven-dried samples were used to measure crude fat using a fat analyzer (Model: SER 148, Italy). 1 g of sample () was weighed accurately by using analytical laboratory balance in a weighed extraction thimble (). The rubber ring was selected, and thimbles were placed in a fat analyzer. 80 mL of petroleum ether was filled into each tube. Then, the fat analyzer was programmed for 5 minutes in immersion, 20 minutes in washing, and 30 minutes in recovery. After completion of the process, the crucible was oven-dried at 105°C for 6 hours and reweighed [14]: 2.4. Antioxidant Activity of Ice Cream Ice cream samples were dried by lyophilization (Martin Christ, Freeze Dryer, Alpha 1-2/LD Plus) for 24 h. An ice cream sample (13 g) was mixed with a mixture of 70 mL methanol/water (80/20, ) and vortexed at high speed for 30 minutes. Then, the mixture was centrifuged (Hettich, EBA 20) for 10 min at 792 rpm. After the centrifugation, the extracts were subsequently filtered through a filter paper (Whatman No. 42; Whatman Paper Ltd, Maidstone, UK). Finally, the prepared extracts were evaporated in a rotary evaporator (Hahnvapor, Model HS-2005 V, Hahnshin Scientific, Korea) at 40°C under vacuum and stored at -18°C until assayed within 1 week [15]. 2.4.1. Total Phenolic Content About 1 mL of the melted ice cream sample was added to a 1.5 mL diluted Folin-Ciocalteu reagent. Samples with the reagent were left for 5 minutes, and then, 1 mL 7.5% sodium carbonate () was added. The samples were vortexed and kept in the dark for 2 hours. The absorbance was measured at 760 nm using a spectrophotometer (Optima, SP-3000, Tokyo, Japan). The calibration curve of gallic acid was plotted to evaluate the activity capacity of the samples. The result was expressed as milligram of gallic acid equivalents (GAE) per gram of ice cream sample (mg GAE/100 g of dry weight) [16]. 2.4.2. DPPH Radical Scavenging Activity About 3 mL of prepared DPPH solution was added to 1 mL of melted ice cream sample. Then, solutions in the test tubes were shaken well. Samples were incubated in the dark for 15 minutes at room temperature. Finally, the absorbance was measured at 517 nm using a spectrophotometer (Optima, SP-3000, Tokyo, Japan) [16]. The DPPH radical scavenging activity (%) was calculated as where is the absorbance of the control and is the absorbance of the sample [16]. 2.4.3. Total Antioxidant Capacity About 0.1 mL of the ice cream sample was added into a test tube and mixed with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tube was capped with an aluminum foil and incubated at 95°C for 90 minutes. Then, the tube was cooled down to room temperature, and the absorbance was measured at 695 nm using a spectrophotometer (Optima, SP-3000, Tokyo, Japan) [16]. 2.5. Sensory Evaluation The acceptability of the coconut milk-based spicy ice cream was evaluated by conducting a sensory evaluation with 30 untrained, both male and female panelists, in the age between 22 and 30 years. The sensory attributes tested were color and appearance, body and texture, aroma and flavor, melting quality, and overall acceptability. A 9-point hedonic scale was used. The values were scored on least preference (1) to most preference (9). 2.6. Statistical Analysis MINITAB version 14 was used to statistically analyze all the results obtained from physicochemical and sensory evaluation. A one-way analysis of variance (ANOVA) was performed, and the significant difference was defined at for the results of all analyses with both control and coconut spicy ice cream. The final results obtained were expressed as the mean of three replicates. Sensory evaluation data were analyzed using the Kruskal-Wallis test. 3. Results 3.1. Selection of the Most Acceptable Spicy Ice Cream Formula Data obtained from sensory evaluation revealed that sample 02, containing 0.018% of spices, is the most acceptable formula. Color and appearance, body and texture, spicy flavor and aroma, and overall acceptability of sample 02 were higher than that of 0.025% spice-incorporated sample 01 and 0.010% spice-incorporated sample 03. Melting quality of sample 02 is lower than that of samples 01 and 03 (Figure 1).
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