Applied Food Research 2 (2022) 100164

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Applied Food Research

journal homepage: www.elsevier.com/locate/afres

Efficacy of plant-based natural preservatives in extending shelf life of

sugarcane juice: Formulation optimization by MOGA and correlation study
by principal component analysis
Bijaya Bharati Bag, Chirasmita Panigrahi∗, Sakshi Gupta, Hari Niwas Mishra
Agricultural and Food Engineering Department, IIT Kharagpur, West Bengal 721302, India

a r t i c l e i n f o a b s t r a c t

Keywords: Sugarcane juice spoils quickly as a result of microbial contamination and enzymatic reactions. To maintain its
Sugarcane juice quality during storage, synthetic preservatives are generally employed. However, their use has been linked to
Natural preservatives potential health hazards. In this regard, some plant based natural preservatives can be utilized as safe alternatives.
Concentration optimization
Lemon juice (LJ), basil leaf extract (BE), mint leaf extract (ME), and lemongrass essential oil (LGEO) were used
MOGA
in this study. LJ, BE and ME were varied from 2 to 8% (v/v) and LGEO was ranged between 1 and 3% (v/v).
PCA
The concentrations of these natural preservatives were optimized by multi-objective genetic algorithm (MOGA)
based on contrasting goals of maximizing soluble solids, phenolic content (PhC) and sensory acceptability score
while minimizing pH, polyphenoloxidase (PPO) activity and bacterial count. The LGEO followed by LJ inhibited
the microorganisms and oxidative enzymes at an appreciable level and ME was the least effective among all. At
the same time, LJ offered more preferable sensory scores than others. The optimized concentration suggested
by MOGA was 6.84% LJ and 1.05% LGEO which resulted in 10.3% rise in PhC, 49.3% PPO inactivation, 2 log
cycle bacterial reduction and 3.5% sensory improvement in the raw juice. The changes in quality parameters
during storage and correlations among them were effectively illustrated by principal component analysis. It was
revealed from microbial safety analysis that sugarcane juice with storage stability of 26 days at 4 ºC could be
prepared by addition of natural preservatives.

Practical applications 1. Introduction

Herbs and spices being composed of many bio-active compounds
and associated with several therapeutic properties are likely to im-
part excellent anti-microbial and anti-browning stability to sugar-
cane juice. Particularly, for the consumers who demand additive-
free minimally processed products with fresh taste, sugarcane
juice preserved using natural plant based additives would be
a healthy deliverable. The outcomes of the optimization study
would facilitate suitable utilities of GA in other food process ap-
plications. Furthermore, determination of correlation among pa-
rameters would be helpful while conducting multiple regression
analysis to forecast the statistical significance of variables.
Sugarcane juice (ScJ) is a very popular sweet drink with its color
ranging from light yellow to dark green based on the variety of sugar-
canes. It is normally consumed fresh as fermentation begins within few
hours of extraction (Kayalvizhi et al., 2016). Due to its rich nutrient
content (mainly sugar), the ScJ spoils fast since microbial (yeasts and
bacteria such as Leuconostoc species) activities immediately onset the
fermentation and other degradation processes which make its long term
storage very difficult (Abhilasha & Pal, 2018; Khare et al., 2012). The
rapid deterioration limits its processing and commercialization. Previ-
ous studies have stated that thermal pasteurization of the ScJ may en-
hance the shelf life and confirm the microbial safety of the juice, but
it can accelerate the undesirable changes in the nutritional and sen-
sory effects of ScJ (Chew et al., 2018). Chemical preservatives have also

Abbreviations: AAD, Absolute average deviation; AMC, Aerobic mesophilic bacteria; ANOVA, Analysis of variance; BE, Basil leaf extract; BHT, Butylated hydrox-
ytoluene; CFU, Colony forming unit; DPPH, 2,2-diphenyl-1-picryl-hydrazyl-hydrate; EO, Essential oil; GAE, Gallic acid equivalent; GA, Genetic algorithm; LGEO,
Lemongrass essential oil; LJ, Lemon juice extract; ME, Mint leaf extract; MOGA, Multi-objective genetic algorithm; NSGA, Non-sorted genetic algorithm; OA, Overall
acceptability; PhC, Phenolic content; PPO, Polyphenoloxidase; PCA, Principal component analysis; RMSE, Root mean square error; ScJ, Sugarcane juice; TA, Titratable
acidity; TSS, Total soluble solids; U, Unit.
∗
Corresponding author.
E-mail address: chirasmitap@iitkgp.ac.in (C. Panigrahi).

https://doi.org/10.1016/j.afres.2022.100164
Received 7 May 2022; Received in revised form 17 June 2022; Accepted 30 June 2022
Available online 6 July 2022
2772-5022/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
B.B. Bag, C. Panigrahi, S. Gupta et al.
been revealed to have adverse health effects (Tribst et al., 2009). At
recent times, consumers demand for more high quality, preservative-
free, minimally processed foods with increased shelf life. This trend has
increased the alternative use of natural products, particularly preserva-
tives obtained from herbs and plant products, in order to substitute the
synthetic chemical compounds in current use (Harvey, 2008).
Since ancient times, herbs and spices are being employed for pre-
venting food deterioration and food borne diseases. Over the past
few decades, the use of herbs and plant products has gained trac-
tion among researchers as food preservatives owing to their antimi-
crobial and antioxidant properties. Moreover these are having fewer
adverse effects, easy availability, lessen toxicological profile, elemen-
tal bioavailability and many additional functional benefits compared
to other available antimicrobial agents (Sánchez-González et al., 2011;
Soković et al., 2013). Specifically, some essential oils (EOs) of plant
origin are dominant food preservatives. The chemical composition of
EOs (thymol, carvacrol, linalool, citral, citronellol, geraniol, eucalyp-
tol, geranyl acetate and limonene) revealed the presence of several
bioactive molecules, which possess antibacterial, antifungal and antiox-
idant activities in vitro (Fisher & Phillips, 2006). One of the candidate
EOs with food-preserving ability is that of lemongrass (Cymbopogon
citrates). Lemongrass extract and essential oil have been used as aids
in food flavoring and preservation due to their pharmacological and
therapeutic activities (Boukhatem et al., 2014). Recent investigations
advocated the prospect of the use of lemongrass essential oil (LGEO)
as an effective inhibitor of bio-oxidation and microbial contamination
(Ekpenyong & Akpan, 2017). In spite of the high competence of the EOs
and their constituents against pathogens and food spoilage microorgan-
isms, this effect in food is only attained with higher concentration of
EOs (Burt, 2004). This fact may result in the alteration of natural taste
and intrinsic flavor of the food by surpassing the thresholds. This prob-
lem may be avoided if essential oils would be possibly combined with
other plant extracts for achieving an additive effect. Such strategy can
overcome the individual limits of each additive and pose a synergistic
effect (Chaves-Lopez et al., 2009).
Normally ScJ is served as a cooling drink with ice, flavored with a
dash of lemon or sizzled with mint, basil leave and salt. All these ad-
ditives have the potential to perform as natural preservatives and also
enhance the flavor of juice (Ramachandran et al., 2017). Citrus fruits are
among the numerous essential horticultural crops, lemon (Citrus limon
L.) being the third most principal citrus crop. Several studies have high-
lighted that lemon is an abundant source of nutrients including citric
acid, vitamin C, minerals and phytochemicals, like, flavonoids which
have copious health promoting properties (Gonzalez-Molina et al., 2010;
Mulero et al., 2012). For this reason, lemon juice is a suitable source
as a food matrix for designing some value-added products. Holy Basil
(Ocimum basilicum) is an extensively grown herb for therapeutic use
known to contain a range of phytochemicals (lithospermic acid, ros-
marinic acid, coumaric acid, vanillic acid, hydroxybenzoacid, proto-
catheuic acid, syringic acid, ferulic acid, caffeic acid) which prove its
ability to be a natural preservative in food (Pandey & Negi, 2018). Sim-
ilarly, studies of Tassou et al. (1995) and Muchuweti et al. (2007) con-
firm the phytochemical constituents of mint (Mentha longifolia) which
contains protocatechuic acid, gallic acid, p-coumaric acid, vanillic acid,
and ferulic acid. These phenolic acids and flavonoids are strong antiox-
idants, free radical scavengers, and metal chelators. There are also re-
ported studies on the antioxidant property of Mentha (Dorman et al.,
2003; Zheng & Wang, 2001).
The current research intends to improve the shelf life of fresh sug-
arcane juice using plant based natural preservatives. Lemon juice, basil
leaf extract, mint leaf extract, and lemongrass essential oil are used in
this study. The aim is to compare the different natural preservatives
on the basis of enzymatic and microbial inhibition and sensory accept-
ability. Optimization of the formulation of natural preservative has been
carried out through multi-objective genetic algorithm. Furthermore, the
2
Applied Food Research 2 (2022) 100164
principal component analysis is performed to visualize the changes in
different quality parameters during storage of preservative added ScJ.
2. Materials and methods
2.1. Samples
Fresh and healthy sugarcane stalks were obtained from local market
of IIT Kharagpur, India. Natural preservatives like lemons, basil leaves,
mint leaves and lemongrass were collected from the agricultural farm
in Agricultural & Food Engineering (AgFE) Department, IIT Kharagpur,
India.
2.2. Extraction and filtration of sugarcane juice
The sugarcane stalks were washed with water to remove dust and
soil residues. Juice was obtained by passing the sugarcane stalks through
power operated stainless steel roller crusher (SYGA ScJ machine; Power-
350 W) and the extracted juice was collected from its outlet. Filtration
of juice was done by sieving (1 mm mesh size) and eight layers of muslin
cloth to remove debris and insoluble matter.
2.3. Preparation of natural preservatives
The selected plant extracts were prepared by aqueous extraction
method and essential oil was prepared using hydro-distillation method.
2.3.1. Lemon juice
Lemons were cut into pieces and lemon pieces were squeezed. The
lemon juice (LJ) was filtered through muslin cloth to remove the seeds
and extraneous matter.
2.3.2. Basil leaf extract
Basil leaves were first washed under running tap water to make it de-
void of any dust and dirt. Then the juice was extracted from the washed
leaves by power operated extractor. The sample to water ratio for ex-
traction was 1:5 (Al-Sum & Al-Arfaj, 2013). The collected basil leaf ex-
tract (BE) was then filtered with muslin cloth and then centrifuged at
605 g/10 min at 4 °C.
2.3.3. Mint leaf extract
Mint leaves were washed first under running tap water. Then,
washed mint leaves were cut into small pieces. The extract was obtained
using a power operated juice extractor with 1:5 leaf to water ratio (El-
Said & Hassan, 2021). After that, mint extract (ME) was filtered through
muslin cloth and shreds of mint were discarded.
2.3.4. Lemongrass essential oil
The lemongrass essential oil (LGEO) was extracted from the lemon-
grass by the hydro-distillation method using a Clevenger’s apparatus
(Lamaty et al., 1987). The sample to water ratio for this procedure was
1:8, i.e. 50 g of lemongrass leaves in 400 ml distilled water (Ranitha
et al., 2014). The recovered oil was dried over anhydrous sodium sul-
fate and stored in dark at 4 °C until use.
2.4. Physico-chemical and microbiological analysis of juice
The physico-chemical and microbiological parameters of the ScJ
samples were determined according to the standard methods and pro-
tocols (Ranganna, 1986; AOAC, 2005).
Total soluble solid (TSS) content is determined by the index of refrac-
tion. This was measured using a refractometer and expressed as degree
Brix (ºBrix). pH values were measured at a temperature of 25 ± 0.5 ºC
using a pH meter (𝜇pH System 361, M/s Systronics, India Ltd., India).
B.B. Bag, C. Panigrahi, S. Gupta et al. Applied Food Research 2 (2022) 100164

Titratable acidity (TA) was determined by titrating against 0.01 NaOH

#Values with different superscript letters are significantly different (p < 0.05). LJ: lemon juice, BE: basil extract, ME: mint extract, LGEO: lemongrass essential oil; P1, P2, P3 and P4 are the samples added with LJ,
using phenolphthalein as indicator (AOAC 942.15).

Sensory acceptability score

Polyphenol content (PhC) was estimated by Folin–Ciocalteu’s
method by taking gallic acid as standard for calibration (Panigrahi et al.,
2020). The absorbance was noted at 760 nm in a UV-Vis spectropho-

7.23 ± 0.07abc
7.93 ± 0.06ab

7.84 ± 0.05ab
8.36 ± 0.05cd

7.85 ± 0.04cd
8.14 ± 0.04bc

7.02 ± 0.12bc

7.46 ± 0.02d
tometer (Lambda 35 UV-Vis, M/s PerkinElmer, Connecticut, USA).

7.95 ± 0.09a

8.07 ± 0.12a
6.83 ± 0.06e
7.8 ± 0.03d

7.5 ± 0.1ab
Polyphenoloxidase (PPO) enzyme activity was measured by adding
5.5 ml of 0.2 M phosphate buffer (pH 6.0) and 1.5 ml of 0.2 M catechol
to an aliquot (0.5 ml) of the sample (Kunitake et al., 2014). Changes in
absorbance values were read at 425 nm in a spectrophotometer at 30 s

AMC (log CFU/ml)

interval. Enzyme activity is calculated as given in Eq. (1),
( )

6.54 ± 0.18abc
6.87 ± 0.07ab
6.75 ± 0.12ab
6.05 ± 0.21cd

5.94 ± 0.15cd
bc

6.32 ± 0.06bc
( )

5.54 ± 0.13d

5.56 ± 0.12d

7.12 ± 0.54a
4.81 ± 0.27e
𝐴𝑏𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑏𝑏𝑙𝑎𝑛𝑘

6.43 ± 0.19
𝑈

7.09 ± 0.1a
6.8 ± 0.1ab
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (1)
𝑚𝑙 0.001 𝑋 𝑡
where, Absample and Abblank are absorbance values for sample and blank,
respectively; t is the reaction time.

PPO activity (U/ml)

Aerobic mesophilic bacteria count (AMC) was determined to exam-
ine the microbiological stability of samples using nutrient agar as the

23.12 ± 0.04ab

13.33 ± 0.16ef
d

17.84 ± 0.13d

19.11 ± 0.09d

22.37 ± 0.11b
24.36 ± 0.24a
14.88 ± 0.14e

14.04 ± 0.05e
20.75 ± 0.14c

12.25 ± 0.12f
21.65 ± 0.1bc
18.59 ± 0.08

24.69 ± 1.9a
growth media (Panigrahi et al., 2018). The incubation temperature and
time were 37 ºC and 24 h to observe the growth. AMC was expressed as
log10 colony forming unit per ml (CFU/ml).

2.5. Sensory evaluation

PhC (mg GAE/100 ml)

A 12-membered panel consisting of faculty and research scholars

41.51 ± 0.11 g
38.35 ± 0.07cd

38.29 ± 0.02cd
38.43 ± 0.12d
b

37.94 ± 0.02b

37.43 ± 0.06a
38.89 ± 0.06e

38.78 ± 0.05e

38.74 ± 0.01e
38.17 ± 0.09c

39.91 ± 0.04f
37.92 ± 0.03
(age: 25–35 years, 5 males and 7 females) of the AgFE Department,

38.22 ± 0.1c
IIT Kharagpur, India was constituted for the sensory evaluation. The
members were selected according to the good health, interest, previ-
ous experience in sensory analysis and familiarity with sugarcane juice
properties. Prior to the test, all the panellists were informed about the
characteristic nature (plant-like flavor and sugary taste) and the sen- Titratable acidity (%citric acid)
sory attributes (color, flavor, taste and overall acceptability) based on
which the analysis was to be done. They were instructed to provide
scores against each attribute using the 9-point hedonic scale (1 = dis-
like extremely, 2 = dislike very much, 3 = dislike moderately, 4 = dis- 0.21 ± 0.03cd
0.19 ± 0.03cd
0.13 ± 0.04cd

0.17 ± 0.03cd

0.18 ± 0.06cd
0.13 ± 0.03cd
0.17 ± 0.03cd
0.11 ± 0.02d
a

0.51 ± 0.02a

0.22 ± 0.03c

0.2 ± 0.03cd
0.42 ± 0.04

0.7 ± 0.03b

like slightly, 5 = like slightly, 6 = like fairly well, 7 = like moderately,

8 = like very well, and 9 = like extremely) provided in the sensory score-
card. The score higher than 5 depicted acceptance of the juice (Pandraju
& Rao, 2020). Initially, the panelists were asked to take two quick sniffs

BE, ME and LGEO, respectively, where, a, b, and c depict the concentration of additives.
for scoring flavor. They were also advised to rinse their mouths with
6.16 ± 0.09 g
5.07 ± 0.06de

5.93 ± 0.08fg
5.34 ± 0.07ef

5.28 ± 0.08ef
4.97 ± 0.09d

4.81 ± 0.03d
3.22 ± 0.06b
a

3.53 ± 0.12a

4.29 ± 0.05c
5.82 ± 0.07f

5.48 ± 0.04f
3.72 ± 0.08

water and swallow it between the samples. Overall acceptability (OA)

was the only sensory parameter considered for optimization and storage
Experiments conducted and juice quality parameters evaluated in the study.

study.
pH
abc

14.13 ± 0.04abc

13.77 ± 0.06abc
13.83 ± 0.06abc
13.53 ± 0.15abc

2.6. . Optimization of preservative concentration

14.27 ± 0.21ab
13.06 ± 0.06bc
13.3 ± 0.03abc

14.1 ± 0.03abc
14.32 ± 0.06a
12.97 ± 0.06c

14.2 ± 0.05ab
13.67 ± 0.06

14 ± 0.06abc
TSS (o Brix)
Responses

Different concentrations of LJ, BE, ME, and LGEO were added to the
sugarcane juice as mentioned in Eqs. (2)–(5). The varying formulations
are listed in Table 1. The best combination was selected on account of
juice quality analysis. The optimization was done by an inbuilt function
LGEO (%)

‘gamultiobj’ in the toolbox of MATLAB R2019a.

The optimization variables and constraints are given below:
0
0
0
0
0
0
0
0
0
1
3
5
0

Maximize: f1 (X) = y1 = TSS; f3 (X) = y3 = TA; f4 (X) = y4 = PhC;

f7 (X) = y7 = OA and minimize: f2 (X) = y2 = pH; f5 (X) = y5 = PPO; f6
ME (%)

(X) = y6 =AMC subject to :

0
0
0
0
0
0
2
5
8
0
0
0
0
Independent variables

𝑥1 _lower (2) ≤ LJ ≤ 𝑥1 _upper (8)(%) (2)

BE (%)

0
0
0
2
5
8
0
0
0
0
0
0
0

𝑥2 _lower (2) ≤ BE ≤ 𝑥2 _upper (8)(%) (3)

LJ (%)

2
5
8
0
0
0
0
0
0
0
0
0
0

𝑥3 _lower (2) ≤ ME ≤ 𝑥3 _upper (8)(%) (4)

Control
Sample
Table 1

code

P1b

P2b

P3b

P4b
P1a

P2a

P3a

P4a
P1c

P2c

P3c

P4c

𝑥4 _lower (1) ≤ LGEO ≤ 𝑥4 _upper (5)(%) (5)

3
B.B. Bag, C. Panigrahi, S. Gupta et al.
The regression analysis was performed using the data analysis tool
in MS Excel v.2013 to derive the equations adequately fitting the exper-
imental data. Seven objective functions (the regression equations with
only significant terms) for MOGA optimization are specified below in
Eqs. (5)–(11).
( ) ( 2 ) Titratable acidity of P2, P3 and P4 samples did not deviate significantly
y1 = − 14.002 − 0.167x1 + 0.053x2 + 0.059x3 − 0.13x4 R ∶ 0.998 (5)
( 2
y2 = 4.122 − 0.17x1 − 0.028x3 − 0.147x4 + 0.024x24 R ∶ 0.967
( )
y3 = − 0.267 + 0.058x1 − 0.025x2 − 0.024x3 − 0.07x4 + 0.01x23 + 0.022x24
( ) ( 2 )
y4 = − 38 − 0.074x1 + 0.134x2 − 0.06x3 + 0.045x21 + 0.033x24 R ∶ 0.993
( ) (
y5 = 20.02 − 0.37x1 + 1.22x2 + 1.72x3 − 4.52x4 − 0.03x21 − 0.172x22 − 0.2x23 +0.6x24
( )
y6 = 6.534 − 0.04x1 + 0.19x2 + 0.22x3 − 0.414x4 − 0.03x22 − 0.03x23 + 0.02x24
( ) ( 2
y7 = − 7.6 + 0.36x1 + 0.14x2 − 0.22x3 − 0.43x4 − 0.04x21 − 0.03x22 − 0.03x23
The MATLAB optimization program is usually a minimization prob-
lem for which, the objective functions to be maximized are multiplied
by (−1). Therefore, Eqs. (3), (5), (7) and (11) have been added with
negative signs.
2.7. Storage study
The optimized formulation of natural preservative solution added
into sugarcane juice was then filled in sterilized glass bottles. The ScJ
samples (added with natural preservatives) were stored at 4 ± 1 °C for
the period of 30 days. Evaluation of each sample was carried out at 7
days interval up to 3 weeks and alternate days thereafter. The shelf life
was determined with respect to physicochemical and microbiological
characteristic changes monitored during storage.
2.8. Statistical analysis
The response parameters were determined three times and values
were presented as mean ± standard deviations of three replications.
The data obtained were analyzed and interpreted by analysis of vari-
ance (ANOVA). Tukey means comparison test was performed to eval-
uate the significance at a probability level of 5%, using SPSS Version
20.0 (IBM SPSS software). For correlation study, principal component
analysis (PCA) was carried out in order to determine the interrelation-
ships among different parameters and changes in sample quality during
storage. Pearson’s correlation matrix and PCA plots were produced in
the statistical software Origin 2019b.
3. Results and discussions
3.1. Effect of natural preservatives addition on quality parameters of
sugarcane juice
The values of different juice quality parameters for different concen-
trations of added preservatives are given in Table 1. Here, P1, P2, P3
and P4 are the ScJ samples added with LJ, BE, ME and LGEO, respec-
tively, where, a, b, and c depict the concentration of additives. Among
Applied Food Research 2 (2022) 100164
the preservative added ScJ samples, TSS was found to be in the range
of 12.97–14.32 o Brix, the maximum was of P2c. However, there was no
significant difference (p > 0.05) in TSS among all the samples includ-
ing control (raw juice). The pH of all the samples differed significantly
(p < 0.05) after addition of preservatives and varied from 3.22 to 6.16.
compared to control irrespective of the preservative concentration and
) its values range within 0.11–0.7% citric acid. Minimum pH of 3.22 and
(6) maximum acidity of 0.7% citric acid were found in the sample P3c (10%
LJ) due to higher proportion of lemon.
( ) In Table 1, PPO activities of samples added with each concentra-
R2 ∶ 0.989
tion of LJ and LGEO were significantly reduced whereas, BE and ME
(7)
at low concentration (2%) were unable to reduce the PPO activity sig-
nificantly. The ScJ supplemented with 5% LGEO imparted the highest
inhibition (50.38%) of enzyme activity followed by 8% LJ (39.73%),
(8) whereas, ME could provide the least resistance to browning (12.3%).
The anti-browning efficiency of LGEO is supported by the study of
) Soares et al. (2013) where they have reported that LGEO shows similar
R2 ∶ 0.919 activity to that of the synthetic antioxidant butyrated hydroxytoluene
(9) (BHT). Eissa et al. (2008) found the highest reduction (92.65%) in PPO
activity in apple juice supplemented with LGEO. Its antioxidant activity
( 2 ) is ascribed to the synergistic actions of monoterpenoid compounds such
R ∶ 0.978
as a-citral, b-citral, myrcene, and eugenol (Rathbai & Kanimozhi, 2013).
(10)
Because of these properties, LGEO has been used extensively to pre-
serve different food substrates, including watermelon juice, apple juice,
)
R ∶ 0.987 pear juice (0.4–2% concentration) and mixed fruit juice (0.5% LGEO)
(11) (Friedman et al., 2004; Massilia et al., 2006; Tyagi et al., 2010). In lemon
juice, flavones and flavanones form the group of major flavonoids, fol-
lowed by hydroxycinnamic acids and quercetin derivatives which con-
tribute to the phenolic content (PhC) of P1 samples and are responsi-
ble for the antioxidant activity of lemon juice (Girones-Vilaplana et al.,
2012). The antioxidant potential of basil is supported by the study of
Jayasinghe et al. (2003), where they identified the main components
of O. basilicum leaf that act as antioxidants. They further revealed that
its potency as a natural antioxidant is due to high prevalence of phe-
nolic compounds which is also be revealed from the highest PhC value
of P2c (Table 1). Here, PhC of all the preservative added samples has
increased significantly (p < 0.05) owing to presence of various types of
phenols and flavonoids in lemon, basil, mint and lemongrass. The rise in
phenolic content by LJ and LGEO are almost comparable at the studied
concentrations. Basil and mint, especially the former at higher concen-
tration seemed to improve the PhC of ScJ slightly more than the rest
two preservatives.
It can be observed from the Table 1 that, AMC values of P1 and P4
samples at all concentrations of additives were significantly (p<0.05)
lower compared to the control. Furthermore, the activities of 2% and 5%
LJ are equivalent to 1% LGEO and 8% LJ similar to 3% LGEO (p<0.05).
AMC was the lowest in samples added with 5% LGEO achieving 2.31 log
reduction, followed by 8% LJ (1.58 log reduction). The order of preser-
vatives in terms of bacterial reduction is: LGEO > LJ > BE > ME. The
presence of significant amount of oxygenated monoterpenes (78.9%)
denoted by citral compounds designates high antimicrobial potential of
lemongrass oil (Tyagi & Malik, 2010). This kind of observation was also
reported by other researchers, like, Celinaa et al., (2018) who found that
the addition of 0.1% LGEO significantly suppressed the microbial load
of pineapple juice during 5 days at 4ºC. The finding related to LGEO is
also supported by Eissa et al., (2008) where they have found the high-
est antimicrobial activity of LGEO (among basil, rosemary, sage, lemon-
grass and clove EO) on apple juice during storage at 4°C for 4 weeks.
The application of LJ was also giving a fair result in this aspect. The an-
timicrobial activity of LJ was tested against various gram positive and
gram negative bacteria and it was found that juice of C. limon displayed
the highest antimicrobial activities, as it inhibited 13 isolates (out of
15) of the bacteria with inhibition zone ranging from 10-30 mm (Hindi
& Chabuck, 2013). Similar result was also reported by Sengun & Kara-
pinar, (2004) where they had found that treatment of carrot samples
4
B.B. Bag, C. Panigrahi, S. Gupta et al.
Table 2
GA parameters applied for optimization.
Parameters Set value
Population type Double vector
Population size 50
Population creation function Constraint dependent
Population selection function Tournament selection
Crossover function Multiple (three) point
Crossover probability 0.8
Mutation function Constraint dependent
Mutation probability 0.3
Number of generations 500
Function tolerance 10−6
with LJ was effective in reducing the counts of inoculated Salmonella ty-
phimurium and caused significant reductions (0.79–3.95 log cfu/g). Ad-
ditionally it can be noticed from Table 1 that, basil extract of higher
concentration (8%) could only pose significant declination in AMC with
reference to raw (control) juice whereas, with 2% and 5% BE, there was
insignificant effect (p>0.05). The antimicrobial property of BE is par-
tially supported by the study of Hossain et al., (2010) where they have
found that methanol extracts (300 𝜇g/disc) of O. basilicum presented
great antibacterial activity against B. cereus, B. subtilis, B. megaterium, L.
monocytogenes, S. aureus, E. coli, and S. typhi. The results of this study
recommended that the natural products derived from O. basilicum may
have probable use in the food as antimicrobial agents. From the result it
can also be observed that (Table 1), the reduction in AMC by the addi-
tion of ME was statistically insignificant (p>0.05) for all the concentra-
tions relative to the control. This means the ScJ supplemented with mint
extract has shown minimal antimicrobial activity even with the highest
concentration of 8%. Similar result was depicted by Mahmood et al.,
(2010) where they had examined the antimicrobial activity of aque-
ous extracts of nine plants including mint, against 20 microbial isolates,
mostly food borne including pathogens (E. coli, Salmonella typhi and Can-
dida albicans). It was found by antimicrobial screening that the extract
of mint showed no antimicrobial activities against many of the tested
isolates. Jain et al., (2010) have evaluated the antibacterial activity of
aqueous leaf extracts of Mentha arvensis (mint), against E. coli and B.
subtilis by agar well diffusion method and found that mint did not show
any activity against both the test organisms.
The sensory acceptability score ranged between 6.83 and 8.36. The
OA scores of LJ, BE and ME at 2% concentration did not deviate signif-
icantly (p > 0.05) from the control. The sample P1b (5% LJ) received
the highest overall acceptability score of sensory evaluation. The lowest
OA score was that of P4c (8% LGEO) which might have been due to the
slight pungent and bitter taste of LGEO. Similar result was also reported
by Celinaa et al. (2018) where the addition of 0.1% LGEO into pineap-
ple juice had a negative impact on the sensorial acceptability. The lower
sensory scores of BE and ME at higher concentrations could be because
of the strong flavor of basil leaf and mint leaf that might be dominating
over the actual flavor of sugarcane juice.
3.2. Optimization of formulation by MOGA
Objective functions were developed from regression analysis as de-
scribed earlier. A multi-objective fitness function was created using
the generated regression models with a function handle as: @multi-
objective. The function was asked to hunt for solutions with maxi-
mum of some parameter values and minimum of others as defined ear-
lier Eqs. (5)–((11)). The various parameter options implemented in the
solver have an influence on GA system design and play a prominent role
on the solution process (Dietz et al., 2008). Such key parameters applied
for GA optimization are given in Table 2.
The genetic algorithm starts with the generation of random popu-
lation based on population size (Np ). Each set of independent variable
5
Applied Food Research 2 (2022) 100164
data (X1 , X2 , X3 and X4 ) was regarded as an individual that were en-
coded as bit strings (“0” and “1”) of length 4. So, the population forms a
16×Np matrix. The population size is an important parameter for decid-
ing the convergence of an algorithm since, very large Np would upset
the efficiency of evolution and if it is very small, it would be easy to fall
into a local optimum, which is not the favourable case. In this study, af-
ter attempting it multiple times, the accurate initial population size (50)
was created. With generations below 50, it was seen that the solutions
were not getting converged into a point even after several iterations. Np
of 50 was set as further increase of the population size beyond it did
not improve the solution accuracy. The type of population was double
vector known to be adequate in case of bound constraints. The individ-
uals were generated using a constraint dependent creation function to
ensure uniform distribution. The constraints were set using the upper
and lower bounds of the concentrations of preservatives in the order:
[LJ, BE, ME, LGEO] with the lower bound being [2, 2, 2, 1] and upper
bound corresponding to [8, 8, 8, 5].
The initialization step (generation of population) was followed by
selection of parents (or mating pool) using the objective functions de-
veloped based on experimental data. The selection of individuals was
done as per the classic non-sorted genetic algorithm (NSGA) -II with the
help of the tournament selection scheme based on crowded-distance op-
erator having a default size of 2. The selected fittest individuals were
subsequently subjected to crossover operation which was done with a
probability value of 0.8 and using a multiple (three) point crossover
function. The next step of operation was mutation. Constraint depen-
dent function was adopted for performing mutation (with a probability
of 0.3) due to its ability of generating more reliable optimized solutions
than other mutation methods. Sites on the bit string for these operations
were randomly chosen. High crossover rates and low mutation proba-
bilities are favored for a better evolution, since, lower crossover proba-
bilities lead to low level of diversity in new generations being evolved
whereas, higher mutation rates cause slower convergence of solutions
(Konak et al., 2006). While running MOGA optimization, the threshold
criterion for the closure of the search algorithm was defined as 500 gen-
erations or a functional tolerance limit of 10−6 , whichever is achieved
earlier. The number of generations was changed numerous times aiming
to acquire optimal solutions in a short computational time. With gener-
ations less than 500, the solution convergence was not found even after
the fixed number of generations reached or till the attainment of toler-
ance limit, whereas in case of above 500, the solution optimality did not
appear to improve. The rise in search space did not lead to more satisfy-
ing results, which therefore, signified that the search space formed was
sufficient for the problem created based on the selected range of con-
centrations (Dietz et al., 2008). The process of getting optimum results
within the solution space was repeated until accomplishing the solution
converged to a point that fulfilled the criteria of all objectives. In other
words, the optimization process was iterated until the population fitness
of a generation reached a static phase. At this point, the objective func-
tion has the most desired (optimal) value and the purpose has almost
been met.
The values for best fitness changed and upgraded across generations
till it converged to a relatively consistent value at around 500 gener-
ations. This ultimately resulted in 7 solutions that fitted the objective
functions to the best possible extent. The optimal set of total 7 solutions
as suggested by MOGA are reported in Table 3. The concept of solution
convergence can be better visualized by a plot that shows the change in
average pareto spread with increasing generations displayed in Fig. 1.
From this graph, convergence could be seen as the solutions being closer
to each other and finally attaining the identical values. The eventually
converged solution set (solution no. 7th in Table 3) was regarded as the
overall finest fit for the concerned multi-objective optimization problem
of the present study. The optimum concentrations of preservatives to be
added in ScJ are 6.84% lemon juice (LJ) and 1.05% lemongrass essential
oil (LGEO). The resulting response parameters were TSS of 13.46 °Brix,
pH of 4.03, titratable acidity of 0.332% citric acid, PhC of 40.543 mg
B.B. Bag, C. Panigrahi, S. Gupta et al. Applied Food Research 2 (2022) 100164

Sensory acceptability score

8.257
7.956
8.162

8.074
8.038
8.04
7.93
Fig. 1. Variation in pareto average spread with generations showing gradual
AMC (log CFU/ml)

convergence of optimal solutions.

GAE/100 ml, PPO activity as 12.69 U/ml, AMC value of 4.8 log CFU/ml
5.763

5.148

and sensory acceptability score of 8.038.

4.99
5.06

4.67
4.82
4.8
PPO activity (U/ml)

3.3. Validation of optimal results

The GA predicted optimal formulation has to be validated with the

actual experiment with those particular concentrations in order to ex-
18.136
15.492
16.75

17.69
19.42
12.83
12.69

amine the method accuracy. The juice was added with 6.84% LJ and
1.05% LGEO and then analysed for quality responses. The comparative
assessment of prediction versus experimentation is given in Table 4. The
PhC (mg GAE/100 ml)

absolute average deviation (AAD) for all responses was found to be less
than 5% signifying the accuracy of prediction (Dang, 2018). Smaller
value of observed root mean square error (RMSE) implies a good perfor-
mance of the model (Soleimani et al., 2013). From this, a good harmony
38.629

41.275

40.543
39.53

39.86

40.69

of simulated data with the actual values is portrayed that illustrates ap-
38.2

preciable reliability of MOGA optimization. It was also observed from

Table 4 that, there was significant difference (p < 0.05) in all the pa-
Titratable acidity (%citric acid)

rameters, except pH between raw and preservative added samples. The

Final solution#LJ: lemon juice, BE: basil extract, ME: mint extract, LGEO: lemongrass essential oil.

addition of natural preservatives caused 4.2% decrease in TSS, 3% re-

duction in pH (insignificant, p > 0.05), increase by 87.64% in acidity,
10.28% rise in PhC, 49.27% PPO inactivation, 2.05 log cycle reduction
in AMC and improved the sensory acceptability score by 3.46% in raw
sugarcane juice.
This is found in other studies also that the use of mixed additive
0.329

0.376

0.347
0.332
0.63

0.31

imparts synergistic effect and proves effective in enhancing the food

0.4

preservative actions of individual additives. Adding LGEO and lime peel

extracts to chicken patties augments the antioxidant and antimicrobial
3.246

4.273

4.167
4.64
4.51

4.62

4.03
Converging pareto optimal solution set obtained from MOGA simulation.

actions of both extracts (Hayam & Ferial, 2013). The antioxidant activ-
pH

ity of LJ in combination with other additives was found to be better

TSS (o Brix)

than the LJ alone. This is in agreement with the finding of Girones-

Vilaplana et al. (2012) where they have found the DPPH scavenging
13.458

13.372
13.27

13.61

13.94

13.46
13.8

potential of LJ added with black chokeberry was better than LJ alone.

LGEO (%)

3.4. Evaluation of quality changes during storage by principal component

1.564

1.053

analysis
1.73

1.09
1.14
0

The storability of preservative added ScJ samples for a period of 30

ME (%)

2.059

days in refrigerated conditions (4 ± 1 ºC) was assessed and its shelf life
2.18

was estimated by visualizing the changes in juice qualities by PCA tech-

0

0
0
0

nique. Fig. 2(a) illustrates the scree plot showing eigenvalues, variance
BE (%)

and cumulative variance corresponding to 7 dimensions. It can be per-

2.33

ceived that the first two components have the eigenvalues >1 (1st com-
0
0
0
0

0
0

ponent PC1 = 6.4, 2nd component PC2 = 1.47). It satisfies the Kaiser-
LJ (%)

6.238

Guttman criterion of selecting the principal components and hence by

6.93
5.32
6.79
7.15

6.84
7.6

this rule, the first two principal components were considered in view
of maximum explication of variability (Njintang et al., 2007). PC1 and
Solutions

PC2 individually explained 82.17% and 15.82% of total variance, re-

Table 3

spectively, and both conjointly accounted for a collective variance of

7†
1
2
3
4
5
6

97.99%. Fig. 2(b) depicts PCA bi-plot that provides a graphical represen-
tation showing correlation of response patterns and connection between
stored samples in a k-dimensional space. In this plot, all the parameters

6
B.B. Bag, C. Panigrahi, S. Gupta et al.
namely, TSS, pH, acidity PhC, PPO, AMC and OA score are denoted as
loadings while the days of storage are symbolized as scores.
Samples lying close to each other in the bi-plot are often character-
ized by similar properties. The clustering of AMC adjacent to acidity and
TSS near to pH signifies their close relations. As depicted in Fig. 2(b),
the scattering of the samples on the PCA bi-plot gradually transformed
over time and the sample points moved towards left along the PC2 axis.
From the 26th day onwards, the qualities seem to have undergone ex-
treme changes as apparent from the position of P26, P28, P29 and P30
that lie very isolated and distant from the sample at 0th day of storage.
This suggested that the quality markedly changed after 24–25 days of
storage. This can also be pointed out that, samples up to 14 days (P0,
P7 and P14) possessed highest pH, PhC, TSS and OA that gradually re-
duced thereafter. In contrast, samples at and beyond 26 days that are
positioned on the top left or left of the plot displayed higher amounts of
7
Applied Food Research 2 (2022) 100164
Fig. 2. . PCA of quality parameters in stored
ScJ (a) scree plot (b) correlation matrix show-
ing Pearson correlation coefficients among dif-
ferent variables (c) PCA bi-plot representing
the distribution of samples and variables.
PPO, acidity and AMC. This clearly indicates the progressive growth of
bacteria over time in turn, leading to conversion of TSS into acids and
secondly, reduction of phenolic content with storage time as a result of
PPO enzymatic browning. Moreover, the clustering of parameters oppo-
site to each other in discrete coordinates of the plot designate their neg-
ative associations. For example, lower the bacterial counts (i.e. AMC),
lower the acidity and higher is TSS. Likewise, the samples with lower
PPO tend to have greater PhC. The mutual relationship among the stud-
ied quality parameters is elaborated further with the help of correlation
coefficients.
3.5. Correlation of quality parameters
The correlation matrix having Pearson’s correlation coefficient (r) is
displayed in Fig. 2(c). The r values evaluate the strength of linkage be-
B.B. Bag, C. Panigrahi, S. Gupta et al. Applied Food Research 2 (2022) 100164

tween different pairs of variables. In Fig. 2(c), the negative correlations

TSS with acidity (r = −0.926, p < 0.05) and AMC (r = −0.949, r < 0.05)
are confirmed. As evident by correlation coefficient values, the AMC has
strong negative relations with pH (r = −0.994, p < 0.01) while it is pos-

Sensory acceptability score

itively associated with acidity (r = 0.993, p < 0.05). This is attributed
to the degradation of sugars by microbes converting soluble compounds
into insoluble particles (even forming some polysaccharides e.g. dextran

8.35 ± 0.08a

8.07 ± 0.12a
from sucrose) leading to juice sedimentation in long run during storage
(Panigrahi et al., 2022).
8.038 In Fig. 2(c), strong correlations are identified among PPO and PhC

3.69
0.34
(r = −0.981, p < 0.05). This is ascribed to the browning phenomenon
in which PPO enzyme catalyzes the oxidation of phenols into brown
AMC (log CFU/ml)

pigments called ‘melanins’ (Queiroz et al., 2008). Such finding is sup-

ported by the report of Persic et al. (2017) in case of apple juice. This
5.07 ± 0.17b

7.12 ± 0.54a

is in accordance with Leng et al. (2009) who reported a proportional

reduction in phenolic content with increase of fruit browning enzyme.
4.96
4.8

0.3

Hence, the correlation analysis efficiently highlighted the interrelation-

ship status of various parameters associated with microbial degradation
PPO activity (U/ml)

and enzymatic browning.

12.524 ± 0.07b

3.6. Shelf life

24.69 ± 1.9a
12.69

0.176

From earlier discussion of PCA, it was found that, the changes in

1.33

characteristics of stored samples were more significant after 26 days. For

better calculative shelf life prediction, the number of microorganisms is
PhC (mg GAE/100 ml)

a determining factor in microbiological safety and is consequently be-

ing recognized as a reliable indication of shelf life. So, it is important to
41.28 ± 0.08b

37.43 ± 0.06a

calculate the maximum number of days that product can be stored be-
fore being spoilt or discarded, i.e. the time taken by microbes to reach
40.543

the critical count limit. According to Food Safety and Standards Reg-
0.742
1.79

ulations (Revised Microbiological Standards for Fruits & Vegetables &

their products, 2018), the upper limit of consumer acceptability in case
Titratable acidity (%citric acid)

of juice for AMC is 6 log CFU/ml. The result of AMC data up to 26th day
(5.91 log CFU/ml) conform the stringent microbiological safety guide-
lines. In light of these findings, it can be ascertained that the sugarcane
juice added with 6.84% lemon juice and 1.05% ginger extract can be
stored up to 26 days under normal refrigeration temperature (4 ± 1 ᵒC).
0.319 ± 0.003b

0.17 ± 0.03a

#Values with different alphabets as superscripts are significantly different (p < 0.05).

4. Conclusion
0.332

0.013

The present investigation has attempted MOGA tool for solving a

4

multi-criteria optimization. As revealed by validation results, the ge-

4.16 ± 0.13a

4.29 ± 0.05a

netic algorithm had an adequate accuracy in optimizing the formulation

of natural preservatives to be added onto ScJ. The optimized concentra-
0.134

tion was found to be 6.84% lemon juice and 1.05% lemongrass essential
4.03

3.2
pH

oil. This caused 10.3% rise in phenolic content, 49.3% PPO inactivation,
destroy 2 log bacterial counts and improved the sensory acceptability
13.67 ± 0.03b

14.27 ± 0.21a

score by 3.5% of the raw juice without preservative. The preservative

TSS (o Brix)

added ScJ could be stored up to 26 days under refrigeration with ensure

13.46

0.212
1.54

microbial safety and appreciable sensory appeal. This provides unique

scope of additional benefits (like, improving organoleptic properties) to
Verification result of the optimal solution.

non-thermal processing of juice applied for its shelf life enhancement.

Raw sample without preservative (Control)

The addition of natural plant based agents to minimally processed juice

can act as an effective hurdle technology intending the anti-browning
and preservation aspect. Above all, the chemical preservatives can be
replaced with such natural preservatives. Since these methods are sim-
ple, inexpensive and convenient, the utilization of the preserved juice
should be encouraged as health/therapeutic drink.

Declaration of Competing Interest

Experimental
Predicted

AAD (%)

The authors have no competing interest.

Table 4

RMSE

CRediT authorship contribution statement

Bijaya Bharati Bag: Conceptualization, Methodology, Data cura-

tion, Writing – original draft. Chirasmita Panigrahi: Formal analysis,

8
B.B. Bag, C. Panigrahi, S. Gupta et al.
Validation, Writing – original draft, Writing – review & editing. Sakshi
Gupta: Data curation, Writing – original draft. Hari Niwas Mishra: Su-
pervision, Writing – original draft, Writing – review & editing.
Acknowledgments
This work is financially supported by the Ministry of Food Processing
Industries, Govt. of India, with the code IIT/SRIC/R/TSJ/2019, dated
28-03-2019.
Ethical statement
Written consent was obtained from all study participants for sensory
evaluation. There was no involvement of animals in this study.
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