Food and Humanity 2 (2024) 100263

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Food and Humanity

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Evaluation of mint (Mentha spicata) hydrodistillation aqueous byproducts

and its utilization in development of bioactive-rich functional drink
Bhagwat Madhura a, b, Kavitha Lakshmipathy a, b, Chidanand D.V. a, b, *, C.K. Sunil c, N. Baskaran d,
Ashish Rawson b, e, R. Dhivya a, b
a
Department of Industry-Academia Cell, National Institute of Food Technology, Entrepreneurship, and Management-Thanjavur, India
b
Centre of Excellence in Non-Thermal Processing, National Institute of Food Technology, Entrepreneurship, and Management-Thanjavur, India
c
Department of Food Engineering, National Institute of Food Technology, Entrepreneurship, and Management-Thanjavur, India
d
Department of Academics and Human Resource Development, National Institute of Food Technology, Entrepreneurship, and Management-Thanjavur, India
e
Department of Food Safety and Quality Assurance, National Institute of Food Technology, Entrepreneurship, and Management-Thanjavur, India

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

Keywords: Distillation aqueous byproducts are discarded despite being rich in bioactive components. In this study, the
Aqueous byproducts aqueous byproducts (hydrolats and flask water fractions) resulting from the hydrodistillation of fresh and dry
Bioactive compounds mint leaves were compared, with a primary focus on their essential oil yield and bioactive properties. Dry mint
Flavored drink
leaves yielded approximately eight times more essential oil than fresh leaves. The hydrolat extracted from dried
Mint
Hydrolat
mint leaves (MHD) exhibited higher phenolics (35.5 ± 1.1d mg GAE/100 mL), flavonoids (7.8 ± 0.29d mg QE/
100 mL) and 2,2-diphenylpicrylhydrazyl scavenging potential (21.13%), compared to the hydrolat derived from
fresh leaves (MHF), which had lower values (7.115 ± 0.9e mg GAE/100 mL, 5.72 ± 0.422d mg QE/100 mL and
6.79%, respectively). Similarly, the flask water fraction from dry mint leaves (FWF2) had a higher concentration
of bioactive compounds compared to that from fresh leaves (FWF1). The hydrolat with higher bioactivity (MHD)
was selected for infusion to develop a flavored drink and its GCMS profile indicated a relatively abundant
presence of D-Carvone. The flavored drink prepared with a 20:100 ratio of hydrolat was preferred for its flavor
intensity, a key sensory parameter for determining the hydrolat amount. Additionally, for Gum Arabic, a 2.5:100
ratio in the drink resulted in a more acceptable consistency score.

1. Introduction fresh or in product (Bokić et al., 2020). It is traditionally added to hot

drinks (tea, medicinal water, and soups) and foodstuffs like paneer for
Bioactive compounds are chemical compounds found in plants and herbal flavors. It offers health benefits due to water-soluble bioactive
natural sources that can provide health benefits, including antioxidants, compounds with antimicrobial properties found in native and hybrid
antidiabetic, antimicrobial, and anti-inflammatory properties upon varieties (Ohtsu et al., 2018).
consumption. Horticultural products like fruits and vegetables contain Distillation is a process that is primarily used to extract essential oils
these beneficial phenolic and flavonoid compounds, but processing can but also produces byproducts that are potentially wasted (Aswandi &
alter their effectiveness (Soquetta et al., 2018). Additionally, these Kholibrina, 2021). Hydrodistillation is one of the types of distillation
critical components are lost when a significant portion of fresh produce that separates elevated volatility inherent compounds by boiling sam­
is discarded. Although various established extraction technologies are ples in water and then condensing the vapors generated. The essential
available, these complexes remain underutilized in product develop­ oil acquired in the adjacent tube of the apparatus due to the difference in
ment (Renard, 2018). densities brings about a distinct layer (Bayan & Küsek, 2018). Similar to
Mint varieties from different regions offer a delightful flavor and steam distillation, hydrodistillation generates byproducts such as
possess herbal and therapeutic qualities beneficial in multiple applica­ hydrolat (condensed aqueous extract with suspended oil mixture), re­
tions. It contains high levels of phenols, flavonoids, and antioxidants, sidual water (flask water fractions for hydrodistillation using Cle­
sensitive to various processing and storage conditions, whether used as venger’s apparatus), and plant material (sample) (Celano et al., 2017).

* Corresponding author at: Department of Industry-Academia Cell, National Institute of Food Technology, Entrepreneurship, and Management-Thanjavur, India.
E-mail address: chidanand@iifpt.edu.in (C. D.V.).

https://doi.org/10.1016/j.foohum.2024.100263
Received 26 October 2023; Received in revised form 18 February 2024; Accepted 21 February 2024
Available online 23 February 2024
2949-8244/© 2024 Elsevier B.V. All rights reserved.
B. Madhura et al. Food and Humanity 2 (2024) 100263

The essential oils are the primary outcome of such distillation, which has 2. Materials and methods
maximum marginal recovery with the elevated market price compared
to byproducts. These aqueous byproducts can be employed directly and 2.1. Materials
have no damaging consequences, as distilled water, a pure solvent, was
used in hydrodistillation (Gavahian et al., 2019). Mint leaves were obtained from the Thanjavur regional market and
A cooling, refreshing, and thirst-quenching effect can be achieved by manually destemmed. The leaves were air dried for 24 h to assess the
mixing the mint-flavored aqueous extract with water and making a differences in distillation byproducts between fresh and dry leaves.
flavored drink (Larionov et al., 2020). Since people are becoming more These leaves were refrigerated in an airtight bag and utilized for
health-conscious, beverages with low-calorie, non-carbonated drinks extraction. Implementation of study, including hydrodistillation and
are gaining popularity. It can add value to widely sold mineral water drink preparation, was conducted at the National Institute of Food
while utilizing the benefits of bioactive compounds (Kapp et al., 2020). Technology, Entrepreneurship and Management-Thanjavur. Laboratory
A mint extract can impart a strong flavor to added items while sup­ grade analysis chemicals were procured from SRL Chemicals, Pvt. Ltd.
plying bioactive compounds. The incorporation of hydrolat in water, The food-grade additives Gum Arabic (Prix Global, Indore) and Potas­
together with food additives, will help in the development of a bioactive sium sorbate (Ases Chemical Works, Jodhpur) were purchased for
compound-rich drink that also hydrates the body (Thiagarajah et al., incorporation into the infused drink. Ozonated water was collected from
2019). Crossfield’s Best Water Doctor, a purification system in the Institute.
There are studies on utilizing hydrolat in soft drinks, but only a few
focused on bioactive-rich and non-carbonated beverages or drinks 2.2. Methods
(Hamedi et al., 2017). Thus, this study is particularly focused on
assessing the aqueous byproducts obtained from distillation and their 2.2.1. Analytic comparison of fresh and dry mint leaves
use in developing a bioactive-rich flavored drink. The hydrodistillation The color values were determined according to the methodology
byproduct with suitable color and lower total soluble solids was chosen outlined by Biswas et al. (2012). The water activity of the leaves was
for infusion. Among the selected byproducts (fresh and dry), the one determined using a digital water activity tester (Aqualab 4TE), which
with the highest bioactive properties (phenols, flavonoids, and antioxi­ calculates the dew point temperature and displays water activity.
dants) was used to make drinks to enhance the benefits of flavor and The comparison of essential oil concentration in both fresh and dry
bioactive compounds. mint leaves was conducted following hydrodistillation. The essential oil
content was measured using graduations on Clevenger’s apparatus,
where a distinct oil top layer emerged and could be measured as the
extraction process neared completion. The essential oil content was
calculated using the formula.,

Fig. 1. Hydrodistillation of fresh and dry mint leaves and aqueous by products.

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B. Madhura et al. Food and Humanity 2 (2024) 100263

mL of essential oil levels, in a two-square factorial design. The quantities were taken as
Essential Oil Content (%) = × 100
Raw material (g) follows:

(Trujillo-Echeverria et al., 2020).

• Control (ozonated water),
• A (20 mL/100 mL hydrolat, 2.5 g/100 mL Gum Arabic),
2.2.2. Extraction of Mint leaves by hydrodistillation
• B (30 mL/100 mL hydrolat, 2.5 g/100 mL Gum Arabic),
Fig 1 illustrates the process carried out during this study. The fresh
• C (20 mL/100 mL hydrolat, 5 g/100 mL Gum Arabic),
(aw = 0.9803 at ± 30 ℃) and dry mint leaves (aw = 0.5986 ± 30 ℃)
• D (20 mL/100 mL hydrolat, 5 g/100 mL Gum Arabic).
were chopped/crushed to easily add to the extraction round bottom
flask of one-litre volume. Around 100 g mint leaves were added to a one-
Gum Arabic was weighed and mixed with measured hydrolat until
litre flask separately, upon which water was added to entirely immerse
clear pale-yellow liquid formed, and the volume was made up to 100 mL
those into water. The leaves to water ratio of 1:3 (Fresh) and 1:5 (Dried)
by Ozonated water.
was sufficient to avoid bumping. Dried leaves absorb water more
quickly; hence, a higher water to leaves ratio was maintained as a pre­
caution. The regularly employed hydrodistillation system (Clevenger’s 2.3. Analysis of aqueous byproducts and flavored drink
apparatus) was fixed on the neck of the round bottom glassware, and
another end was affixed to the condenser. The upper condenser opening 2.3.1. Color
was closed with cotton to avoid the escape of elevated volatile fractions For color identification, the Hunter Lab colorimeter (Hunter Asso­
(Clevenger, 1928). The extraction was performed at 60 ℃ for 2 hrs. ciates Laboratory, USA) was utilized to provide information about color
Temperature-time was chosen based on earlier research that directed an values L* (lightness), a* (greenness), and b* (yellowness). The hue
optimal degree of bioactive chemicals (Psarrou et al., 2020). The ex­ parameter reflected perceptions of colors such as green, red, and yellow,
tracts were stored in an ambered glass vial in refrigerated conditions to while chroma offered insights into hue intensity or purity. The browning
prevent the loss of bioactive components due to light and temperature. index plays a pivotal role in enzymatic and non-enzymatic browning in
food products, impacting their flavor, color, and texture. The color
2.2.3. Recovery of hydrodistillation aqueous byproducts (flask water changes (ΔE), browning index (BI), hue angle and chroma of the samples
fractions and hydrolats) were measured using the formula (Johnson et al., 2023),
Following the distillation process, hydrolat was obtained by ΔE = [(L*-L*0)2 + (a*-a0*)2 + (b*-b0*)2]1/2
removing the essential oil layer from fresh leaves (MHF) and dry leaves
(MHD). The residual water in the flask was recovered through a strainer BI = 100*
X − 0.31
on which leaves were retained and flask water fractions were collected 0.17
for fresh leaves (FWF1) and dry leaves (FWF2). These samples were
cooled and stored in an ambered glass bottle in the refrigerator. A X = (a* + 1⋅75 L*) (5⋅645 L*+ a* − 3⋅012b*)
control sample was prepared by grinding leaves in a grinder using 10 mL Hue angle = tan-1 (b*/a*)
of water and filtering the mixture.
Chroma = [ (a*)2 + (b*)2]1/2
2.2.4. Qualitative GCMS profile of selected hydrolat for drink
The chemical profile of the hydrolat selected for drink was obtained
by Gas Chromatography-Mass Spectrometry (GC-MS) (Agilent Tech­
2.3.2. pH
nologies, Inc., USA) analysis using the purge and trap method. The
The determination of sample acidity or alkalinity was carried out by
hydrolat was stored at refrigerated condition, and a 5 mL sample was
using a digital pH meter (Eutech Instruments Pvt. Ltd., Singapore)
transferred to loop for analysis. The sample was subjected to purge and
(Lakshmipathy et al., 2022).
trap and reconditioned after each desorption. Conditions maintained
were - Column (DB-624-(30 m x 250 µm x 1.4 µm) (or) equivalent), Flow
2.3.3. Total soluble solids
rate (1.0 mL/min), Injection volume (1 µl), Initial temperature (40 ℃),
The total soluble solids (% Brix) in hydrolats were measured using a
Injector temperature (200 ℃), Sparging temperature (11.4 min), Loop
digital refractometer (Milwaukee, Romania - MA871) ranging from 0 to
temperature (112 ℃), AUX-1 temperature (115 ℃), Interface tempera­
85% Brix. The reading was obtained by placing a few drops of the
ture (250 ℃), ion source temperature (230 ℃), Carrier gas as Helium
sample on the prism and pressing the button. The display provided the
and Purge and Trap split mode (Sonmezdag et al., 2017).
reading value along with the temperature surrounding the instrument
(Trujillo-Echeverria et al., 2020).
2.2.5. Developing hydrolat infused flavored drink
The hydrolat is a water medium with slightly suspended oil mole­
2.3.4. Specific gravity
cules and was stabilized by Gum Arabic (natural emulsifier and stabi­
The specific gravity of the sample was determined as the ratio of its
lizer). As a preservative, water-soluble potassium sorbate (0.005%) was
density to that of water. It was assessed by a pycnometer (10 mL), which
added in limits (Safety, 2011). The proportion of Gum Arabic and
is volumetric glassware. The pycnometer, along with the sample, was
hydrolat in water was decided by following the formulation according to
weighed after recording the empty pycnometer weight (Lakshmipathy
Rezvani et al. (2012) with slight changes. The proportion was deter­
et al., 2022).
mined post-randomization, considering two factors: the quantity of Gum
The specific gravity was calculated as-
Arabic (g/100 mL) and the chosen hydrolat (g/100 mL), each at two

(Weight of sample with pycnometer – Weight of empty pycnometer)

Density of sample (ϱs) =
Total volume of sample taken(10 mL)

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B. Madhura et al. Food and Humanity 2 (2024) 100263

Density of sample panellists conducted sensory analysis, evaluating parameters such as

Specific gravity =
Density of water appearance, color, flavor intensity, taste, consistency, and overall
acceptability in the drink samples, and average intensity scores were
2.3.5. Opacity of drink calculated for each attribute at the completion of the evaluation process
The opacity of the drink was assessed using a spectrophotometer to (Almoselhy, 2023).
determine its nature (opaque or transparent). A solution was prepared
by mixing 1 mL of the drink with 100 mL of distilled water, and the 2.4. Microbial analysis
absorbance at 660 nm was recorded. An absorbance value of 1 signifies
opaque, while a value close to 0 indicates transparency (Rezvani et al., Microbial analysis involved the use of plate count agar, lactose tri­
2012). phenyl tetrazolium chloride agar, and chloramphenicol yeast glucose
agar to determine the total aerobic mesophilic plate count, Escherichia
2.3.6. Total phenolic content (TPC) coli count, and yeast and mold counts, respectively. Through the pour
The determination of the TPC of the samples was assessed through a plate method, a 1 mL portion of a sequentially diluted sample was
modified Folin–Ciocalteu (FC) method (Lakshmipathy et al., 2023). The introduced into a sterile Petri dish, and approximately 20 mL of medium
FC reagent was diluted in distilled water at a 1:10 v/v ratio, and a 7.5% was poured, allowing it to solidify. Following this, plates designated for
sodium carbonate solution was prepared. Subsequently, 5 mL of the total aerobic mesophilic plate count and Escherichia coli were incubated
diluted FC reagent and 4 mL of sodium carbonate were introduced to at 37 ± 2 ◦ C for 20–24 h, while those containing chloramphenicol yeast
1 mL of the sample. After shaking the mixture for 10 min, it was left at glucose agar were incubated at 25 ± 3 ◦ C for 3 days (Amany et al.,
30 ◦ C for 40 min. The resulting solution was analyzed at 760 nm using a 2012).
UV spectrophotometer (Shimadzu Corporation, Japan-UV 1800). Gallic
acid served as the standard for constructing the calibration curve 2.5. Statistical analysis
(equation y = 0.0172x+0.0626, R2 = 0.98). The TPC was represented as
milligrams of gallic acid equivalent per 100 millilitres (mg All findings were expressed as mean values ± standard deviation
GAE/100 mL). derived from triplicate measurements. Group comparisons were per­
formed using IBM SPSS software version 27.0.1.0. Specifically, an in­
2.3.7. Total flavonoid content (TFC) dependent t-test was applied for fresh and dry mint leaves due to the
The TFC was determined using an aluminium chloride (AlCl3) smaller sample size (fewer than three). All comparisons were conducted
method with minor changes, and the results were expressed in Quercetin using one-way analysis of variance (ANOVA), followed by a post-hoc
equivalents (mg QE/100 mL) (Lakshmipathy et al., 2023). Briefly, Tukey’s test, with a significance level set at 0.05.
2.7 mL of the sample was combined with 0.15 mL of a 5% sodium nitrate
solution for 6 min. Subsequently, 0.15 mL of a 10% AlCl3 solution was 3. Results and discussion
added to the mixture for another 6 min, followed by the introduction of
2 mL of a 4% sodium hydroxide solution. The reaction mixture was then 3.1. Analysis of fresh and dry mint leaves
left undisturbed for 20 min. A standard Quercetin solution was
employed to establish the calibration curve (represented as 0.0131y = Table 1 demonstrates that dry mint leaves extraction through
0.0142x with an R2 value of 0.999), and the absorbance of the prepared hydrodistillation was found to be more effective, resulting in a higher
sample was measured at 510 nm using a UV spectrophotometer. yield of essential oils and bioactive compounds compared to fresh mint
leaves. The essential oil content (%) obtained through hydrodistillation
2.3.8. Antioxidant activity (DPPH) for fresh and dry leaves was 0.14% and 1.14%, respectively. The dif­
The ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radi­ ferences in essential oil content may be attributed to variations in
cals was evaluated using a method adapted from Osae et al., 2019, with distillation time and experimental factors, including sample source
minor modifications. Briefly, 1 mL of the sample was blended with 3 mL (Beigi et al., 2018). (Gharib and da Silva, 2013) found that essential oil
of methanol DPPH solution (60 mM) at a concentration of 0.2 mM, content for fresh and dry leaves of the same mint type was of 0.314% and
followed by vigorous vortexing. The mixture was left undisturbed for 0.756% when hydrodistilled for 3 h. Additionally, the L* , a* , and
30 min at room temperature (25 ◦ C) in darkness. The mixture was left b* values of the leaves underwent significant changes upon drying, with
undisturbed for 30 min at room temperature (25 ◦ C) in darkness. Then, L* decreasing from 40.26 ± 0.28a to 24.6 ± 0.006b, a* shifting from
the absorbance was measured at 517 nm using a UV spectrophotometer, − 9.62 to − 0.027, and b* decreasing from 25.76 to 7.3. These color
with a blank consisting of 3 mL of DPPH solution and 1 mL of methanol changes are likely associated with the breakdown of chlorophyll com­
(80%). The DPPH radical scavenging activity was then determined using pound (Therdthai & Zhou, 2009).
an equation (Lakshmipathy et al., 2023),
The Percent DPPH Inhibition was calculated as - 3.2. Aqueous byproducts from hydrodistillation of mint

(Absorbance of control − Absorbance of sample)

%DPPH Inhibition = 3.2.1. Physicochemical properties
Absorbance of sample
Table 1 presents information on the physicochemical analysis of
× 100 aqueous byproducts obtained from the hydrodistillation of mint. In
terms of the color parameters, the L* value of the MHD sample (9.69
2.3.9. Descriptive sensory evaluation of a drink ± 0.01b) exhibited higher lightness than all other samples and was
Sensory evaluation in this study adhered to ethical standards, with comparable to the control (10.6 ± 0.08a). The a* and b* values of
approval and consent obtained from institutional and/or national hydrolats indicated a color profile, implying a greenish and bluish hue,
research committees, following the guidelines of the 1964 Helsinki in contrast to FWF, which exhibited positive a* (red) and b* (yellow)
Declaration and its later amendments or equivalent ethical standards. A values. The total color Difference (ΔE) was significantly higher
group of twenty-five semi-trained professional panellists, consisting of (p < 0.05) in hydrolats compared to FWF. The difference can only be
10 women and 15 men aged 20 to 30, participated voluntarily, with an recognized by a trained examiner, whereas hydrolats can be easily
understanding of the sample safety. Utilizing a 9-point hedonic scale distinguished with ΔE values around and above 5 (Sadowska et al.,
(ranging from 1 for "dislike extremely" to 9 for "like extremely"), 2016). Meanwhile, the hue angle was in the range of 60–80 suggests a
yellowish-green color. Whereas, the BI representing brown color is an

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B. Madhura et al. Food and Humanity 2 (2024) 100263

Table 1
Physicochemical and Biochemical properties of mint juice(control), hydrolats and flask water fractions.
Controly MHF‡ MHD§ FWF1¶ FWF2yy
a e b c
Color L* 10.6 ± 0.08 6.29 ± 0.05 9.69 ± 0.01 9.31 ± 0.07 8.49 ± 0.02d
a* 0.29 ± 0.06c -0.15 ± 0.06d -0.15 ± 0.04d 1.09 ± 0.04b 1.86 ± 0.03a
b* 3.95 ± 0.03a -1.03 ± 0.05d -0.58 ± 0.03c 3.39 ± 0.04b 3.35 ± 0.07b
ΔE - 6.62 ± 0.06a 4.47 ± 0.32b 1.73 ± 0.22d 2.72 ± 0.11c
BI 2.10 ± 0.43c -1.78 ± 0.71d -1.1 ± 0.26d 8.3 ± 0.32b 15.34 ± 0.22a
Hue angle 62.01 ± 0.01a 81.71 ± 0.06ab 75.49 ± 0.06bc 72.17 ± 0.01c 60.95 ± 0.00d
Croma 3.96 ± 0.02a 1.04 ± 0.04d 0.59 ± 0.02e 3.56 ± 0.02c 3.83 ± 0.07b
pH 5.76 ± 0.2d 7.8 ± 0.04b 8.1 ± 0.01a 6.4 ± 0.02c 5.7 ± 0.01d
TSS* 1.1 0.1 0.2 1.7 4.4
Specific gravity 0.960 ± 0.004c 0.964 ± 0.004c 0.94 ± 0.0002d 0.98 ± 0.0001a 0.97 ± 0.0006b
TPC‡‡ 464.5 ± 11.9a 7.115 ± 0.9e 35.53 ± 1.1d 189.4 ± 1.25c 394.6 ± 7.43b
TFC§§ 308.6 ± 1.06c 5.72 ± 0.422d 7.883 ± 0.29d 359.4 ± 10.3b 888.5 ± 26.2a
DPPH¶¶ 258.2 ± 14.2b 6.798 ± 0.6d 21.13 ± 0.5d 67.1 ± 0.86c 332.2 ± 2.5a

*Values are mentioned as mean ± standard deviation and different letters in superscript indicates significant difference among means
†Control is Mint juice, ‡MHF: Mint Hydrolat from fresh leaves hydrodistillation, §MHD: Mint Hydrolat from dry leaves hydrodistillation, ¶FWF1: Flask water fractions
after fresh leaves hydrodistillation, ††FWF2: Flask water fractions after dry leaves hydrodistillation, ΔE: Total color difference, BI: Browning Index, *TSS: Total Soluble
Solids, ‡‡TPC: Total phenolic content as Gallic Acid Equivalent mg/100 mL, §§TFC: Total flavonoid content as Quercetin Equivalent mg/100 mL, ¶¶ DPPH: Percent
DPPH inhibition.

essential parameter in processes involving enzymatic or non-enzymatic displayed an even higher concentration at 394.6 mg GAE/100 mL. In
browning. The BI of FWF was significantly higher (p < 0.05) compared comparison, a previous study on residual waters collected during the
to hydrolats, possibly due to enzymatic browning by polyphenol oxi­ steam distillation of similar plants from the same family reported total
dase, leading to the darkening of the extract when exposed to molecular phenolics ranging from 152 to 443 mg GAE/100 mL (Celano et al.,
oxygen (Kashfi et al., 2020). 2017). The TFC of FWF1 was nearly 359.43 mg QE/100 mL, which was
The pH range of the hydrolat was found to be neutral to slightly significantly lower (p < 0.05) than FWF2 (888.542 mg QE/100 mL).
alkaline, demonstrating a significant difference (p < 0.05) compared to Additionally, the percent DPPH inhibition scavenging potential was
the flask water fractions, which tended more towards an acidic pH. 67.1% for FWF1 and 332.2% for FWF2. Overall, FWF2 demonstrated
Furthermore, the specific gravity of the samples closely resembled that superiority in terms of various bioactive compounds attributed to water
of water, aligning with the criteria for suitability in drink development absorption during the distillation process, as previously mentioned.
(Lakshmipathy et al., 2022). Notably, the hydrolats exhibited lower total
soluble solids, indicating a less dense and transparent nature compared 3.3. GCMS profile of hydrolat (MHD) selected for drink
to the flask water fractions. Overall, the color values, total soluble solids,
and pH of the hydrodistillation byproducts collectively suggest that According to the internal NIST library, 20 compounds were found
hydrolat could be preferable for drink infusion compared to flask water during the GCMS analysis of hydrolat selected for infusion. D-Carvone
fractions. (31.19%), Isobutyl isovalerate (20.02%), Oleic acid (16.5%), and 9,12-
Octadecadienoic acid (14.16%) were the most abundant chemicals
3.2.2. Bioactive properties identified is represented in Fig. 3. D-carvone, a monoterpene present in
Table 1 presented information regarding the phenolics, flavonoids, essential oils, exhibits multiple biological activities, including antimi­
and DPPH radical scavenging activity of mint hydrolats, which varied crobial, antioxidant, and anticancer properties (Johri, 2011). Isobutyl
depending on the divergent components (Benabdallah et al., 2016). The isovalerate (Isovaleric acid) was found impact directly on the smooth
control mint sample used for extraction underwent analysis for bioactive muscles in the colon, leading to muscle relaxation (Blakeney et al.,
compounds in the form of mint juice. The results showed total phenolics 2019). Oleic acid, predominant in olive oil, benefits health by improving
of 464.54 ± 11.95 mg GAE/100 mL, total flavonoids of 308.63 pancreas and liver function, reducing the risk of gastric-duodenal ulcers,
± 1.06 mg QE/100 mL, and DPPH scavenging activity of 258.2 modifying lipid profiles, reducing inflammation, oxidative stress, coag­
± 14.23%, respectively. When the same mint variety was boiled in water ulation, and enhancing glucose homeostasis and blood pressure (Picci­
to create a concentrated liquid, it yielded a total phenolic content nin et al., 2019). 9,12-Octadecadienoic acid effectively lowers
ranging from 188 to 326 µg/mL, approaching the total phenolic content cholesterol, improves insulin sensitivity, reduces blood pressure, and
observed in our experimental control (Rita et al., 2016). The proportion helps prevent heart disease (Rajaram, 2014). Supplementary Fig 1 and
of bioactive chemicals decreases after isolating essential oil from Table 2 gives a detailed list of compounds identified with retention time
hydrolat. The Hydrolat obtained from hydrodistillation of dry leaves (min), MS Ion Fragments (m/z), relative abundance (%), chemical
demonstrated a five-fold greater phenolic content, a three-fold higher category, molecular formula and molecular weight. According to a prior
percentage of DPPH scavenging activity, and a slightly higher flavonoid GC study by investigators, some of the chemicals identified in our results
content. The increased bioactive properties of dry leaves may be are also present in the mint essential oil of the same species (Abu Goukh
attributed to their tendency to rehydrate in water, potentially leading to ABA & El Hassan GM, 2015).
enhanced extraction of bioactive compounds during hydrodistillation
(Sellami et al., 2011).
3.4. Byproduct utilization in bioactive-rich flavored drink
Another considerable aqueous byproduct was Flask Water Fraction
(FWF), which is obtained in larger quantities from fresh leaves
Essential oils are frequently utilized as flavoring agents and additives
compared to dry leaves during hydrodistillation. When subjected to
in various foods, including beverages. While hydrolats are less
analysis for bioactive components, it was observed that a substantial
commonly used for drink infusion, they are often consumed directly as
amount of these components persisted in the water even after a 2-hour
flavored water. However, this direct consumption can result in an
distillation period. The FWF1, derived from fresh mint leaves, exhibi­
overpowering flavor and reduced stability due to the dispersion of
ted a TPC of approximately 189.41 mg GAE/100 mL, while FWF2
essential oils (D’Amato et al., 2018). To address these challenges, a

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B. Madhura et al. Food and Humanity 2 (2024) 100263

Table 2
Gas Chromatography Mass Spectrometry Chemical Profile of Hydrolat (MHD) infused in drink.
Compound Retention Time MS Ion Fragments Chemical category Molecular Molecular Weight Relative abundance
(min) (m/z) Formulae (kDa) (%)

Butyraldehyde 3.24 44, 43, 72 Aldehyde C4H8O 72 0.64

Eucalyptol 9.44 43, 81, 108 Monoterpenoid C10H18O 154 1.22
Piperidine 9.73 84, 85, 57 Heterocyclic amine C5H11N 85 0.36
2-Butenedioic acid 10.86 98, 45, 53 Organic acid C4H4O4 116 0.79
Cyclohexanone 16.50 55, 42, 98 Ketone C6H10O 98 0.41
p-Menth-8-en-2-ol, 17.98 43, 41, 39 Cyclohexanol Acetate C12H20O2 196 0.36
acetate
D-Carvone 18.17 82, 54, 108 Terpenoids C10H14O 150 31.09
Dodecanal 18.37 41, 57, 55 Fatty aldehyde C12H24O 184 0.59
Piperazine 19.13 44, 29, 30 Hexahydro pyrazine C4H10N2 86 0.73
Carveol 19.81 119, 91, 134 Monoterpenoid alcohol C10H16O 152 3.32
2,4- 20.42 44, 105, 91 Phenethylamine C11H17N 163 0.40
Dimethylamphetamine
p-Hydroxy amphetamine 21.43 44, 77, 107 Amphetamines C9H13NO 151 0.75
2-Methyl-E-7-octadecene 21.91 57, 43, 69 Alkene C19H38 266 0.32
Tetradecane 23.26 57, 43, 71 Alkane C14H30 198 0.72
Metanephrine 23.58 44, 46, 93 Catecholamines C10H15NO3 197 0.54
9-Eicosene 23.89 57, 55, 43 Aliphatic hydrocarbons C20H40 280 2.19
Oleic Acid 24.43 41, 55, 43 Monounsaturated fatty C18H34O2 282 16.53
acid
Isobutyl isovalerate 25.70 85, 57, 56 Fatty acid ester. C9H18O2 158 20.02
p-Menth-8(10)-en-9-ol 26.46 81, 55, 95 Terpineol C10H18O 154 4.87
9,12-Octadecadienoic 27.04 67, 81, 82 Polyunsaturated fatty C18H32O2 280 14.16
acid acid

3.5. Physicochemical analysis of drink combinations

Table 3
Drink combinations based on experimental design.
Table 4 presented the results of the physicochemical analysis con­
Combinations Levels Gum Arabic Hydrolat ducted on the prepared drink combinations. The specific gravity for all
A 0 0 2.5 20 combinations fell within the range of 0.96 to 0.97, showing no signifi­
B 0 1 2.5 30 cant differences (p < 0.05). In terms of color analysis, the lightness
C 1 0 5 20
reading (L*) increased consistently with the addition of gum Arabic in
D 1 1 5 30
all drink samples compared to the control. The yellowness (b*) exhibited
an upward trend in drink combinations B, C, and D as the amount of
bioactive-rich flavored drink was prepared by incorporating Gum Arabic Gum Arabic increased. The decrease in a* readings suggested a shift
to effectively blend dispersed oil particles homogeneously, along with towards greenness in all combinations, which aligns with a study con­
the addition of potassium sorbate for preservation. The mixture is then ducted by (Lachowicz et al. (2018) on chokeberry juice with guar gum
diluted with ozonated water to create a well-balanced drink with addition.
desirable physicochemical properties, including color, pH, TSS, specific Furthermore, the ΔE, a discriminant parameter, was determined for
gravity, and opacity. Table 3 shows the experimental design with levels the products. This parameter is crucial for the industry as it reflects the
for drink preparation. consumer’s ability to distinguish between the colors of two products. It

Table 4
Physicochemical and bioactive properties of bioactive-rich flavoured Drink.
Controly A‡ B§ C¶ Dyy

Color L* 2.81 ± 0.07e 11.8 ± 0.075d 12.18 ± 0.006c 14.41 ± 0.07b 15 ± 0.01a
a* -0.17 ± 0.06a -0.37 ± 0.03b -0.47 ± 0.012c -0.59 ± 0.012d -0.79 ± 0.006e
b* -0.01 ± 0.036c -0.14 ± 0.03d 0.08 ± 0.036b 1.14 ± 0.00a 1.16 ± 0.015a
ΔE - 9.05 ± 0.15d 9.34 ± 0.07c 11.51 ± 0.014b 12.09 ± 0.083a
BI -4.56 ± 1.43e -2.25 ± 0.17d -2.76 ± 0.06c -2.96 ± 0.06b -3.8 ± 0.03a
Hue angle 3.36 ± 0.08b 20.72 ± 0.10a 80.34 ± 0.06c 62.64 ± 0.01d 55.74 ± 0.03d
Croma 0.17 ± 0.05e 0.39 ± 0.01d 0.47 ± 0.01c 1.28 ± 0.01b 1.40 ± 0.01a
pH 6.84 ± 0.053a 5.73 ± 0.321b 6.7 ± 0.021a 4.5 ± 0.37c 5.57 ± 0.34b
TSS‡‡ 0.23 ± 0.06e 1.73 ± 0.057d 1.93 ± 0.057c 2.93 ± 0.057b 3.23 ± 0.058a
Specific gravity 0.963 ± 0.007a 0.96 ± 0.009a 0.96 ± 0.00014a 0.976 ± 0.003a 0.977 ± 0.0009a
Opacity 0 0.001 0.002 0.003 0.004
TPC§§ Not tested 49.57 ± 0.183d 53.41 ± 0.06c 59.51 ± 0.150b 67.88 ± 0.03a
TFC¶¶ Not tested 5.94 ± 0.07c 6.27 ± 0.08c 11.72 ± 0.12b 18.80 ± 0.45a
DPPHyyy Not tested 47.56 ± 0.62d 54.06 ± 0.6b 49.71 ± 0.62c 61. 0.25a
Total Plate count (log CFU/mL) 2.47x101 2.30 x101 2.09 x101 2.18 x101 1.98 x101
Yeast and Mold count (log CFU/mL) Nil Nil Nil Nil Nil
Escherichia coli (log CFU/mL) Nil Nil Nil Nil Nil

* *Values are mentioned as mean ± standard deviation and different letters in superscript indicates significant difference among means
†Control is ozonated water, ‡A: 20 mL/100 mL hydrolat, 2.5 g/100 mL Gum Arabic, §B: 30 mL/100 mL hydrolat, 2.5 g/100 mL Gum Arabic, ¶C: 20 mL/100 mL
hydrolat, 5 g/100 mL Gum Arabic, ††D: 20 mL/100 mL hydrolat, 5 g/100 mL Gum Arabic, ΔE: Total color difference, BI: Browning Index, ‡‡TSS: Total Soluble Solids,
§§TPC: Total phenolic content as Galic Acid Equivalent mg/100 mL, ¶¶TFC: Total flavonoid content as Quercetin Equivalent mg/100 mL, †††DPPH: Percent DPPH
inhibition

6
B. Madhura et al. Food and Humanity 2 (2024) 100263

is generally accepted that a human can only perceive a color difference β-Citronellol, and 2,4-Di-tertbutylphenol (Elnour et al., 2018). Likewise,
when ΔE is equal to or greater than 5 units (Sadowska et al., 2016). The the DPPH radical scavenging activity of combination D (56.61%)
color difference of the drink combinations was consistently higher in all exhibited a more significant difference (p < 0.05), followed by B
combinations, ranging from 9 to 12.05. The hue angle was expressed in (54.06%), C (49.7%), and A (47.56%), respectively. This increase is
degrees from 0◦ to 360◦ , with 0◦ (red) denoting a position on the likely linked to the higher phenolic and flavonoid compounds, as studies
+a* axis. It then rotated counterclockwise to 90◦ (yellow) for the have shown a proportional relationship between antioxidant activity
+b* axis, 180◦ (green) for − a* , 270◦ (blue) for − b* , and circled back to and bioactive content (Lakshmipathy et al., 2022, 2023).
360◦ = 0◦ (Johnson et al., 2023). All the drink combinations showcased
redness and yellowish hues. Chroma, representing color intensity, was 3.7. Sensory evaluation
more significant in combinations C and D compared to B, A and the
control. Both the chroma and hue angle of all samples exhibited a sig­ Fig. 2 illustrated a sensory evaluation through a chart encompassing
nificant difference (p < 0.05). The BI, a crucial parameter in processes all sensorial parameters and various drink combinations, including a
involving enzymatic or non-enzymatic browning, exhibited negative control group. The infusion of Hydrolat (MHD) into drink combinations
values, signifying differences in all drink combinations. This observation resulted in variations in acceptance based on sensory parameters
aligns with the findings of A Eissa (2014) study on Pear juice. compared to the control (ozonated water) and all four combinations (A,
Regarding pH, combinations A, C, and D showed a significant B, C, D). Drinks were compared with water to determine which pa­
decrease (p < 0.05) from the control, except combination B. The decline rameters were deemed superior by panellists after mint infusion. The
in pH was associated with the increased addition of Gum Arabic, appearance and consistency of water were preferable compared to the
resulting in a shift towards acidity. This trend aligns with observations drinks. Regarding other parameters, combination D exhibited accept­
from previous studies by (Haji Ghafarloo et al., 2020). in yogurt-based able color, combinations A and C showed better flavor intensity and
Iranian drinks. Notably, combinations A and D exhibited similar pH combination A was preferred for taste. Overall, the acceptability of drink
values. Furthermore, adding Gum Arabic led to a consistent increase in A surpassed others, considering its favourable performance across
total soluble solids and opacity of the drink. This increase was attributed multiple parameters. The incorporation of hydrolat minimally influ­
to the formation of emulsions between dispersed oil droplets in hydro­ enced sensory characteristics, and panellists expressed a preference for
lats and the added emulsifier. The addition of Gum Arabic was also lower hydrolat formulations for enhanced flavor. In accordance with
found to contribute to the increase in opacity (Rezvani et al., 2012). D’Amato et al. (2018), hydrosols demonstrated negligible changes in
sensory attributes when introduced to food products. The quantity of
3.6. Phenolics, flavonoids and antioxidants in drink combinations Gum Arabic employed influenced the drink’s consistency, appearance,
and overall acceptability, with higher amounts correlating with reduced
The ready-to-serve functional drink was developed with different acceptability for certain parameters. In conclusion, sensory evaluations
combinations of mint hydrolat and Gum Arabic. Among the four drink indicated a preference for drinks with lower hydrolat content, specif­
combinations, those with higher proportions of both Gum Arabic and ically combinations A and C.
hydrolat, as per the design, exhibited significantly increased (p < 0.05)
levels of bioactive compounds when compared to the other combina­ 3.8. Microbial analysis of the drink
tions (Table 4). Overall, combination D exhibited significantly higher
(p < 0.05) levels of phenolics (67.88 ± 0.03a mg GAE/100 mL), flavo­ The total plate count of the different functional drink combinations
noids (18.80 ± 0.45a mg QE/100 mL) than other combinations (C, B, A). ranged from 2.53x101 to 1.98x101 log CFU/mL, respectively (Table 4). It
This rise in TPC and TFC could be attributed to the incorporation of a was observed that the combination D (1.98x101 log CFU/mL) with high
higher quantity of hydrolat in this specific combination. As evident in hydrolat and Gum Arabic resulted in a maximum reduction of meso­
Table 2, this hydrolat contains various bioactive compounds such as D- philic organisms when compared with other combinations. This reduc­
carvone, carveol, eucalyptol, p-Menth-8(10)-en-9-ol, and piperidine, tion may be attributed to the presence of bioactive compounds in the
which played a pivotal role in enhancing these phenolics and flavonoids. hydrolat, including D-carvone, P-menth-8(10)-en-9-ol, carveol, and
Additionally, Gum Arabic contributed to the elevation of TPC and TFC as eucalyptol, which exerted antimicrobial effects against the growth of
it consists of inherent bioactives like Hydroquinone, Isovitamine C, aerobic mesophilic microorganisms (Jirovetz et al., 2009). Likewise,

Fig. 2. Sensory analysis of drink combinations.

7
B. Madhura et al. Food and Humanity 2 (2024) 100263

Fig. 3. GC m/z graph for abundant compounds identified in mint hydrolat of dry leaves (MHD).

neither yeasts, molds, nor Escherichia coli were undetectable in the Ethics/ Informed Consent Statement
control group or the other combinations under examination. Similar
results were observed in a functional beverage consisting of carrot The study adhered to ethical guidelines, and participants provided
blends (Amany et al., 2012). Earlier study by Shahbazi (2015) observed informed consent.
the beneficial impact of mint extract on Escherichia coli bacteria which
aligns with the current findings. CRediT authorship contribution statement
In accordance with the FSSAI standards in India, the total plate count
should not surpass 50 CFU/mL, and the total yeast count should not Madhura Bhagwat: Writing – review & editing, Writing – original
exceed 2 CFU/mL for non-carbonated water-based beverages (non- draft, Software, Resources, Methodology, Data curation, Conceptuali­
alcoholic) (Safety, 2011). The findings from present study suggest that zation. Kavitha Lakshmipathy: Writing – review & editing, Writing –
the prepared drinks are suitable for consumption. original draft, Software, Methodology, Data curation. Chidanand D V:
Writing – review & editing, Visualization, Validation, Supervision,
4. Conclusion Project administration, Investigation, Funding acquisition, Formal
analysis, Conceptualization. Sunil C K: Writing – review & editing,
In conclusion, the study explored the potential utilization of aqueous Visualization, Validation, Supervision, Investigation, Formal analysis.
byproducts from mint hydrodistillation, specifically hydrolats, in the Baskaran N: Writing – review & editing, Visualization, Validation, Su­
development of a bioactive-rich flavored drink. The physicochemical pervision. Ashish Rawson: Writing – review & editing, Visualization,
analysis revealed that hydrolats exhibited favourable properties for Validation, Supervision, Formal analysis. Dhivya R: Writing – original
drink infusion, showcasing neutral to slightly alkaline pH, lower total draft, Methodology.
soluble solids, and distinct color profiles. Furthermore, the bioactive
compounds present in hydrolats, such as phenols, flavonoids, and anti­
oxidants, were successfully incorporated into the drink formulations. Declaration of Competing Interest
The sensory analysis highlighted the preference for drink combinations
with lower hydrolat content, emphasizing the importance of balancing The authors declare no potential conflict of interest.
flavor enhancement with overall acceptability. Based on sensory eval­
uations, drinks with lower hydrolat content (combinations A and C)
Acknowledgement
were preferred. The flavored drink being studied contains bioactive
compounds with proven health benefits and can also serve as a
The authors would like to acknowledge and thank, Ministry of Food
refreshing, hydrating drink with its mint infusion, potentially helping
Processing Industries, Government of India (File No: Q-11/11/2020-
those with hyperdipsia. Overall, the study demonstrates the potential of
R&D) for funding our work.
incorporating mint hydrolats into bioactive-rich flavored drinks, a sus­
tainable and beneficial approach to both flavor enhancement and
bioactive compound enrichment in the drinks. Appendix A. Supporting information

Supplementary data associated with this article can be found in the

online version at doi:10.1016/j.foohum.2024.100263.

8
B. Madhura et al. Food and Humanity 2 (2024) 100263

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