Implementation of plant extracts for cheddar-type cheese production in conjunction with FTIR and Raman spectroscopy comparison

Abstract

Plant extracts have demonstrated the ability to act as coagulants for milk coagulation at an adequate concentration, wide temperatures and pH ranges. This research is focused on the use of different vegetative extracts such as Citrus aurnatium flower extract (CAFE), bromelain, fig latex, and melon extract as economical and beneficial coagulants in the development of plant-based cheddar-type cheese. The cheddar-type cheese samples were subjected to physicochemical analysis in comparison to controlled cheese samples made from acetic acid and rennet. The fat, moisture, protein, and salt contents remained the same over the storage period, but a slight decline was observed in pH. The Ferric reducing antioxidant power (FRAP) increased with the passage of the ripening period. The FTIR and Raman spectra showed exponential changes and qualitative estimates in the binding and vibrational structure of lipids and protein in plant-based cheeses. The higher FTIR and Raman spectra bands were observed in acid, rennet, bromelain, and CAFE due to their firm and strong texture of cheese while lower spectra were observed in cheese made from melon extract due to weak curdling and textural properties. These plant extracts are economical and easily available alternative sources for cheese production with higher protein and nutritional contents.

Full text

Food Chemistry: X 22 (2024) 101256
Available online 28 February 2024
2590-1575/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
Implementation of plant extracts for cheddar-type cheese production in
conjunction with FTIR and Raman spectroscopy comparison
Usman Mir Khan
a
, Aysha Sameen
b
,
*
, Eric Andrew Decker
c
, Muhammad Asim Shabbir
a
,
Shahzad Hussain
d
, Anam Latif
e
, Gholamreza Abdi
f
,
*
, Rana Muhammad Aadil
a
,
*
a
National Institute of Food Science and Technology, University of Agriculture, Faisalabad 38000, Pakistan
b
Department of Food Science and Technology, Government College Women University, Faisalabad 38000, Pakistan
c
Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
d
Department of Food Science and Nutrition, College of Food and Agriculture, King Saud University, Riyadh 11451, Saudi Arabia
e
Institute of Food Science and Nutrition, University of Sargodha, Sargodha 40100, Pakistan
f
Department of Biotechnology, Persian Gulf Research Institute, Persian Gulf University, Bushehr 75169, Iran
ARTICLE INFO
Keywords:
Plant extracts
Cheddar-type cheese
Physicochemical properties
Antioxidant activity
Spectroscopy
ABSTRACT
Plant extracts have demonstrated the ability to act as coagulants for milk coagulation at an adequate concen-
tration, wide temperatures and pH ranges. This research is focused on the use of different vegetative extracts such
as Citrus aurnatium ower extract (CAFE), bromelain, g latex, and melon extract as economical and benecial
coagulants in the development of plant-based cheddar-type cheese. The cheddar-type cheese samples were
subjected to physicochemical analysis in comparison to controlled cheese samples made from acetic acid and
rennet. The fat, moisture, protein, and salt contents remained the same over the storage period, but a slight
decline was observed in pH. The Ferric reducing antioxidant power (FRAP) increased with the passage of the
ripening period. The FTIR and Raman spectra showed exponential changes and qualitative estimates in the
binding and vibrational structure of lipids and protein in plant-based cheeses. The higher FTIR and Raman
spectra bands were observed in acid, rennet, bromelain, and CAFE due to their rm and strong texture of cheese
while lower spectra were observed in cheese made from melon extract due to weak curdling and textural
properties. These plant extracts are economical and easily available alternative sources for cheese production
with higher protein and nutritional contents.
1. Introduction
Cheese is considered the major fermented dairy product with a
nutritional constituent of the dairy industry which serves as a tremen-
dous source of milk protein and fat, which is essential for a healthy
lifestyle (Cifelli, 2021). Chymosin is the main milk-coagulating protease
in animal rennet, it has been a well-known source of milk clotting for
centuries. However, due to higher rennet prices, religious restrictions
(Halal and Haram), vegetarian diet concerns, or restrictions on recom-
binant animal rennet, other protease milk clotting substitutes were
sought after (Ab Mutalib & Hakim, 2023). Additionally, to satisfy the
requirements of the consumers, animal rennet must also provide safety
regarding their usage (Zain et al., 2023). Furthermore, cheese and other
dairy products must be safe for consumption so that the ow of zoonotic
toxoplasmosis in food chain can be encountered efciently. Also, even if
it is raw milk, the animal must be checked for any history of infection of
their outbreak reports (Almuzaini AM, 2023). However, the rate of
transmission depends on diverse sources and the varying percentages
found in dietary materials (Javed & Alkheraije, 2023). The programs for
food safety should be put in place to guarantee food safety and inform
the public of the benets of implementing food safety precautions
(Alshaikh et al., 2023; Kukina et al., 2024).
Plants and compounds of plant origin have been proven to be
benecial for the health and well-being of humans and animals as they
also provide the bacterium for microbial production of rennet (Nkosi
et al., 2023; Abbas and Alkheraije, 2023; Rehman et al., 2023). Plants
can provide multiple health aspects in body as well as showed promising
functional aspects when used in certain foods (Bangulzai et al., 2022; Al-
Hoshani et al., 2023; Abduallah et al., 2023; Saleh et al., 2023). There
are various plant species, each of which depends on an element that is a
* Corresponding authors.
E-mail addresses: dr.ayshasameen@gcwuf.edu.pk (A. Sameen), abdi@pgu.ac.ir (G. Abdi), muhammad.aadil@uaf.edu.pk (R.M. Aadil).
Contents lists available at ScienceDirect
Food Chemistry: X
journal homepage: www.sciencedirect.com/journal/food-chemistry-x
https://doi.org/10.1016/j.fochx.2024.101256
Received 21 December 2023; Received in revised form 13 February 2024; Accepted 25 February 2024

Food Chemistry: X 22 (2024) 101256
2
part of the same plant but has a distinct number and kind of enzymes.
Many different plants have aspartic proteases (APs), which play a role in
plant senescence, responses to pathogens and stress, protein storage
mechanisms, and protein breakdown throughout plant development
(Folgado & Abranches, 2020). The plants productivity could be possibly
increased through the introduction of hybrids and cultivars deemed
prolic from different regions. These kinds vary and provide higher
yields of enzymes and other bioactive components in conjunction with
dense planting with specic improvements in genetic aspects (Adelaide
et al., 2023; Djulardi et al., 2024).
These plant extracts provide specic milk clotting activity (MCA),
proteolytic activity (PA), and functional properties such as avor and
texture enhancement in food products (Gupta et al., 2022). Plant pro-
teases provide an essential part in cheese ripening at an early stage
concerning their primary activity in milk coagulation as well as anti-
oxidant properties and signicant control over lipid oxidation (Sharma
et al., 2023). Cheese casein micelles are hydrolyzed by the remaining
coagulant, creating vital substrates for bacterial microora and their
decomposition enables the production of tastes as the cheese ripens
(Nadi et al., 2024).
Citrus aurnatium ower extract (CAFE) showed MCA/ PA over a wide
temperature range (35–70 ◦C) in milk. The MCA / PA ratio was sufcient
to cause milk coagulation like the commercial rennet (Khan et al., 2019).
Fig latex contains cin enzyme whose application, mechanism of action,
and physical properties still need to be explored. The research interest in
cin is increasing due to its proteolytic extract to have active fragments
production of antibiotics, milk clotting, promiscuous activity, and meat
tenderization. The plant extracts of gs have gained importance related
to the health perception of consumers (Shabani et al., 2018; Tahir et al.,
2023). Bromelain has a higher MCA/PA ratio with an optimum pH range
between 6 and 7 at 70 ◦C when used for milk coagulation. The milk
coagulation activity of bromelain showed stability over a wide range of
pH thus it is effective over the entire gastrointestinal tract (Singh et al.,
2023). It is safe and non-toxic but there is a need to explore its potential
in food products to have health advantages (Banerjee et al., 2018; Imran
& Alsayeqh, 2022). The melon extract from the fresh sarcocarp portion
showed a higher MCA/PA ratio at optimum pH and temperature but
MCA declined at lower temperature and pH levels (Khan et al., 2023).
The MCA / PA activities of plant extracts depend on the coagulant
type, amount of coagulant used, and specic enzymatic activity. These
plant extracts demonstrated variation in their characterization which
depends upon their hydrolyzing capacity against diverse substrates over
a wide range of temperatures and pH, and these extracts contained
several proteases at an adequate concentration that were used for milk
coagulation over broad pH ranges. Some of the attributes of these
vegetative coagulants were expressed as chymosin-like characteristics,
and they represented MCA and PA properties that were fairly like those
of rennet (Ferragina, 2015).
Thus, this study focused on the use of CAFE, Cucumis melo L. (melon)
extract, Ficus carica (g) latex, and bromelain from Annanas comosus
(pineapple) in their optimized MCA / PA activity with optimum time
and temperature treatments to develop cheddar-type cheese and to
evaluate the plant extracts effects on antioxidant activity. Raman and
FTIR spectroscopy of acetic acid and animal rennet coagulated cheese
was carried out to have more information about the microstructure
composition and interactions of cheese components such as casein, fat,
and other molecules to have a better understanding of the texture and
rheological aspects.
2. Materials and methods
2.1. Collection and preparation of plant extracts
The extraction of plant extracts was performed at the Department of
Food Science, University of Massachusetts, Amherst, MA, USA. CAFE,
melon extract, g latex, and bromelain as natural plant extracts were
extracted by using the hot continuous extraction methods (Soxhlet)
according to the method described by Khan et al. (2023). CAFE was
prepared by blending citrus owers with a cold buffer of Tris-HCl (20
mmol/L; 7.2 pH). The blend was ltered and stored till further use. The
fresh melon extract was prepared by homogenization and centrifugation
of melon mesocarp slice in a Beckman centrifuge (ThermoScientic
CL10, Centrifuge, USA) and the extract was maintained at 4 ◦C or frozen
till usage. Fig latex was obtained from the g plant and no further
processing was applied to the crude g latex. The crude bromelain
extraction was carried out by peeling, cutting, and grounding pineapple
fruit. The ltrate was mixed with 0.1 M phosphate buffer and centri-
fuged at 3500 rpm for 15 min in the Beckman centrifuge (Thermo-
Scientic CL10, Centrifuge, USA). Then, it was ltered and stored at 4 ◦C
till application. The bovine milk (10 L for each treatment) was procured
from the Equine and Livestock Research and Education Farm of the
University of Massachusetts, MA, USA. Acetic acid and animal rennet
type II 9042–08-4 were purchased from Sigma Aldrich, MA, USA. The
milk was standardized and homogenized to the standardized fat and
solid-not-fat (SNF) contents typically required for cheddar-type cheese
development. All other chemical reagents were purchased from Sigma
Aldrich, USA. All treatments were analyzed in triplicates for standard-
ized readings.
2.2. Treatments and experimental design
The cheddar-type cheese was produced at the food pilot plant of the
Department of Food Science, University of Massachusetts, Amherst, MA,
USA. The experiment included the use of CAFE, melon extract, F. carica
extract (g latex), and pineapple extract (bromelain) at their optimized
higher milk MCA and PA ratios (2.50 MCU/mg; approximately 35 mL/L
at 45 ◦C). The MCA / PA ratio depends on the ability of plant extracts to
demonstrate coagulation at optimal time and temperature treatments,
pH, type of plant, protein content and amino acid proling. Therefore,
concentrations of the plant extracts were chosen based on their opti-
mized MCA / PA ratio to have higher milk coagulation according to the
methods mentioned in the study of Khan et al. (2023). The developed
cheddar-type cheese was compared with acetic acid and rennet-
coagulated controlled cheddar-type cheese samples. A total of six
cheddar-type cheese production trails were planned (Table 1).
The cheese samples were evaluated for their physicochemical
parameter analysis, antioxidant activities (ferric reducing antioxidant
power (FRAP) and free radical scavenging activity (2,2-Diphenyl-1-
picrylhydrazyl commonly known as DPPH) and rheological aspects
concerning Fourier Transform Infrared Spectroscopy (FTIR), and Raman
spectroscopy techniques.
2.3. Development of cheddar-type cheese
The cheddar-type cheese was developed by slight modication in the
cheese production methods described by Fox et al. (2017). Fresh buffalo
milk (10 L for each treatment) was purchased from the Equine and
Livestock Research and Education Farm of the University of Massa-
chusetts, MA, USA for each trial. Five liters of buffalo milk was used in
each treatment to make the cheddar-type (smaller scale) cheese at the
food pilot plant of the Department of Food Science, University of
Table 1
Concentrations of treatments for cheddar-type cheese production.
Trials Plant extracts Concentrations (mL/L)
T
1
(Control) Acetic acid 10
T
2
(Control) Rennet 0.5
T
3
CAFE 35
T
4
Fig latex 35
T
5
Bromelain 35
T
6
Melon extract 35
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
3
Massachusetts, Amherst, USA. Milk was rst pasteurized at 65 ◦C for 30
min. Then it was inoculated with 2 % of starting cultures added to all
treatments (Lactococcus lactis subsp. Cremoris and Lactococcus lactis
subsp. Lactis). The controlled treatments were coagulated for 45 min at
33 ◦C with rennet (0.002 %) and acetic acid (2 %) at their optimum level
until the milk was coagulated while the plant extracts were used at their
optimum coagulation concentration levels (optimized MCA/PA ratio)
for milk coagulation. Then the rmed curd was cut, and stirred, and the
whey was separated from the curd. Then, to further separate the whey
from the curd, it was heated and cooked at 38 ◦C for 15 to 20 min. The
cheese blocks are then pressed to remove extra whey. The cheese blocks
were waxed with food-grade wax made from microcrystalline wax to
protect them from microbes and molds. First, the coating wax is heated
in temperature-controlled containers. The coating wax is applied to the
product using a straightforward dipping method that can be done
manually or with the use of mechanical devices (overhead handlers/
conveyor belts). The cheese surface must be waxed in a very careful,
clean and thorough way so that there is no chance of environmental
contamination. Salting was done at the rate of 2.5 % then by pressing the
cheese blocks whey was removed. The cheeses were then stored and
ripened for two months at 10 ◦C.
2.4. Physicochemical properties of cheddar-type cheese
The physicochemical properties such as pH, moisture, salt, fat, and
protein contents of plant extracts-based cheddar-type cheese samples
were compared with the controlled cheese samples by the methods of
AOAC (Poitevin, 2016).
2.5. Antioxidant activity of cheddar-type cheese
The antioxidant activity of cheddar-type cheese developed from
CAFE, melon extract, g latex, and bromelain were measured by mod-
ications in the method explained by Fardet & Rock (2018). The water-
soluble cheddar-type cheese extract was diluted in a phosphate buffer
(0.1 M) of pH 7 and their antioxidant activity was tested using FRAP and
DPPH assay (Himed-Idir et al., 2021).
2.5.1. Ferric reducing antioxidant power assay (FRAP assay)
The antioxidant activity of cheddar-type cheeses (made from CAFE,
C. melo L. (melon sarcocarp), and F. carica (Ficin)) was evaluated using
FRAP assay. A ferric complex of 2,4,6-tris(2-pyridyl)-s-triazine and Fe3
+was used to conduct the assay (dissolving 0.31 g of TPTZ, 0.54 g Fe
Cl
3
⋅6H
2
O in 100 mL of acetate buffer of 3.6 pH). After 4 min of incu-
bation at room temperature in the dark, the 20 µL of cheese extracts
were dissolved with 2 mL of ferric complex and then the absorbance was
recorded at 593 nm with a spectrophotometer (Thermosher Scientic
Spectrophotometer, Evolution Series 201/220, USA) (Chapeau et al.,
2016).
2.5.2. Free radical scavenging activity (DPPH assay)
The DPPH assay was evaluated by slight modication in the DPPH
assay method as described by V´
azquez-García et al. (2021). The extracts
(50 µL) were dissolved in 2 mL of a methanol solution containing 0.06
mmol/L DPPH. The samples were incubated in the dark for 60 min, after
which the reading from the spectrophotometer was at 517 nm. A Trolox
calibration curve was prepared (0.02–1 mmol/L), and data were
expressed in Trolox equivalent antioxidant capacity (mmol TEAC/kg).
The below-mentioned equation was used to calculate the DPPH-
scavenging activity.
% DPPH −scavenging activity =Absorbance (control) − absorbance (sample)
Control absorbance
×100
where Trolox was used for control absorbance and blank reading was
recorded without DPPH addition.
2.6. Fourier Transform Infrared spectroscopy (FTIR) and Raman
spectroscopy
The cheese sample analysis was done by IR Presige-21 Shimadzu
FTIR, Massachusetts, USA with slight modications in the method of
Tarapoulouzi et al. (2020). While Raman spectroscopy was investigated
by the method described by Yaman et al. (2022) and Zhang (2020).
2.6.1. FTIR spectroscopy
The fresh cheese samples were measured for 28 days of storage
period. The direct measurement of the cheese sample was performed by
taking slices of cheese (about 0.5 gm each sample) from different areas
of the cheese samples. The cheddar-type cheese samples were then
pressed with a high-pressure clamp to ensure good contact between the
Table 2
Effect of plant extracts on physicochemical parameters of cheddar-type cheese.
Treatments Fat content Protein content Moisture content pH Salt contents
Week 0 Week 4 Week 9 Week 0 Week 4 Week 9 Week 0 Week 4 Week 9 Week
0
Week
4
Week
9
Week
0
Week
4
Week
9
T
1
36.32
±
0.14
b
33.56
±
0.11
b
29.12
±
0.11
c
25.70
±
0.14
d
24.11
±
0.09
b
20.39
±
0.13
b
32.97
±
0.13
b
31.66
±
0.18
a
26.49
±
0.17
b
5.52
±
0.10
c
5.52
±
0.11
b
5.49
±
0.07
c
2.46
±
0.19
a
2.41
±
0.06
c
2.38
±
0.19
a
T
2
37.01
±
0.20
a
33.12
±
0.10
b
30.10
±
0.12
b
25.31
±
0.11
d
23.89
±
0.12
c
20.92
±
0.16
b
33.56
±
0.18
a
31.64
±
0.14
a
27.50
±
0.19
a
5.53
±
0.11
b
5.52
±
0.10
b
5.51
±
0.13
a
2.43
±
0.10
d
2.41
±
0.12
c
2.38
±
0.16
a
T
3
37.57
±
0.19
a
34.12
±
0.16
a
31.04
±
0.17
a
27.53
±
0.16
b
25.33
±
0.13
a
25.41
±
0.17
a
32.63
±
0.13
b
30.42
±
0.12
b
26.09
±
0.16
b
5.52
±
0.12
d
5.52
±
0.12
b
5.50
±
0.13
b
2.45
±
0.13
b
2.43
±
0.14
a
2.37
±
0.17
b
T
4
36.34
±
0.13
b
34.56
±
0.15
a
30.13
±
0.13
b
26.08
±
0.10
c
23.91
±
0.14
c
20.30
±
0.14
b
33.42
±
0.15
a
29.70
±
0.11
b
25.50
±
0.14
c
5.54
±
0.15
a
5.53
±
0.18
a
5.50
±
0.11
b
2.44
±
0.13
c
2.43
±
0.13
a
2.34
±
0.12
e
T
5
36.17
±
0.11
b
34.87
±
0.17
a
29.97
±
0.12
c
29.84
±
0.18
a
25.80
±
0.18
a
18.80
±
0.09
d
33.66
±
0.19
a
29.54
±
0.10
b
25.41
±
0.13
c
5.53
±
0.13
b
5.52
±
0.13
b
5.49
±
0.10
c
2.43
±
0.11
d
2.42
±
0.11
b
2.36
±
0.13
c
T
6
36.02
±
0.10
b
34.00
±
0.15
a
30.67
±
0.14
b
25.48
±
0.12
d
23.38
±
0.11
c
19.02
±
0.11
c
32.77
±
0.11
b
29.65
±
0.11
b
24.02
±
0.11
d
5.54
±
0.17
a
5.53
±
0.15
a
5.50
±
0.12
b
2.44
±
0.12
c
2.43
±
0.15
a
2.35
±
0.10
d
Different small alphabets show signicant differences among the different treatments (P <0.05).
T
1
=Controlled cheese with acid; T
2
=Controlled cheese with rennet, T
3
=Cheese prepared with CAFE; T
4
=Cheese prepared with bromelain, T
5
=Cheese prepared
with g latex; T
6
=Cheese prepared with melon extract.
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
4
diamond crystal and the sample. The FTIR scanning process was per-
formed on 400–4000 cm
−1
wavenumber with the 4 cm
−1
resolution. The
signal was improved by adding 10 times measurements for each spec-
trum. A single laser beam was used to measure the absorption of the
spectrum in comparison to the background level of air. Three spectra
were collected for each sample of cheese to record precise observations.
The wavenumber of the amide functional group of the samples was
compared with the current standard to determine the functional group.
2.6.2. Raman spectroscopy
The cheese samples were cut into 50 µm thick slices. A 96-well plate
was lled with cheese samples. The Raman spectra of the cheese sample
were recorded at 0 and 28 days of storage period. The instrument used to
record Raman spectra was the Metrohm MIRA-M1 laser Raman spec-
trometer (ProttezRaman-d3; Enwave Optronics Inc., Massachusetts,
USA) with modications of the method described by Yaman et al. (2022)
and Zhang (2020). The laser with an excitation wavelength of 785 nm
and 450 mW with an integration time of 100 s was used implementing
Orbital Raster Scan (ORS) technology. A spectral range of 300 to 3000
cm
−1
with a resolution of 1 cm
−1
was set to operate the spectrometer at
4.5 s. The spectrum was obtained by choosing eight points (located on
the different corner or middle positions of the cheese sample). The
average spectrum of each sample was used in the chemometric analysis.
Raman spectroscopy technology was used to detect the spiral intensity
on acquisition time and to examine the increased dispersion of repli-
cated spectra. The Raman peaks were compared with the standard
Raman peaks for a better understanding of the textural and rheological
aspects of the microstructure composition of the cheddar-type cheese.
2.7. Statistical analysis
Statistical analysis was performed to determine the signicance level
of the data obtained from each parameter of cheddar-type cheese sam-
ples using a Completely Randomized Design (CRD) (Montgomery,
2017). The signicant difference comparisons were performed by
Duncan’s Multiple Range (DMR) Test (SAS 9.1 Statistical Software).
Table 3
Effect of plant extracts on antioxidant activity of cheddar-type cheese.
Coagulants Antioxidant activity
FRAP* (mmol Fe
2+
/g) DPPH* (Trolox equivalent µM/g)
0 day 20 days 40 days 60 days 0 day 20 days 40 days 60 days
Acid 3.12 ±0.21
f
5.33 ±0.43
f
8.30 ±0.14
f
11.29 ±0.18
f
421.03 ±14.11
f
535.03 ±13.12
f
686.02 ±16.11
f
711.01 ±19.10
f
Rennet 4.33 ±0.45
e
9.32 ±0.58
e
13.32 ±0.30
e
16.28 ±0.27
e
836.04 ±15.12
e
956.03 ±17.14
e
1098.03 ±18.12
e
1131.02 ±21.11
e
CAFE 7.38 ±0.19
ab
14.45 ±0.29
c
29.51 ±0.23
b
38.55 ±0.34
b
2143.08 ±27.12
b
3304.07 ±33.10
b
4421.05 ±40.14
b
5013.03 ±47.14
b
Bromelain 6.29 ±0.17
c
11.37 ±0.13
d
23.46 ±0.59
c
29.51 ±0.38
c
1054.06 ±20.14
c
1164.05 ±25.11
d
1267.04 ±26.10
c
1376.02 ±31.17
c
Fig latex 7.39 ±0.20
a
17.47 ±0.15
a
36.52 ±0.29
a
42.57 ±0.29
a
2243.09 ±24.13
a
3406.06 ±38.12
a
4674.04 ±40.13
a
5219.03 ±49.13
a
Melon extract 5.26 ±0.64
d
13.30 ±0.11
b
21.36 ±0.40
d
28.44 ±0.32
cd
931.04 ±16.13
d
1042.03 ±21.12
c
1189.03 ±29.11
d
1256.02 ±33.17
d
Fig. 1. FTIR spectra comparison of cheddar-type cheese developed from acetic acid, rennet, and plant extracts.
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
5
3. Results and discussions
3.1. Physicochemical analysis of cheddar-type cheese
Cheddar-type cheese samples were analyzed for physicochemical
properties such as pH, fat, moisture, protein, and salt contents at 63 days
of storage. Results showed no signicant differences (P <0.05) among
pH, moisture, fat, and salt contents but a signicant difference was
observed in the protein content of cheese made from plant extracts as
compared to acid and rennet-based cheeses due to higher protein con-
tents of plant extracts (Table 2).
The fat contents remained the same over the storage period and a
slight decline was observed due to the lipolysis of fat during 9 weeks of
storage period. The fat contents of control T
1
decreased from 36.32 ±
0.14 to 29.12 ±0.11 while T
2
showed a slight decrease from 37.01 ±
0.20 to 30.10 ±0.12 fat contents after 9 weeks of storage. There was a
slight decline in the fat contents of T
3
from 37.57 ±0.19 to 31.04 ±0.17
while fat contents in T
4,
T
5
and T
6
showed same decline from 36.34 ±
0.13 to 30.13 ±0.13, 36.17 ±0.11 to 29.97 ±0.12 and 36.02 ±0.10 to
30.67 ±0.14 respectively, after 9 weeks of storage. The leakage of fat
globules with moisture occurred on the outer surface of cheese during
storage at room temperature (Alinovi & Mucchetti, 2020). At room
temperature, the fat globules showed signs of leaking from the cheese
matrix, making cheese hard and compact with less free space for fat
globules to ll in the curd matrix. Moreover, the higher fat content led to
a fatty texture of cheddar-type cheeses, with intense fatty aroma and
avor development but the decrease in fat may also be due to clotting
time and if curd is not rm enough then it leads to a reduction in curd
rmness and cheese yield (Cao & Mezzenga, 2020).
The protein content of plant extracts was higher and showed a sig-
nicant difference between acid and rennet-coagulated cheddar-type
cheeses due to the higher protein content of the plant extracts. The
protein contents of controls T
1
and T
2
were 25.70 ±0.14 and 25.31 ±
0.11 respectively, which were similar to protein content of T
6
(25.48 ±
0.12). The protein contents in T
3
, T
4
, and T
5
were 27.53 ±0.16, 26.08
±0.10 and 29.84 ±0.18 respectively, which were higher in protein
content as compared to controlled ones (Table 2). The type of protease
that is present in the plant extract determines its protein content, but the
diversity and activation of these enzymes dene the nature of proteo-
lytic activity. Plant extracts showed properties to enhance the protein
content, but it also depends on plant type, origin, and extrac-
tion methods used to extract these plant extracts (Khan et al., 2023;
Shabani et al., 2018). The protein contents in all cheese samples tend to
decline during storage due to proteolysis during cheese ripening and
storage conditions. The starter cultures and other microorganisms also
contribute to proteolysis of protein which leads to breakdown of protein
into smaller peptides and amino acids (Feeney et al., 2021). Such pep-
tides and amino acids further contribute to the avor and maturation of
the cheese and lead to a decline in the total nitrogen protein contents.
Some other factors such as improper storage conditions and contami-
nation aspects may contribute to the decline of the protein contents in
certain conditions (Paximada et al., 2021).
The results showed that no signicant difference was observed
among different concentrations of moisture content in all cheese sam-
ples (Table 2). The processing, storage, and ripening conditions affect
the moisture retention in cheese. The cheese is considered a viscoelastic
solid with a casein protein network entrapped with moisture and fat.
Thus, moisture contents also prevent fat from leaking outside of cheese
boundaries and overcooking of curd closes the holes and makes the curd
brittle which decreases the moisture retention capability of the curd, and
continuous decreases were observed during storage (Lamichhane et al.,
2018). The rmness is related to cheese moisture and if entrapped fat
globules leak from the moisture which can make the cheese much harder
to have elasticity. Moisture regulation contributed to restoring the
textural properties upon a 50 % ratio in cheese samples, but other
storage and processing condition factors were also important for the
better quality of cheese (Alinovi & Mucchetti, 2020).
The plant extracts-based cheddar-type cheese samples showed the
same trend in pH, which is related to controlled ones made by using
rennet, and no signicant difference was observed in all treatments
(Table 2). There was a decrease in pH from 6 to 5.52 during the cheese
production process, which is necessary to continuously monitor the pH
during curd formation, whey drainage, and ripening for the proper
maturation of cheese samples. These plant enzymes exhibit a drop in pH
after 2 months of storage when cheese samples were added with addi-
tives to support texture rmness (Grossmann & McClements, 2021). The
higher time temperature treatments during the curdling process may
lead to acidic pH, which may cause a problem in the rheological prop-
erties of cheese during maturation (Yano & Fu, 2022).
The salt contents tend to remain constant in ripening but a slight
decline in the salt contents was observed due to the leak out of moisture
contents from cheese samples after pressing or withering in storage
conditions. This decrease in the trend of salt contents in cheeses showed
no changes in the composition or quality of cheese and it also masked
the bitterness that occurred by the vegetative coagulants by higher
addition of salts or brine solutions to the cheese. The increase in the salt
content increases the rmness but decreases springiness and cohesive-
ness during the texture analysis of cheese analogs (Rocchetti et al.,
2021).
Table 4
FTIR spectra comparison of cheddar-type cheeses with standard FTIR spectra
ranges.
Observed Wavenumber
(cm
−1
)
Standard wavenumber
(cm
−1
)
Amide
types
Specic marker
groups
Acetic acid (3350 ≤
3300)
3300 Amide A N
–
H
Rennet (3320 ≤3300)
CAFE (3379 ≤3300)
Bromelain (3349 ≤
3300)
Fig latex (3370 ≤
3300)
Melon extract (3370 ≤
3300)
Acetic acid (2920 <
3100)
3100 Amide B N
–
H
Rennet (2925 <3100)
CAFE (2923 <3100)
Bromelain (2919 <
3100)
Fig latex (2924 <
3100)
Melon extract (2893 <
3100)
Acetic acid (1645) 1600–1690 Amide I C
–
–
O
Rennet (1630)
CAFE (1746 >1690)
Bromelain (1746 >
1690)
Fig latex (1638)
Fig latex (1741 >
1690)
Melon extract (1713 ≥
1690)
– 1480–1575 Amide II C
–
N, N
–
H
Bromelain (1128 <
1229)
1229–1301 Amide III C
–
N, N
–
H
Fig latex (1162 ≤
1229)
Melon extract (1133 <
1229)
– 625–767 Amide IV O
–
C
–
N
Acetic acid (950 ≥
800)
640–800 Amide V C
–
–
O
Rennet (994 ≥800)
Fig latex (1069 >800)
– 537–606 Amide VI N
–
H
– 200 Amide VII Skeleton torsion
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
6
3.2. Antioxidant activity
The plant coagulants expressed a higher antioxidant potential during
ORAC analysis. Therefore, the plant-based cheese samples were
explored for their antioxidant potential. The antioxidant activity of these
extracts was evaluated by using 2 methods (FRAP and DPPH assay).
3.2.1. Ferric reducing antioxidant power (FRAP) assay
The results showed that FRAP values for g latex (42.57 ±0.29) and
CAFE (38.55 ±0.34) were higher than those of bromelain (29.51 ±
0.38) and melon (28.44 ±0.32) after 60 days. The acid and rennet
expressed the lowest FRAP activity values of 11.29 ±0.18 and 16.28 ±
0.27 respectively, as compared to vegetative coagulants after 2 months
of ripening (Table 3). The FRAP activity increased with the passage of
the ripening period of cheeses while no signicant effect was observed at
0 days, but it tended to increase during the ripening stage. The FRAP of
g latex and CAFE was higher due to the presence of phenolic com-
pounds in their nal form. The use of plant extracts in cheese enhanced
the antioxidant activity due to the presence of higher total phenolic
compounds (Asala et al., 2022). Thus, the increase in FRAP value was
greater for cin and CFE than the endogenous phenols of milk proteins
while proteolysis was similar for each treatment, but it did not signi-
cantly affect the overall cheese composition. As a result, the increase in
antioxidant activity of all cheddar-type cheeses led to chemical changes
Fig. 2. Raman spectra comparison of cheddar-type cheese developed from acetic acid, rennet, and plant extracts.
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
7
like proteolysis, which revealed the hidden antioxidant amino acid with
sulfur (Granato et al., 2017). The FRAP ability of some amino acids did
not show any antioxidant properties in vivo but contributed towards the
antioxidant properties in vitro stage. This solemnly depends on the
method of extraction of extracts and their use in the food product
(Ch´
avez-Servín et al., 2018).
3.2.2. Free radical scavenging activity (DPPH) assay
The antioxidant activity of cheddar-type cheeses made from CAFE,
melon extract, g latex, and bromelain was measured by DPPH assay.
The higher DPPH activity values were observed with g latex (5219.03
±49.13) and CAFE (5013.03 ±47.14) while the bromelain (1376.02 ±
31.17) and melon extract (1256.02 ±33.17) showed lower DPPH ac-
tivity values. The rennet and acid-coagulated cheddar-type cheese had
the lowest antioxidant activity values of 711.01 ±19.10 and 1131.02 ±
21.11 respectively, after 60 days of ripening period. DPPH value
expressed as Trolox equivalent µM/g, which is obtained from the Trolox
solution with an antiradical capacity equivalent to that of the dilution of
cheese extract (Table 3).
The antioxidant activity was decreased during cheese maturation in
all cheese samples, which was dependent on the origin of free radicals
and temperature used for different assays, but there was no signicant
effect on the overall composition of cheese samples (Melini et al., 2019).
But this longer storage, different processing conditions, types of cheese,
Fig. 2. (continued).
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
8
amount of extracts used, proteolysis and higher avor compounds pro-
duction of cheese affected their antioxidant activities which led to a
decline in antioxidant activity after 2 month storage period (Lone et al.,
2023; Yang et al., 2021). The antioxidant activity of ethanolic extracts,
total phenolic content (TPC), and avonoids varies among all plant
extracts depending on the plant material, their solid-solvent ratio, and
extraction methods of plant extracts. There is a dire need to evaluate the
extract on their antioxidant potential in both in vivo and in vitro potential
for a better future of the antioxidant potential of such vegetative co-
agulants as functional food ingredients (Latif et al., 2021).
Different small alphabets show signicant differences among the
different treatments (P <0.05).
3.3. Fourier Transform Infrared spectroscopy (FTIR)
The amide types observed in spectra of cheese samples made from
vegetative coagulants showed signicant differences from acid and
rennet amide types. This difference was due to the higher protein con-
tent and presence of vegetative protein with milk protein in the cheese
samples which led to the shift of the spectra band to a higher place
(Fig. 1).
The decrease in fat constituents and the slight increase in protein
contents of cheese made from plant extract and thus amides showed
higher wavenumbers than standards (Tarapoulouzi et al., 2020). The
FTIR spectra showed the types of amides which are easy-to-identify
protein group markers, which were detected by FTIR due to specic
functional groups, and then comparing them with standard amide
functional groups (Table 4).
The FTIR spectra of plant-based cheese show that proteins are pre-
sent at 1700–1600 cm
−1
and that lipids and carbohydrates are present at
1300–600, 1750, and 3000–2800 cm
−1
. The amide I band spectra were
observed with a range of 1600 to 1700 cm
−1
for acid and rennet while
plant extracts showed amide I band at a little higher range of 1700 to
1750 cm
−1
. The water absorbs in the region of 3000 to 3600 cm
−1
and
strongly above 1650 cm
−1
. The moisture affected the multiple N
–
H
bonds (Amide A and Amide B) in 2500 to 3500 cm
−1
regions. At the
initial stage, spectra masking was predominant due to free water. The
moisture of cheese affected the spectra of refrigerated cheese samples as
it masked or modied strong broad brands (Pax et al., 2019). Strong
bands were observed for all cheese samples from 2900 to 3500 cm
−1
range of spectra. The secondary structure of the protein in cheese made
with acid and rennet was reected in the IR spectrum due to the region
of amide I (1600–1690). The strong broad bands at 2700 to 3300 cm
−1
and the amide I band in the region between 1600 and 1700 cm-1 were
either masked or modied by the moisture in cheese, which had an
impact on the spectra of microtome-frozen cheese samples. This
outcome supported the ndings by Alkhalf Maha and Mirghani (2017)
that water absorbs strongly between bands at 3000 and 3600 cm
−1
while
bands at 1650 cm
−1
lead to affect the textural properties of the cheese
depending upon the storage conditions for ripening.
The next important aspect was whether the FTIR spectrum-based
prediction of cheese total solids was a more precise estimation of fat
and protein (as well as other components in cheese) retention in
cheese curd or simply a different representation of the constant pro-
portion of its fat and protein contents (Mota et al., 2022). Finally, the
lower ability of FTIR calibration to forecast fat recovery than pro-
tein given higher energy value of fat as compared to protein which ex-
plains the decreased accuracy of recorded energy of FTIR spectrum
compared to cheese solid (Fan et al., 2023).
Additionally, non-uniform cheese sample slices, voids in the cheese
matrix, non-homogeneity in the fat and bound moisture in the protein
matrix were the primary cause of the difference in the amide spectra
from the standard spectra of all the cheese samples. Thus, it could be
linked with β structure while the amide spectrum was higher in cheese
made from plant extract due to higher vibrational stretching of the
carbonyl groups and bands thus representing helical and random
Fig. 2. (continued).
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
9
portions of proteins (Ferragina et al., 2013).
The numerous N
–
H bonds are also impacted by the moisture bands.
Despite all efforts to obtain uniform samples, non-homogenized milk
was used, which resulted in the non-homogeneity of the cheese samples
used in FTIR tests. Another likely explanation could be the presence of
holes in the cheese matrix as well as the non-homogeneity of fat and
binding moisture in the protein matrix (Leite et al., 2019). The lower
absorbance was at the start and higher bandwidths were due to the
decrease in fat as a result of lipolysis during the storage of cheese. As a
result, when the amount of fat in cheese was reduced, the absorbance at
bands associated with fat also decreased whereas bands related to pro-
tein showed the opposite trend. FTIR results can be combined with
electrophoresis results (SDSD-PAGE) to study the protein characteris-
tics, chemical groups, and related compounds (Schreuders et al., 2021).
3.4. Raman spectroscopy
Raman spectroscopy revealed rich components and molecular vi-
bration information of cheese samples. The higher bands were observed
in bromelain and CAFE due to their rm and strong texture of cheese
while the lowest spectra were observed in cheese made from melon
extract due to weak curdling and textural properties (Fig. 2).
The spectra shift to the higher bands after 28 days of storage due to
proteolysis and this activity of lower to sudden higher change of spectra
bands still needs to be evaluated. The appearance of the cheese sample
was similar with eight points of randomly selected data and the
complexity and uctuation in this data occurred, but high consistency
was observed in overall spectra (Chawanji et al., 2022). The observed
cheese sample spectra were compared with the standard spectra bands
(Table 5).
The higher Raman above 1700 cm
−1
peaks that attributed to the
C
–
–
O ester stretching of fatty acid molecules. The Raman peaks above
1600 cm
−1
showed the characteristics of amide I and unsaturated fatty
acids with C
–
–
O stretching vibration. The weaker bands above 1300
cm
−1
were attributed to the amino acid phenylalanine and the lowest
bands were observed with CH
2
deformation, twisting, and vibration
which was expressed as carbohydrates and lipids (Sha et al., 2020). Fig
latex showed the spectra bands in a similar range of acid. Rennet
expressed the spectra with medium to high bands after 28 days with
continuous bands spectra. The vegetative extracts showed higher bands
of spectra due to the higher protein content and presence of vegetative
protein with milk protein in the cheese samples which led to the shift of
the spectra band to a higher place. But it also depends upon the type of
plant, extraction method, and processing conditions used for the
extraction (Nasiri & Hanian, 2022). Carbohydrate-related vibrational
modes with distinctive characteristics can be seen in the spectral region
between 1100 and 950 cm
−1
where plant protease resides with the
vibrational and rotational bonds of lipids and protein amides. These
amides showed strong vibrational bonds thus comparable to each other
these can be separated for identication of amid bond types and their
contribution in the curd formation (Genis et al., 2021). The vibrational
modes are related to the vibrational mode of the β-1–4 glycosidic bond
Fig. 2. (continued).
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
10
and are ascribed to CO stretching. The CC and COH deformation modes
(1120–1064 cm
−1
), and COC deformation (950–870 cm
−1
) showed no
rotational vibrations for the plant protease neither the vibrations were
observed for Acetic acid and rennet coagulated cheese samples. The
difference is simple which is based on the presence of a band in 1700 to
1800 cm
−1
that is characteristic of lipids that show lower variations in
acetic acid and rennet coagulated one cheese (1600 to 1745 cm
−1
).
Using the correct marking by resampling approach, the uncertainty
calculation during sample classication was carried out, allowing the
building of a more reliable classication model and reducing the
Table 5
Raman spectra comparison of cheddar-type cheese with standard Raman spectra
bands.
Observed wavenumber (cm
−1
) Standard
wavenumber
(cm
−1
)
Marker group assignment
Acetic acid (0 days: 2545 ≥
2500; 28 days: 2890 ≤
3000)
2500–3000
ν
ass
(CH
2
)
Rennet (0 days: 2590 ≥2500;
28 days: 2950 ≤3000)
CAFE (0 days: 2625 ≥2500;
28 days: 2901 ≤3000)
Bromelain (0 days: 2603 ≥
2500; 28 days: 2880 ≤
3000)
Fig latex (0 days: 2545 ≥
2500; 28 days: 2890 ≤
3000)
Melon extract (0 days: 2527 ≥
2500; 28 days: 2981 ≤
3000)
Acetic acid (0 day: 1935 ≤
2000; 28 days: 2490 ≤
2500)
2000–2500
ν
S
(CH
3
)
Rennet (0 days: 1899 ≤2000;
28 days: 2401)
CAFE (0 days: 1995 ≤2000;
28 days: 2345)
Bromelain (0 days: 2010; 28
days: 2487)
Fig latex (0 days: 2234; 28
days: 2445)
Melon extract (0 days: 1874 ≤
2000; 28 days: 2401)
– 1850–2000
ν
S
(CH
2
)
Acetic acid (0 days: 1754; 28
days: 1825)
1750–1850
ν
(C
–
–
O)
ester
Rennet (0 days: 1764; 28 days:
1848)
CAFE (0 days: 1763; 28 days:
1803)
Bromelain (0 days: 1790; 28
days: 1843)
Fig latex (0 days: 1782; 28
days: 1833)
Melon extract (0 days: 1753;
28 days: 1822)
Acetic acid (0 days: 1660; 28
days: 1734)
1650–1750
ν
(C
–
–
O) amide I;
ν
(C
–
–
C)
Rennet (0 days: 1678; 28 days:
1741)
CAFE (0 days: 1690; 28 days:
1735)
Bromelain (0 days: 1677; 28
days: 1746)
Fig latex (0 days: 2234; 28
days: 1732)
Melon extract (0 days: 1664;
28 days: 1712)
Acetic acid (0 days: 1610; 28
days: 1632)
1600–1650
ν
(C
–
C)
ring
Rennet (0 days: 1603; 28 days:
1638)
CAFE (0 days: 1611; 28 days:
1628)
Bromelain (0 days: 1601; 28
days: 1619)
Fig latex (0 days: 1611; 28
days: 1635)
Melon extract (0 days: 1620;
28 days: 1631)
– 1450–1600 δ(CH
2
)
Acetic acid (0 days: 1321; 28
days: 1425)
1300–1450
τ
(CH
2
)
Rennet (0 days: 1310; 28 days:
1432)
Table 5 (continued )
Observed wavenumber (cm
−1
) Standard
wavenumber
(cm
−1
)
Marker group assignment
CAFE (0 days: 1307; 28 days:
1434)
Bromelain (0 days: 1309; 28
days: 1427)
Fig latex (0 days: 1313; 28
days: 1436)
Melon extract (0 days: 1305;
28 days: 1446)
Acetic acid (0 days: 1132; 28
days: 1298)
1130–1300
ν
(C
–
O) +
ν
(C
–
C) +δ
(C
–
O
–
H)
Rennet (0 days: 1135; 28 days:
1294)
CAFE (0 days: 1142; 28 days:
1287)
Bromelain (0 days: 1155; 28
days: 1296)
Fig latex (0 days: 1134; 28
days: 1277)
Melon extract (0 days: 1195;
28 days: 1283)
Acetic acid (0 days: 1093; 28
days: 1123)
1090–1130
ν
(C
–
O) +
ν
(C
–
C) +δ
(C
–
O
–
H)
Rennet (0 days: 1103; 28 days:
1127)
CAFE (0 days: 1091; 28 days:
1120)
Bromelain (0 days: 1094; 28
days: 1122)
Fig latex (0 days: 1134; 28
days: 1277)
Melon extract (0 days: 1195;
28 days: 1283)
Fig latex (0 days: 1082; 28
days 1086)
1080–1090
ν
(C
–
O) +
ν
(C
–
C) +δ
(C
–
O
–
H)
Melon extract (0 days: 1083;
28 days: 1089)
Acetic acid (0 days: 1023; 28
days: 1063)
1019–1070 Ring-breathing
(phenylalanine);
ν
(C
–
C)
ring
Rennet (0 days: 1021; 28 days:
1068)
CAFE (0 days: 1028; 28 days:
1043)
Bromelain (0 days: 012 ≤
1019; 28 days: 1076 ≥
1070)
Fig latex (0 days: 1026; 28
days: 1052)
Melon extract (0 days: 1030;
28 days: 1055)
Acetic acid (0 days: 942; 28
days: 1001)
938–1010 δ(C
–
O
–
C) +δ (C
–
O
–
H)
+
ν
(C
–
O)
Rennet (0 days: 957; 28 days:
1005)
Fig latex (0 days: 992; 28 days:
1007)
Melon extract (0 days: 928 ≤
938; 28 days: 1003)
– 850–925 δ(C
–
C
–
H) +δ (C
–
O
–
C)
– 620–825 δ(C
–
C
–
O)
– 450–600 Glucose
– 230–384 Lactose
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
11
likelihood of misclassication (de S´
a Oliveira et al., 2016).
The bands at 1600 to 1650 cm
−1
were assigned to ring vibration of
aromatic amino acids and observed more clearly in the Raman spectra
than in the FTIR spectra. The most prominent band were in the region of
1650 cm
−1
and above 1800 cm
−1
that is assigned to
α
-helix segments
(Smith et al., 2017).
An increase in β-sheet structures was observed in the plant-based
cheese during ripening that could be deduced from the intensity in-
crease at 1650 cm
−1
, continuing up to the end of the storage period
(Zhang et al., 2023). The aromatic bands of the side chain also change in
intensity over the entire ripening period. This increase in unordered
structures, possible increase in turns, and changes in side-chain aromatic
amino acid bands also took place during ripening in the cheese before
being frozen when placed in the refrigeration temperature. The spectra
(which are not included) also indicated that β-sheet structures increased
over the rst week of storage at the expense of a decrease in
α
-helices
structure. Similar spectral changes were also observed when cheese was
frozen under liquid nitrogen vapors (Li Vigni et al., 2020). During 3
months of ripening, the Raman spectra showed exponential changes in
the binding and vibrational structure of lipids and protein in the plant-
based cheeses that expressed signicant changes in freezing processes
and shelf life at the end of ripening (Li et al., 2022).
Conclusion
The plant extracts proved better, economical, and easily available
commercial sources for milk coagulation as a replacement for animal
and microbial rennet. These plant extracts showed antioxidant potential.
The antioxidant potential depends upon the type of plant, extraction
method, and time and temperature treatment during milk coagulation.
The acetone mixtures performed better than methanol ones in the
polyphenol extraction method. In most cases, the number of extraction
stages had a statistically signicant impact on both the phenolic
extraction yield and the antioxidant capacity of plant extracts. The CAFE
and bromelain showed higher coagulation activity and cheese with
better textural properties. While higher antioxidant activity was
observed in the cheese made from CAFE and g latex. Further studies are
needed to investigate the naturally derived plant extracts and their
polyphenols to understand their physiological effects on the human
body. The FTIR and Raman spectroscopy of cheddar-type cheese
observed the qualitative estimates of protein and fat complex, and future
study is needed to implement these spectroscopy techniques for better
textural, rheological, and sensorial aspects. Future research must be
focused on the involvement of amino acids in avor development, and
how to produce cheese with better avor. Additionally, this study will
provide new opportunities in the food science research eld to charac-
terize and modify plant extracts to obtain active bio-peptides for
numerous health benets such as anti-photo-aging, antioxidant, anti-
cancer, anti-hypertensive and cholesterol-lowering effects.
Ethical approval
Not applicable.
Authors contributions
Usman Mir Khan: Conceptualization, methodology, software, and
writing-original draft. Aysha Sameen: Supervision, Methodology,
writing-review, and editing. Eric Andrew Decker: Resources, method-
ology, writing-review, and editing. Muhammad Asim Shabbir: Writing
review, and editing. Shahzad Hussain: Writing review, and editing.
Anam Latif: Writing review, and editing. Gholamreza Abdi: Funding,
Conceptualization, writing review, and editing. Rana Muhammad
Aadil: Supervision, methodology, conceptualization, writing review,
and editing.
Funding
The authors received no funding to conduct this study and to assist
with the preparation of this manuscript.
CRediT authorship contribution statement
Usman Mir Khan: Writing – review & editing, Writing – original
draft, Software, Resources, Project administration, Methodology, Data
curation, Conceptualization. Aysha Sameen: Supervision, Project
administration, Methodology, Investigation, Conceptualization. Eric
Andrew Decker: Writing – review & editing, Writing – original draft,
Supervision, Methodology, Investigation. Muhammad Asim Shabbir:
Writing – review & editing, Supervision, Methodology, Investigation,
Data curation. Shahzad Hussain: Visualization, Validation, Investiga-
tion, Funding acquisition, Conceptualization. Anam Latif: Writing –
review & editing, Visualization, Data curation. Gholamreza Abdi:
Writing – review & editing, Project administration, Investigation,
Funding acquisition, Conceptualization. Rana Muhammad Aadil:
Writing – review & editing, Visualization, Validation, Supervision,
Project administration, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgment
The authors are thankful to the National Institute of Food Science
and Technology, University of Agriculture, Faisalabad, Pakistan. The
authors are grateful to the Higher Education Commission (HEC) of
Pakistan for providing the HEC Indigenous scholarship and HEC Inter-
national Research Support Initiative (IRSIP) opportunity to carry out the
research in the USA. The authors are also thankful to the Department of
Food Science, University of Massachusetts, Amherst, USA for facilitating
this research. The authors also appreciate the support from the Re-
searchers Supporting Project number (RSPD2024R1073), King Saud
University, Riyadh, Saudi Arabia.
References
Ab Mutalib, S. R., & Hakim, M. H. H. M. (2023). Enzyme, the cheese case. In Innovation of
food products in halal supply chain worldwide (pp. 79–85). Elsevier.
Abbas, A., & Alkheraije, K. A. (2023). Immunomodulatory effects of Carica papaya
extract against experimentally induced coccidiosis in broiler chickens. Pakistan
Veterinary Journal, 43(3), 628–632.
Abduallah, A. M., Ahmed, A. E., Bajaber, M. A., & Alalwiat, A. A. (2023). Evaluation of
the antidiabetic effects of methanolic extracts of neem (Azadirachta indica) seeds on
the streptozotocin-induced wistar rats. Pakistan Veterinary Journal, 43(4), 792–798.
Al-Hoshani, N., Zaman, M. A., Al Syaad, K. M., Salman, M., Rehman, T. U., &
Olmeda, A. S. (2023). Assessment of repellency and acaricidal potential of nigella
sativa essential oil using rhipicephalus microplus ticks. Pakistan Veterinary Journal,
43(3), 606–610.
Alinovi, M., & Mucchetti, G. (2020). Effect of freezing and thawing processes on high-
moisture mozzarella cheese rheological and physical properties. LWT Food Science
and Technology, 124, Article 109137.
Alkhalf Maha, I., & Mirghani, M. E. S. (2017). Detection of formaldehyde in cheese using
FTIR spectroscopy. International Food Research Journal, 24, 496–500.
Asala, T. M., Rowaiye, A. B., Salami, S. A., Baba-Onoja, O. M., Abatan, M. O.,
Ocheja, B. O., Ada, G., & Ogu, A. M. (2022). The antioxidant and hematopoietic
effects of the methanolic extract fractions of Ocimum basilicum in acetaminophen-
induced albino rats. International Journal of Veterinary Science, 11(3), 289–294.
Banerjee, S., Ranganathan, V., Patti, A., & Arora, A. (2018). Valorisation of pineapple
wastes for food and therapeutic applications. Trends in Food Science & Technology, 82,
60–70.
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
12
Bangulzai, N., Ahmed, S. F., Kashif, M., Fatima, M., Ahmed, M., & Mushtaq, N. (2022).
Antifungal activity of essential oils extracted from different plants against
Penicillium digitatum causing green mold of citrus. Internatioanl Journal of
Agriculture and Biosciences, 11(2), 75–83.
Cao, Y., & Mezzenga, R. (2020). Design principles of food gels. Nature Food, 1(2),
106–118.
Chapeau, A. L., Silva, J. V. C., Schuck, P., Thierry, A., & Floury, J. (2016). The inuence
of cheese composition and microstructure on the diffusion of macromolecules: A
study using uorescence recovery after photobleaching (FRAP). Food Chemistry, 192,
660–667.
Ch´
avez-Servín, J. L., Andrade-Montemayor, H. M., V´
azquez, C. V., Barreyro, A. A.,
García-Gasca, T., Martínez, R. A. F., Ramírez, A. M. O., & de la Torre-Carbot, K.
(2018). Effects of feeding system, heat treatment and season on phenolic compounds
and antioxidant capacity in goat milk, whey and cheese. Small Ruminant Research,
160, 54–58.
Chawanji, A., Holroyd, S. E., & Nickless, E. (2022). Raman confocal microscopy to assess
changes in cheddar cheese during maturation. International Journal of Dairy
Technology, 75(4), 882–891.
Cifelli, C. J. (2021). Looking beyond traditional nutrients: The role of bioactives and the
food matrix on health. Nutrition Reviews, 79(Supplement_2), 1–3.
de S´
a Oliveira, K., de Souza Callegaro, L., Stephani, R., Almeida, M. R., & de
Oliveira, L. F. C. (2016). Analysis of spreadable cheese by Raman spectroscopy and
chemometric tools. Food Chemistry, 194, 441–446.
Djulardi, A., Triani, H. D., & Yuniza, A. (2024). Study of avocado seed and banana peel
processing as corn element substitution in japanese quail (Coturnix-coturnix
japonica) ration. International Journal of Veterinary Science, 13(1), 42–50.
Fan, M., Wei, T., Lu, X., Liu, M., Huang, Y., Chen, F., Luo, T., Fan, Y., Liu, R., & Deng, Z.
(2023). Comprehensive quality evaluation and interaction mechanism of processed
cheese analogues prepared from starch, protein, and oil from plant sources. Journal
of the Science of Food and Agriculture, 103(13), 6595–6604.
Feeney, E. L., Lamichhane, P., & Sheehan, J. J. (2021). The cheese matrix: Understanding
the impact of cheese structure on aspects of cardiovascular health–a food science and
a human nutrition perspective. International Journal of Dairy Technology, 74(4),
656–670.
Ferragina, A. (2015). New phenotypes predictions obtained by innovative infrared
spectroscopy calibrations and their genetic analysis in dairy cattle populations. PhD
dissertation, Universita di Padova. Acces at https://www.research.unipd.it/handle/
11577/3424294.
Ferragina, A., Cipolat-Gotet, C., Cecchinato, A., & Bittante, G. (2013). The use of fourier-
transform infrared spectroscopy to predict cheese yield and nutrient recovery or
whey loss traits from unprocessed bovine milk samples. Journal of Dairy Science, 96
(12), 7980–7990.
Folgado, A., & Abranches, R. (2020). Plant aspartic proteases for industrial applications:
Thistle get better. Plants, 9(2), 147.
Fox, P. F., Guinee, T. P., Cogan, T. M., McSweeney, P. L. H., Fox, P. F., Guinee, T. P.,
Cogan, T. M., & McSweeney, P. L. H. (2017). Salting of cheese curd. Fundamentals of
Cheese Science, 251–277.
Genis, D. O., Sezer, B., Durna, S., & Boyaci, I. H. (2021). Determination of milk fat
authenticity in ultra-ltered white cheese by using Raman spectroscopy with
multivariate data analysis. Food Chemistry, 336, Article 127699.
Granato, D., Nunes, D. S., & Barba, F. J. (2017). An integrated strategy between food
chemistry, biology, nutrition, pharmacology, and statistics in the development of
functional foods: A proposal. Trends in Food Science & Technology, 62, 13–22.
Grossmann, L., & McClements, D. J. (2021). The science of plant-based foods:
Approaches to create nutritious and sustainable plant-based cheese analogs. Trends
in Food Science & Technology, 118, 207–229.
Gupta, A. K., Pathak, U., Tongbram, T., Medhi, M., Terdwongworakul, A.,
Magwaza, L. S., Mditshwa, A., Chen, T., & Mishra, P. (2022). Emerging approaches
to determine maturity of citrus fruit. Critical Reviews in Food Science and Nutrition, 62
(19), 5245–5266.
Himed-Idir, H., Mouhoubi, K., Siar, E., Boudries, H., Mansouri, H., Adjeroud, N.,
Madani, K., & Boulekbache-Makhlouf, L. (2021). Effect of rosemary (Rosmarinus
ofcinalis L.) supplementation on fresh cheese: Physicochemical properties,
antioxidant potential, and sensory attributes. Journal of Food Processing and
Preservation, 45(1), e15057.
Imran, A., & Alsayeqh, A. (2022). Anticoccidial efcacy of Citrus sinensis essential oil in
broiler chicken. Pakistan Veterinary Journal, 42(4), 461–466.
Khan, U. M., Aadil, R. M., Shabbir, M. A., Shahid, M., & Decker, E. A. (2023). Interpreting
the production, characterization and antioxidant potential of plant proteases. Food
Science and Technology, 43, e84922.
Khan, U. M., Ahmad, I., Inayat, S., Amin, H. M. A., & Selamoglu, Z. (2019).
Physicochemical properties of Cheddar cheese made from Citrus reticulata Blanco
crude owers extract. Turkish Journal of Agriculture-Food Science and Technology, 7
(6), 856–860.
Kukina, N. V., Makarenko, Y. P., Melnyk, L. M., Zavoloka, Y. M., & Sidnenko, M. V.
(2024). Financial dominant of activation of investment opportunities of agro-food
production entities of Ukraine. International Journal of Agriculture and Biosciences, 13
(1), 1–17.
Lamichhane, P., Kelly, A. L., & Sheehan, J. J. (2018). Symposium review: Structure-
function relationships in cheese. Journal of Dairy Science, 101(3), 2692–2709.
Latif, A., Issa Khan, M., Rakha, A., & Ali Khan, J. (2021). Evaluating the therapeutic
potential of white button mushroom (Agaricus bisporus) against DMBA-induced
breast cancer in Sprague Dawley rats. Journal of Food Biochemistry, 45(12), e13979.
Leite, A. I. N., Pereira, C. G., Andrade, J., Vicentini, N. M., Bell, M. J. V., & Anjos, V.
(2019). FTIR-ATR spectroscopy as a tool for the rapid detection of adulterations in
butter cheeses. LWT Food Science and Technology, 109, 63–69.
Li Vigni, M., Durante, C., Michelini, S., Nocetti, M., & Cocchi, M. (2020). Preliminary
assessment of Parmigiano Reggiano authenticity by handheld Raman spectroscopy.
Foods, 9(11), 1563.
Li, Y., Gao, Y., Ling, N., Shen, Y., Zhang, D., Ou, D., Zhang, X., Jiao, R., Zhu, C., & Ye, Y.
(2022). Rapid and simple quantitative identication of Listeria monocytogenes in
cheese by isothermal sequence exchange amplication based on surface-enhanced
Raman spectroscopy. Journal of Dairy Science, 105(12), 9450–9462.
Lone, A. B., Bhat, H. F., Kumar, S., Manzoor, M., Hassoun, A., Aït-Kaddour, A.,
Mungure, T. E., Aadil, R. M., & Bhat, Z. F. (2023). Improving microbial and lipid
oxidative stability of cheddar cheese using cricket protein hydrolysates pre-treated
with microwave and ultrasonication. Food Chemistry, 423, Article 136350.
Melini, F., Melini, V., Luziatelli, F., Ficca, A. G., & Ruzzi, M. (2019). Health-promoting
components in fermented foods: An up-to-date systematic review. Nutrients, 11(5),
1189.
Montgomery, D. C. (2017). Design and analysis of experiments (9th ed.). Wiley, Hoboken,
NJ, USA: John Wiley & Sons.
Mota, L. F. M., Giannuzzi, D., Bisutti, V., Pegolo, S., Trevisi, E., Schiavon, S., Gallo, L.,
Fineboym, D., Katz, G., & Cecchinato, A. (2022). Real-time milk analysis integrated
with stacking ensemble learning as a tool for the daily prediction of cheese-making
traits in Holstein cattle. Journal of Dairy Science, 105(5), 4237–4255.
Nadi, W. G., Taher, E. M., Awad, A. A. N., & Ahmed, L. I. (2024). Lactoferrin’s potential
application in enhancing yoghurt’s microbial and sensory qualities, with emphasis
on the starter culture activity. Journal of Dairy Research, 1–6.
Nasiri, M., & Hanian, S. (2022). Enterococcus faecalis and enterococcus faecium in
pasteurized milk: Prevalence, genotyping, and characterization of virulence traits.
LWT Food Science and Technology, 153, Article 112452.
Nkosi, D. V., Bekker, J. L., & Hoffman, L. C. (2023). Physical hazards in wild ungulate
meat harvested for food purposes: A systematic review. International Journal of
Veterinary Science, 12(3), 272–283.
Pax, A. P., Ong, L., Vongsvivut, J., Tobin, M. J., Kentish, S. E., & Gras, S. L. (2019). The
characterisation of mozzarella cheese microstructure using high resolution
synchrotron transmission and ATR-FTIR microspectroscopy. Food Chemistry, 291,
214–222.
Paximada, P., Howarth, M., & Dubey, B. N. (2021). Double emulsions fortied with plant
and milk proteins as fat replacers in cheese. Journal of Food Engineering, 288, Article
110229.
Poitevin, E. (2016). Ofcial methods for the determination of minerals and trace
elements in infant formula and milk products: A review. Journal of AOAC
International, 99(1), 42–52.
Rehman, T. U., El-Mansi, A. A., Alhag, S. K., Al-Shuraym, L. A., Saeed, Z., Arif, M.,
Rashid, M., Bhutta, Z. A., & Zaman, M. A. (2023). Antiparasitic activity of
methanolic and ethyl acetate extracts of Azadirachta indica against haemonchus
contortus. Pakistan Veterinary Journal, 43(1), 199–203.
Rocchetti, G., Michelini, S., Pizzamiglio, V., Masoero, F., & Lucini, L. (2021). A combined
metabolomics and peptidomics approach to discriminate anomalous rind inclusion
levels in parmigiano reggiano PDO grated hard cheese from different ripening stages.
Food Research International, 149, Article 110654.
Saleh, M., Ramadan, M., Elmadawy, R., Morsi, M., & El-Akabawy, L. (2023). The efcacy
of alcoholic extracts of Morus macroura (mulberries), Lepidium sativum (garden
cress seeds) and diclazuril against E. stiedae in experimentally infected rabbits.
International Journal of Veterinary Science, 12(6), 869–878.
Schreuders, F. K. G., Sagis, L. M. C., Bodn´
ar, I., Erni, P., Boom, R. M., & Van Der
Goot, A. J. (2021). Small and large oscillatory shear properties of concentrated
proteins. Food Hydrocolloids, 110, Article 106172.
Sha, M., Zhang, D., Zhang, Z., Wei, J., Chen, Y., Wang, M., & Liu, J. (2020). Improving
Raman spectroscopic identication of rice varieties by feature extraction. Journal of
Raman Spectroscopy, 51(4), 702–710.
Shabani, R., Shahidi, S., & Rafe, A. (2018). Rheological and structural properties of
enzyme-induced gelation of milk proteins by cin and Polyporus badius. Food
Science & Nutrition, 6(2), 287–294.
Sharma, S., Kumar, A., Kumar, S., Katare, A. K., Bhat, H. F., Hassoun, A., Aït-Kaddour, A.,
Aadil, R. M., Mungure, T. E., & Bhat, Z. F. (2023). Withania somnifera fruit extract is
effective in controlling microbial growth and lipid oxidation and improves the
functional value of cheese. Food Chemistry Advances, 2, Article 100204.
Singh, B. P., Bangar, S. P., Alblooshi, M., Ajayi, F. F., Mudgil, P., & Maqsood, S. (2023).
Plant-derived proteins as a sustainable source of bioactive peptides: Recent research
updates on emerging production methods, bioactivities, and potential application.
Critical Reviews in Food Science and Nutrition, 63(28), 9539–9560.
Smith, G. P. S., Holroyd, S. E., Reid, D. C. W., & Gordon, K. C. (2017). Raman imaging
processed cheese and its components. Journal of Raman Spectroscopy, 48(3),
374–383.
Tahir, Z., Khan, M. I., Ashraf, U., Adan Ijaz, R. D. N., & Mubarik, U. (2023). Industrial
application of orange peel waste; a review. International Journal of Agriculture and
Biosciences, 12(2), 71–76.
Tarapoulouzi, M., Kokkinofta, R., & Theocharis, C. R. (2020). Chemometric analysis
combined with FTIR spectroscopy of milk and Halloumi cheese samples according to
species’ origin. Food Science & Nutrition, 8(7), 3262–3273.
V´
azquez-García, R., Cardador-Martinez, A., Orihuela-L´
opez, M. A., Ramos-
Hern´
andez, L. S., & Martín-del-Campo, S. T. (2021). Preliminary study of extended
ripening effects on peptides evolution and dpph radical scavenging activity in
mexican goat cheese. Catalysts, 11(8), 967.
Yaman, H., Aykas, D. P., Jim´
enez-Flores, R., & Rodriguez-Saona, L. E. (2022). Monitoring
the ripening attributes of Turkish white cheese using miniaturized vibrational
spectrometers. Journal of Dairy Science, 105(1), 40–55.
U.M. Khan et al.

Food Chemistry: X 22 (2024) 101256
13
Yang, W., Hao, X., Zhang, X., Zhang, G., Li, X., Liu, L. U., Sun, Y., & Pan, Y. (2021).
Identication of antioxidant peptides from cheddar cheese made with Lactobacillus
helveticus. LWT Food Science and Technology, 141, Article 110866.
Yano, H., & Fu, W. (2022). Effective use of plant proteins for the development of “new”
foods. Foods, 11(9), 1185.
Zhang, Z.-Y. (2020). Rapid discrimination of cheese products based on probabilistic
neural network and Raman spectroscopy. Journal of Spectroscopy, 2020, 8896535.
Zhang, Z.-Y., Jiang, M.-Q., & Xiong, H.-M. (2023). Optimized identication of cheese
products based on Raman spectroscopy and an extreme learning machine. New
Journal of Chemistry, 47(14), 6889–6894.
U.M. Khan et al.