Production, Purification and Characterization of Antioxidative and

Antidiabetic peptides from fermented cheese whey with Anti-inflammatory

activity

ABSTRACT
Whey protein, a significant co-product derived from dairy processing, represents a notable
environmental pollutant due to its high BOD and COD values with high nutritions. Therefore,
fermentation of cheese whey to produce antioxidative and antidiabetic peptides is an innovative
approach that can enhance the value of whey as by-product. Fermentation of cheese whey with
L. helveticus MTCC 5463 (V3, GQ253959) culture was conducted for different time intervals
(0, 12, 24, 36 and 48 h) at 37°C. Using the OPA technique, proteolytic activity was carried out
to maximize the growth conditions (incubation time, i.e. 0, 12, 24, 36, 48 h and inoculation
rate, i.e. 1.5%, 2.0%, and 2.5%) at 37°C for the generation of maximum peptides. The findings
revealed that highest amount of peptides (8.41 mg/ml) were produced at 2.5% inoculation rate
after 48 h of incubation. The fermented cheese whey inhibited the production of %NO induced
by LPS, while reduced the production concentrations of IL-6, IL-1β and TNF-α in RAW 267.4
cells. The antidiabetic and antioxidative activities of fermented cheese whey fractions (3 kDa
and 10 kDa permeates and retentates) were also performed. Molecular weight of fermented
whey peptides was determined using SDS-PAGE, and twelve peptide sequences were identified
through 2D gel electrophoresis. Moreover, various functional groups were analysed using FTIR
analysis.

Key words: Cheese whey, Lactobacillus, Peptide, Antidiabetic, antioxidative, anti-

inflammatory, molecular docking

Introduction
Since long, whey has become recognized as one of the most valuable secondary bioresources
derived from cheese production. Approximately 24,000,000 tons of cheese are produced
worldwide annually, resulting in an estimated 21,600,000 tons of cheese whey. (Lopes et al.,
2019). High nutritional and technical value of whey is due to whey proteins, which are a unique
component in whey. Around 20% of the total milk protein constitutes whey protein, with casein
comprising the majority at about 80% of the total protein content in milk (Patel, 2015; Davoodi
et al., 2016; Bhat et al., 2016).
Traditionally, whey is categorized as either acid whey or sweet whey depending on its
processing conditions. Many of researchers has been focused on acid whey and sweet whey.
Acid whey is the liquid that is extracted from the making process of dairy products of acid
types such as; strained yogurt, Greek yogurt, acid-curdled cottage cheese, and ricotta cheese.
Sweet Whey is the whey liquid obtained from the manufacture of rennet types of cheese like
Swiss and Cheddar cheese. The main differences between the two types of whey protein in
their protein content, acidity, mineral composition (Pushpa et al., 2018) and the specific
composition of the whey protein fraction (Parmar et al., 2016; Macwan et al., 2016). Whey
proteins exhibit a globular structure, featuring a prominent presence of α-helix motifs, with a
balanced distribution of acidic/basic and hydrophobic/hydrophilic amino acids along their
polypeptide chains (Madureira et al., 2007).

Whey proteins are rich in essential amino acids, especially sulphur containing amino acid such
as cysteine and methionine, which significantly contribute to their high nutritional value.
(Yasmin et al., 2013) and it also contains a notable amount of branched chain amino acid
(BCAA) such as leucine, isoleucine, valine. They exhibit high-quality protein characteristics
with a protein efficiency ratio (PER) of 3.4 exceeding that of casein (2.8) and comparable to
egg albumin (Nath et al., 2015). The primary constituents of whey proteins include
immunoglobulin (IG), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), and bovine serum
albumin (BSA), representing 15%, 20%, 50%, and 10% of the whey fraction, respectively. All
of these major proteins, with the exception of BSA and IG, are synthesized within the mammary
gland's epithelial cells.

In addition to these, whey also contains numerous minor proteins, referred to as low abundance
proteins, such as lactoferrin (LF), lactoperoxidase (LP), proteose peptone (PP), osteopontin
(OPN), lysozyme (LZ), among others; lactoferrin (LF) and lactoperoxidase (LP) are the most
abundant minor proteins (Santos et al., 2012; Mollea et al., 2013).

Moreover, whey proteins exhibit techno-functional qualities that are necessary for usage in
food applications, such as high solubility, water absorption, gelatinization, and emulsifying
capacities (Solak and Akin, 2012). Studies have shown Whey proteins have numerous
biofunctional properties (Brandelli et al., 2015). Due to its anti-inflammatory, antioxidant, anti-
cholesterol, anti-obesity, blood pressure-lowering, and appetite-suppressing effects, whey
protein appears promising as a potential functional food. Whey proteins also possess potential
as a functional food ingredient capable of modulating body weight by eliciting satiety signals
that impact both short-term and long-term food intake (Sharma 2019).

Traditionally, unprocessed whey has been employed as feed for animals, such as pigs but it
should be used after specific processing for the feeding of calves and sheep. Liquid whey is
major biohazards in environment due to its high organic loading contents and its elevated
volume. On the other hand, liquid whey has bioactive components specially whey protein.
Lactose is the major component of whey and which have many applications in the dairy and
food industries (Dominici et al., 2022). Minerals such as calcium, magnesium, and phosphorus,
sodium, potassium, and vitamin B12, B6 are present in whey solution (Macwan et al., 2016)

Researchers have studied potential strategies to valorise cheese whey. The biotransformation
of whey can be accomplished utilizing biotechnological tools, i.e., microbial fermentation and
enzymatic treatment is the one method to produce peptides as bioactive substances from raw
whey. In recent years, there has been extensive research on bioactive peptides, driven by they
are utilized as food additives to maintain food quality and microbiological safety (Najafian,
2023) and their potentiality as functional ingredients that can confer beneficial health effects
on consumers. Bioactive peptides are protein constituents that remain inactive within the
protein structure until they are released through processes such as enzymatic hydrolysis,
microbial fermentation, and gastrointestinal digestion (Karami and Akbari-Adergani, 2019;
Zaky et al., 2022; Chakrabarti et al., 2018; Daliri et al., 2017). Fermented food is a good source
of bioactive compounds compared to most unfermented food. In bacterial fermentation,
microorganisms like bacteria, fungi, or yeasts are used in a medium supplemented with protein
substrates, to produce bioactive peptides bacterial hydrolases are used to breakdown of proteins
into smaller peptide fragments. Microbial fermentation offers specific advantages that are
distinct from alternative peptide delivery methods. At the same time, production of
hydrolysates can be an interesting approach to enhance the nutritional content of whey protein
and also protect the environment from their pollutants. Valorisation of cheese whey can be
achieved by the production of ethanol using lactose converting microorganisms. Fermented
cheese whey beverage produced by kefir grains has been studied by Magalhães et al., 2010.
Rosa et al., 2023 studied impact of different probiotic strains (Lactobacillus acidophilus, La-
05; Lactobacillus acidophilus La-03; Lacticaseibacillus casei-01 and Bifidobacterium Bb-12)
on the physicochemical characteristics, biological activity, antioxidant capacity and bioactive
peptides of fermented cheese whey milk beverage during storage (4ºC, 30 days).
This study aims to assess the in vitro antioxidant, antidiabetic, and anti-inflammatory
characteristics and possess maximum peptide production of cheese whey fermented with L.
helveticus MTCC 5463 (V3, GQ253959) as well as the identification of antioxidative and
antidiabetic peptides through RP-HPLC and mass spectrophotometry.

Material and Methods

Sample and culture collection
Lactobacillus helveticus MTCC 5463 (V3, GQ253959) was obtained from the Culture
Collection of the Department of Dairy Microbiology at the SMC College of Dairy Science,
Kamdhenu University, located in Anand, Gujarat, India. Cheese whey samples were collected
from Vidya Dairy, Anand, Gujarat, India and immediately chilled in an icebox during
transportation to the Microbiology Laboratory at SMC College of Dairy Science and processed
up on arrival.

Preparation of fermented cheese whey

After the collection of fresh cheese whey, it was filtered using muslin cloth to eliminate cheese
particles. Subsequently, it was sterilized at 121°C (250°F) under 15 psi (pounds per square
inch) pressure for 15 minutes and after cooling stored at 5 ± 1°C. Propagation from
reconstituted skim milk to cheese whey medium was performed at an inoculation rate of 2%
(v/v) of active L. helveticus (V3) lactic culture, followed by incubation for durations of 0, 12,
24, 36, and 48 hours at 37°C.

Evaluation of anti-diabetic activities of fermented cheese whey

a. ɑ-glucosidase inhibition activity

α-glucosidase inhibition activity was assessed through the measurement of the release of p-
nitrophenol (pNP) resulting from the hydrolysis of p-nitrophenyl-α-D-glucopyranoside
(pNPG) by the alpha-glucosidase enzyme. Methodology for measurement of α-amylase
inhibition of fermented cheese whey was suggested by Shukla et al., (2022). ɑ-glucosidase
inhibition was measured in different time periods (0, 12, 24, 36 and 48h). Absorbance of the
formed product which is characterized by a yellow colour was measured at 405 nm using a
UV-Visible spectrophotometer.

b. ɑ-amylase inhibition activity

The α-amylase inhibition activity of fermented cheese whey was performed using the 3,5-
dinitrosalicylic acid (DNSA) as per the method suggested by Shukla et al., (2022). Absorbance
was measured at 540 nm using a UV-Visible spectrophotometer.

The inhibition activity of α-amylase of fermented cheese whey was performed using the 3,5-
dinitrosalicylic acid (DNSA) as per the method suggested by Shukla et al., (2022). UV-Visible
spectrophotometer was used to detect the absorbance at 540 nm.

c. Lipase inhibition activity

Lipase inhibitory activity was assessed following the method proposed by Shukla et al., (2022),
measuring the release of 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl oleate (4-
MU oleate) substrate. Using a UV-Visible spectrophotometer, the absorbance of 4-
methylumbelliferone (4MU) was determined at 260 nm.

Assessment of antioxidant activity

The ABTS free radical scavenging activity of fermented cheese whey was assessed following
the method outlined by Yan et al., (2018). Absorbance was recorded at 734 nm using a UV-
Visible spectrophotometer.

Assessment of proteolytic activity

The extent of proteolysis was studied using the O-phthalaldehyde (OPA) method (Hati et al.,
2015; Solanki et al., 2017). Various inoculation rates (1.5, 2.0, and 2.5%) and incubation
duration (0, 12, 24, 36, and 48 h) were used for the sterilized cheese whey.

Electrophoresis (SDS PAGE) analysis of fermented cheese whey

Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to
the protocol outlined by Laemmli (1970), with slight modifications. The separating gel was
prepared using a 12% bis-acrylamide gel, while the stacking gel was prepared using a 5%
acrylamide/bis-acrylamide gel.

Two-dimensional gel electrophoresis (2D-PAGE) of fermented cheese whey

According to Yang et al., (2014), peptides generated from fermented cheese whey were isolated
using 2D gel electrophoresis, with slight adjustments made to the electrical parameters. The
proteins are separated based on two distinct properties in a two-step process: isoelectric
focusing (IEF) for the first dimension and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) for the second dimension.

Isoelectric focusing
Proteins are separated based on their isoelectric points (pI) in the IEF step. This step was
performed as per the protocol of Panchal et al., (2020). In the second step, SDS-PAGE is
employed to separate the proteins based on their molecular mass, following the method
proposed by Carrasco-Castilla et al., (2012) and Laemmli (1970).

Peptide fractionation using RP-HPLC

Cheese whey was fermented using V3 culture with 2% rate of inoculation and incubated for 48
h at 370C. Peptide production in the fermented cheese whey (3 kDa and 10 kDa: permeate and
retentate of water-soluble extracts) was quantified using RP-HPLC (Shimadzu LC-20, Japan),
in accordance with the methodology outlined by Chopada et al., (2022)

Separation, purification, and characterization of antioxidant and anti-diabetic peptides

Fresh cheese whey was inoculated with L. helveticus (V3) at an inoculation rate of 2.5% and
fermented at 37 °C for 48 hours. Peptides were subsequently separated using reverse-phase
high-performance liquid chromatography (RP-HPLC) and extracted using the method
described by Gibbs et al., (2004).

RPLC/MS identification and characterization of isolated peptides

Liquid chromatography
An ACQUITY UPLC-BEH -C-18 liquid chromatography column (2.1 × 150 mm, 1.7 μm,
WATERS) was employed for liquid chromatography. The column elution settings were
optimized following the method by Parmar (2017). The 2D-PAGE fraction of V3 fermented
cheese whey was digested with trypsin and analysed using an LC/MS instrument.

Peptide identification and data analysis

The mass spectra were analyzed using ProteinPilot software (version 5.0.2) for the
identification of proteins and peptides. The resulting peptide sequences were then associated
to the antidiabetic and antioxidative peptide database (BIOPEP-UWM™:
http://www.uwm.edu.pl/biochemia/index.php/en/biopep) to confirm their antidiabetic and
antioxidative properties.
Fourier Transform-Infrared Spectroscopy (FTIR) evaluations of fermented and
unfermented cheese whey
Fourier transform-infrared spectroscopy (FTIR) of the freeze-dried cheese whey samples was
analysed by following the method of León-López et al., (2020); Khakhariya et al., (2023).
FTIR spectroscopy was performed on the freeze-dried fermented cheese whey powder using
Bruker's Invenio-R apparatus. After applying a loading force, the samples were placed close to
the diamond crystal. An empty cell spectrum was used as a back-ground. Sample was placed
on the sample holder of the instrument and scanned at 400 - to 2200-cm−1 wavelenths with a
4 cm−1 resolution at ambient temperature.

Cell culture study

The murine macrophage cell line (RAW 264.7) was used in this study and obtained from the
National Cell Science Center (Maharashtra, India). Dulbecco's modified Eagle's medium
(DMEM) and Penicillin/Streptomycin (P/S) were supplied by Gibco (Thermofisher Scientific,
USA). Cusabio Biotech (China) provided the lipopolysaccharide. ELISA kits for TNF-α, IL-6,
& IL-1β were supplied by Elabscience (United States of America-USA) and Fetal Bovine
Serum (FBS) was provided by MP Biomedicals. The cells were cultured in DMEM containing
10% foetal bovine serum (FBS), P/S, and maintained at 37ºC in an atmosphere with 5% CO2.
The cells were cultivated and subcultured in tissue culture flasks until they achieved around
80% confluence.

Production of Nitric oxide (NO)

NO synthesis activity was assessed using the Griess procedure, following the methodology
described by Khare et al., (2020). RAW 264.7 cells were seeded in a 48-well plate (2105 cells)
and cultivated for 24 h. Furthermore, confluent macrophages were treated with 1µg/ml LPS
and V3strain used for cheese whey fermentation at doses of 2, 1, and 0.5 mg/mL. Cells were
kept for a further 16 hours in a humidified CO2 incubator. Nitrite were evaluated using the
Griess reagent, and optical density absorption was measured at 540 nm. Cytokines were
analysed using cell-free supernatants.
Measurement of TNF-α, IL-6 and IL-1β cytokines
TNF-α, IL-6, and IL-1β levels in culture supernatants were measured using a commercial
ELISA kit and following the manufacturer's instructions (Elabscience, USA).

Statistical analysis
All experimental data were analyzed using statistical software and designs with a significance
level of 5.0%, following the statistical procedure outlined by Steel and Torrie (1980). Results
from cell culture experiments were expressed as mean ± SEM. Comparison between different
groups was conducted using one-way or repeated measures ANOVA followed by Tukey’s post
hoc test, with statistical significance set at p< 0.05. Data analysis and visualizations were
performed using GraphPad Prism 8.0 software.

Results and Discussions

Antidiabetic activities of fermented cheese whey
Assessment of α-glucosidase inhibition activity
Figure 1a shows the fermented cheese whey's α-glucosidase inhibitory action. During the
incubation period from 0 to 48 h, the α-glucosidase inhibitory activity of the fermented cheese
whey ranged from 5.14% to 65.39%. The extended incubation period was associated with a
considerable increase in inhibitory activity after 48 h, the highest inhibition activity of α-
glucosidase (65.39%) was observed in the fermented cheese whey sample.

Lacroix and Li-Chan. (2013) evaluated that the α-glucosidase activity was inhibited by 36%,
33%, and 24% for peptic hydrolysates of bovine whey protein isolate, β-lactoglobulin (β-LG),
and α-lactalbumin (α-LA), respectively, at a concentration of 2.5 mg/ml. In contrast, the non-
hydrolyzed whey fractions did not demonstrate similar inhibitory effects. Akan, (2021) showed
antidiabetic potentials (inhibition activity of α-glucosidase) of donkey and camel milk peptides
released from whey proteins. Whey fractions from camel and donkey milk were hydrolyzed
using porcine pepsin and pancreatin enzymes at 37°C. Values for the inhibitory activity of α-
glucosidase were calculated as camel whey (32.84%) and donkey whey (89.38%) respectively.
However, the exact mechanism by which peptides inhibit α-glucosidase action is still unclear.
known. Lacroix and Li-Chan. (2013) suggested that peptides may exert inhibitory activity via
hydrophobic interactions, potentially by binding to the active site of the enzyme.

Assessment of α-amylase inhibition activity

The α-amylase inhibitory activity of fermented cheese whey is depicted in Figure 1a. From 0
to 48 h incubation, the α-amylase inhibitory activity of the fermented cheese whey varied
between 5.70% and 67.86%. It was noted that α-amylase inhibition activity improved
considerably with the incubation period from 0 to 48h. Maximum α-amylase inhibition activity
of (67.86%) was observed in the fermented cheese whey sample, after 48h incubation.

A comparative examination of whey fermented with Lactobacillus species from cow and
buffalo milk was carried out by Vankudre et al. (2015). The maximum -amylase inhibition
was observed in the product fermented by L. delbrueckii. They also observed that the α-amylase
inhibitory activity of cow milk whey (46.59%) was higher than buffalo milk whey (32.29%).
Kinariwala and Hati (2020) investigated the α-amylase inhibition activity of three milk-based
beverages prepared using different cultures namely, Lactobacillus fermentum (M2),
Lactobacillus fermentum (M7), and Lactobacillus paracasei (M11). The highest alpha-amylase
inhibitory activity was observed on the 0th day for both flavored (51.49%) and without
flavoured products (41.28%). Results showed decrease of α-amylase activity inhibition from
day 0th day to 9th day, which indicates that diabetic patients get benefit more from consuming
products when it is fresh. The inhibition ranged from 41.28% to 3.27% for the fermented milk
(flavored product) and 51.49% to 30.48% for the beverage (without flavored product).
From existing literature, the mechanisms behind α-amylase inhibition also remain unclear.
Molecular docking and molecular dynamics simulations of α-amylase and inhibitory peptides
can provide a deeper understanding of the binding sites and mechanisms underlying the
inhibitory peptide function (Li et al., 2024). Possibly, the amino acid residues and their
interaction through hydrogen bonds also influence α-amylase inhibition.
Ngoh and Gan (2016, 2018) reported the presence of 6 to 16 amino acid residues within the
α-amylase inhibitory peptides, sourced from pinto beans. These peptides identified in this study
exhibit a combination of hydrophilic residues (such as cysteine, methionine, and histidine)
along with hydrophobic residues (including alanine, leucine, proline, glycine, phenylalanine,
and valine). Mudgil et al., (2018) also examined peptides from camel milk with α-amylase
inhibitory properties have informed the presence of similar amino acid residues, including
leucine, lysine, cysteine, methionine, glycine, and phenylalanine.

Assessment of lipase inhibition activity

The lipase inhibitory activity of fermented cheese whey is shown in Figure 1a. During the
incubation period from 0 to 48 h incubation, the lipase inhibitory activity of fermented cheese
whey ranged between 4.90% and 63.38%. The inhibitory activity significantly increased with
the duration of the incubation period, reaching its highest after 48 hours and the fermented
cheese whey sample displayed the highest lipase inhibition activity (63.38%) after 48h of
incubation.

Similar results were found by Mudgil et al., (2018) found in their study that whole camel milk
protein hydrolysates had increased pancreatic lipase inhibition than unhydrolyzed camel whey
protein. Gil-Rodríguez and Beresford, (2021) also investigated the potential of fermented milk
produced with the bile salt hydrolase (BSH) positive strains was examined for potential lipase
inhibitory activity in vitro. All samples exhibited moderate activity, 7 samples out of the 16
samples displayed inhibition levels that were pointedly higher than those of the non-fermented
skim milk (NFSM) control. The maximum levels of lipase inhibition were observed for milk
samples fermented with LAB culture L. plantarum SC70 (37.2%) and SC80 (35.5%). Other
fermented milks that showed inhibition levels significantly higher than the control (>30%)
included those produced with L. acidophilus SC63 (31.9%) and SC1 (32.8%) L. brevis SC35
(33.9%), and P. pentosaceus SC57 (31.7%) and SC30 (32.3%).

Evaluation of antioxidant activity of fermented cheese whey

The antioxidant activity of fermented cheese whey is shown in Figure 1a. The fermented cheese
whey exhibited antioxidant activity ranging from 6.03 % to 64.82% over 0 to 48h incubation
at 37°C. According to our results, longer incubation times were associated with higher
antioxidant activity. The highest antioxidant activity of (64.82%) was observed in the
fermented cheese whey sample after 48 h fermentation.

Similar findings were reported by Dineshbhai et al., (2022), who investigated the antioxidant
activity of WPC-80 fermented with L. fermentum (KGL4) strain with various incubation time
periods (12, 24, 36, and 48 h). They observed antioxidant activity ranging from 66.72% to
78.29% during the incubation period of 0 to 48 hours. They also studied others activities like
Superoxide free radical scavenging activity (SOFRSA) and hydroxyl free radical scavenging
activity (HFRSA). KGL4 showed the highest ABTS activity (78.29%) SOFRSA (55.45%) and
HFRSA (57.42%), after 48 hours of incubation at 37 °C.

Virtanen et al., (2007) studied antioxidant activity in milk whey during fermentation with LAB
at 37°C for 24 h. In this study, 25 LAB strains were screened for their antioxidant capability.
The ABTS radical scavenging activities exhibited variability among the strains, with inhibitory
rates ranging from 3% to 53%. They observed the ABTS activity was significantly increased
with increasing incubation time along with LAB cultures in the study.

Dalaka et al., (2023) investigated the antioxidant properties of sweet whey (SW) obtained from
bovine, ovine, and caprine milk, both before and after in vitro simulated gastrointestinal
digestion. They reported digested SW showed higher antioxidative effect than undigested SW.
However, antioxidant mechanisms of whey protein peptides have been attributed due to the
synergistic action of sulfhydryl groups, the chelation of iron and specific amino acids that
scavenge free radicals (Tong et al., 2000). The antioxidant activity of whey protein fractions
also involves the chelation of transition metals by lactoferrin and serum albumin. Some amino
acids, including cysteine, tyrosine, methionine, histidine, lysine, tryptophan, and proline, have
been demonstrated to exhibit antioxidant activity by scavenging the free radicals and also
experienced that that these amino acids might also have prooxidant activity in specific
conditions (Vavrusova et al., 2015).
Corrochano et al., (2019) reported the whey fractions i.e., α-LA have antioxidant properties
and after in vitro gastrointestinal digestion of WPI, due to releases of the highest amount of
tryptophan from α-lactalbumin, gastric digested WPI showed highest antioxidant properties.

Optimization of peptide production in fermented cheese whey

Proteolytic activity of cheese whey fermented with the V3 culture was quantified by measuring
the quantity of free amide groups using spectrophotometry at a wavelength of 340 nm. Total
proteolytic activity ranged between 1.49 (rate of inoculation 1.5% in 0 h) to 8.41 mg/ml (rate
of inoculation 2.5% in 48 h of incubation). Proteolytic activity of V3 showed a significant
increase from 1.49 to 8.41 mg/ml over 48h, with the highest activity at 2.5% inoculation at 48h
(p<0.05). As shown in Figure 1b, the proteolytic activity of fermented cheese whey increased
significantly (p < 0.05) in response to longer incubation periods and different inoculation rates
during the fermentation process.

According to our research, the extended incubation periods and varied inoculation rates applied
to cheese whey resulted in an increase in proteolytic activity. Our result is similar to Ahtesh et
al., (2016), who evaluated proteolytic activity of whey protein concentrate during fermentation
by Lactobacillus helveticus strains (881315, 881188, 880474 and 880953). 4% WPC solution
was fermented with 4 different L. helveticus strains at 37°C for 0, 4, 8, and 12 h. Proteolytic
activity was significantly affected (p <0.05) by strains in the range of 0.1 to 0.5
(spectrophotometer absorbance) after 12 h of incubation. This work also supports our data in
terms of significantly increase (p<0.05) proteolytic activity of Lactobacillus culture with the
time of incubation ranged from 7.09 to 7.82 mg/ml after 48h of fermentation. Pescuma et al.,
(2008) performed whey fermentation by LAB and analysed the proteolytic activity through
OPA method. L. delbrueckii subsp. bulgaricus CRL 454, Lactobacillus acidophilus CRL 636,
and S. thermophilus CRL 804 were used for fermentation of reconstituted whey powder at the
temperature of 37 or 42 °C for 1 day. L. delbrueckii subsp. bulgaricus CRL 454 exhibited
higher proteolytic activity (90 mg Leu/ml), followed by L. acidophilus CRL 636 (36 mg
Leu/ml) and S. thermophilus CRL 804 (30 mg Leu/ml) after 24 h of incubation.

SDS-PAGE analysis of cheese whey (Fermented and unfermented)

In our current study, SDS-PAGE analysis of unfermented cheese whey revealed a higher
number of protein bands compared to fermented cheese whey. Due to strong proteolytic activity
of V3 strain in cheese whey during fermentation, protein break into small peptides. Protein
bands ranging within the range of approximately 10–180 kDa were observed in unfermented
cheese whey, whereas bands ranging from 10–166 kDa were detected in fermented cheese
whey. Protein bands were not visible in the permeate samples (Figure 4a).

Dineshbhai et al., (2022) showed the SDS PAGE analysis of fermented WPC-80 by
combination of Lactobacillus and yeast strains. They revealed that unfermented WPC-80
protein bands were ranged in between 10 to 180 kDa while fermented WPC-80 had a range in
between 10 to 55kDa. In our study also, protein bands were observed in the range of 10 to 166
kDa after fermentation of cheese whey protein with V3 culture. Pescuma et al., (2008)
performed whey fermentation by LAB and analysed the SDS-PAGE profile of whey protein
fractions. S. thermophilus CRL 804, L. delbrueckii subsp. bulgaricus CRL 454 and
Lactobacillus acidophilus CRL 636 were used for fermentation of reconstituted whey powder
at 37 or 42 °C for 24 h. They also reported bacterial strains were able to break down the main
fractions of whey proteins, with α-lactalbumin breaking down more (2.2–3.4 times) than β-
lactoglobulin.

Electrophoresis on a 2D gel
A total of 28 spots were observed on a 2D gel electrophoresis analysis of fermented cheese
whey generated by the V3 strain. Proteolytic activity of the lactic acid bacteria was shown by
the protein spots, which were about between 10 and 80 kDa.

Chopada et al., (2022) examined the protein profile of fermented WPC-80 using a combination
of L. paracasei (M11) and S. cerevisiae (WBS2A) and identified a total of 28 spots in the 2D-
PAGE of the fermented whey protein. These spots had molecular weights ranging between 10
and 70 kDa, attributed to the proteolytic activity of L. paracasei (M11) and S. cerevisiae
(WBS2A). Mansinhbhai et al., (2021) exhibited the protein profile of Whey protein concentrate
70% (WPC-70) hydrolyzed with alcalase enzyme at pH 8.5, 65°C. No spots were detected in
the 2D SDS PAGE of hydrolyzed WPC-70 compared to the unhydrolyzed WPC. They also
reported that native whey protein was fully hydrolyzed by the alcalase enzyme.

RP-HPLC analysis for peptide production

The proteolytic activity, various antidiabetic effects, and antioxidant activity of the 3 and 10
kDa fractions are shown in Figure 1c. The RP-HPLC chromatogram of 3 and 10 kDa permeate
and retentate from fermented cheese whey produced using V3 is depicted in Fig. 3a-e,
respectively. The 10 kDa permeate showed higher ABTS activity, α-glucosidase inhibition
activity, α-amylase inhibition activity, and lipase inhibition activity, i.e., 57.01%, 57.84%,
60.89%, and 56.73%, respectively, compared to the 3 kDa permeate. Similarly, the 10 kDa
retentate exhibited higher ABTS activity, α-glucosidase inhibition activity, α-amylase
inhibition activity, and lipase inhibition activity, i.e., 63.84%, 65.23%, 67.71%, and 61.85%,
respectively, compared to the 3 kDa retentate.

Peptide characterisation of 3 and 10 kDa peptides from fermented cheese whey was carried out
using RP-HPLC, as illustrated in Figure 3a–e. Maximum height of peaks in 10 kDa permeate
compared to 3 kDa (permeate and retentate) and 10 kDa retentate fractions were obtained,
suggesting that maximum peaks were in the range of 3-10 KDa. In the chromatogram of the 10
kDa retentate, a maximum of 57 peaks was observed, with retention times ranging from 17.12
to 53.31, compared to the chromatograms of the 10 kDa permeate (49 peaks), 3 kDa retentate
(46 peaks), and 3 kDa permeate (35 peaks) (Figure 3a-e).
Mansinhbhai et al., (2021) evaluated the antioxidant activity of WPC-70 hydrolyzed with
alcalase enzyme at pH 8.5 and 65°C. From all the samples, 10 kDa retentate expressed
excessive antioxidant activity (87.88%) than the other permeates. While on the other hand,
lowest antioxidant activity showed by the 3 kDa permeate (79.81%) samples during the study.
FTIR analysis
FT‐IR spectra of unfermented cheese whey and fermented whey peptides are presented in
Figure 5. FTIR as a rapid method of monitoring the distribution of molecular weights during
the enzymatic degradation of food processings by products This technique is based in the
intrinsic vibrational frequencies of the chemical bonds existing within the molecules. FTIR
spectrometry can be used to study the structure and content of proteins and polypeptides. Figure
5 displays FTIR spectra of the freeze-dried powder of cheese whey with V3 culture (Red line)
and freeze-dried powder of unfermented cheese whey powder (Grey line).
In the range from 1800 to 400 cm-1, the much more significant peaks were identified. Among
them, the ranges of 510.09 cm-1 and 1020.09 cm-1 had the largest peaks, with integrated areas
with substantial differences between the samples.

Effect of fermented cheese whey on anti-inflammatory activity in RAW264.7

Glycomacropeptide (GMP) makes up around 20–25% of the protein fraction of whey protein
making it the third most abundant protein after β-lactoglobulin and α-lactalbumin (Thoma et
al., 2006). Research suggests that whey protein, along with its peptides, including GMP,
exhibits immunomodulatory properties (Koh et al., 2022; Olsen et al., 2023). GMP appears to
exert its anti-inflammatory action by reducing the activity of the nuclear factor-κB (NF-κB)
and mitogen-activated protein kinase (MAPK) pathways (Requena et al., 2009). MAPK and
NF-κB pathways are prominent inflammatory signaling pathways known for their involvement
in the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-8 (Xiao et al.,
2020). GMP shows an anti-inflammatory mechanism by binding to inflammatory LPS derived
from pathogenic bacteria. This interaction limits the inflammatory response by preventing LPS
from adhering to cell receptors. Additionally, GMP has the ability to bind to bacteria directly,
preventing their adherence to epithelial cells and reducing inflammation in the process (Olsen
et al., 2023). Chun et al., (2019) investigated the anti-inflammatory properties of Maillard
reaction products derived from whey protein isolate (WPI) and sugar through an in vitro
approach. Galactose (GWA) and WPI were combined in an aqueous solution at 65°C for 24
hours to generate a glycated conjugate. Then this conjugate was fermented with using
Lactobacillus gasseri 4M13 culture to produce the fermented product (F-GWA). On LPS-
stimulated RAW264.7 cells, the fermented product (F-GWA) showed an anti-inflammatory
activity. It also decreased LPS-stimulated elevations in TNF-α and cyclooxygenase-2 gene
expression levels, as well as LPS-induced nitric oxide generation. Sansi et al., (2023)
investigated the anti-inflammatory effects of peptides whey protein hydrolysates (WPH) and
casein protein hydrolysates (CPH) combined with LPS. At dosages ranging from 50 to 550
mg/ml, the CPH and WPH peptides fractions had no cytotoxic effect on the HT-29 cell line.
Cell viability was high for both the peptides, with cell survival rate reaching 80% and with no
statistically significant differences were found. CPH and WPH peptide fractions demonstrated
moderate cytotoxicity while exhibiting cell selectivity in the investigation. In our study,
fermented cheese whey demonstrated stronger anti-inflammatory effect on RAW 264.7 cell.

Isolated peptide identification and characterisation using RP-LC/MS

Using mass spectrometry analysis of the 2D-PAGE study, a total of 12 peptide sequences
produced by the V3 strain were discovered and are reported in Table 2. Several peptide
sequences were verified by the using the databanks of bovine whey protein (Bos taurus). The
pool of peptides identified in the fermented cheese whey was analyzed using BIOPEP software
to identify antioxidant and anti-diabetic peptides. Most of the identified peptides consisted of
di-peptides (38 peptides) and tri-peptides (29 peptides). The majority of the identified peptide
sequence exhibited antidiabetic properties (12 peptides) and antioxidant activities (8 peptides),
with 8 peptides demonstrating both inhibitory effects (Table 3 and Table 4).

In our studies, different novel peptides sequences were identified from 2D-PAGE, which have
a high peptide ranker
In our studies, various novel peptide sequences with high peptide ranker (>0.5) scores were
identified from the 2D-PAGE analysis i.e., LDQWLCEK, DDQNPHSSNICNISCDK,
ALCSEKLDQWLCEK, CLLLALALTCGAQALIVTQTMK, GLLCALLPGVRGR,
LDLSPWASR, SKSSMFQNRTLK, QISACRLRQR, SIYAVFGAEINLKGIPVYR,
QSLRAVSWAKLAR, KLFVPALLSLGALGLCLAAPR and VGINYWLAHK. In the present
research, sequences of GLLCALLPGVRGR were partly coordinated with LP (Rice bran), LL
(Rat intestine), AL (Milk), VR (Casein derived) and RG (Human dipeptidyl peptidase IV
inhibitor), confirmed its antidiabetic activity on online-based BIOPEP database. Sequences of
CLLLALALTCGAQALIVTQTMK were partly coordinated with LA (Bella et al., 1982), LL
(Bella et al., 1982) from rat small intestinal and AL (Nongonierma et al., 2013) from milk and
GA (Hikida et al., 2013 ), LI (Lan et al., 2015), LT (Lan et al., 2015), MK (Lan et al., 2015),
QA (Lan et al., 2015), QT (Lan et al., 2015), TM (Lan et al., 2015), VT (Lan et al., 2015)
respectively from human dipeptidyl peptidase IV inhibitor and confirmed its antidiabetic
activity and similarly were also partly coordinated with LTC (Tian et al., 2015), CGA (Tian et
al., 2015) informed from whey protein (β-lactoglobulin) and confirmed for their antioxidative
activity on online-based BIOPEP database. One peptide sequence with a high ranker value of
0.98, KLFVPALLSLGALGLCLAAPR, closely matched SL, as reported by Nongonierma et
al., (2013) in lactoferrin, indicating its potential antidiabetic properties.

Conclusions
The antidiabetic and antioxidative characteristics of dromedary cheese whey enhanced as it
was fermented with V3 strain. The α-glucosidase, α-amylase, and lipase inhibition rates were
65.39%, 67.86%, and 63.38%, respectively. At a 2.5% inoculation rate at 37ºC for 48 hours,
the highest proteolytic activity was 8.41 mg/ml while the antioxidant inhibition activity was
64.82%. SDS-PAGE and 2D-PAGE study on fermented cheese whey revealed protein bands
in the ranges of 10-166 kDa and 10-80 kDa respectively.

From 2D-PAGE analysis, the ALCSEKLDQWLCEK peptide sequence was matched with the
antidiabetic inhibitor fractions of LCSEKLDQWL (α-lactalbumin), AL (Milk), WL (Milk
protein) and EK (Milk), where DDQNPHS SNICNISCDK was matched with the antioxidative
inhibitor fraction of QNPHSSNICN (Bovine α-lactalbumin) in the BIOPEP databases. Numerous
techniques were found to be comparable, although specific descriptions of the nature of these
interactions may be obtained using FTIR and HPLC. Furthermore, it has been concluded that,
fermented cheese whey may be used as functional ingredients for various food formulations
and pharmaceutical industry.