Food Chemistry 400 (2023) 133988

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Green solvent processing: Effect of type of solvent on extraction and quality

of protein from dairy and non-dairy expired milk products
Hina Kamal a, b, Asgar Ali a, b, *, Cheng Foh Le a
a
Centre of Excellence for Postharvest Biotechnology (CEPB), School of Biosciences, University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor Darul
Ehsan, Malaysia
b
Future Food Beacon of Excellence, University of Nottingham, Loughborough, LE 12 5RD, United Kingdom

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

Keywords: The present study was designed to study the effect of green solvent processing in two folds, (i) to extract valuable
Green solvent protein from dairy and non-dairy expired milk products and (ii) to compare extraction efficiency and quality of
Extraction efficiency extracted protein using conventional (CS) and green solvents (GS). Ethyl acetate, ethanol, isopropanol, n-heptane
Renewable protein
and cyclopentyl methyl ether (CPME) were selected as the GS for the possible substitution of hexane and ethyl
N-heptane
Protein recovery
ether. For each respective solvent, protein recovery, structural and functional modifications were studied. Pro­
tein yield was extracted most effectively by GS n-heptane in dairy milk (5.33 ± 0.01%) with a protein purity of
Chemical compounds studied in this article:
Hexane (PubChem CID: 8058)
39.73 ± 0.90%. Non-dairy milk and product had similar protein yield when treated with CS and GS. Total mean
n-Heptane (PubChem CID: 8900) of extraction efficiency, structural and functional modifications across all samples showed GS solvents were
Isopropanol (PubChem CID: 3776) statistically more effective than CS.
Ethanol (PubChem CID: 702)
Ethyl ether (PubChem CID: 3283)
Bicinchoninic Acid (PubChem CID: 71068)
Cyclopentyl methyl ether (PubChem CID:
138539)
Ethyl acetate (PubChem CID: 8857)
Copper (II) sulfate pentahydrate (PubChem
CID: 24463)
Ammonium Sulfate (PubChem CID: 6097028)

1. Introduction Markedly known, milk is an important source of high-quality protein

Amid the current pandemic together with disrupt supply chains
worldwide, consumers are prompted to stock pile. This has led to a
direct increase in throwing away food more frequently, with consumers
having concerns of product quality and safety approaching expiration
date. Expired products are often disposed due to lack of methods to
recover valuable components (Kamal, Le, Salter, & Ali, 2021). Thus, it
becomes a major disposal source of environmental pollution and valu­
able resources loss. Dairy waste has become a major global concern,
especially at the retail and household levels. It is estimated approxi­
mately as third highest group in terms of dollar value of food wasted,
equaling to 17–20% of total value food waste (United Nations Envi­
ronment Programme, 2021).
(Maqsood et al., 2019) both in reference to its high nutritional quality
(amino acids) and bioavailability (Mudgil et al., 2019). Solvents are
conventionally used in extraction as a complete process or as an essen­
tial pre-requisite (Richardson, 2001). In other words, solvent extraction
is indispensable. However, the application of organic solvents (for
example hexane, acetone, petroleum ether, ethanol) in food industry is
diminishing (Tan et al., 2021). Moreover, principally due to food safety
concerns among researchers, food scientist and technologist, there is a
shift towards the use of “green solvents”, along with green technologies.
According to Clarke, Tu, Levers, Bröhl, & Hallett, (2018) the term
“green” is used to describe solvents that are in accordance to the Envi­
ronmental, Safety and Health (ESH) characteristics. In simpler context, a
green solvent is one that (a) originate from a renewable biomass

* Corresponding author at: Centre for Postharvest Technology, School of Bioscience, Faculty of Science and Engineering, University of Nottingham Malaysia, Jalan
Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia.
E-mail address: asgar.ali@nottingham.edu.my (A. Ali).

https://doi.org/10.1016/j.foodchem.2022.133988
Received 7 February 2022; Received in revised form 21 July 2022; Accepted 22 July 2022
Available online 27 August 2022
0308-8146/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
H. Kamal et al. Food Chemistry 400 (2023) 133988

resource (vegetable oil, wood, fruits etc), (b) environment friendly, in
reference to green house effect and environmental pollution, (c)
biodegradable and (d) low toxicity levels (Li, Fan, Wu, Jiang, & Shi,
2019; Yara-Varón et al., 2016).
The current research work is in accordance with the efforts set up by
the United Nations (UN) Sustainable Development Goals (SDGs) Target
12.3 to shrink the global food waste generated per capita along the food
supply chain up to 50% near to 2030 (Kamal et al., 2021). Although, the
current study is following sustainability but in a more holistic and
multidisciplinary manner such that, recycling of expired dairy products
using green solvents is an ideal practical solution for an integrated
sustainable eco-system. This study primarily focuses on (a) green solvent
reaction efficiencies in protein yield and (b) analyzing the structural and
functional quality of the recycled dairy and non-dairy protein. The
quality of the extracted protein will thus highlight the commercial
valorization potential of recycled protein for instance as co-products in
production of snack food to produce texture or for overall nutritional
fortification. The use of extracted protein can also be in non-food tech­
nical products for instance as an adhesive or coating agent.
2. Material and method
2.1. Material
2.1.1. Chemicals
All the reagents and chemicals used were of analytical grade. The
conventional solvents (CS) namely hexane, ethyl ether, along with
Amicon ultra centrifugal filter unit (10KDa) with ultracel-regenerated

Table 1
Experimental design for the extraction of protein from dairy and non dairy expired milk samples using One Factor at a Time (OFAT) model.
No. of Sample Sample Solvent Name Solvent Solvent Concentration Sample/solvent Salt Name Salt Concentration
Run Name Type Type (%) ratio (%)

1 Cow Milk Dairy n-heptane GS 100 1:1 Ammonium 0.9

Sulfate
2 Cow Milk Dairy CPME GS 100 1:1 Ammonium 0.9
Sulfate
3 Cow Milk Dairy Ethyl Acetate GS 100 1:1 Ammonium 0.9
Sulfate
4 Cow Milk Dairy Ethanol GS 100 1:1 Ammonium 0.9
Sulfate
5 Cow Milk Dairy Isopropanol GS 100 1:1 Ammonium 0.9
Sulfate
6 Yogurt Dairy n-heptane GS 100 1:1 Ammonium 0.9
Sulfate
7 Yogurt Dairy CPME GS 100 1:1 Ammonium 0.9
Sulfate
8 Yogurt Dairy Ethyl Acetate GS 100 1:1 Ammonium 0.9
Sulfate
9 Yogurt Dairy Ethanol GS 100 1:1 Ammonium 0.9
Sulfate
10 Yogurt Dairy Isopropanol GS 100 1.1 Ammonium 0.9
Sulfate
11 Soy Milk Non Dairy n-heptane GS 100 1:1 Ammonium 0.9
Sulfate
12 Soy Milk Non Dairy CPME GS 100 1:1 Ammonium 0.9
Sulfate
13 Soy Milk Non Dairy Ethyl Acetate GS 100 1:1 Ammonium 0.9
Sulfate
14 Soy Milk Non Dairy Ethanol GS 100 1:1 Ammonium 0.9
Sulfate
15 Soy Milk Non Dairy Isopropanol GS 100 1:1 Ammonium 0.9
Sulfate
16 Tofu Non Dairy n-heptane GS 100 1:1 Ammonium 0.9
Sulfate
17 Tofu Non Dairy CPME GS 100 1:1 Ammonium 0.9
Sulfate
18 Tofu Non Dairy Ethyl Acetate GS 100 1:1 Ammonium 0.9
Sulfate
19 Tofu Non Dairy Ethanol GS 100 1:1 Ammonium 0.9
Sulfate
20 Tofu Non Dairy Isopropanol GS 100 1.1 Ammonium 0.9
Sulfate
21 Cow Milk Dairy Hexane CS 100 1:1 Ammonium 0.9
Sulfate
22 Cow Milk Dairy Ethyl ether CS 100 1:1 Ammonium 0.9
Sulfate
23 Yogurt Dairy Hexane CS 100 1:1 Ammonium 0.9
Sulfate
24 Yogurt Dairy Ethyl ether CS 100 1:1 Ammonium 0.9
Sulfate
25 Soy Milk Non Dairy Hexane CS 100 1:1 Ammonium 0.9
Sulfate
26 Soy Milk Non Dairy Ethyl ether CS 100 1:1 Ammonium 0.9
Sulfate
27 Tofu Non Dairy Hexane CS 100 1:1 Ammonium 0.9
Sulfate
28 Tofu Non Dairy Ethyl ether CS 100 1.1 Ammonium 0.9
Sulfate

2
H. Kamal et al.
cellulose membrane and Whatman 1-phase separator filter paper
(Whatman, Maidstone, UK) was procured from Merck KGaA, Darmstadt,
Germany. Bicinchoninic Acid (BCA), copper (II) sulfate pentahydrate,
ammonium sulfate, glutaraldehyde, bovine serum albumin (BSA) pro­
tein standard and green solvents (GS) namely, isopropanol, ethanol,
ethyl acetate, n-heptane and cyclopentyl methyl ether (CPME) were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.1.2. Sample collection
Dairy milk products (cow milk and yogurt) and non-dairy milk
products (soy milk and tofu) were selected. Expired samples including
UHT-treated homogenized cow milk and yogurt by Summerfield Hybrid
Allied Dairy Company Sdn Bhd, soy milk by Homesoy Lam Soon Group
and fresh tofu by Taufu Lembut CF were collected from a local market
shop in Semenyih on special request, as the expired samples are
generally brought back by the producer. All the expired samples were
seven days past the “use by date” and were free from any visible physical
damage at the time of extraction. Initial pH (Table S-1 of the supple­
mentary Material) was conducted at the start of extraction as a pre-
caution to indicate reference to food safety. Control or non-expired
(fresh) samples were bought from Tesco Semenyih Malaysia to view
any significant pH changes in expired samples. The samples were
refrigerated at 4 ◦ C till extraction.
2.2. Method
2.2.1. Experimental design
The experiment followed OFAT (one factor at a time) design
(Table 1). For each respective solvent, protein recovery (protein yield
and protein purity), structural characterization and functional proper­
ties (water holding capacity, foaming capacity and stability, emulsifying
capacity and stability) were studied. All the parameters were analyzed in
triplicate. Ethyl acetate, ethanol, isopropanol, n-heptane and cyclo­
pentyl methyl ether (CPME) were selected as the GS for the possible
substitution of commonly used CS namely, hexane and ethyl ether.
Briefly, for CS extraction method, sample weighing 100 mL (100 mg
for tofu and yogurt) was mixed for 15 min at 600 rpm with 100% (ab­
solute) CS in a ratio of 1:1. Ammonium sulfate (0.9%) was added to the
above mixture and mechanically stirred for another 15 min under same
conditions. The mixture was set aside for 2 h at 4 ◦ C to allow protein
precipitation, before centrifugation at 6,600 × g for 15 min at 4 ◦ C.
Organic solvent fraction was carefully removed and collected sepa­
rately. Collected protein extracts were further centrifuged using Amicon
ultra centrifugal filter units (10 kDa) at 6,600 × g for 15 min at 4 ◦ C, to
remove excess salt impurities. Table 1, clearly illustrates that during
extraction, solvent to sample ratio, solvent concentration, salt type and
concentration were kept constant to highlight the prime efficiency and
quality of each solvent (CS or GS) in the extraction of protein.
Furthermore, the same extraction method was applied using GS.
2.2.2. Protein recovery
The BCA assay was performed to calculate protein content in the
extracted protein samples, according to Baba et al. (2021) and Non­
gonierma et al. (2015). Bovine serum albumin (BSA) was used as a
protein standard of known concentration (25–2000 µg/mL). The
absorbance at 562 nm (A562nm) was measured with a UV–vis Multi­
mode Spectrophotometer (Variosken Flash Multimode Reader, Ther­
moFisher Scientific, USA). Protein yield (Equation (1)) and purity
(Equation (2)) was calculated and expressed as percentage (%) as
follows:
Protein yield = (mass of protein in extracted sample/initial known mass of protein)x100
3
Food Chemistry 400 (2023) 133988
Protein purity = (mass of protein in extracted sample/mass of sample)x100
(2)
2.2.3. Secondary structure characterization
2.2.3.1. Fourier transform infrared Spectroscopy (FTIR) analysis. The
FTIR spectra of control and extracted proteins was recorded using an
infrared spectrometer (PerkinElmer Frontier IR/NIR systems, Waltham,
MA USA) as described by Ye et al. (2017). The FTIR spectrum of each
sample was scanned from 4000 to 500 cm − 1 after subtracting the
background in the absorbance mode.
2.2.3.2. Variable pressure scanning electron Microscopy (VPSEM) ana­
lysis. Variable pressure scanning electron microscope imaging (VPSEM,
FEI Quanta 400F FESEM Oxford-Instruments INCA 400 with X-Max
Detector, The Thermo Scientific™ Quanta™, USA) was applied to
examine the morphology and surface appearance of extracted protein.
The method adopted was in accordance with Mimouni et al. (2010) with
some modifications. Briefly, the extracted protein samples were sub­
jected to (i) chemical fixation with 3% glutaraldehyde in 100 mM
phosphate buffer (pH 7) for 15 min, followed by (ii) washing with
deionized water (3 times), (iii) dehydration using ethanol for 5 min and
lastly (iv) dried using CO2. For analysis, the VPSEM utilizes “differential
pumping” with several stages to obtain the desired elevated pressure in
the specimen chamber while simultaneously maintaining a satisfactory
pressure for stable operation. Samples were placed on the carbon double
sided tape mounted onto the electron microscopy stubs and examined in
a Sirion SEM at 10 kV with 1.5 nm resolution.
2.2.4. Functional modifications
2.2.4.1. Water holding capacity (WHC). The water holding capacity
(WHC) was determined according to Zielińska et al. (2018) with minor
modifications. Extracted protein sample (0.5 g) was mixed with 20 mL of
distilled water and stirred at 600 rpm for 30 min using an IKA RW 20
(IKA® Works (Asia) Sdn Bhd) stirrer. The samples were then centrifuged
at 10,000 × g, 15 min and 25 ◦ C. After centrifugation, the weight of the
precipitate was compared with the initial weight. The results were
expressed as gram of absorbed water per gram of sample.
2.2.4.2. Emulsifying activity and stability. Emulsifying activity (EA) and
stability (ES) were analyzed by a method described by Zielińska et al.
(2018) and Wu et al. (2009) with minor modifications. Briefly, 300 mg
of extracted protein sample was mixed with 30 mL of deionized water
(1% protein w/v). This mixture was stirred at 600 rpm for 30 min using
an IKA RW 20 (IKA® Works (Asia) Sdn Bhd) stirrer. This results in sol­
ubilization of extracted protein, which was then mixed and homoge­
nized with 10 mL of sunflower oil in 1:1 ratio using an Ultra-Turrax
homogenizer (Ultra-Turrax T25, IKA, Staufen im Breisgau, Germany)
at 20,000 rpm for 1 min. The samples were then subjected to centrifu­
gation at 3500 × g, 5 min, 25 ◦ C and the volume of the individual layers
were noted. EA (Equation (3)) was calculated and expressed as per­
centage (%) as follows:
EA(%) = (Ve /V)x100 (3)
where:
Ve = Volume of the emulsified layer.
V = Total volume.
(1)
H. Kamal et al. Food Chemistry 400 (2023) 133988

ES was evaluated by heating the emulsion for 30 min at 80 ◦ C. After
30 min, samples were centrifuged at 3500 × g, 5 min, 25 ◦ C and the
volume of the individual layers were noted. Similarly, ES (Equation (4))
was calculated and expressed as percentage (%) as follows:
ES(%) = (V30 /Ve )x100
where:
Ve = Volume of the emulsified layer.
V30 = Volume of the emulsified layer after heating.
2.2.4.3. Foaming capacity and stability. The foaming capacity (FC) and
stability (FS) was calculated by the methods described by Guo et al.
(2015). Summarily, 0.2 g of extracted protein sample was suspended in
20 mL of distilled water (1% protein w/v) and homogenized using an
Ultra-Turrax homogenizer (Ultra-Turrax T25, IKA, Staufen im Breisgau,
Germany) at 16000 rpm for 2 min. The whipped sample was at once
transferred into a graduated cylinder, and the total volume was deter­
mined at 0 and 30 min. FC (Equation (5)) was calculated and expressed
in percentage (%) as follows:
FC(%) = [(V0 − V)/V ] × 100
where:
V = Volume before whipping (mL),
V0 = Volume after whipping (mL).
However, FS (Eq. (6)) was calculated and expressed in percentage
(%) after 30 min as follows:
FS(%) = (V30 − V0 ) × 100
where:
V30 = Volume before whipping (mL),
V0 = Volume after whipping (mL).
2.2.5. Statistical analysis
The experiment followed OFAT (one factor at a time) design. All the
parameters were analyzed in triplicate. Analysis of variance (ANOVA)
and Tukey’s test were used to study the data from protein recovery,
along with functional (water holding capacity, foaming capacity and
(4)
stability and emulsifying activity and stability) tests. Means separation
using Tukey’s test (p < 0.05) was carried out when a significant dif­
ference was detected. All statistical analysis was carried out using SPSS
version 21 (SPSS Inc., Chicago, IL, USA, 2012).
3. Results and discussion
3.1. Protein recovery
Solvent extraction efficiency and reproducibility were evaluated
both quantitatively and qualitatively via calculating protein yield and
protein purity respectively. Protein recovery from dairy and non-dairy
milk samples for each solvent is tabulated in Table 2. Extraction using
GS, with n-heptane, CPME, isopropanol and ethanol recovered
comparatively higher protein yields, with 5.33 ± 0.01% (cow milk),
(5) 5.18 ± 0.00% (soy milk), 4.84 ± 0.05% (cow milk), 4.78 ± 0.02% (cow
milk) and 4.58 ± 0.12% (yogurt) respectively. Relatively, between GS
and CS extractions, overall the highest protein yields were among GS
samples, with n-heptane as the main contender (Table 2). In reference to
sample groups, highest protein yield via GS extraction was calculated in
cow milk samples, followed by soy milk, tofu and yogurt. Compara­
tively, extraction using CS, with hexane recovered better protein yield
than ethyl ether. Tofu samples extracted with hexane yielded the highest
protein 4.97 ± 0.08% among all sample groups.
Moreover, the results from the protein purity analysis were observed
(6)
to be in accordance with protein yield (Table 2). Overall, solvent pro­
cessing either via GS or CS depicts lower yield outcomes with protein
purity < 50%. Albeit, ammonium sulfate (0.9%) was added to aid the
precipitation of protein. Nonetheless, the current results are in contrast
with existing solvent induced protein recover studies (Mæhre, Dalheim,

Table 2
Recovery of protein from dairy and non dairy expired milk samples.
No. of Run Sample Name Sample Type Solvent Name Solvent Type Protein Yield % Protein Purity %

1 Cow Milk Dairy n-heptane GS 5.33 ± 0.01a 39.73 ± 0.90a

2 Cow Milk Dairy CPME GS 4.82 ± 0.07b 35.92 ± 0.12b
3 Cow Milk Dairy Ethyl Acetate GS 3.76 ± 0.03c 28.02 ± 0.18c
4 Cow Milk Dairy Ethanol GS 4.78 ± 0.02b 35.63 ± 0.11b
5 Cow Milk Dairy Isopropanol GS 4.84 ± 0.05b 36.07 ± 0.03b
6 Yogurt Dairy n-heptane GS 3.76 ± 0.13c 28.02 ± 0.09c
7 Yogurt Dairy CPME GS 4.27 ± 0.08bc 31.82 ± 0.07bc
8 Yogurt Dairy Ethyl Acetate GS 4.58 ± 0.12b 34.13 ± 0.05b
9 Yogurt Dairy Ethanol GS 4.06 ± 0.10bc 30.26 ± 0.21bc
10 Yogurt Dairy Isopropanol GS 4.12 ± 0.18bc 30.71 ± 0.16bc
11 Soy Milk Non Dairy n-heptane GS 4.24 ± 0.05bc 31.60 ± 0.03bc
12 Soy Milk Non Dairy CPME GS 5.18 ± 0.00a 38.61 ± 0.14a
13 Soy Milk Non Dairy Ethyl Acetate GS 3.82 ± 0.07c 28.47 ± 0.22c
14 Soy Milk Non Dairy Ethanol GS 4.08 ± 0.02bc 30.41 ± 0.11bc
15 Soy Milk Non Dairy Isopropanol GS 3.01 ± 0.05d 22.43 ± 0.15d
16 Tofu Non Dairy n-heptane GS 5.12 ± 0.06a 38.16 ± 0.04a
17 Tofu Non Dairy CPME GS 4.68 ± 0.16b 34.88 ± 0.11b
18 Tofu Non Dairy Ethyl Acetate GS 4. 22 ± 0.04bc 31.45 ± 0.09bc
19 Tofu Non Dairy Ethanol GS 3.25 ± 0.12d 24.22 ± 0.07d
20 Tofu Non Dairy Isopropanol GS 3.35 ± 0.08d 24.97 ± 0.13d
21 Cow Milk Dairy Hexane CS 4.76 ± 0.07b 35.48 ± 0.2b
22 Cow Milk Dairy Ethyl ether CS 3.12 ± 0.05d 23.25 ± 0.27d
23 Yogurt Dairy Hexane CS 3.22 ± 0.21d 24.01 ± 0.03d
24 Yogurt Dairy Ethyl ether CS 3.55 ± 0.17c 26.46 ± 0.12c
25 Soy Milk Non Dairy Hexane CS 4.11 ± 0.22bc 30.63 ± 0.18bc
26 Soy Milk Non Dairy Ethyl ether CS 4.72 ± 0.05b 35.18 ± 0.07b
27 Tofu Non Dairy Hexane CS 4.97 ± 0.08b 37.04 ± 0.01b
28 Tofu Non Dairy Ethyl ether CS 4.17 ± 0.16bc 31.08 ± 0.09bc

Values are presented as means of three replications. Across rows and columns, means followed by same letter(s) were not significantly different using Tukey’s test at 5
% level.

4
H. Kamal et al.
Edvinsen, Elvevoll, & Jensen, 2018; Richardson, 2001). This variance in
protein recovery is due to the fact that in this study commercial ho­
mogenized UHT treated samples were analyzed which explains that
protein recovery is co-dependent on the structure and composition of the
sample (Rizzo & Baroni, 2018). In other words, solvent extraction of raw
milk will have higher protein yield and protein purity than homogenized
UHT treated sample (Kamal et al., 2018). Protein recovery is also inter-
reliant on the pre-treatments. According to Zisu et al. (2010) a pre-
treatment of fat removal aids to yield higher protein content. This can
be observed especially in non-dairy samples, where soy milk and tofu,
although natively containing higher protein content, analyzed to have
less protein recovery.
3.2. Secondary structure characterization
Variable pressure scanning electron micrographs of high protein
yielding expired dairy and non-dairy extracted protein samples at
1600× magnification is revealed in Fig. 1. Panel A and B for each sample
group reflects extracted protein using GS and CS respectively. GS
extracted cow milk protein depicts a less dense, porous matrix with
numerous irregular (large and small) corpuscular structures. In com­
parison, CS extracted cow milk protein viewed a fairly less porous
structure with a dense network of aggregated protein. According to Li
et al. (2018) the differences in structural uniformity highlights the
interaction of micro and macro-structural characteristics. In other
words, a dense network of aggregated proteins with smaller overall
corpuscular structure, probably indicate higher protein fractions in the
intact state. This was observed in sample groups other than cow milk, i.
e., yogurt, soy milk and tofu. Furthermore, it was inferred from Fig. 1
that very dense, compact and relatively uniform pattern of agglomerated
protein mass indicates a lower protein yield as evident from the results
mentioned in section 3.1.
Additionally, the FTIR microspectroscopy studied the identification
of the characteristic amide I bands from extracted protein for each
sample group. However, only the normalized FTIR spectra of high
yielding protein, including (A) dairy and non-dairy (C) expired samples
is depicted in Fig. 2. Thus, allowing confirmation for the expected
structural changes due to extraction method using both GS and CS
Fig. 1. Variable pressure scanning electron micrographs of high yielding expired dairy and non dairy extracted protein samples at 1600x magnification. The white
arrows in each panel (1A, 1B, 2A, 2B, 3A, 3B, 4A and 4B) indicate porosity level and surface texture.
5
Food Chemistry 400 (2023) 133988
individually. As evident, among all sample groups, the FTIR spectra
revealed common characteristic features attributable to the main com­
ponents (lipids, water and protein). This indicates towards the results
tabulated for protein extraction efficiency specific to protein purity
(Table 2), where the extracted protein from each sample group was
concluded to have protein purity <50% (Section 3.1). Also, spectra of
samples were mostly measured on wet basis (liquid state), hence in the
high wave number region occurring from 3600 to 3100 cm− 1 the bands
observed represents (0-H) stretching bonds of water molecules in the
sample (Tarapoulouzi, Kokkinofta, & Theocharis, 2020). In particular,
the prominent bands in 1650–1450 cm− 1 region indicate the amide I and
II modes of protein (Van der Ven et al., 2002). The amide I vibration is
caused primarily by the stretching of the C–– O bonds, whereas the amide
II vibration is caused by deformation of the N–H bonds and stretching
of the C–N bonds (Li et al., 2019). Amide I band (C– – O) represents the
vital secondary protein structure and change in the band shape (in­
tensity) and position (shift placement) highlights the different protein
conformations (Tarapoulouzi et al., 2020). Therefore, the amide I band
was the main peak used in this study for determining differences in
protein secondary structure.
According to Fig. 2(A), the amide I band in spectra of cow milk
extracted via n-heptane (GS) and hexane (CS) individually revealed the
most intense amide I band within the range of 1660–1550 cm− 1, indi­
cating the main protein conformation in the form of β-sheet structures.
Similarly, major change in amide I band shape is observed in extracted
protein from GS-yogurt (ethyl acetate) and CS-yogurt (ethyl ether) with
no major shift in the position of the amide I band. Moreover, for non-
dairy extracted proteins as depicted in Fig. 2(C) the alternations
occurring in GS-soy milk (CPME), CS-soy milk (ethyl ether), GS-tofu (n-
heptane) and CS-tofu (hexane) with respect to shape and position of
amide I band reflect the possible denaturation of protein that contrib­
uted to different β-sheet structures. These results are in accordance with
a previously observed denaturation pattern in soy milk protein (Kumar,
Kumar, Jayachandran, & Rao, 2021). Particularly GS-soy milk (CPME)
from 1668 to 1450 cm− 1, shows increase aggregated β-sheet structures,
thus suggesting the co-existence of smaller number of β-sheet structures.
This is in accordance with the variable pressure scanning electron mi­
crographs, where GS-soy milk (CPME) depicted a denser microstructure
H. Kamal et al. Food Chemistry 400 (2023) 133988

Fig. 2. Normalized FTIR spectra of high protein yielding dairy (A) and non dairy (C) expired samples, whereas (C) showcases control samples for non-dairy analysis.
The broken line in (A) indicates the range of functional groups for both lipids and protein (1700-1250 cm-1).

resulting from smaller corpuscular structure and increment in aggre­ resulting into aggregation of protein. Aggregated β-sheet protein struc­
gated β-sheet structures. According to Ong et al. (2020) lower pH (4.6) is tures than increases the hardness, thus providing further evidence that
also a possible contributing factor in protein recovery such that it de­ secondary structures are vital to comprehend and formulate product
creases electrostatic repulsion by increasing hydrophobic interaction structure and function.

Fig. 3. Functional modifications of high protein yielding dairy and non dairy expired samples via CS and GS extraction, where (A) water holding capacity (WHC), (B)
emulsifying activity (EA) and stability (ES) and (C) foaming capacity (FC) and stability (FS).

6
H. Kamal et al. Food Chemistry 400 (2023) 133988

3.3. Functional modifications

An overview of (a) solvent properties, (b) extraction efficiency, (c) economic efficiency, (d) denaturation and structural modifications, (e) functional modifications and (f) ecological effect as sourced from the current

Toxicity
Proteins are responsible for many of the functional properties (sol­

index
ubility, water binding, thermal stability, emulsifying, foaming, gelation)

5
5

5
5
that influence consumer acceptance of food products; therefore, they
play a key role in food processing and product development (Buβler

mutagenic reprotoxic
et al., 2016). In the preceding discussions, section 3.2 identifies a
dependent structural–functional relationship, where protein function­

Carcinogenic
ality is associated closely with the changes occurring in secondary and
territory structure (Foegeding & Davis, 2011). Our goal in this study was

(CMR)
to understand and identify-three main functional modifications namely

No

No
No

No
No
(i) water holding capacity (WHC), (ii) emulsifying activity (EA) and

2
stability (ES), followed by (iii) foaming capacity (FC) and stability (FS)
occurring due to extraction via GS and CS individually. Fig. 3 illustrates

Structural
changes
the functional modifications of high protein yielding dairy and non-

order
dairy expired samples via CS and GS extraction.

6
4

1
5
According to Fig. 3(A), soy milk extracted proteins (both via CS and
GS) showed significantly higher WHC among all sample groups, fol­
lowed by yogurt, cow milk and tofu. However, among the conventional

Modification
Functional
and green solvent processing, overall GS processing depicted higher
WHC, with CPME (soy milk 277.29 ± 0.19), followed by ethyl acetate

order
(yogurt 155.17 ± 0.22) and n-heptane (cow milk 129.74 ± 0.07).

6
4

1
5
Moreover, ethanol and isopropanol extracted protein had least WHC.
CPME and n-heptane were found to be efficient in tabulating higher

Economic
efficiency
emulsifying properties (both EA and ES). Fig. 3(B) illustrates CPME
extracted soy milk protein with 37.84 ± 0.04 (EA) and 26.61 ± 0.16 as

order
the highest among all sample groups. Cow milk extracted via n-heptane

6
4

3
7
with 27.59 ± 0.11 showed highest EA among dairy samples. However,

efficiency order
the stability of the emulsion (ES) was found to be least effective in cow
milk (15.81 ± 0.27) and highest in tofu (31.04 ± 0.18). The increase in

Extraction
emulsifying properties is due to facilitation in the diffusion of protein at
oil–water interface and can be attributed to structural modifications as

4
7

1
5
discussed in section 3.2. Contrary to water holding and emulsifying re­
research study and published literature (Clarke, Tu, Levers, Bröhl, & Hallett, 2018; Yara-Varón et al., 2016).

sults, foaming analysis (FC and FS) as marked in Fig. 3(C) showed a poor
Polarity

correlation between green solvent processing and foaming capacity and

Index

stability. Ideally, an increase in WHC means a higher protein-water

0.1

2.8

3.9
4.4

0.1
–

–
interaction, which then facilitates improved foaming capacity. Howev­
er, this trend was not observed in either of sample groups. Foaming
capacity (FC) was highest in hexane extracted proteins for both soy milk
–23.3
Point
Flash

− 3.3
11.7
− 45
(◦ C)

6.5

8.9
(9.72 ± 0.16) and cow milk (5.81 ± 0.41). Also, ethanol was observed to

7
be equally effective especially in tofu with 2.84 ± 0.24 foaming stabil­
ity. Comparatively, the functional modifications studied were found to
Point (◦ C)
Boiling

be less than the cited literature (Lu, Chen, Wang, Yang, & Qi, 2016; 98.42
68.5

34.5

73.0
73.9

72.6
105.3

Sharma, Oey, & Everett, 2014). Nonetheless, the results are in accor­
dance to preceding protein recovery and structural modifications results
(section 3.1 and 3.2). The existing structural and compositional forma­
Weight (g/mol)

tion differences among dairy and non dairy groups are responsible for
Molecular

the variance in functional properties. In other words, protein function­

100.21
74.12

100.2
86.2

60.1
88.1

46.1

ality has classically been viewed from a single colloidal structure’s

perspective, whereas the extracted samples from expired, commercial
homogenized food matrix suggest a complex and direct correlation be­
tween protein functionality, size, shape, hydrophilic-hydrophobic bal­
(C2H5)2O
Chemical
Formula

C2H5OH
C6H12O

C4H8O2

ance of amino acid (AA), along with degree of hydrolysis (DH),

C3H8O
C6H14

C7H16

denaturation and protein purity (Kamal et al., 2018).

Type

3.4. Experimental green solvent (GS) substitution

GS

GS
GS

GS
GS
CS

CS

In this section we delineate if it is possible to substitute CS like

Cereal crop
Cereal crop

Cereal crop

hexane and ethyl ether with green solvents like ethyl acetate, ethanol,
Pine wood
Petroleum

Chemical

Chemical
synthesis

synthesis

isopropanol, n-heptane and CPME. The experimental parameters

Source

considered here are based on the appropriate comparisons performed in

section 3.1, 3.2 and 3.3 correspondingly, along with additional eco­
nomic and ecological parameters. Accordingly, Table 3 illustrates an
Isopropanol
Ethyl ether

n-heptane
acetate

overview of (a) solvent properties, (b) extraction efficiency, (c) eco­

Ethanol
Hexane
Solvent
Table 3

CPME

Ethyl

nomic efficiency, (d) denaturation and structural modifications, (e)

functional modifications and (f) ecological effect as sourced from the

7
H. Kamal et al.
current research study and published literature.
The importance and need of the potential of GS processing is gaining
momentum both in academia and industry particularly due to climate
change initiatives worldwide (Yara-Varón et al., 2016). Therefore, sol­
vents from renewable sources (biomass) that are environment friendly
are replacing frequently preferred CS like hexane (Li et al., 2019).
Hexane is one of the most used solvents to extract due to its low polarity,
stability, higher evaporation rate in reference to energy consumption
(Iloki-Assanga et al., 2015). In this context, Table 3 cites energy required
to evaporate 1 kg of solvent (kWh) and consequently CO2 mass gener­
ation according to Yara-Varón et al. (2016). Albeit, from a single
perspective of energy consumption as tabulated in Table 3, hexane re­
quires less energy (E) to be evaporated than all the evaluated GS.
Nonetheless, CPME and ethyl acetate require 9 and 5% more E,
respectively. However, by and large GS processing offers better physical
and chemical properties as required especially for product formulation
in food industry as well as lower toxicity in comparison to fossil sourced
solvents (Clarke et al., 2018).
4. Conclusion
This study aimed to evaluate (a) sustainable solvent recovery of
valuable protein from dairy and non-dairy expired waste, (b) to compare
extraction efficiency and quality of extracted protein using conventional
(CS) and green solvents (GS) and (c) highlighting the commercial
valorization potential of recycled protein as co-products in production of
food and non-food technical products. The results highlighted, in com­
parison GS depicted a higher protein recovery than CS. However, the
overall protein recovery was low. Microstructure revealed major
changes in the structural characterization of extracted protein. GS
extracted protein was found to be less detrimental in structural changes
co-relating to functional properties. Lastly, in comparison to non-treated
samples, extracted proteins showed diminished functional stability. This
means that replaced GS are promising due to their properties to extract
and their low toxicity, which is very important for the food industry. In
addition, GS provides lower carbon foot print, thus inducing more
sustainability.
CRediT authorship contribution statement
Hina Kamal: Formal analysis, Investigation, Methodology, Data
curation, Writing – original draft. Asgar Ali: Supervision, Validation,
Project administration, Funding acquisition, Writing – review & editing.
Cheng Foh Le: Formal analysis, Software, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgement
The authors duly acknowledge the Future Food Beacon, University of
Nottingham, for the financial support provided (Grant No. IAHB0013).
8
Food Chemistry 400 (2023) 133988
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.133988.
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