LWT - Food Science and Technology 140 (2021) 110692

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LWT
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Extraction of ultra-low gossypol protein from cottonseed: Characterization

based on antioxidant activity, structural morphology and functional
group analysis
Manoj Kumar a, *, Jayashree Potkule a, Sharmila Patil b, Sujata Saxena a, P.G. Patil a,
V. Mageshwaran c, Sneh Punia d, Eldho Varghese e, Archana Mahapatra f, Nandita Ashtaputre a,
Charlene D.’ Souza a, John F. Kennedy g
a
Chemical and Biochemical Processing Division, ICAR – Central Institute for Research on Cotton Technology, Matunga, Mumbai, 400019, India
b
Quality Evaluation and Improvement Division, ICAR – Central Institute for Research on Cotton Technology, Matunga, Mumbai, 400019, India
c
ICAR – National Bureau of Agriculturally Important Microorganisms, Mau, 275103, India
d
Department of Food Science and Technology, Chaudhary Devi Lal University, Sirsa, India
e
Fishery Resources Assessment Division, ICAR – Central Marine Fisheries Research Institute, Kochi, 682 018, India
f
Transfer of Technology Division, ICAR – Central Institute for Research on Cotton Technology, Matunga, Mumbai, 400019, India
g
Chembiotech Laboratories, Advanced Science and Technology Institute, Kyrewood House, Tenbury Wells, Worcs, WR15 8FF, UK

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

Keywords: Cottonseed can be considered as a potential candidate to act as an important protein source. However, toxic
Cottonseed protein isolate compound “gossypol” is the main hurdle in utilization of this potential source of protein. The current study aims
Extraction to optimize the extraction of cottonseed protein isolate (CSPI) from defatted cottonseed meal (CSM) using Box-
Functional characterization
Behnken design (BBD) with ultra-low gossypol concentration (ULGC, <0.1 μg/mg or 100 ppm) which is under
the allowable limits of 600 ppm as per United Nations Food and Agriculture Organization and World Health
Organization for application in foods. Results indicate that obtained optimal conditions for the recovery of
protein from CSM were alkali: sample ratio (33:1), NaCl (0.15 mol/L), Na2SO3 (0.27%) and time (147.5 min) at
pH 11 with predicted value of 93.6% protein recovery. Wet-lab confirmed that experimental yield of CSPI was
88.5% with 29.0 ppm of free gossypol concentration. CSPI was analysed for the physicochemical characteristics
and found to have balanced ratio of essential and non-essential amino acids using HPLC. Functional group
analysis, antioxidant activity, molecular weight distribution and microstructure confirmed various structural
features of the isolated protein. Hence, the resultant CSPI can be utilized as a feed supplement for monogastric
animals and food supplement for human beings.

1. Introduction crushed by screw-pressing method, 5% by solvent extraction method

Cotton (Gossypium hirsutum L.) is produced in more than 80 countries
and is the most prominent source of fiber for the textile industry (He,
Zhang, & Cao, 2018). Apart from fiber, cottonseed consists of valuable
by-products such as hull (27%), linters (8%), oil (18%) and CSM (45%)
(Mageshwaran, D’souza, Kambli, & Kathe, 2015). India is the largest
producer of cotton with an annual production of 5.80 million tons from
26.06 million tons of world cotton production (CCI Mumbai, 2019). The
cottonseed production during 2017–18 is 12 million tons (AICOSCA,
Mumbai, 2018). Of the total cottonseed produced, 75% is directly
and 20% is fed to cattle directly. After the extraction of oil from cot­
tonseed, it is left with meal or cake which is a rich source of protein. CSM
contains about 45–55% crude protein with a balanced amino acid
composition (Bertrand, Sudduth, Condon, Jenkins, & Calhoun, 2005).
The presence of gossypol, however, makes the cottonseed protein
harmful for human consumption, rendering it underutilized. Gossypol
[1,1′ ,6,6′ ,7,7′ -hexahydroxy-5,5′ -diisopropyl-3,3′ -dimethyl-(2,2′ -
binaphthalene)-8,8′ dicarboxaldehyde] is a polyphenolic compound
produced in pigment glands of seed, stem, leaves and flower buds as a
natural defense mechanism against insect, pests and toxic to most of the

* Corresponding author.
E-mail addresses: manojkumarpuniya114@gmail.com, manoj.kumar13@icar.gov.in (M. Kumar).

https://doi.org/10.1016/j.lwt.2020.110692
Received 17 August 2020; Received in revised form 26 November 2020; Accepted 30 November 2020
Available online 8 December 2020
0023-6438/© 2020 Elsevier Ltd. All rights reserved.
M. Kumar et al.
monogastric animals viz. Poultry, fisheries and piggeries (Bernard,
2016; Bolek, Tekerek, Hayat, & Bardak, 2016; Gadelha, Fonseca, Oloris,
Melo, & Soto-Blanco, 2014; Kumar, 2019a; Mageshwaran et al., 2015).
Mainly, two types of gossypol are present in cottonseed-free gossypol
and bound gossypol. Free gossypol is more harmful and can lead to
toxicity and contribute towards the anti-sterility effects in animals and
human beings. Though the bound gossypol is non-toxic, it decreases
bio-availability and nutritive value of lysine, an essential amino acid by
binding with epsilon amino group of lysine and reduces the digestibility
of CSM (Saki et al., 2012). According to FDA (Food and Drug Admin­
istration), cottonseed protein (CSP) and their food products can be
considered as edible if they contain less than 0.045% and 0.8% of free
gossypol and bound gossypol, respectively (Ma et al., 2018). The
reduction of gossypol from cottonseed thus becomes important to use it
as an alternative source of food-grade protein.
Several new processes and solvents have been tried to remove the
gossypol from cottonseed so that it can be used for edible purposes
without any adverse effect. Efforts are also being made to develop
glandless cotton varieties with very low gossypol content. However
glandless cotton varieties have not been so popular due to their low
resistance to insects, pests and the low yield on account of the absence of
gossypol (Palle et al., 2012). Alternatively, numerous methods have
been attempted for extraction of protein from CSM with high recovery
and the minimum level of gossypol. The solvent extraction using
aqua-based and alkali-based solvent system are the most widely used
methods by researchers (He, Zhang, & Olk, 2015; Mageshwaran et al.,
2015; Saki et al., 2012). Aqua- and enzyme based extraction methods
often suffer from low protein recovery as plant cell protein is hardly
soluble in water due to the presence of the hydrophobic group and
disulphide bond in protein molecules (Kumar et al., 2019b). On the
other hand, alkali-assisted extraction of protein increases the surface
charge of protein which leads to an enhanced solubility of the protein
(Cui et al., 2017). Salts are commonly used to isolate peripheral pro­
teins; hence the combination of alkali and salt assisted protein extraction
results in an optimum yield of protein (Zheng, Gao, Wu, & Yin, 2019, pp.
271–293). Besides high protein recovery, the alkali-salt extraction has
been reported to reduce gossypol poisoning in the cottonseed-based food
products (Gadelha et al., 2014; Karishma, Lakshmi, Suneetha, Chinna, &
Krishna, 2016). However, there is no systematic study on alkali-assisted
extraction of protein from CSM. Furthermore, the use of CSP as an
ingredient in various food products depends on its nutritive value as well
as functional properties (Chen et al., 2014; Ma et al., 2018; Tong, Luo, &
Li, 2015). The present study has been undertaken with the intent to
develop a protocol for alkali-assisted extraction of CSM protein with the
ULGC which can be utilized as an ingredient for the food industry.
Protein extraction from plant matrices like CSM by alkali-assisted
method requires process optimization regarding its operational param­
eters such as the concentration of alkali to sample ratio, time and con­
centration of salts. The optimization was done based on a second order
model connecting the response generated form a Box-Behnken design
(BBD) and the input factors. Compared to other experimental designs,
BBD was found to be superior for fitting the quadratic model because of
its higher efficiency and lower cost of experimentation.
The CSPI obtained using optimized process conditions was further
characterized for its total and free gossypol, antioxidant activity, total
protein content, amino acid profile using high performance liquid
chromatography (HPLC), structural morphology using scanning electron
microscopy (SEM), functional groups [Fourier transform infrared (FTIR)
spectroscopy analysis], molecular weight using sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This appears
to be the first report on an optimal alkali-assisted extraction of CSP with
ULGC and its functional characterization for potential application in
food and feed industry.
2
LWT 140 (2021) 110692
2. Material and methods
2.1. Materials
The de-oiled CSM was generously supplied by Clean Cotton Impex,
Pitchampalayam, Tiruppur, Tamil Nadu, India. Groundnut oil was ob­
tained from the local market, Mumbai, India. Dialysis bags were pro­
cured from Himedia Laboratories, Mumbai, India. Ammonium sulphate,
bovine serum albumin, 2, 2-diphenyl1-picrylhydrazyl, Tris base were
purchased from Genex Life Science Pvt. Ltd., Mumbai, India. Acryl­
amide, bis-acrylamide, sodium dodecyl sulphate, N,N,N,N tetrametyle­
thylenediamide, β-mercaptoethanol, ammonium persulphate were
purchased from Himedia Laboratories, Mumbai. Standard protein ladder
(12 kDa–78 kDa) was purchased from Merck Specialities Pvt. Ltd.,
Germany. All reagents and chemical used in the current study were of
analytical grade.
2.2. Sample preparation
CSM was ground in a domestic grinder and screened through 50
mesh sieves to obtain a sample with same characteristics. The powdered
CSM was then mixed vigorously to ensure uniformity and kept at 4 ◦ C
temperature until extraction and analysis.
2.3. Protein extraction experiments
2.3.1. Alkali extraction followed by ammonium sulphate precipitation and
purification by a dialysis membrane
CSPI was obtained from defatted CSM using alkaline extraction
method with some modifications (Boye & Barbana, 2012, pp. 85–114;
Du et al., 2018; Ma et al., 2018; Zhang, Cui, Yin, Li, & Zhou, 2009). CSM
was added in alkali solution at pH 11 and 1:30 ratio [v w− 1] (0.1 mol/L
KOH and varying concentration of NaCl and Na2SO3) to extract the
protein. The solution was placed on an orbital shaker (Gallenkamp
orbital shaker incubator, UK) for varying time, then centrifuged (Beck­
man coulter centrifuge, USA) at 27,000 g for 15 min at 4 ◦ C temperature.
The supernatant was collected and precipitated with 85% ammonium
sulphate (Das purkayastha, Dutta, Barthakur, & Mahanta, 2015) ac­
cording to the volume of the supernatant (the saturation levels were
calculated with help of EnCor Biotechnology Inc.). Continuous mixing
was required to induce protein precipitation at 4 ◦ C for 1 h. Cottonseed
protein (CSP) was then recovered by centrifuging at 27,000 g at 4 ◦ C
temperature for 15 min. CSP pellet was dissolved in distilled water and
purified by using dialysis method (dialysis membrane-150, size- 37.7 ×
25.4 mm, capacity- 5 mL cm− 1, Himedia Laboratories, Mumbai). The
membrane containing CSP extract was submerged in a beaker contain­
ing distilled water for 2 h with continuous magnetic stirring. Dialyzed
protein sample was then lyophilized using lyophilizer (VirTis SP Scien­
tific, USA) to recover CSPI (Kramer, Shende, Motl, Pace, & Scholtz,
2012; Siong, Mailer, Blanchard, & Agboola, 2010; Zhang et al., 2009).
The flow diagram showing CSPI extraction from CSM is depicted in
Fig. 1.
2.4. Experimental layout and statistical analysis
2.4.1. Single-factor experiments
Experiments were conducted in triplicate to interrogate the effect of
four processing parameters (alkali to sample ratio, extraction time, the
concentration of sodium chloride and sodium sulphite) on protein re­
covery from CSM (Fig. 2). For conducting the single-factor experiments,
varying alkali to sample ratio [10:1, 20:1, 30:1, and 40:1 (v w− 1)], time
(60, 120, 180, and 240 min), concentration of NaCl (0.05, 0.10, 0.15,
and 0.20 mol/L), concentration of Na2SO3 (0.10, 0.20, 0.30, and 0.40%)
was used. The range for the single factor analysis was chosen on the basis
of review of literature in the different crops such as rapeseed for the
extraction of protein (Das purkayastha et al., 2015). The maximum
M. Kumar et al.
Fig. 1. Flow diagram showing extraction of CSPI from CSM.
Fig. 2. Bar graph for the % recovery of purified protein from CSM (values are
mean of 3 independent experiments). Where, PAS1–PAS4 = varying alkali to
sample ratio (10:1, 20:1, 30:1, and 40:1 [v w− 1]), PT1–PT4 = Varying time (60,
120, 180, and 240 min), PNC1–PNC4 = varying concentration of NaCl (0.05,
0.10, 0.15, and 0.20 mol/L), PNS1–PNS4 = (varying concentration of Na2SO3
(0.10, 0.20, 0.30, and 0.40%). The maximum protein recovery (PAS3, PT2,
PNC2 and PNS2) in all the four experiments was considered as central point for
optimizing the experimental factors for maximum response in response surface
methodology using Design Expert 12.0 software. Key to short form: PAS: pro­
tein recovery when alkali: sample ratio is variable; PT: protein recovery when
time is variable; PNC: protein recovery when NaCl is variable; PNS: protein
recovery when Na2SO3 is variable. Values are expressed as means of triplicate
measurements: Means with different alphabets are significantly different at p
< 0.05.
protein recovery was achieved at 30:1 [v w− 1] alkali to sample ratio,
120 min, 0.10 mol/L, NaCl and 0.20%. These four experimental condi­
tions were considered as central point for optimizing the experimental
factors for maximum response in response surface methodology using
design expert software.
3
LWT 140 (2021) 110692
2.5. Characterization of cottonseed meal (CSM) and cottonseed protein
isolate (CSPI)
CSM and CSPI were analysed for their total protein content, free and
total gossypol content, antioxidant activity, amino acid profile, FTIR
spectroscope and surface morphology.
2.5.1. Total protein contents
The total protein content CSM and CSPI were measured according to
AOAC method (2000) with some modifications. 0.3 g of protein sample,
3 g catalyst mixture (potassium sulphate + copper sulphate) and 10 mL
of concentrated H2SO4 was poured in digestion tubes. Run samples for
30 min at 350 ◦ C and 90 min at 420 ◦ C for digestion (Kelplus-kelvac
nitrogen analyzer, Chennai), remove assembly and allow to cool.
Transfer tube in distillation unit (Kelplus- Distyl EMS, Chennai) at one
place and indicator containing 20 mL boric acid funnel under other
suction straw and run program for distillation and then titrated using
0.1 mol/L HCL. Total protein content was calculated according to
standard Kjeldahl method.
Protein % = Titration reading * Normality (N) * 1.4007 * 6.25/ Weight of
sample (W)
Where, 1.4007 = atomic weight of nitrogen, 6.25 = protein – nitrogen
conversation factor.
2.5.2. Free gossypol (FG)
Free gossypol content of CSM and CSPI was determined according to
AOCS official method number Ba 7–58 (1958). Transfer 1 g of protein
sample in 250 mL Erlenmeyer flask and cover the bottom of the flask
with glass beads. Add 50 mL 70% acetone and shake vigorously on a
mechanical shaker for 1 h. It was then filtered using Whatman #1 filter
paper. Sample aliquot A- 1 mL sample aliquot, add 0.10 mL 10%
aqueous thiourea, 0.05 mL 1.2 mol/L HCl and dilute up to 25 mL with
aqueous isopropyl alcohol. Prepare a reagent blank A containing a 1 mL
of 70% acetone equal to that of the sample aliquot and add 0.10 mL 10%
aqueous thiourea dilute with aqueous isopropyl alcohol. Sample aliquot
B- 1 mL sample aliquot, add 0.10 mL 10% aqueous thiourea, 0.05 mL,
1.2 mol/L HCl and 2 mL of distilled aniline. Prepare a reagent blank B
containing 1 mL 70% acetone and add 0.10 mL 10% aqueous thiourea
and 2 mL of distilled aniline. Sample aliquot B and reagent blank B was
placed in a boiling water bath for 30 min, allowed to cool at RT and
dilute with aqueous isopropyl alcohol. Read the absorbance of sample
aliquot A and B at 440 nm by using reagent blank A and B. The con­
centration of gossypol is calculated with following formula:
Corrected Absorbance = [Absorbance solution B - Absorbance solution A]
G = Corrected absorbance * calibration factor
Free gossypol (%) = 5 G / W* V
Where, G = mg gossypol in the sample aliquot, W = sample weight, V =
volume of the sample.
2.5.3. Total gossypol (TG)
Total gossypol content of CSM and CSPI was determined according to
AOCS official method number Ba 8–78 (1997). 0.5 g of sample was
dispersed in 10 mL of complexing (2 mL 3-amino-1-propanol + 10 mL
glacial acetic acid and dilute up to 100 mL with dimethylformamide)
reagent in 50 mL volumetric flask and prepared a reagent blank con­
taining 10 mL of complexing reagent and heat in boiling water bath for
30 min, allowed to cool at RT and dilute with aqueous isopropyl
alcohol-hexane mixture and then filtered. Sample aliquot A- 1 mL
sample aliquot and dilute to volume 25 mL with aqueous isopropyl
alcohol-hexane mixture. Prepare a reagent blank A containing blank
solution equal to that of the sample aliquot and dilute up to 25 mL with
aqueous isopropyl alcohol-hexane mixture. Sample aliquot B- 1 mL
M. Kumar et al. LWT 140 (2021) 110692

sample aliquot and add 2 mL of distilled aniline. Prepare a reagent blank
B containing blank solution equal to that of the sample aliquot and 2 mL
of distilled aniline. Sample aliquot B and reagent blank B was placed in a
boiling water bath for 30 min, allowed to cool at RT and dilute up to 25
mL with aqueous isopropyl alcohol-hexane mixture. Read the absor­
bance of sample aliquot A and B at 440 nm by using reagent blank A and
B. The concentration of gossypol is calculated with following formula:
Total gossypol (%) = 5 G / W* V
Where, G = mg gossypol in the sample aliquot, W = sample weight, V =
volume of the sample.
2.6. Box-Behnken design and statistical analysis
2.6.1. Box-Behnken design (BBD)
The BBD and statistical analysis for alkali extraction of protein were
performed according to Kumar et al. (2019c,d) with modification in the
input factors. The input factors (alkali solution to sample ratio, incu­
bation time, NaCl and Na2SO3 concentrations) and response (protein
recovery) were subjected to ANOVA and regression analysis to find out
the significance of the performed model. The significance of linear,
interactive and quadratic terms was also established using regression
and ANOVA analysis.

2.5.4. Antioxidant activity
Antioxidant activity of CSM and CSPI was assessed by 2, 2-diphenyl-
1-picryl-hydrazyl (DPPH) assay as described by Hendel, Larous, and
Belbey (2016). 100 μL (10 mg/mL) of CSM, CSPI solution was added into
the 5 mL 0.004% DPPH methanol solution. The absorbance A1 recorded
at 517 nm after 30 min reaction at RT and prepared control to measure
the absorbance A0. The clearance rate (I%) of DPPH could be calculated
by the following formula:
I% = [(A0 − A1)/ A0] × 100
Where, A1: Absorbance of sample and A0: Absorbance of control against
methanol.
2.6.2. Model prediction and validation of optimized conditions
Second-order regression model was fitted to experimental data to
obtain the predictive model. The accuracy of the model was analysed
based on the criteria given by Myer and Montgomery (2002). The model
fitness was assessed using adjusted R2. The 3D graph of response surface
curve was used to visualize the change in response, at different levels of
the input variables and to flag the response at optimum input points.
Validation of the optimum response was also done. The generation of
experimental design and statistical analysis were performed using
Stat-Ease software (Design-Expert 12.0, licensed to ICAR-CMFRI).
2.7. Statistical analysis

Data on percentage recovery of protein from CSM under different

2.5.5. Fourier transform infrared (FTIR) spectroscope determination
conditions were subjected to one-way ANOVA separately for varying
CSM and CSPI samples were ground with potassium bromide at a
alkali to sample ratio (10:1, 20:1, 30:1, and 40:1 [v w− 1]), Varying time
1:100 ratio (w w− 1) and pressed into potassium bromide pellet at high
(60, 120, 180, and 240 min), varying concentration of NaCl (0.05, 0.10,
pressure. Fourier transform infrared spectrophotometer (IR Prestige 21,
0.15, and 0.20 mol/L), (varying concentration of Na2SO3 using PROG
Shimadzu, Japan) was used to record FTIR spectra of protein samples in
GLM of SAS software package, version 9.4 (SAS Institute, Cary, North
the range of 400–4000 cm− 1 at room temperature. The measuring res­
Carolina, USA). Further, Tukey’s Honest Significant Difference (HSD)
olution was 4 cm− 1 with scan number of 49 and recorded with the help
test was used to identify the pair-wise significant differences among
of IR solution software.
various treatments which were significant, as per ANOVA and presented
in Fig. 2. The treatment pairs which are represented by same alphabets
2.5.6. High-performance liquid chromatography (HPLC)
are not statistically significant. Statistical significance of the variables
CSM and CSPI samples were characterized by HPLC using an Agilent
viz., total protein content, antioxidant activity, free gossypol concen­
HPLC system separation module (Agilent Technologies, Inc. USA)
tration and total gossypol concentration were assessed using Kruskal-
equipped with a column (Agilent ZORBAX Eclipse Plus C18 stationary
Wallis test, a nonparametric one-way ANOVA for independent samples
phase column, 2.1 × 150 mm, 3.5 μm) rapid resolution method and a
using PROC NPAR1WAY of SAS and given in Fig. 4 as the assumptions of
prostar DAD detector. 100 mg of protein was hydrolysed with 10 mL of
parametric ANOVA were not met for these variables.
6 mol/L HCl at 110 ◦ C by microwave digesters and evaporated to collect
hydrolysed solution (200 μL). 5 μL samples were taken from hydrolysed
3. Results and discussion
solution and diluted with 500 μL of distilled water. 20 μL sample solu­
tion was injected in the column and protein and peptide were eluted
Achieving ULGC in obtained CSPI becomes an integral process to
using a borate buffer (Supplementary Information Fig. S2) pH 8.2. The
obtain food-grade protein from CSM. In this study, food-grade protein
flow rate was 1.5 mL min− 1. The UV detection was measured at 338 nm
was extracted from defatted CSM using alkali-salt exaction method.
and a reference wavelength of 390 nm.

3.1. Preliminary experiments

2.5.7. SDS-PAGE
Protein profile was determined using SDS-PAGE (Amersham Bio­
In order to determine the preliminary range of extraction variables
sciences AB, Sweden) according to the modified Laemmli method
for process optimization through response surface methodology, single-
(Brunelle & Green, 2014; Kumar, Ganesan, Selvaraj, & Rao, 2014; Singh
factor experiments were conducted. The effect of four extraction vari­
& Kaur, 2019; Singh & Sogi, 2018). PAGE was done on a 5% stacking
ables namely; alkali to sample ratio, extraction time, concentration of
and 12% resolving gel in an SDS-PAGE running buffer system. 10
NaCl and Na2SO3 were optimized with respect to protein recovery from
mg/mL sample was diluted with sample buffer (4:1 ratio) and boiled at
defatted CSM. The first experiment was conducted with the single factor
95 ◦ C for 90 s 20 μL of protein sample was loaded in each lane of the gel.
of alkali to sample ratio in the range of 10:1 to 40:1 [v w− 1]. The other
Run electrophoresis and stained with Coomassie blue staining solution
three parameters, including extraction time, concentration of NaCl, and
to reveal the protein bands.
Na2SO3 were set constant at 120 min, 0.1 mol/L, and 0.2%, respectively.
The next single factor was analysed according to the optimal result of a
2.5.8. Scanning electron microscopy (SEM)
previous single factor experiment. Color of extracted protein plays a
Surface morphology of CSM and CSPI was studied by SEM (Philips
critical role in the acceptability of any food product in food industry. We
XL30 SEM, Netherlands) at 10 kV accelerating voltage and 500X
found that color of cottonseed protein also varied with the change in the
magnification. The samples were placed on the sample holder and then
extraction conditions (Supplementary information Fig. S1). The varia­
vacuum sputtered with the gold-palladium mixture to improve its con­
tions in the color of extracted protein may occurred due to the presence
ductivity and viewed under SEM.
of salts (NaCl and Na2SO3) in the extraction solvent. Das purkayastha

4
M. Kumar et al. LWT 140 (2021) 110692

et al. (2015) reported NaCl breaks interaction between protein and Table 1
polyphenolic compounds while, Na2SO3 play important role to protect BBD and experimental data for protein recovery (%) from cottonseed meal for
polyphenols like gossypol from oxidation during protein extraction. alkali extraction.
Input Input Input Factor (C) Input Response
3.1.1. Influence of extraction solvent (alkali) to solid ratio Run Factor (A) Factor (B) Factor (D)
It was observed that as alkali to solid ratio increased from 10:1 to Alkali to Time NaCl Na2SO3 Protein
30:1 [v w− 1], there was a significant increase in protein recovery from sample (minutes) concentration (%) recovery
CSM. However, protein recovery declined with further increase in the ratio (M) (%)
ratio above 30:1 [v w− 1]. The range of alkali to solid ratio was thus 1 30 120 0.1 0.2 85.3
chosen as 20:1 to 40:1 [v w− 1] for further process optimization using 2 40 120 0.1 0.1 75.1
RSM. 3 20 120 0.1 0.3 71.4
4 40 60 0.1 0.2 54.6
5 30 120 0.15 0.3 88.2
3.1.2. Influence of extraction time 6 20 120 0.1 0.1 73.5
Extraction time is one of the most important parameters to obtain 7 30 120 0.1 0.2 91.2
high recovery of protein from CSM. The yield of protein from CSM 8 40 120 0.15 0.2 82.9
9 30 180 0.1 0.3 80.9
enhanced with increase in incubation period from 60 to 120 min then
10 30 180 0.15 0.2 81.1
decreased significantly after 180 min of incubation. The other extraction 11 30 60 0.15 0.2 64.7
parameters-alkali to sample ratio, NaCl and Na2SO3 concentration were 12 30 120 0.1 0.2 95.0
kept constant as 30:1 [v w− 1], 0.1 mol/L and 0.2% respectively 13 30 120 0.05 0.3 82.8
throughout the experiment. The highest recovery of 81% was obtained 14 40 120 0.1 0.3 77.7
15 30 180 0.1 0.1 71.2
at 120 min of extraction time, followed by a significant decrease of 2- 16 20 180 0.1 0.2 53.2
folds (Fig. 2). 17 20 120 0.15 0.2 69.0
18 40 120 0.05 0.2 71.7
3.1.3. Influence of sodium chloride (NaCl) 19 40 180 0.1 0.2 82.6
20 30 60 0.05 0.2 76.8
Effect of NaCl on protein recovery from CSM was investigated at the
21 30 60 0.1 0.3 69.0
levels ranged from 0.05 to 0.20 mol/L. The other extraction parameters- 22 30 60 0.1 0.1 76.5
alkali to sample ratio, extraction time and Na2SO3 concentration were 23 20 120 0.05 0.2 78.8
kept constant as 30:1 [v w− 1], 120 min and 0.2% respectively 24 20 60 0.1 0.2 76.1
throughout the experiment. 0.1 mol/L NaCl concentration resulted in 25 30 120 0.1 0.2 91.1
26 30 120 0.05 0.1 89.3
maximum protein recovery of 85.4%. There was a subsequent decrease 27 30 120 0.15 0.1 79.2
in protein yield above 0.1 mol/L NaCl concentration. Based on the re­ 28 30 120 0.1 0.2 91.6
sults, the preliminary range of NaCl concentration was selected as 29 30 180 0.05 0.2 69.7
0.05–0.15 N.

3.1.4. Influence of sodium sulphite (Na2SO3)
The effect of Na2SO3 concentration on protein recovery followed a
parabolic curve, showing an increase in protein recovery up to 0.2%
concentration and gradual decrease thereafter (Fig. 2). The other
extraction parameters-alkali to sample ratio, extraction time, and NaCl
concentration were kept constant as 30:1 [v w− 1], 120 min and 0.1 mol/
L respectively throughout the experiment. The concentration range of
0.1–0.3% was thus used for further optimization of process parameters
using RSM.
3.2. Model fitting
RSM was used to optimize protein extraction from CSM. BBD with
four independent input factors at three levels was carried out to study
the effect on protein recovery. Based on the results of preliminary single
factor experiments, the range of extraction variables was selected as
20:1–40:1 [v w− 1] (alkali to sample ratio), 60–180 min (extraction
time), 0.05–0.15 mol/L (concentration of NaCl) and 0.1–0.3% (con­
centration of Na2SO3). A total of 29 experiments were according to BBD
and mean values were used in data analysis (Table 1). The predicted
model for protein recovery was tested for fitness and adequacy by
ANOVA and results are shown in Table 2.
3.3. Effect of independent variables on protein recovery
Experimental data showed that the protein recovery from CSM var­
ied from 53.2% to 95%, based on the process conditions (Table 1). In
most of RSM experiments, protein recovery was found to be above 65%
by alkaline extraction at pH 11. The findings are consistent with the
results of He, Cao, Cheng, Zou, and Hunt (2013); Ma et al. (2018); Zhang
et al. (2009). Similar protein recovery in the range of 58–99% using
alkali extraction method was reported for watermelon seed (Wani, Sogi,
Grover, & Saxena, 2006) red pepper seed (Firatligil-Durmus & Evranuz,
2010), peanut (Rustom, Lopez -Leiva, & Nair, 1991), tomato seeds
(Mechmeche et al., 2016; Liadakis, Tzia, Oreopoulou, & Thomopoulos,
1995), grass pea (Feyzi, Milani, & Golimovahhed, 2018), winged bean
(Indah & Helmy, 2015) and custard apple seed (Vaidya, Saxena, &
Omani, 2017). Zhu, Zhao, Zhang, and Liu (2019) obtained 81.36%
protein yield by alkaline extraction of Haematococcus pluvialis at pH 11
and acid precipitation. In contrast, alkali extraction method was
observed to yield low protein recovery than that of enzyme and
microwave-assisted extraction method in rice bran (Yilmaz & Dilek,
2016) and sesame (Phongthai, Lim, & Rawdkuen, 2016).
After regression analysis of data and fitting RSM for all independent
and dependent variables, a quadratic regression model was obtained.
The regression equation in terms of coded variables showing the effect of
extraction variables on the protein recovery (%) is given below:
Protein Recovery (%) = 90.42 + 1.89A + 1.74B–0.3275C + 0.4533D +
12.72AB + 5.23AC + 1.15AD + 5.87BCE + 4.30BD + 3.87CD – 11.73A2 –
13B2 – 3.28C2 – 3.19D2
The coefficients and other statistics depicted fair suitability of model
in predicting protein recovery from CSM (Table 2).
ANOVA of the model fitted to protein recovery data (Table 2) showed
that effect of A, B, interactions effect AB, AC, BC, BD, CD and four
quadratic terms A2, B2, C2 and D2 were significant model terms (p <
0.05) affecting the yield of the protein. No significant interaction was
found between A and D. The alkali-to-sample ratio had most significant
linear effect followed by extraction time. AB and BC showed profound
mutual effect and positively influencing recovery of protein from CSM.
The effect of extraction conditions on protein recovery from CSM can
also be seen in the 3-D surface plots and contour graphs (Fig. 3a–f). The
effect of alkali to sample ratio (A) and extraction time (B) on protein
recovery from CSM at a fixed concentration of sodium chloride and

5
M. Kumar et al. LWT 140 (2021) 110692

Table 2
Model summary statistics for protein recovery.
Source Sum of squares DF Mean square F-value P-value Regression coefficient

Model 2796.90 14 199.78 47.07 <0.0001**

A 42.90 1 42.90 10.11 0.0067** 1.89
B 36.19 1 36.19 8.53 0.0112* 1.74
C 1.29 1 1.29 0.3032 0.5905ns − 0.3275
D 2.47 1 2.47 0.5810 0.4586ns 0.4533
AB 646.94 1 646.94 152.42 <0.0001** 12.72
AC 109.41 1 109.41 25.78 0.0002** 5.23
AD 5.24 1 5.24 1.24 0.285ns 1.15
BC 137.95 1 137.95 32.50 <0.0001** 5.87
BD 73.96 1 73.96 17.43 0.0009** 4.30
CD 60.06 1 60.06 14.15 0.0021** 3.87
A2 891.85 1 891.85 210.12 <0.0001** − 11.73
B2 1096.98 1 1096.98 258.45 <0.0001** − 13.00
C2 69.60 1 69.60 16.40 0.0012** − 3.28
D2 66.09 1 66.09 15.57 0.0015** − 3.19
Residual 59.42 14 4.24
Lack of fit 24.41 10 2.44 0.2789 0.9538ns
Pure error 35.01 4 8.75
Corrected total 2856.32 28
R2 0.979
R2 (Adjusted) 0.958
R2 (Predicted) 0.932
Adequate precision 25.378
CV% 2.66

Key to short forms: A = alkali to sample ratio; B = time; C = NaCl concentration; D = Na2SO3 concentration; AB = alkali to sample ratio × Time; AC = alkali to sample
ratio × NaCl concentration; AD = alkali to sample ratio × Na2SO3 concentration; BC = Time × NaCl concentration, BD = Time × Na2SO3 concentration; CD = NaCl
concentration × Na2SO3 concentration; A2, B2, C2 and D2 are representing the quadratic terms for alkali to sample ratio, time, NaCl concentration, Na2SO3 con­
centration, respectively; CV = Co-variance and values shown are regression coefficient of respective term, * = significant at p < 0.05, ** = significant at p < 0.01, ns =
non-significant.

sodium sulphite at its central level can be seen in Fig. 3a. Similarly, the
interaction between A and C, A and D, B and C, B and D, C and D can be
observed in Fig. 3b, c, 3d, 3e and 3f respectively. Results also coincide
with single factor experiment.
It is evident from response surface plots (Fig. 3) that high protein
recovery can be obtained at high alkali to sample ratio and high
extraction time. The highest protein recovery of 95% was observed at
30:1 [v w− 1] alkali to sample ratio and 120 min extraction time. Alkali
to sample ratio increases protein solubility with an increasing alkaline
condition, but a high concentration of alkali may lead to degradation
and denaturation of protein (Das purkayastha et al., 2015). Protein
extraction increases with an increase in retention time. Lower retention
time gives low protein yield due to less solubility of proteins as acidic
and neutral amino acid present in the protein were not fully ionized.
Protein extraction above 120 min gives a negligible difference in protein
recovery, hence 120 min was a suitable time interval for protein
extraction. The significant quadratic effect between factor A and B
revealed that increasing of alkali to sample ratio and extraction time
increased the protein recovery up to certain level and decreased there­
after. Zhang et al. (2009) reported 70% cottonseed protein extraction
using 0.1 mol/L KOH, 12 [w v− 1] (solvent-to-flour ratio), 60 (temper­
ature), 12.5 pH and 40 min (time interval) and Das purkayastha et al.
(2015) reported 95% rapeseed protein extraction by optimizing solvent
to solid ratio, extraction time, NaCl, and Na2SO3. Though NaCl con­
centration had non-significant (p > 0.05) negative linear relationship on
protein recovery, its quadratic effect with alkali to sample ratio is pos­
itive and significant (p < 0.05). The similar positive quadratic effect was
observed among all independent variables.
3.4. Optimization and validation of processing input factors
The optimum processing conditions (alkali to sample ratio, extrac­
tion time, NaCl concentration and Na2SO3 concentration) for the
extraction of protein from CSM was determined using design-expert
software. The main criterion for optimization was to obtain a higher
yield of protein. The optimum processing conditions obtained were
alkali: sample ratio: 33:1 [v w− 1], NaCl concentration: 0.15 mol/L,
Na2SO3 concentration: 0.27% and extraction time: 147.5 min at pH 11.
The predicted value of protein recovery obtained from RSM was 93.6%
and it was confirmed by conducting lab experiments. The experimental
yield of CSM protein was 88.5%, within the predicted interval. The re­
sults demonstrated the validation of the RSM model in predicting opti­
mum conditions for extraction of CSM protein with maximum recovery.
3.4.1. Characterization of CSPI using various analytical methods
3.4.1.1. Total protein content, free and total gossypol content. Results
showed that the total protein content of alkaline extracted CSPI was
higher (93.1 ± 6.7%) than that of CSM (56.8 ± 5.4%) (Fig. 4.). A lower
total protein content of 49.6% in alkali assisted extraction of soy protein
isolate and 50.7% in glanded and 59.4% in glandless cottonseed protein
isolate (He, Zhang and Olk (2011). The variation in the resultant may be
due to the variation in experimental conditions and meal used. The
combination of alkali and salts assisted in the solubilization of the
protein from plant matrix to extraction solvent and resulted in increased
protein content in CSPI (Zhang, Sanders, Xiao, & Bruins, 2015).
As evident from Fig. 4, total protein content, free and total gossypol
content varied significantly (p < 0.05). Free and total gossypol content
of CSM was 700 ± 34.6 ppm and 20,900 ± 195.9 ppm respectively. CSPI,
however, showed a low level of free and total gossypol content i.e. 29 ±
1.3 ppm and 270 ± 21.0 ppm, depicting about 95% and 98% reduction
in gossypol levels respectively. Reduction in gossypol content by alkali
and salt treatment was due to weak bonding between gossypol and
protein (Das purkayastha et al., 2015). Addition of Na2SO3 prevents
oxidation of phenolic compounds and improves the color and flavour of
protein (Aider & Barbana, 2011). A similar reduction in gossypol con­
tent of alkaline extracted CSM was studied. Zhang, Wang, Dai, He, and
Ma (2018) reported that microbial fermentation technique also helps to
degrade free gossypol in CSM and improves the nutritional value of
cottonseed protein.
3.4.1.2. Antioxidant activity. Antioxidant activity of CSM and CSPI was

6
 M. Kumar et al.
7

LWT 140 (2021) 110692

Fig. 3. Response surface analysis of protein recovery (%) from cottonseed meal for the effect of alkali to sample ratio and extraction time (a), alkali to sample ratio and NaCl concentration (b), alkali to sample ratio and
Na2SO3concentration (c), extraction time and NaCl concentration (d), extraction time and Na2SO3concentration (e), NaCl concentration and Na2SO3concentration (f) on recovery of protein. The darker shades (red)
depict the maximum recovery of the protein at specific level of the input factors whereas lighter shade (blue) indicates the minimum protein recovery. Yellow and green shades depict intermediate protein recovery. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
M. Kumar et al.
Fig. 4. (A) Total protein content, (B) antioxidant activity, (C) free gossypol and (D) total gossypol concentration in cotton seed meal (CSM) and cotton seed protein
isolate (CSPI). Values are expressed as means of triplicate measurements: Means with different alphabets are significantly different at p < 0.05.
measured in terms of DPPH radical scavenging activity (RSA). DPPH
assay depends on decolourization of DPPH solution in the presence of
antioxidants. Ascorbic acid was used as reference standard for DPPH
assay. Results showed that RSA increased with increasing concentration
of sample from 10 to 50 μg. Antioxidant activity differed significantly (p
< 0.05) among CSM, CSPI and ascorbic acid at 50 μg concentration with
ascorbic acid exhibiting the highest (0.956%) and CSPI the lowest value
(0.043%) (Fig. 4). CSM showed higher RSA than CSPI which may be due
to the presence of secondary metabolites/phenolics in CSM, contrib­
uting to higher antioxidant activity than CSPI which contains only
protein (Adhikari, Poojary, & Vishnumurthy, 2015). The higher total
gossypol content of 20,900 ± 195.9 ppm in CSM compared to CSPI (270
± 21.0 ppm) also supports higher antioxidant of CSM compared to CSPI
as gossypol acts as strong antioxidant.
3.4.1.3. Scanning electron microscopy. Morphological characteristics of
the three samples (CSM and CSPI) were studied by SEM and images are
shown in Fig. 5. The surface of the CSM was more porous and amorphous
in structure. This may be due to the presence of polysaccharides such as
cellulose, hemicellulose, fiber (He et al., 2015, 2017). The extracted
protein sample from CSM showed large variation in shape and size and
Fig. 5. Scanning electron microscopy images of the cottonseed meal (CSM) and
cottonseed protein isolate (CSPI).
8
LWT 140 (2021) 110692
have a flatter and tighter microstructure with sharp angles (He et al.,
2017). The flatter or smoother surface of CSPI may be due to the pres­
ence of alkali (0.1 M KOH) which leads to the formation of gelatinous
lamellar structure (Zhang et al., 2018). The similar protein structure was
also reported by Saad, Gaiani, Mullet, Scher, and Cuq (2011) in wheat
protein displaying its suitability for application in foods. There were no
open pores observed on the CSPI microstructure. The minor difference in
the microstructure of proteins from other sources may be attributed to
the extraction procedure and concentration of alkali used (He et al.,
2017; Zhang et al., 2018).
3.4.1.4. Fourier transform infrared (FTIR) spectroscope determination.
FTIR is a powerful technique used to study the secondary structure and
major functional group of proteins. CSPI and CSM spectrum were
observed in the range of 500–4000 cm− 1 as shown in Fig. 6a. The peaks
at 1658.78, 1546.91, 1432.25 and 3234.46 cm− 1 were correspond to
amide I (C–– O, C–N stretching), II (N–H bending), III & V (C–N in pri­
mary amide) and amide A bands (free O–H and N–H groups in the
protein) of proteins respectively (Saad et al., 2011; Zhao, Liu, Zhang, &
Zhu, 2019). The sharp peak at 617.22 cm− 1 peaks was attributed to
N–C– – O in amides for CSPI, respectively. The absence of a sharp peak at
617.22 cm− 1 in CSM and indicated the purity of CSPI due to presence of
prominent peak at 617.22 cm− 1. A sharp peak at 2927.94 cm− 1 in CSM is
due to symmetrical and asymmetrical stretching of C–H groups. The
difference between CSPI and CSM spectrum revealed that alkaline
extraction of protein improved the spectrum of CSPI over defatted CSM.
Similar FTIR spectroscope has been reported by Liu, Wang, Wu, He, and
Wan (2018) for raw cottonseed meal and Chen et al. (2019) for cot­
tonseed protein. FTIR spectrum of CSPI is similar to the major spectral
features of soybean protein isolate (He et al., 2013; Ma et al., 2018).
3.4.1.5. Qualitative functional analysis of CSPI. The amino acid profile is
considered as the most crucial factor for determining the quality of any
protein. Hence, extracted CSPI was further analysed for its amino acid
profile. It was found that all the essential amino acids were present in the
extracted protein for which the standards have been used. The overall
profile of amino acid can be seen in supplementary information Fig. S2
and Fig. 6b. Optimized CSPI was found to contain 17 amino acids on the
basis of amino acid standards used. The CSPI was reported to have
M. Kumar et al.
Fig. 6. (A) Comparison of Fourier transform infrared spectroscopy profile of
CSM and CSPI (red line- CSM, green line- CSPI). CSPI and CSM spectrum were
observed in the range of 500–4000 cm− 1. (B) HPLC chromatogram of optimized
protein extract. Optimized CSPI was found to contain 17 amino acids on the
basis of amino acid standards used (relative content of amino acids has been
depicted in the supplementary information Fig. S2). Key to short forms: CSM-
cottonseed meal; CSPI- cottonseed protein isolate. (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web
version of this article.)
balanced ratio of identified amino acids as compared to commercial soy
protein and other plant-based protein (Gorissen et al., 2018). The results
coincide with previously reported amino acid composition of CSM by
Bertrand et al. (2005) and other research findings (Saad et al., 2011;
Bressani, Elias, & braham, 1966; Singhal & Mudgel, 1984; Alam et al.,
2018). Variation in results may be due to difference in extraction con­
dition and experimental material. This could be attributed to the dif­
ference in the sample material and extraction conditions used.
3.4.1.6. SDS-PAGE. Protein profile of CSM and CSPI was studied by
using SDS-PAGE. All samples were separated by 4–12% gradient poly­
acrylamide gel electrophoresis. There were 6 and 8 polypeptide bands
on SDS-PAGE of CSM (L2) and CSPI (L3) respectively as presented in
supplementary information Fig. S3 as arrow heads. In the case of CSM
and CSPI (L2 and L3, respectively), the polypeptide bands were ranged
from 55 kDa to 12.3 kDa. Similar results were reported by He et al.
(2018) and He (2016) in cottonseed protein. The reported bands may
have further polypeptide bands which looks unresolved in the
SDS-PAGE. The results reported by He et al. (2018) confirmed as many
as 18 proteins have appeared in one SDS-PAGE band after mass spec­
troscopy analysis of water and alkali-soluble portion of CSM protein.
Furthermore, the molecular mass distribution of CSM protein and isolate
was more concentrated in the range of 22–12 kDa, which is associated to
cottonseed storage globulin. The major and minor bands were also found
9
LWT 140 (2021) 110692
in the range of 49 kDa–59 kDa and 35–38 kDa respectively (King, 1980;
Liadakis et al., 1995; Ma et al., 2018). These proteins are important in
the physiology of cotton plant as these proteins play a key role in energy
metabolism, storage function, transcription, translation and embryo­
genesis (He et al., 2018). More recently, Singh and Kaur (2019) reported
the SDS-PAGE profile of cotton lines of two Gossypium species.
4. Conclusion
In the present study, we optimized alkali-assisted extraction (alkali:
sample ratio 33:1 [v w− 1], NaCl concentration 0.15 mol/L, 0.27%
Na2SO3 and time 147.5 min) has been proven to be an efficient method
for extraction of protein from the CSM. Alkali-assisted extraction not
only gives a high recovery (88.5%) but also resulted in ULGC (29.0 ppm)
with excellent structural features. Hence, eco-friendly and cost-effective
nature of the current method of extraction of protein from CSM. Further,
the resultant CSPI can be utilized as a feed supplement for monogastric
animals and food supplement for human beings as the gossypol content
is under the allowable limits of 600 ppm. The cytotoxicity and geno­
toxicity of the obtained CSPI on rat model need to be further evaluated.
CRediT authorship contribution statement
Manoj Kumar: Conceptualization, Methodology, Supervision,
Investigation, Writing - original draft, Project administration, Funding
acquisition, discussed the results and contributed to the final manu­
script. Jayashree Potkule: Investigation, Writing - original draft, dis­
cussed the results and contributed to the final manuscript. Sharmila
Patil: Writing - review & editing, Visualization, Formal analysis, Soft­
ware, discussed the results and contributed to the final manuscript.
Sujata Saxena: Conceptualization, Methodology, Supervision. P.G.
Patil: Project administration, and Funding acquisition, discussed the
results and contributed to the final manuscript. V. Mageshwaran:
Conceptualization, Methodology, Supervision, discussed the results and
contributed to the final manuscript. Sneh Punia: Writing - review &
editing, Visualization, Formal analysis, Software, discussed the results
and contributed to the final manuscript. Eldho Varghese: Writing -
review & editing, Visualization, Formal analysis, Software, discussed the
results and contributed to the final manuscript. Archana Mahapatra:
Writing - review & editing, Visualization, Formal analysis, Software,
discussed the results and contributed to the final manuscript. Nandita
Ashtaputre: Investigation, Writing - original draft, discussed the results
and contributed to the final manuscript. Charlene D.’ Souza: Investi­
gation, Writing - original draft, discussed the results and contributed to
the final manuscript. John F. Kennedy: Writing - review & editing,
Visualization, Formal analysis, Software.
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.
Acknowledgement
The laboratory facilities provided by ICAR-Central Institute for
Research on Cotton Technology, Mumbai and funding received from the
Science for Equity, Empowerment and Development Division Depart­
ment of Science and Technology, Government of India, New Delhi
(Grant No. SP/YO/476/2018) is highly acknowledged. The software
support (Design Expert 12.0) was provided by ICAR-CMFRI is also duly
acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
M. Kumar et al.
org/10.1016/j.lwt.2020.110692.
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