Journal of Cereal Science 117 (2024) 103889

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Journal of Cereal Science

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Inactivation of lipase and lipoxygenase in whole wheat flour using

atmospheric cold plasma and steam treatments: Kinetics, mechanism, and
impact on its compositional properties
Snehasis Chakraborty a, b, Manoj Kumar Pulivarthi a, Anu Suprabha Raj a,
Shivaprasad Doddabematti Prakash a, Hemanth Bommina a, Kaliramesh Siliveru a, *
a
Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA
b
Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India

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

Keywords: This study aims to utilize two separate techniques, viz., atmospheric cold plasma (voltage: 5–25 kV; time: 1–10
Lipolytic enzymes min) and steam treatments (at 100 ◦ C for 1–4 min), to inactivate lipase and lipoxygenase in whole wheat flour
Voltage sensitivity (WWF) and assess the impact on its compositional properties. The cold plasma exposure of WWF at 25 kV/6 min
nth order inactivation
resulted in 97.5% and 100% inactivation in lipase and lipoxygenase activity, respectively, whereas corre­
Secondary structure
sponding inactivation values after the steam treatment (100 ◦ C/4 min) were 90.6% and 99.7%. The inactivation
Reactive species
Optical emission spectra order (n) of lipase and lipoxygenase during cold plasma treatment was 1.2 and 1.0, respectively. In the optical
emission spectra of cold plasma-induced air, the band heads within 328–426 nm confirmed the transition be­
tween various reactive nitrogen species (second positive, first negative, second positive, and mono-positive
systems). The band-head for OH radicals was recorded at 314 nm. The oxygen molecular band at 739 nm and
singlet oxygen transition line at 778 nm were also observed. The cold plasma and steam treatments resulted in a
more random coil in lipase’s secondary structure. The cold plasma treatment at 25 kV/6 min didn’t impact the
particle size, density, color profile, and proximate composition of the flour.

1. Introduction Lipase activity in WWF primarily resides in the bran fraction of the
grain, leading to a release of non-esterified fatty acids that can diminish
Whole wheat flour (WWF) retains the natural proportions of the sensory quality and functional properties. Higher concentrations of
entire wheat grain - the bran, germ, and endosperm. It packs signifi­ these fatty acids can affect dough mixing, reducing the binding capacity
cantly higher amounts of essential nutrients like vitamins, minerals, and of gluten, and thereby compromising its gas-holding capacity and elas­
antioxidants compared to regular wheat flour due to its concentrated ticity (Rose et al., 2008). Conversely, enzymatic lipid oxidation is
content in the outer parts of the grain (Gómez et al., 2020). Notably, facilitated by lipoxygenase (EC 1.13.11.12) in WWF. Lipoxygenase tar­
WWF contains more enzymatic activity, lipids, and antioxidants than gets the methylene group between double bonds in polyunsaturated
wheat flour, impacting its end-use and storage properties. For instance, fatty acids, especially non-esterified ones, initiating a chain reaction
widely used in noodle-making, WWF can lead to rancidity in semi-dried resulting in hydroperoxides and the formation of volatile compounds
noodles due to its higher moisture content, which compromises its like epoxy-aldehydes, ketones, and lactones (Poudel and Rose, 2018).
acceptance (Guo et al., 2020). Lipids within WWF undergo breakdown This oxidation process can significantly impact the nutritional quality
processes known as hydrolytic rancidity, which can then progress to and consumer acceptance of WWF and its products by diminishing
oxidative rancidity, impacting the flour’s overall quality. Hydrolytic essential fatty acids and affecting overall product quality (Doblado-­
rancidity, triggered enzymatically by lipase (EC 3.1.1.3), breaks down Maldonado et al., 2012). A set of thermal treatments have been explored
triacylglycerols into non-esterified fatty acids, diglycerides, mono­ to inactivate lipolytic enzymes in WWF and wheat kernel, such as dry
glycerides, and eventually glycerol (Doblado-Maldonado et al., 2013). heating (Rose et al., 2008) and microwave heating of WWF (Sun et al.,

* Corresponding author. Department of Grain Science and Industry, Kansas State University, 209 Shellenberger Hall, 1301 Mid Campus Drive, Manhattan, KS
66506, USA.
E-mail address: [email protected] (K. Siliveru).

https://doi.org/10.1016/j.jcs.2024.103889
Received 22 December 2023; Received in revised form 22 February 2024; Accepted 7 March 2024
Available online 16 March 2024
0733-5210/© 2024 Elsevier Ltd. All rights reserved.
S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

2023), steaming (Poudel and Rose, 2018) and superheated steam between reactive oxygen and nitrogen species caused by cold plasma
treatment of wheat kernel (Guo et al., 2020; Jia et al., 2021). Expectedly, and these enzymes. Further, the physical attributes (particle size, color
dry heating was employed on WWF, but wet steam treatment was profile, and density) and other compositional properties (moisture
employed on the kernels, not on the WWF. For instance, Guo et al. content, water activity, protein, ash, and damaged starch content) were
(2020) found that subjecting wheat grain to superheated steam at 190 ◦ C compared for these two selected cold plasma and steam-treated enzy­
for 10 s resulted in lipase and lipoxygenase inactivation. Poudel and matically stable WWF.
Rose (2018) observed significant inactivation in lipase and lipoxygenase
activity when the wheat kernels were steamed for 90 s. De Almeida et al. 2. Materials and methods
(2014) also observed inactivation of lipase after steaming the wheat
kernel. These studies affirmed the potential of steam treatment of wheat 2.1. Whole wheat flour
kernels for lipolytic enzyme inactivation. However, heat treatment
negatively influences the functional properties of WWF. This triggered The organic hard red winter wheat (Triticum aestivum) supplied from
exploring the option of non-thermal processing for stabilizing lipolytic Eden Foods Clinton, Michigan, USA (lot #23621) was used. A batch of
enzymes in WWF. 1.5 kg of wheat was taken twice and passed through a roller mill
Cold plasma, a non-thermal technology harnessing energetic reactive (CHOPIN Technologies Labmill, Villeneuve-la-Garenne CEDEX, France).
species like free radicals, excited ions, atoms, and electrons within From the roller mill, the average break, sizing, and reduction flour
ionized gas, interacts with food’s physical, chemical, and biological el­ fractions were 24.0, 13.4, and 35.8% w/w, respectively. The bran, fine
ements (Doddabematti Prakash et al., 2023b). When a gas receives en­ bran, and shorts fractions were 5.0, 8.0, and 8.3% w/w, respectively;
ergy via heating or electromagnetic fields, it transitions into a plasma these fractions were passed through a rotor mill (Cross Beater Mill SK
state, exhibiting electrical conductivity and fluid-like characteristics 300, Retsch GmbH, Haan, Germany), leading to a 93.7% yield. The bran,
(Moutiq et al., 2020). The dynamic plasma species create an oxidative germ, and white flour, all derived from the same batch of wheat grains
environment, leading to surface modifications in the treated substance post-milling, were subsequently combined to create whole wheat flour
and facilitating microbial and enzyme inactivation in grain-based (WWF).
products (Jaddu et al., 2022). There are few studies that explored at­
mospheric cold plasma (ACP) treatments on wheat flour or WWF. For 2.2. Experimental design
instance, Chaple et al. (2020) explored the impact of dielectric barrier
discharge plasma treatment, one type of ACP, on the functional prop­ A full factorial design (2 factors and mixed levels) was used for the
erties of WWF. They revealed that the cold plasma processes (80 kV for cold plasma treatment of WWF. The root mean square (rms) voltage
5–30 min) enhanced the hydration and pasting viscosity of the WWF due (Vrms, kV) and treatment time (t, min) were the two independent vari­
to partial disorganization of the starch structure. Janić Hajnal et al. ables. The Vrms varied at 5 levels (5, 10, 15, 20, and 25 kV), whereas the
(2019) employed ACP treatment to reduce Alternaria toxins in wheat treatment time varied at 8 levels (1, 2, 3, 4, 5, 6, 8, and 10 min). The cold
flour. The most significant reduction in Alternaria toxins occurred with plasma experiments were conducted at all possible combinations of Vrms
cold plasma treatment at a 6 mm distance from the source, lasting 3 min and time. This led to 5 × 8 = 40 experimental runs as per the full-
at 2.1 kV root-mean-square voltage (Vrms). This resulted in reductions of factorial design. In a separate experiment, WWF was exposed to steam
60.6%, 73.8%, and 54.5% for alternariol, alternariol monomethyl ether, at 100 ◦ C and the exposure time was varied for 1, 2, 3, and 4 min. The
and tentoxin, respectively. Misra et al. (2015) evaluated the influence of activity of lipase and lipoxygenase in the WWF were the two responses
ACP treatment on the rheological and functional properties of dough measured after each cold plasma or steam treatment. The enzymatic
from wheat flour. They observed the viscosity of the dough increased stability in food products is often associated with 90% inactivation (10-
with voltage (60–70 kV) and exposure time (5–10 min) of ACP. The fold or 1 log cycle reduction in initial activity) of the target enzyme
secondary structure of gluten protein was altered by cold plasma activity in response to various processing conditions (Peng et al., 2017).
exposure. Bahrami et al. (2016) found that applying ACP at 20 kV for 2 Thus, the cold plasma and steam-treated WWF with <10% residual ac­
min had no effect on the concentration of total non-starch lipids, tivity in the most resistant lipolytic enzyme were taken further to
non-polar, and glycolipids in wheat flour. However, the treatment did analyze other quality attributes such as particle size, color profile,
decrease total free fatty acids and phospholipids in a dose-dependent density, moisture content, water activity, protein, ash, and damaged
manner. starch content.
The potential of cold plasma treatment in the inactivation of lipolytic
enzymes in various grain-based products is also explored. For instance, 2.3. Cold plasma treatment
Sutar et al. (2021) observed a 35% reduction in lipase enzyme activity
after subjecting wheat flour to 60 W-30 min cold plasma treatment. A batch-mode multipin-plane plasma reactor (Model: LCP-MP,
Tolouie et al. (2018) employed ACP to inactivate lipase and lip­ Ingenium Technologies, Odisha, India) assembly, which operates in
oxygenase in wheat germ. According to Wang et al. (2022), the exposure open-air, was used to treat WWF (Fig. 1). The system operates up to Vrms
of Mizhi millet to 25 kV for 12 min resulted in a 57% and 73% decrease of 30 kV (equivalent to a peak-to-peak voltage of 84 kV). The system
in lipoxygenase and lipase activity, respectively. Zhou et al. (2024) comprises a high-voltage step-up transformer, an electronic control
obtained 64.5% and 29.1% inactivation in lipase and lipoxygenase ac­ panel, and a cold plasma reactor, all manufactured by Ingenium Tech­
tivity, respectively, in lightly milled rice while employing argon as the nologies, Odisha, India. The power transformer (Model: LCP-LPS40;
input gas for cold plasma treatment. However, no study has explored the dimension 50 cm × 50 cm × 50 cm) features a stainless steel-304 cart
impact of cold plasma treatment on the inactivation of lipolytic enzymes and epoxy glass holder. Its output termination utilizes a wide-diameter
in WWF. Therefore, this study aims to study the effect of cold plasma ceramic bushing, standing at a height of 40 cm. The transformer in­
treatment on the inactivation kinetics of lipolytic enzymes (lipase and corporates a custom-designed, high break-down voltage, oil-cooled step-
lipoxygenase) in WWF. For comparison, the inactivation of lipolytic up transformer with exceptional dielectric strength. The cold plasma
enzymes in steam-treated WWF was also analyzed. The minimum in­ power supply has a digital metering system and operates on a standard
tensity required to achieve a minimum of 90% inactivation in lipase and single-phase input supply of 100–110 V, 60 Hz, and 15 A. Generating a
lipoxygenase activity in WWF was selected from both cold plasma and high voltage sinusoidal output of up to 40 kV (with a maximum current
steam treatments. The changes in the secondary structure of the resistant of 12 mA) and 1200 W power, this system is designed for efficient
enzyme in WWF after cold plasma and steam treatments were explored. performance. The accompanying control panel (Model: LCP-LPS;
Additionally, the secondary goal was to examine the connections dimension 35 cm × 30 cm × 25 cm) is constructed from powder-

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S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

Fig. 1. The schematic showing the pin-to-plate atmospheric cold plasma treatment assembly employed on whole wheat flour.

coated steel. Employing an intelligent, fast digital power control topol­ operated at 100 ◦ C steam. The pouches were treated inside the steam for
ogy, the control panel effectively prevents arcing during plasma treat­ the desired time (1–4 min). After the treatment, the pouches were
ment. It also integrates an in-built digital meter for monitoring current, removed, and the surface was blotted, followed by air-cooling. Once the
voltage, and power, providing a convenient reference for both input and pouches reached room temperature (20 ± 2 ◦ C), WWF was taken out for
output parameters during direct use. The control panel draws power further analysis.
from the input line (110 V at 60 Hz). The line power supply is switched
via a semiconductor switching element whose output is fed to the step-
up transformer to result in the high voltage required for ionization. The 2.5. Enzyme assay
control board also samples the voltage and current signals from the step-
up transformer. It applies a proprietary control algorithm for controlling The assessment of lipase and lipoxygenase activity followed the
the atmospheric plasma discharge in the load reactor. The cold plasma method outlined by Poudel et al. (2017). Initially, 1 g of WWF was
reactor (Model: LCP-MP) has a dimension of 40 cm × 25 cm × 20 cm. combined with 5 mL of deionized water, vortexed thoroughly, and
The assembly involves an acrylic support plate that securely holds a horizontally shaken for 30 min. Subsequently, centrifugation was per­
perforated stainless-steel plate and multiple pin emitters. Nylon bolts formed at 4500×g for 15 min to obtain the supernatant, constituting the
and washers are utilized for fastening these components. The acrylic crude lipase extract. The substrate solution was prepared by combining
support plate is positioned on two vertical supporting blocks, secured in 31.8 mg of p-nitrophenyl palmitate (Sigma-Aldrich), 34 mg of sodium
place by stainless steel Allen bolts. These vertical supporting blocks dodecyl sulfate, and 2 g of Triton X-100 in deionized water to a final
additionally serve as hosts for a ground stainless steel electrode (exposed volume of 200 mL. This solution underwent heat treatment at 65 ◦ C for
area of 25 cm × 20 cm), facilitated by a sliding mechanism. The high- 15 min to eliminate turbidity. For the assay, 0.5 mL of the crude enzyme
voltage cable from the step-up transformer is linked to a stainless-steel extract was mixed with 1.25 mL of Tris-Cl buffer (0.1 mol/L, pH 8.2) and
bolt in this arrangement. There were 88 pins (11 × 8 pins, each 27 1.25 mL of the substrate solution. The assay mixture was then incubated
mm long; grid spacing 20 mm × 20 mm) placed 5 cm above the plate at 37 ◦ C for 15 min, after which absorbance was measured at 400 nm. A
(discharge gap). The reactor is operated within the limits of the standard curve was constructed using p-nitrophenol in Tris-Cl buffer
discharge strength of the air trapped in the discharge volume, including (ranging from 0 to 0.1 mM). Lipase activity, assessed using p-nitrophenyl
inside the Petri plate. 20 g whole wheat flour was placed inside the palmitate as the substrate, was denoted as U/g. A single unit (U) was
polystyrene petri dishes (100 mm × 15 mm) with a lid and spread defined as the micromoles of p-nitrophenol liberated per hour.
gently. The Petri dish was placed at the center of the exposed area on the 1 g of flour was mixed with 5 mL of pH 6.5 phosphate buffer and
ground plate. The height of the sample inside the Petri dish was 2 mm. horizontally shaken. After centrifugation at 5000×g for 15 min at 4 ◦ C,
The vertical gap between the bottom edge of the pin and the top surface the resulting supernatant served as the crude enzyme extract. The sub­
of the flour inside the Petri dish was 4.7 cm. The gap between the sample strate solution was prepared by emulsifying 1.57 mL of linoleic acid
surface to the lid was 1.2 cm. During the plasma treatment, the sample (Sigma-Aldrich) and 1.57 mL of Tween 20 in 50 mL of distilled water,
inside the Petri dish vibrated and floated up in the empty area. However, then adjusting the volume to 2 L with pH 6.5 phosphate buffer. 100 μL of
the lid was closed to ensure no spillover of the sample. crude enzyme extract was mixed with 2.9 mL of the substrate solution at
In the control panel, the Vrms were set between 5 and 25 kV, whereas 30 ◦ C. After 2.5 min, 1 mL was transferred to 4 mL of 0.1 mol/L sodium
the treatment time was set within 1–10 min. The come-up times to reach hydroxide to stop the enzymatic reaction and enhance solution clarity
5, 10, 15, 20, and 25 kV were 17, 21, 26, 29, and 32 s. The voltage- by forming a Na-salt with linoleic acid. Lipoxygenese activity was
current-power profile for cold plasma treatment of WWF at 15 kV for measured in units (U/g), with one unit (U) representing the increase in
3 min has been presented in Fig. S1. The voltage was maintained at the absorbance at 234 nm per minute during the reaction.
set value ± 1 kV throughout the treatment, and the current ranged be­
tween 0.7 and 0.9 mA. This trend was true for all the experiments. The
2.6. Sensitivity analysis
temperature of the WWF during and after the treatment was measured
by a digital infrared thermometer temperature gun (Lasergrip 774,
Four experimental runs were executed at the extremities of the cold
Etekcity Corporation, Anaheim, California, USA). The maximum tem­
plasma treatment range, encompassing conditions at 5 kV/1 min, 5 kV/
perature rise in the sample during cold plasma treatment was 2.1 ◦ C,
5 min, 25 kV/1 min, and 25 kV/5 min. Sensitivity indices regarding the
whereas the initial temperature was 18.4 ± 1.1 ◦ C (Fig. S2).
enzyme’s inactivation, specifically based on voltage (SIV) and treatment
time (SIt), were computed following Eqs. (1) and (2), respectively.
2.4. Steam treatment ⃒ [∑
⃒1
∑ ]⃒
Y25kV Y5kV ⃒⃒
SIV = ⃒⃒ − ⃒ (1)
σ 2 2
20 g of WWF was packed inside a stomacher pouch and sealed while
removing the maximum air. The pouch was placed inside the autoclave

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S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

⃒ [∑ ∑ ]⃒
⃒1 Y5 min Y1 min ⃒⃒ While exploring the secondary structure of lipase inside the amide
SIt = ⃒⃒ (2)
band (1600-1700 cm− 1), the deconvolution of FTIR spectra was per­
− ⃒
σ 2 2
formed using Origin Pro 2023b software (Origin Lab Corporation,
In this context, Y represents the inactivation of either the lipase or lip­ Northampton, MA, USA). The ‘Peak Analyzer’ tool was used where 2nd
oxygenase enzyme, while σ stands for the standard deviation of Y values. derivative method was used for peak finding. The Savitzky-Golay
The enzyme displaying the lowest values for SIV and SIt was identified as smoothing method was employed for this purpose. The area under the
the target enzyme for stabilizing the entirety of the WWF. Gaussian fitted curve was recorded, and the corresponding peak wave­
number was identified. Depending on the peak wavenumber, the sec­
2.7. Kinetic modeling ondary structure of composition was assigned.

In investigating enzyme inactivation for each voltage of cold plasma 2.10. Optical emission spectroscopy
treatment, an initial evaluation involved testing a first-order (n = 1) or
log-linear kinetic equation. This first-order model, presented as Eq. (3), The setup for optical emission spectroscopy (OES) is depicted in
describes enzyme inactivation kinetics, where Ai represents initial Fig. 1. The procedure detailed in Misra et al. (2015) has been followed in
enzyme activity, A denotes remaining enzyme activity after t minutes of this study. A 5-mm collimating lens captured the light emitted from the
treatment at a specific voltage (Vrms), and k signifies the rate constant electrical discharge within the discharge space over the sample. This
(min− 1). lens directed the light into an optical fiber with a 600 μm core diameter.
[ ] The collimating lenses were positioned 6 cm from the plate’s edge. This
A transferred the light via the optical fiber to an Ocean Optics
ln = − kt when n = 1 (3)
Ai High-Resolution spectrometer (HR200191, Ocean Optics Inc., Florida,
Simultaneously, an nth order kinetic model (Eq. (4)) was examined, USA). Spectral recordings were taken with an integration time of 1 s,
relating residual activity (A/Ai) to the rate constant (k, Un− 1⋅min− 1), averaging 10 spectra to maximize the signal-to-noise ratio. The spec­
inactivation order (n), treatment time (t, min), and Ai. trum is shown between the wavelengths of 200 and 800 nm. Each
spectrum underwent correction for dark current and background noise
A [ ]1
via subtraction using Ocean View software (Ocean Optics, FL, USA), and
= 1 + A1−i n (n − 1)kt 1− n when n ∕
=1 (4)
Ai the resulting spectra were reported.
Determining a fixed value of n across the entire voltage-time range
was accomplished by minimizing the sum of square errors (SSE) be­ 2.11. Statistical analysis
tween calculated and predicted residual activity values using an algo­
rithm developed by Chakraborty et al. (2015). Upon establishing the All the experiments were conducted and analyzed in triplicates. The
inactivation order (n), Eq. (4) became a function of the rate constant, algorithm for finding out the inactivation order was addressed using
residual activity, and treatment time. Subsequently, rate constant values Microsoft Excel (Microsoft Office system, USA). Origin Pro 2023b soft­
(k, Un− 1⋅min− 1) were computed at each voltage (Vrms). The enzyme’s ware (Origin Lab Corporation, Northampton, MA, USA), was utilized for
sensitivity to the set RMS voltage (SV) was quantified by formulating a conducting nonlinear curve fitting to determine k in relation to RMS
new equation (Eq. 5). The slope of ln k plotted against voltage (Vrms, kV) voltage. Additionally, OriginPro 2023b software was also employed for
was utilized to calculate the voltage-sensitivity (SV, [kV]− 1) of the deconvolution of FTIR spectra, analysis of variance (ANOVA), and
enzyme (Eq. (5)). executing Tukey’s HSD test.
( )
lnk = ln kref − SV Vrms − Vrms,ref (5) 3. Results and discussion

where, kref represents k at a reference voltage (Vrms,ref).

3.1. Sensitivity of lipase and lipoxygenase towards cold plasma treatment

2.8. Physicochemical properties The sensitivity indices with respect to voltage (SIV) for lipase and
lipoxygenase were 1.570 and 1.576, respectively. On a similar note, the
The moisture, protein, and ash levels in the WWF were assessed sensitivity indices with respect to treatment time (SIt) for lipase and
following the AACC methods (AACC, 2010). Color analysis employed a lipoxygenase were 0.340 and 0.393, respectively (Table 1). The higher
MiniScan EZ 4000 Portable Spectrophotometer (HunterLab) to measure the sensitivity indices, the changes in the response by that factor is
L* (lightness), a* (red/green), and b* (blue/yellow) values. Particle size higher. Therefore, lipoxygenase in WWF is more sensitive toward inac­
analysis utilized an Air Jet Sieve E200 LS (Hosokawa Alpine) with sieve tivation by cold plasma treatment than lipase. In this sense, lipase
sizes ranging from 25 to 250 μm. The geometric mean particle size, bulk inactivation should be targeted in the WWF. A slightly higher sensitivity
density, and tapped density of the WWF were measured as per the
protocol described by Doddabematti Prakash et al. (2023a). The fatty
Table 1
acid value in the WWF was measured gravimetrically as per the protocol The sensitivity indices of lipase and lipoxygenase in whole wheat flour towards
described by Jia et al. (2021). inactivation by pin-to-plate atmospheric cold plasma treatment.
Treatment Condition Residual Enzyme Activity (%)
2.9. FTIR spectra of lipase
Voltage (kV) Time (min) Lipase Lipoxygenase
5 1 97.7 ± 3.6d 95.4 ± 4.3d
Fourier transform infrared (FTIR) spectroscopy was performed for 5 5 89.4 ± 2.2b 82.3 ± 4.7b
the 500 mg pure lipase from wheat (L3001, Sigma Aldrich) treated by 25 1 50.8 ± 2.0b 43.1 ± 4.8c
cold plasma (at 25 kV for 6 min) and steam treatments (100 ◦ C/4 min). 25 5 11.3 ± 2.6a 1.1 ± 0.9a
The procedures followed for these treatments have been discussed in Sensitivity index based on voltage (SIV) 1.570 1.576
Section 2.3 and 2.4. FTIR spectroscopy of the lipase sample was con­ Sensitivity index based on time (SIt) 0.340 0.393
ducted within 380–4000 cm− 1 using a PerkinElmer FTIR spectrometer The alphabets (a, b, & c) in the superscripts denote that the mean values are
(Spectrum 400, Universal ATR Sampling Accessories, PerkinElmer, statistically different across the various cold plasma treatment conditions at p <
Shelton, CT, USA). The spectrum data (transmittance, %) were identified 0.05.
and analyzed using PerkinElmer Spectrum Software with a scan of 32. The enzyme activity in the untreated sample was considered 100%.

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S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

of lipoxygenase than lipase in whole wheat kernel towards steam lipoxygenase activities are 90.2, 69.2, and 20.2%, respectively. As ex­
treatment was reported by Poudel and Rose (2018). An opposite trend pected, for a fixed Vrms, a higher enzyme inactivation was observed
has been reported during atmospheric plasma treatment (Tolouie et al., when the sample was exposed for a longer time. For example, at 15 kV of
2018), microwave and conventional heat treatments of wheat germ (Xu Vrms, the lipase inactivation was 21.2, 40.8, 52.9, 60.8, and 67.8% after
et al., 2016). The varying sensitivity of lipase and lipoxygenase could 2, 4, 6, 8, and 10 min of cold plasma treatment, respectively. Similarly,
stem from differences in their susceptibility to the specific conditions at 20 kV of Vrms, the lipoxygenase activity was 56.1, 31.4, 19.8, 10.6,
created by cold plasma treatment. Factors like enzyme structure, sta­ and 5.5% after 2, 4, 6, 8, and 10 min of cold plasma treatment,
bility, unique activation energies, and susceptibility to changes induced respectively (Fig. 2). For all the cold plasma treatment conditions, the
by cold plasma (such as electrical fields or reactive species generated) inactivation of lipoxygenase was higher than lipase in WWF. For
could influence their responses. instance, after 5 kV/3 min, 15 kV/5 min, 25 kV/1 min exposure of cold
plasma, there was 7.3, 46.9, 49.3% inactivation of lipase and 10.7, 59.6,
56.9% of lipoxygenase inactivation in WWF, respectively.
3.2. Impact of cold plasma treatment on lipolytic enzyme activity Tolouie et al. (2018) observed 50% inactivation in lipoxygenase
activity in wheat germ after 25 min of ACP treatment at 24 kV. They
The cold plasma exposure significantly affected the lipase and lip­ observed an opposite trend in the resistance between lipase and lip­
oxygenase activity in WWF. Within the domain of 5–25 kV/1–10 min of oxygenase activities in wheat germ. On the other hand, Xu et al. (2016)
cold plasma treatment, the lipase activity reduced to 2.5% when reported that lipase was more resistant to heat inactivation than lip­
exposed at 25 kV/6 min (Fig. 2). At that condition, lipoxygenase was oxygenase in wheat germ. Cold plasma generates key chemical reactive
completely inactivated in WWF. At the lowest intensity of cold plasma species, namely reactive oxygen species (ROS) and reactive nitrogen
such as 5 kV/1 min, lipase and lipoxygenase activity in WWF were species (RNS). They act by oxidizing sulfur-containing amino acids and
reduced by 2.3% and 4.6%, respectively. With increased Vrms, enzyme disrupting disulfide bonds, ultimately causing the inactivation of lipo­
inactivation increased for a fixed treatment time. For instance, after 2 lytic enzymes in WWF. The distinct biochemical structures, vulnerability
min of treatment, the residual lipase activity was 94.6, 78.8, and 31.2% of active sites, presence of sulfur-containing residues, and specific mo­
at 5, 15, and 25 kV of Vrms, respectively. The corresponding lecular targets, collectively contribute to the varying sensitivity of lipase
and lipoxygenase enzymes in WWF to cold plasma treatment (Misra
et al., 2016).

3.3. Impact of steam treatment on lipolytic enzyme activity

Steaming at 100 ◦ C significantly inactivated lipase and lipoxygenase

in WWF. After 1, 2, 3, and 4 min of steam treatment, the residual activity
of lipase was 55.4, 30.9, 17.2, and 9.4%, respectively. The extent of
lipoxygenase inactivation was 50.3, 75.3, 87.8, and 99.7% after 1, 2, 3,
and 4 min of steam treatment (Fig. S3). Lipase was more resistant than
lipoxygenase in WWF after steam treatment. A similar trend was ob­
tained by Xu et al. (2016) while treating wheat germ by microwave and
heat treatment. Poudel and Rose (2018) observed 81% and 63% inac­
tivation in lipase and lipoxygenase activities in the wheat kernel after
steaming in a boiling water bath for 90 s. Wang et al. (2020) observed
complete inactivation of lipase activity in buckwheat grains after a su­
perheated steam treatment for 5 min at 170 ◦ C. Steam treatment’s
ability to inactivate lipase and lipoxygenase enzymes in whole wheat
flour arises from a combination of heat-induced denaturation, disrup­
tion of active sites, moisture effects, and the resultant aggregation of
proteins, collectively rendering the enzymes non-functional. Combining
heat and moisture, like in steam treatment, can cause protein aggrega­
tion, where the unfolded or denatured enzymes clump together. This
aggregation further impedes the enzymes’ ability to function properly,
contributing to their inactivation (Guo et al., 2020).

3.4. Enzyme inactivation kinetics

The inactivation of lipase in WWF during cold plasma treatment was

described by nth-order inactivation kinetics. For log-linear inactivation
kinetics (n = 1.0), the corresponding sum of the square of errors (SSE) in
residual activity fractions was 0.327; whereas for n = 1.2, the SSE was
the minimum at 0.282 (Fig. S4). Within the domain of n = 0–2, the SSE
for lipase was a maximum of 0.851 for n = 2. The corresponding R2 for
the nth-order model fitting for lipase varied between 0.85 and 0.95. On
the other hand, the log-linear kinetics (n = 1.0) was the best-fit model
for lipoxygenase inactivation by cold plasma treatment. The corre­
sponding R2 while fitting first-order kinetics for lipoxygenase inactiva­
tion data was around 0.90–0.97. The SSE for lipoxygenase inactivation
Fig. 2. The changes in residual activity of lipase and lipoxygenase activity in at n = 1 and 1.2 were 0.352 and 0.402, respectively.
whole wheat flour during cold plasma treatment at various combinations of The inactivation rate constant for lipase was 0.027, 0.071, 0.129,
RMS voltage and treatment time. 0.272, and 0.647 Un− 1⋅min− 1 when the Vrms were 5, 10, 15, 20, and 25

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S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

kV, respectively (Table 2). In the case of lipoxygenase, the inactivation treated lipase also. However, there are two specific instances where
rate was slightly higher than lipase. For instance, at 5, 10, 15, 20, and 25 some additional peaks were observed for cold plasma and steam-treated
kV of Vrms, k value for lipoxygenase was 0.040, 0.088, 0.194, 0.292, and lipase samples. The intensity of the peak at 569 cm− 1 for untreated
0.854 min− 1, respectively. For the steam treatment, the lipase and lip­ lipase aligns with the C–O stretching of the acetyl group present on the
oxygenase inactivation rates were 0.554 and 0.692 min− 1, respectively. catalyst’s surface. Moreover, the cold plasma and steam-treated lipase
The higher the inactivation rate is, the more sensitive the enzyme is. showed additional peak at 656 and 655 cm− 1 respectively. This suggests
Thus, in line with the trend obtained in the sensitivity analysis, the ki­ the enzyme’s engagement in forming an enzyme-substrate complex. The
netic data also confirmed that the lipase was more resistant than lip­ cold plasma and steam-treated lipase showed additional peak at 3638
oxygenase in WWF during both cold plasma exposure and steam and 3622 cm− 1 respectively. These peaks indicate the presence of
treatment. hydrogen-bonded hydroxyl groups within the enzyme’s structure.
The changes in rate constant values for both lipolytic enzymes varied
systematically with the Vrms. A logarithmic relation between rate con­ 3.5.2. Secondary structure of lipase
stant and Vrms was prominent during cold plasma treatment of WWF. The secondary structure of lipase is comprised of α-helices, β-sheets,
The ln k vs Vrms for both lipase and lipoxygenase enzymes yielded a β-turns, and random loops or coils, which collectively determine it’s
straight line with an R2 of 0.98, as per Eq. (5). The corresponding activity and stability. In the FTIR spectra between 1600 and 1700 cm− 1,
sensitivity of the enzyme to voltage (SV) was 145.7 V for lipoxygenase the β-sheet, random coil, α-helix, and β-turn show the peak at
and 154.2 V for lipase (Fig. S5). This refers to the change in Vrms required 1628–1642, 1643–1650, 1650–1659, and 1660-1699 cm− 1, respec­
to induce a modification in k-value of the enzyme. Indirectly it repre­ tively. While exploring the proportion of secondary structure of lipase
sents how sensitive the enzyme is towards the cold plasma treatment. It enzyme obtained from the FTIR spectra of amide I region (1600–1700
can be imagined analogous to activation energy in classical thermal cm− 1), the untreated lipase had 41.3% β-sheet, 21.7% random coil,
kinetics. In the literature, a change in the rate of enzyme inactivation as 25.4% α-helix, and 11.6% β-turn (Table S1). There was a significant
a function of voltage has been reported. Segat et al. (2016) employed the impact of cold plasma and steam treatments on the secondary structure
Weibull model to describe the inactivation of alkaline phosphatase and of lipase enzyme. For instance, after the cold plasma treatment at 25 kV/
observed the positive influence of voltage on the inactivation rate. 6 min, the proportions for β-sheet, random coil, α-helix, and β-turn were
Pankaj et al. (2013) observed the sensitivity of rate constant to voltage 17.9, 54.4, 14.4, and 13.3%, respectively. On the other hand, steam
while inactivating tomato peroxidase and polyphenoloxidase by ACP treatment at 100 ◦ C for 4 min resulted in the proportion of 14.3%
treatment. The impact of Vrms in the cold plasma treatment of WWF is β-sheet, 58.3% random coil, 14.9% α-helix, and 12.5% β-turn in the
significant. Voltage determines the energy level within the plasma sys­ lipase enzyme. Surowsky et al. (2013) concluded that cold plasma
tem, which influences the production and concentration of ROS and treatment resulted in the loss of enzyme’s helical structure. Segat et al.
RNS. Higher Vrms can lead to increased production of these reactive (2016) observed that the enzyme’s inactivation was linked to the
species, which, in turn, can more effectively inactivate enzymes present degradation or reduction of its α-helical and β-sheet secondary struc­
in the WWF. tures. Baltacıoğlu et al. (2015) corroborated that the thermal treatment
of mushroom polyphenol oxidase resulted in aggregated β-sheet and
3.5. Mechanism of lipase inactivation random coil in its secondary structure after thermal treatment.
The impact of cold plasma on the secondary structure of lipase pri­
3.5.1. FTIR spectra of lipase marily involves oxidative stress caused by ROS and RNS generated
The Fourier-transform infrared (FTIR) spectrum of untreated lipase during the treatment. Oxidative stress can lead to various modifications
unveils three prominent vibrations in the peptide group range of 1800 to in the enzyme, such as oxidation of amino acid side chains, cross-linking
1200 cm− 1 (Fig. S6). Specifically, the functional group segment span­ of protein molecules, and changes in the hydrogen bonding within the
ning from 1600 to 1800 cm− 1 serves as a vital identifier for lipase. protein structure. These modifications can potentially disrupt the sec­
Within this range, the peaks stem from vibrations in ester and carboxylic ondary structure of lipase, affecting its folding pattern, stability, and
groups. Distinctly, the precise peak at 3276 cm− 1 reflects the asym­ ultimately, its activity. Moreover, when lipase is subjected to steam
metric stretching of carboxylic acid and alkane groups. Whereas the treatment, the heat and moisture can induce alterations in its secondary
peaks at 2159 and 2026 cm− 1 correspond to the symmetric stretching of structure. Elevated temperatures can disrupt the weak interactions (such
these groups. These are in line with the FTIR spectra obtained for lipase as hydrogen bonds and hydrophobic interactions) that maintain the
from various sources such as wheat extract (Pradima et al., 2019) and secondary structure of lipase. This disruption can lead to changes in the
Candida rugosa (Natalello et al., 2005). folding pattern of lipase, affecting its stability and activity.
Most of the peaks obtained in the FTIR spectra of untreated lipase
mimicked for cold plasma (25 kV/6 min) and steam (100 ◦ C/4 min) 3.5.3. Optical emission spectroscopy (OES)
The emission spectrum spanning 280–800 nm confirmed the cold
plasma setup as a source for reactive nitrogen species (RNS) and reactive
Table 2
oxygen species (ROS). The spectra unveiled robust emissions between
The inactivation rate of lipase and lipoxygenase in whole wheat flour during
atmospheric cold plasma and steam treatments. 328 and 426 nm, originating from transitions within nitrogen’s second
positive system (N2(C–B)) and first negative system (N2+ (B-X)) (Fig. 3).
Voltage (Vrms, kV) Inactivation rate (k × 10− 2, min− 1
± 95% CI)
Distinct band heads of the N2(C3Πu → B3Πg) second positive system were
Lipase Lipoxygenase observed at 335, 352, 356, 374, and 398 nm, while the spectral emission
∑ 2∑+
n = 1.2 n = 1.0 of nitrogen mono-positive ion N2(B2 + u → X g ) appeared at 378 nm

5 2.7 ± 0.4 aB
4.0 ± 0.6aC
and 426 nm with relatively lower intensities. The detection of the hy­
10 7.1 ± 1.1bB 8.8 ± 1.1bC droxyl molecular band, A2(Σ+ → X2Π), was recorded at very low in­
15 12.9 ± 1.1cB 19.4 ± 1.4cC tensity, marked by a band-head at 314 nm. Additionally, the oxygen
20 27.2 ± 0.9dB 29.2 ± 1.0dB molecular band at 739 nm, linked to O (2s22p2(1D)4p → 2s22p2(1D)3d)
25 64.7 ± 1.5eB 85.4 ± 1.2eC
vibrations in a magnetic dipole transition, and the singlet oxygen tran­
Steam (100 ◦ C) 55.4 ± 0.8B 69.2 ± 1.0C sition line at 778 nm arising from O (2s22p33p5P → 2s22p33s5S) vibra­
CI, confidence interval; n, inactivation order. The alphabets in small letters (a, b, tions, were observed. A minor peak at 314 nm was identified as the OH
& c) and capital letters (B, & C) in the superscripts denote that the mean values radical transition. These findings aligned with previous studies,
are statistically different across the column and rows, respectively, at p < 0.05. corroborating spectral similarities reported by different researchers

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S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

Fig. 3. Optical emission spectra obtained over the whole wheat flour during cold plasma (25 kV/6 min) treatment. ROS, reactive oxygen species; RNS, reactive
nitrogen species.

(Misra et al., 2015; Moutiq et al., 2020). various mechanisms by which plasma-generated reactive species induce
Air contains 106 electrons per m3; therefore, when high voltage or an structural modifications and enzyme activity loss.
electromagnetic field is applied to the air, there will be a sudden flow of
charged species. The plasma system had 11 × 8 = 88 pins spaced in a 20 3.6. Impact of cold plasma and steam treatments on other attributes
mm gap. These produce a non-uniform electric field within the captured
air in between those pins. Electrons within the air gain energy from the The WWF had the geometric mean diameter (dgw) of 88.7 μm,
applied electric field and undergo collisions with other gas molecules. whereas the d50 of 92.58 μm (Table 3). The corresponding d90 of the
Electrons can collide with molecular nitrogen (N2) or oxygen (O2), WWF was 185.85 μm. The particle size of the WWF after cold plasma
causing them to become ionized or excited. The drifting of charged treatment did not change from the untreated WWF. However, the steam-
particles in the direction of the electric field resulted in a cascade of treated (100 ◦ C/4 min) flour showed a slightly higher diameter like dgw
reactions. These reactions result in the formation of various species such of 97.7 μm, d50 of 101.99 μm, and d90 of 200.49 μm. Hong et al. (2023)
as nitrogen ions (N+), oxygen ions (O+), excited nitrogen molecules found that dry heating of wheat flour led to increased particle size. It
(N2˙), excited oxygen molecules (O2˙), and other radicals or reactive might be due to the clustering of starch granules and protein aggregation
species. Air contains 78% of N2, which is easy to ionize and there was a induced by steaming (Hu et al., 2017). The color profile of untreated
blue discharge due to the electron shift. The vivid spectral signatures WWF had a lightness (L*) of 80.07, greenness (a*) of 3.39, and yel­
pointed to dynamic collisions between electrons and molecular nitrogen lowness (b*) of 13.14. Upon cold plasma treatment at 25 kV/6 min, the
in the surrounding air. WWF did not change the color profile. The cold plasma and steam
ROS and RNS can inactivate enzymes through oxidative modifica­ treatments did not influence the color profile, as it appears from Fig. S7.
tion, free radical attack, disulfide bond disruption, and protein frag­ The instrumental color profile of steam-treated WWF was lightness (L*)
mentation. ROS, like superoxide (O2•− ), hydroxyl radicals (•OH), and of 76.96, greenness (a*) of 3.59, and yellowness (b*) of 13.56. A slight
hydrogen peroxide (H2O2), can oxidize specific amino acid residues change in color profile in steam-treated samples might be due to little
within the enzyme’s active site. The radicals like peroxynitrite browning after treatment. This is in line with the observation made by
(ONOO− ) and nitrogen dioxide (NO2•), can directly interact with the Guo et al. (2020) while treating WWF with superheated steam. The
enzyme, leading to modifications or damage to essential functional untreated, cold plasma, and steam-treated WWF showed fatty acid
groups or amino acids within the enzyme’s structure. ROS can break values of 114.5 ± 6.5, 69.5 ± 6.1, and 76.5 ± 7.2 mg NaOH/100 g,
down disulfide bonds within enzymes, which are crucial for their respectively. The cold plasma and steam-treated sample had a minimum
structural stability (Misra et al., 2016). The excited atomic oxygen and (<10%) lipase and lipoxygenase activity which was reflected in the fatty
nitride oxide in a plasma system are primary factors causing enzyme acid values. Jia et al. (2021) observed a significant decrease in fatty acid
activity loss. The oxygen atoms in plasma could extract hydrogen from value after superheated steam treatment. The untreated WWF had a bulk
the protein’s backbone, initiating radical sites. These sites triggered density of 258.2 kg/m3 and tapped density of 50.8 kg/m3. The density
processes leading to polymer chain cleavage and volatile compound values of the WWF did not change after cold plasma and steam treat­
formation upon reacting with oxygen atoms or molecules (Kylián et al., ments. Jaddu et al. (2022) observed no difference in bulk densities in
2008). The hydroxyl radicals (OH•), superoxide anion radicals (O2− •), cold plasma-treated little millet flour. The steam-treated WWF had a
hydroperoxy radicals (HOO•), and nitric oxide (NO•) generated from slightly higher water activity (0.44) than the untreated (0.43) and cold
plasma sources can cause chemical alterations in amino acid side chains plasma-treated (0.43) flour. The protein, ash, and damaged starch
like cysteine, phenylalanine, tyrosine, and tryptophan, resulting in content of the WWF did not change (p > 0.05) after cold plasma and
enzyme activity loss. The oxidation by oxygen-containing plasma’s steam treatments. However, the steam-treated flour showed a moisture
active species (leading to the decomposition of C–H, C–N, and N–H of 9.8%, whereas the untreated and cold plasma-treated samples had a
bonds into CO2, NO2, and H2O) contributed to the loss of β-structure in moisture content of 10.9% and 10.7%, respectively. Misra et al. (2015)
enzymes (Hayashi and Yagyu, 2008). These collectively emphasize the did not see any change in moisture content in cold plasma-treated wheat

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S. Chakraborty et al. Journal of Cereal Science 117 (2024) 103889

Table 3 analysis, Writing – review & editing. Shivaprasad Doddabematti

The various quality attributes of whole wheat flour after cold plasma and steam Prakash: Formal analysis, Writing – review & editing. Hemanth
treatments. Bommina: Formal analysis, Resources. Kaliramesh Siliveru: Supervi­
Attributes Whole wheat flour, after various treatments sion, Writing – review & editing.
Cold plasma treatment Steam treatment Untreated
(25 kV/6 min) (100 ◦ C/4 min) Declaration of competing interest
Particle diameter (di, μm)
dgw, (μm) 89.41 ± 0.86b 97.70 ± 1.55a 88.70 ± The authors declare that there are no known conflicts of interest.
1.34b
d10 (μm) 40.42 ± 0.94b 50.38 ± 3.58a 41.97 ± Data availability
1.71b
d50 (μm) 93.72 ± 0.64b 101.99 ± 0.69a 92.58 ±
8.24b
Data will be made available on request.
a a
d90 (μm) 189.10 ± 2.10 200.49 ± 14.05 185.85 ±
0.73a Acknowledgements
Color profile
a b
Lightness (L*) 79.69 ± 0.28 76.96 ± 0.50 80.07 ±
This paper is contribution number 24-147-J from the Kansas State
0.68a
Redness (a*) 3.42 ± 0.09ba 3.59 ± 0.03a 3.39 ± University Agricultural Experiment Station.
0.07b
Yellowness (b*) 13.04 ± 0.51b 13.56 ± 0.07a 13.14 ± Appendix A. Supplementary data
0.12b
Proximate and Other Parameters
Bulk density (kg/ 262.50 ± 3.25a 264.50 ± 1.14a 258.23 ±
Supplementary data to this article can be found online at https://doi.
m3) 3.33a org/10.1016/j.jcs.2024.103889.
Tapped density 49.68 ± 1.49a 51.27 ± 0.16a 50.89 ±
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