\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Atmospheric Cold Plasma Treatment of Soybean Oil with Hydrogen Gas}



\author[1]{Ximena Yepez}%
\author[1]{Haci Baykara}%
\author[2]{Lei Xu}%
\author[3]{Kevin Keener}%
\affil[1]{Escuela Superior Politecnica del Litoral}%
\affil[2]{Purdue University}%
\affil[3]{University of Guelph}%


\vspace{-1em}



  
  \date{May 10, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
High voltage atmospheric cold plasma (HVACP) treatment generates
reactive gas species that induce inter and intramolecular reactions in
soybean oil. The goal of this study is to analyze the effect of HVACP
treatment on the chemical structure of soybean oil in a hydrogen gas
environment at atmospheric pressure. HVACP was used to treat soybean oil
(15g), for up to 6h by triplicate. Plasma generated reactive gas species
interact with the sample producing three distinct fractions identified
as a liquid, gel, and solid. Fatty acid profile, FTIR, 1H-NMR/13C-NMR,
GPC, thermal properties and peroxide value, were used to characterize
the chemical structure. Results indicated a lower content of
polyunsaturated fatty acids, increased content of saturated fatty acids,
and the presence of isomers. An insoluble portion was observed in the
solid fraction and increase with treatment time up to 42\% in the 6h
treated samples. Plasma species may cause two main reactions:
polymerization and hydrogenation.%
\end{abstract}%



\sloppy


\textbf{ABSTRACT}

High voltage atmospheric cold plasma (HVACP) treatment generates
reactive gas species that induce inter and intramolecular reactions in
soybean oil. The goal of this study is to analyze the effect of HVACP
treatment on the chemical structure of soybean oil in a hydrogen gas
environment at atmospheric pressure. HVACP was used to treat soybean oil
(15g), for up to 6h by triplicate. Plasma generated reactive gas species
interact with the sample producing three distinct fractions identified
as a liquid, gel, and solid. Fatty acid profile,
FTIR,\textsuperscript{1}H-NMR/\textsuperscript{13}C-NMR, GPC, thermal
properties and peroxide value, were used to characterize the chemical
structure. Results indicated a lower content of polyunsaturated fatty
acids, increased content of saturated fatty acids, and the presence of
isomers. An insoluble portion was observed in the solid fraction and
increase with treatment time up to 42\% in the 6h treated samples.
Plasma species may cause two main reactions: polymerization and
hydrogenation.

Keywords: cold plasma, soybean oil, hydrogenation, polymerization, oil
modification

\textbf{INTRODUCTION}

As a food ingredient, soybean oil is used mainly as cooking oil or salad
dressing. The high content of unsaturated fatty acids makes liquid
soybean oil susceptible to oxidation and limits its range of
applications. The partial hydrogenation of soybean oil reduces the
polyunsaturated fatty acid content leading to a semi-solid fat that is
less susceptible to oxidation. However, this process isomerizes the
monounsaturated fatty acid, generating 25-40\% of trans fatty acids
(Gunstone, Harwood, \& Harwood, 2007). The consumption of trans fatty
acids is linked to an increased risk of cardiovascular diseases, and
nowadays, partially hydrogenated oils (PHO) are not allowed to be used
as food ingredient (FDA, 2015).

Vegetable oils are also an essential source for the production of
lubricants and polymers because of their availability and low cost.
Diverse technologies have been studied to modify vegetable oils to
obtain epoxides, polyurethanes, polyamides, polyols, and many other
structures with valuable physical and chemical properties (Miao, Wang,
Su, \& Zhang, 2014). The polymerization reactions occur mainly in the
carboxylic end of fatty acids and the double bonds of triglycerides.

Additionally, cold plasma has been studied as a processing aid to
functionalize organic compounds (Pankaj et al., 2017). Collisions of
electrons with molecular or atomic gases generate a series of reactions
that lead to the formation of ions, radicals, and other excited plasma
species that are highly reactive. This technology has been used to add
chemical groups (deposition) to organic structures, and it has been
studied as a method to modify edible films, polymers, or graphene
(Ostrikov, Neyts, \& Meyyappan, 2013). Nitrogen-doped graphene has been
synthesized using ammonia or nitrogen gas (Wang, Maiyalagan, \& Wang,
2012). Hydrogen plasma species can saturate a monolayer of carbon atoms
tightly packed, by exposure to a low-pressure cold plasma (Burgess et
al., 2011). Plasma hydrogenation of graphene converts a highly
conductive material into an insulator, by the deposition of hydrogen
atoms on its structure. These are examples of the versatility of this
technology to form new products by adding specific atoms or molecules.
The reaction products can be controlled by gas composition, input
energy, frequency, or treatment time.

High voltage atmospheric cold plasma (HVACP) is a technology that has
been studied to decontaminate food products and to modify food chemical
structures. This technology is designed to treat a sample contained
within a fixed gas volume under a dielectric barrier discharge high
electric field (Misra et al., 2019). The soybean oil treatment
temperature is maintained lower than 50\selectlanguage{ngerman}°C, which is considered `cold' in
comparison with thermal plasma that can reach temperatures of
2000-3000°C. The treatment of soybean oil using cold plasma is a
potential processing technology to reduce the formation of trans fatty
acids (Yepez \& Keener, 2016).

The goal of this study is to analyze the effect of HVACP treatment on
the chemical structure of soybean oil in a hydrogen gas environment at
atmospheric pressure. Fatty acid composition is measured to determine
changes in the unsaturation degree. FTIR, \textsuperscript{1}H-NMR,
and\textsuperscript{13}C-NMR and GPC techniques were used to acquire
information related to structural modifications. Thermal properties of
all the samples were also investigated.

\textbf{MATERIALS AND METHODS}

\textbf{2.1. Setup and experimental design}

HVACP system employs a transformer that converts an energy input of 120
VAC/60 Hz, into an energy output of 0-130 kV. This device has a
dielectric barrier discharge configuration, as described elsewhere
(Yepez \& Keener, 2016). A plasma state is formed between two aluminum
electrodes. Briefly, electrodes were positioned at the top and bottom of
the plasma chamber, allowing the sample to receive a direct exposure of
the electric field (Fig.1a, 1b). Dielectric layers (4mm thickness,
polypropylene) were added to maintain a uniform glow, free from arcing.
The box (polypropylene, 175 x 275 x 44 mm) used as a plasma chamber was
flushed with hydrogen gas (common grade, 99.8\% purity) for 5 min with a
flow rate of 1 L/min to allow a uniform composition. Then, sealed with a
high barrier film (Cryovac®, Sealed Air Corporation, NJ). The
experiments were carried out inside a hood to extract and neutralize the
reactive species generated through the HVACP treatment. A nitrogen gas
blanket was adapted to cover the electrodes entirely and diminish the
risk of flammability of hydrogen gas. All experiments were conducted at
80 kV.

Soybean oil was procured from a local store, and samples (15 g) were
treated in a glass petri dish for 0, 2, 4, and 6 hours by triplicate.
Treated samples were collected from 3 points of the plasma chamber: 1)
Liquid, inside the petri dish; 2) Gel, outside the petri dish; and, 3)
Solid, from the top of the plasma chamber (Fig. 1c). Samples were
collected after treatment and maintained at -18°C for further analysis.
A partially hydrogenated soybean oil was used as a reference for the
comparison of the treated and untreated samples.

Fig. 1 -- about here

\textbf{2.2. Fatty Acid Composition}

Soybean oil was derivatized to fatty acid methyl esters (FAME) as
described by Kiefer (Kiefer, 1997), then analyzed with GC according to
AOAC Official Method 996.06 (AOAC, 2005). A gas chromatograph (GC-2010,
Shimadzu) was used with a polar ionic liquid capillary column SLB-IL60
30 m x 0.25 mm x 0.20 \selectlanguage{greek}μ\selectlanguage{english}m (Sigma-Aldrich, St. Louis, MO), and a flame
ionization detector (FID) at 280\selectlanguage{ngerman}°C. Helium was used as the carrier gas
with a flow rate of 1 mL/min. The column temperature was programmed from
150°C to 280°C at a heating rate of 5°C/min. A volume of 1\selectlanguage{english}?L was
injected, with a split mode of 50:1. Supelco 37-component FAME mix
10mg/mL (Sigma-Aldrich, St. Louis, MO, USA) was used as an external
fatty acid standard to identify the components. Dodecanoic acid
(Sigma-Aldrich, St. Louis, MO) was added as an internal standard.
SLB-IL60 column allows separating oleic acid (C18:1) with the double
bond in 9c or 11c position in the hydrocarbon chain, being 11c a small
fraction of monounsaturated fatty acids (MUFA).

The insoluble fraction in hexane was quantified by the amount of sample
used for fatty acid composition analysis. During derivatization of the
triglycerides into fatty acid methyl esters, a fraction of the treated
sample did not dissolve completely. The filtered (0.2
\(\mu\)m) samples were injected into the GC, and the
insoluble part was quantified as reported elsewhere (AOAC, 2005).

\textbf{2.3. Fourier Transform Infrared Spectroscopy (FTIR)}

A Nicolet 470 spectrophotometer (Thermo Fisher Scientific, MA) equipped
with a Mercury Cadmium Telluride (MCT) detector was used to collect
FT-IR spectra with a resolution of 4cm\textsuperscript{-1}, and 128
scans. Samples were deposited directly in the Attenuated Total
Reflectance (ATR) crystal covering the surface with a thin film.
Duplicate absorbance mode spectra were recorded from
4000-800cm\textsuperscript{-1} and processed with
Omnic\textsuperscript{TM} software.

\textbf{2.4. Gel Permeation Chromatography (GPC)}

Size exclusion chromatography data was collected on a Hewlett-Packard
1260 infinity series instrument equipped with three PLgel 10
\(\mu\)m mixed-B columns, and a refractive index detector.
Polystyrene standards of molecular weight ranging from 1-200 kg/mol were
utilized to calibrate the instrument. The mobile phase was
tetrahydrofuran with a flow rate of 1mL/min. Vegetable oil samples were
diluted in tetrahydrofuran to a concentration of 1mg/mL, then filtered
(0.45\(\mu\)m) and analyzed.

\textbf{2.5. Nuclear Magnetic Resonance Spectroscopy (NMR)}

Analyses were performed on a Bruker Avance 500 MHz spectrometer. Spectra
were acquired at 300K, observing \textsuperscript{1}H at 500.128 MHz
and\textsuperscript{13}C at 125.75 MHz. Data were processed with Topspin
software package version 1.3. The samples were dissolved in
CDCl\textsubscript{3}. Spectra were acquired using 16 scans, 32 K data
points, 90 pulse angles (10.5 \(\mu\)s), relaxation delay 1
s, acquisition time 2.47 s, and spectral width 13.2 ppm (6613.7 Hz).
Spectra were accurately phased, and their baseline adjusted accordingly.
Tetramethyl silane (TMS) was used as internal standard, and chemical
shifts were reported in ppm.

\textbf{2.6. Thermal Analysis}

Thermal stability was determined by using a TGA 55 (TA instruments, New
Castle, DE). Samples (12 +- 2 mg) were heated from room temperature up to
600degC, at a rate of 10 degC/min under nitrogen gas. Differential scanning
calorimetry (DSC) experiments were performed on a Discovery Series (TA
instruments, New Castle, DE), calibrated with indium and sapphire.
Samples 10 +- 2 mg were weighed in hermetic pans, and an empty pan was
used as a reference. Melting point (T\textsubscript{m}) was measured
according to AOCS official method Cj 1-94 (Firestone, 2009). The
enthalpies (\selectlanguage{greek}Δ\selectlanguage{english}H\textsubscript{m}) of the melting point of each sample
were also reported.

\textbf{2.7. Peroxide Value}

Peroxide value (peroxide milliequivalent/ kg of oil) was tested
according to the International Dairy Federation standard method 74A
(Chew \& Nyam, 2016). A sample of oil (20 mg) was dissolved with 3:2
(v/v) dichloromethane/methanol solution. Peroxides react with iron(II)
chloride and ammonium thiocyanate, followed by absorbance measurements
at 560 nm, as explained by Chew \emph{et al.} (Chew \& Nyam, 2016).

\textbf{2.8. Fat extraction}

Separation of insoluble material from treated samples of soybean oil was
applied using about 220 mL of hexane in a Soxhlet apparatus. The sample
(100-300 mg) was introduced in cellulose thimbles, and the extraction
was continued for five hours. The insoluble material was dried at 70\selectlanguage{ngerman}ºC.

\textbf{2.9. Statistical Analysis}

Data analysis was performed using SAS (version 9.3) for Windows (SAS
Institute Inc, 2008). Analysis of variances was conducted using the GLM
procedure. A Tukey test was used to determine significance differences
at 95\% level of confidence (p\textless{}0.05).

\textbf{RESULTS AND DISCUSSION}

Soybean oil samples were exposed to the direct cold plasma between the
electrodes. The intense treatment drove out two of the sample outside
the glass petri dish. Therefore, three fractions were analyzed from each
treated sample (for 2h, 4h, and 6h): liquid, gel, and solid (Fig. 1c).
Liquid fraction (94.5 \%) was collected from the petri dish and had less
contact with reactive plasma species. Gel fraction (2.5 \%) was
collected from the bottom of the box, and it escaped from the petri dish
due to the intense treatment. Solid fraction (3 \%) was collected from
the top of the box, and it was the sample with higher exposure to plasma
treatment.

\textbf{3.1 Fatty acid composition}

An increase in saturated and a decrease in unsaturated fatty acids were
observed in all treated samples. Solid fractions had higher exposure to
reactive plasma species (especially H radicals and H\textsuperscript{+})
(El-Zeer et al., 2013) and showed more considerable changes in their
composition. A significant increase in stearic acid (C18:0) was observed
in solid fractions from 4.5 to 10.4\%. The total saturated fatty acids
increased from 16.7\% to 22.5\%. Linolenic acid decreased from 8.4
±0.4\% to 1.5±0.5\%, and linoleic acid from 53.4±1.8\% to 17.7±2.6\%.
Results from the fatty acid composition are shown in Fig. A1 in the
appendix data section. According to the results, it can be said that the
decrease in unsaturated fatty acids cannot be directly related to the
increase of saturated fatty acids.

Oleic acid (C18:1-9c) decreased significantly over treatment time from
20.6\% to 13.8\%. Two new peaks were detected in the chromatogram of
HVACP treated samples, corresponding to elaidic acid (C18:1-9t) and
linoelaidic acid (C18:2-9t,12t), that reached 0.8\% and 2.6\% as the
highest values for 6h treated samples, respectively. Traditional partial
hydrogenation of soybean oil may form 25-40\% of trans fatty acid
(Jovanovic, 1998), mostly as elaidic acid (C18:1-9t). Trans isomers are
formed through incomplete hydrogenation, as a product of the
absorption-desorption of unsaturated fatty acids from the catalyst
surface, and it is isomerized to its lower energy positional structure.
The fatty acid composition of a traditional PHO with an iodine value of
84 is shown in Fig. A1 (appendix data section), with a total trans fatty
acid content of 31.8\%. Even though HVACP treatment using hydrogen gas
can reduce IV to the level of a traditional PHO, it has a different
fatty acid composition and a remarkable lower content of trans fatty
acids in the range of 2.0-2.7\%. Total trans fatty acid content is much
lower for HVACP treated samples, mostly formed by linoelaidic acid
(C18:2-9t,12t). In contrast with PHO that mainly has elaidic acid
(C18:1-9t). The mechanism of the formation of trans isomers in HVACP
treatment is not well understood because there is not a catalyst surface
where the double bonds are opened, neither absorption-desorption.

Yepez and Keener reported the absence of trans fatty acids in cold
plasma treatment of soybean oil that reached an iodine value of 92,
using a modified atmosphere of nitrogen-hydrogen gas for a 12h treatment
time (Yepez \& Keener, 2016). In this study, a modified atmosphere of
pure hydrogen gas was used as an approach to accelerate the reactions.
The gel fraction treated for 4h achieved a similar iodine value as the
12h sample treated with nitrogen-hydrogen gas; however, it showed a 2\%
content of trans fatty acids. Therefore, soybean oil treated with
hydrogen gas may increase the isomerization of cis- into trans- double
bonds due to the higher reactivity of hydrogen species and energy
applied by cold plasma treatment (Dijkstra, 2006). It is known that the
bond dissociation energy of a nitrogen molecule requires more energy
than a hydrogen molecule does.

In summary, the fatty acid composition for the 6h solid fraction formed
after HVACP treated sample showed a saturated fatty acid content of
38.8\%, the monounsaturated fatty acids (MUFA) 24.7\%, and the
polyunsaturated fatty acid (PUFA) with a 36.5\%. Including a total trans
fatty acid content of 4.8\%. The iodine value of a 6h solid sample is
86.6, calculated from the fatty acid composition. In contrast, the fatty
acid composition of a PHO with a similar iodine value has a saturated
fatty acid content of 22.5\%, 57.3\% of MUFA, 20.4\% of PUFA, including
31.8\% of trans fatty acids. HVACP treatment produced a fatty acid
composition of soybean oil with a different profile compared to a PHO.

A portion of the samples treated with HVACP under hydrogen gas
atmosphere was insoluble in hexane. This insoluble portion was filtered
before the gas chromatography analysis; therefore, the reported fatty
acid composition corresponds to the soluble portion of the samples. The
amount of insoluble portion of the solid fraction increased consistently
with a prolonged treatment time, reaching up to 41.6\% with respect to
the solid fraction treated for 6h. It is suggested that the formation of
insoluble material may be correlated to the reduction of unsaturated
fatty acids and subsequently polymer formation.

Fig. 2 -- about here

It has been reported that HVACP can degrade the
H\textsubscript{2}molecules forming H\textsuperscript{+},
H\textsubscript{3}\textsuperscript{+}, and H\selectlanguage{english}\textsuperscript{*} species
by 0.0001\%, 0.1\%, and 1 \%, respectively (El-Zeer et al., 2013). Since
the H\selectlanguage{english}\textsuperscript{*} radicals are more abundant in the process, it
can be suggested that H\selectlanguage{english}\textsuperscript{*} radicals are the most active
species and can be responsible for any possible reaction. Indeed, the
H\textsuperscript{+} and H\selectlanguage{english}\textsuperscript{*} radicals are the species
that lack of electrons and their stabilities are similar. So, both
species can be responsible for the mechanism suggested. \selectlanguage{english}Schneider
(Schneider, 2008) reported an updated mechanism for lipid peroxidation,
mentioning the formation of conjugated systems from isolated double
bonds. The suggested mechanism depends on bis-allylic radical stability.
Behr \emph{et al.} also presented the formation of a conjugated system
from linoleic acid by a catalytic reaction leading to five different
structural isomers (Behr, Witte, \& Bayrak, 2013). As aforementioned,
HVACP treated H\textsubscript{2} molecules form H radicals and cations,
and these species can react with unsaturated lipids converting isolated
double bonds into conjugated ones. Fig. 2 illustrates a possible
mechanism for the formation of a conjugated system via interaction
between one of the bis-allylic hydrogens (see hydrogens in red color) in
the structure of linolenic acid with H radicals. Linolenic acid has
given as an example for the fatty acids which contain bis-allylic
hydrogens. The triglyceride that we have studied had 8.4 \% of linolenic
acid, and, as aforementioned, its quantity decreased to 1,5 \% after
HVACP treatment. This can happen in linoleic acid as well.

Figure 3 -- about here

\textbf{3.2. NMR}

Peak assignments of the triglyceride structure of soybean oil in
the\textsuperscript{1}H-NMR spectra are shown in Fig. 3a (each proton in
a different chemical environment has been numbered from 1-10 in blue),
corresponding to: \textbf{1)} Methyl terminal group, ? (ppm)
0.8-0.9;\textbf{2)} Methyl terminal group for linolenic acid, ? (ppm)
0.9-1.0;\textbf{3)} Methylene protons
-CH\textsubscript{2}-\textbf{CH\textsubscript{2}} -CH\textsubscript{2}-,
? (ppm) 1.2-1.4; \textbf{4)} Methylene protons
--C(=O)-CH\textsubscript{2}-\textbf{CH\textsubscript{2}}
-CH\textsubscript{2}- ? 1.5-1.7; \textbf{5)} Methylene protons
-CH\textsubscript{2}-\textbf{CH\textsubscript{2}} -CH=CH-, ?
1.9-2.1;\textbf{6)} Methylene protons
--C(=O)-\textbf{CH\textsubscript{2}}
-CH\textsubscript{2}-CH\textsubscript{2}-? 2.2-2.3; \textbf{7)} Allylic
group\textbf{=} CH\textbf{-CH\textsubscript{2}} -CH=, ? (ppm)
2.7-2.9;\textbf{8)} Glycerol protons n-1 and n-3, doublet: ? (ppm)
4.0-4.3;\textbf{9)} Glycerol protons n-2, ? (ppm) 5.2-5.3; \textbf{10)}
Olefinic protons CH\textsubscript{2}\textbf{-CH=CH-}
CH\textsubscript{2}, ? (ppm) 5.3-5.4. As can be seen in Fig. 3a,
untreated, and treated samples have the same number of peaks with
different intensities as well as the partially hydrogenated sample of
soybean oil (PHO). This result supports the proposed mechanism shown in
Fig. 3 where all the allylic protons with a chemical shift at 2.7-2.9
ppm, have a decreasing tendency concerning treatment time. Soybean
oil-treated for 6h showed a reduction of double bonds (peak 10) from
7.4\% to 4.7\% and an increasing percentage of single bonds (peak 3)
from 53.5\% to 59.5\%. This reduction can be attributed not only to
hydrogenation but also to dimerization by the Diels Alder reaction
(Koehler, Anseth, \& Bowman, 2013; Smith, 2017) between the conjugated
double bonds formed after the HVACP treatment with the double bond of
another triglyceride's fatty acid unit. So, each Diels Alder reaction
(Smith, 2017) decrease two double bonds. The proposed mechanism for
Diels Alder reaction is presented in Fig. 4. Ionescu and Petrovic
patented the cationic polymerization of soybean oil (Ionescu \&
Petrovic, 2009). They have reported that the polymerization initiates
with the formation of conjugated double bonds on the hydrocarbon chain
of linoleic and linolenic acid units (Ionescu \& Petrovic, 2009). Once
the conjugated double bonds are formed, the polymerization goes through
a pericyclic reaction like Diels-Alder (Aragon\selectlanguage{ngerman}ès et al., 2016; Ionescu
\& Petrovic, 2009; Smith, 2017). The results that we have obtained are
consistent with the results published by the authors aforementioned. We
have proposed a similar polymerization/dimerization reaction with one
difference, radical aided conjugated double bond formation, and
following Diels-Alder reactions (Fig. 2 and 4) (Smith, 2017).

Moreover, peaks 5 and 7 of the \textsuperscript{1}H-NMR spectra related
to single bonds close to double bonds will reduce if double bonds are
modified, as it occurs with treated samples. The reduction of peak
integration area for 5 and 7 may correlate with the increased amount of
peak 3, because of possible hydrogenated double bonds or Diels-Alder
reaction (Smith, 2017) proposed (Fig. 4). The glycerol moiety did not
show significant modifications, peaks 8 and 9. Suggesting that treated
samples maintain a triglyceride structure.

Figure 4 -- about here

Oxidation products include hydroperoxides (chemical shift \selectlanguage{english}? (ppm) 5.7,
8.3-8.9), aldehydes (chemical shift \selectlanguage{english}? (ppm) 9.5-9.8), alcohols (chemical
shift \selectlanguage{english}? (ppm) 3.43-3.62), epoxides (chemical shift \selectlanguage{english}? (ppm) 2.63, \selectlanguage{english}? (ppm)
2.88-2.90, \selectlanguage{english}? 3.1), ketones (chemical shift \selectlanguage{english}? (ppm) 6.08, \selectlanguage{english}? 6.82), or
conjugated double bonds (chemical shift \selectlanguage{english}? 5.7-6.4) (Gunstone et al.,
2007). Treated samples showed an increased peak only for conjugated
double bonds, as can be seen in the expanded spectra in figure 3a,
corresponding to \selectlanguage{english}? (ppm) 5.7-6.3. Moreover, spectra from 2D-NMR (HSQC)
of the treated sample was used to identify a cross-peaks between
J-coupled protons in the spin of the conjugated double bonds and each
carbon involved in this network. From this analysis, two points were
identified on treated samples: (\selectlanguage{english}? (ppm) 6.2;126.3) and (\selectlanguage{english}? (ppm)
5.9;128.7), as can be seen in figure 3c. These peaks are related to the
'double bonds' region of the hydrocarbon chain in
the\textsuperscript{13}C-NMR spectra. These results suggest that double
bonds can migrate to a conjugated arrangement during HVACP treatment
(Fig. 2), forming a ring structure that can result in the dimerization
of triglycerides (Fig. 4) (Biswas et al., 2007).

The assignments of carbon resonances of the triglyceride molecules of
soybean oil are characterized by four main signals: carbonyl carbons (\selectlanguage{english}?
(ppm) 170-174), olefinic carbons (\selectlanguage{english}? (ppm) 120-140), glyceride backbone
carbons (\selectlanguage{english}?(ppm) 60-80), aliphatic and methyl carbons (\selectlanguage{english}? (ppm) 10-40), as
shown in Fig. 3b. The carbon spectra for untreated and treated samples
(6h, gel fraction) showed the same number of peaks, and it indicates
that the primary structure of soybean oil treated with HVACP is
maintained as a triglyceride. A shift to the left was observed in the
spectra (Fig. 3b), mostly in the olefinic carbons. A shift may indicate
changes in the electron density near the carbon that affect the whole
molecule, with a higher shielding of the external magnetic field. These
changes may be related to the formation of the modified double bond of
the dimer.

\textbf{3.3. Gel permeation chromatography (GPC)}

The GPC analysis results showed that untreated soybean oil has a single
peak at a retention time of 25.3 min, corresponding to the soybean oil
triglycerides, as shown in Fig. 5. A second peak (retention time at
24.4min) was observed for 2h, 4h, and 6h treated liquid samples, with an
increased area of 25.2+-2.3\%, 29.9+-1.6\%, and 32.2+-1.6\%, respectively.
Corresponding to an average number molecular weight (\(\overset{\overline{}}{\text{Mn}}\))
of 1569+-17.3, 1626+-12.2, and 1705+-12.2g/mol, respectively. This result
indicates the formation of a dimer, as explained in the proposed
Diels-Alder (Smith, 2017) reaction between the triglyceride molecules
forming a dimer (Fig. 4). It is clearly can be proposed that HVACP
treatment may induce polymerization of soybean oil, forming dimers and
oligomers (Ionescu \& Petrovic, 2009; Zhao, Yang, Tao, \& Xu, 2014). The
\(\overset{\overline{}}{\text{Mn}}\)obtained in GPC is an almost double mass of n
untreated triglyceride, which means the dimerization.

Figure 5 -- about here GPC

\textbf{3.4. Thermal properties}

The effect of possible hydrogenation and dimerization/polymerization
reactions can influence the thermal properties, including melting point
and thermal degradation temperatures. Results from DSC and TGA are shown
in Fig. A2, in the supplementary data section. Untreated soybean oil has
a melting point of -28+-0.4degC, while PHO is solid at room temperature
with a melting point of 32.3+-0.3degC. HVACP treated samples showed a
melting point of -27.6+-0.3, -23.5+-0.4 and -4.9+-0.3degC for liquid, gel,
and solid fractions treated for 6h, respectively (Fig. 6). HVACP treated
samples increased the melting point but not at the level of a PHO,
because of the high content of PUFA (36.6\%) compared to a PHO that has
a PUFA content of 20.4\%. Moreover, elaidic acid in PHO behaves like a
saturated fatty acid because the trans isomers create a linear
hydrocarbon chain, increasing the melting point. HVACP treated samples
have a lower content of trans fatty acids, and consequently, they have a
lower melting point. The reason for the increase and broadening in
melting points observed in Fig. A2a comparing to the untreated oil might
be due to the possible amorphous dimers/polymers formed, as suggested in
Fig. 4.

Additionally, a reduction of melting enthalpy was also observed in
treated samples, from 59.1+-3.3 J/g to 9.7+-3 J/g for the untreated and
treated 6h-solid fractions, respectively. A reduction of the endothermic
peak is linked with less amount of crystals or a reduction of crystals
size.

Thermal degradation involves scission, side group elimination, and
depolymerization phenomenon (Crompton, 2012). Samples were exposed to a
temperature up to 600degC, where 100\% of sample degradation was achieved
(Fig. A2b). The onset temperature of untreated soybean oil was
357+-1.5degC. This temperature increased with treated samples, reaching
369+-1.2degC for the 6h-solid fraction. Therefore, a 12-14degC shift was
observed in treated samples through the whole thermal degradation
process, which might be attributed to the formation of dimers/polymers
proposed (Fig. 4). In contrast, PHO showed an onset temperature of 345
degC. Hence, it was characterized by a thermal degradation shifted to a
lower temperature, which means that saturated triglycerides require less
energy to be degraded compared to untreated soybean oil that has more
double bonds and requires more energy to degrade.

\textbf{3.5. FTIR}

Untreated and treated samples (6h) were analyzed with FTIR (Fig. 6).
Fig. 6a shows the hydrogen stretching region, including the range of
2800-3050 cm\textsuperscript{-1}. Liquid, gel, and solid fractions
showed an increase in the number of single bonds (2930
cm\textsuperscript{-1}, 2860 cm\textsuperscript{-1} symmetric and
asymmetric stretching bands), and a reduction of double bonds (3010
cm\textsuperscript{-1}, =C-H stretching band). Suggesting that HVACP
treated samples as previously explained, might have undergone possible
hydrogenation or dimerization/polymerization reaction. Further
information about bond modifications is included in the fingerprint
region (Fig. 6b), in the range of 800-1600 cm\textsuperscript{-1}. The
peak observed at 914 cm\textsuperscript{-1} corresponds to the
cis-olefinic group, which shows a reduction from untreated to treated
samples (Guillen \& Cabo, 1997). However, an increase of trans- double
bonds bending vibration was also observed in the 968
cm\textsuperscript{-1} bands, with a higher peak for PHO. These results
are in accordance with the fatty acid composition.

Fig. 6 -- about here

Hexane was used to separate the liquid oil from the insoluble fraction
by using Soxhlet extraction. The extracted insoluble fraction (Solid-F
in Fig. 6) was characterized by a reduced content of double bonds (peak
at 3010 cm\textsuperscript{-1}) and a higher content of single bonds.
This is probably due to the Diels-Alder (Smith, 2017; Yao et al., 2008)
reaction between the conjugated double bonds formed by HVACP treatment
in one triglyceride with the one double bond of another triglyceride
molecule. Therefore, two double bonds are disappearing by the reaction
and a reduction in absorbance of the mentioned peak. Besides of an
increase of absorbance in the peak observed at 1465
cm\textsuperscript{-1} that corresponds to bending vibration of the
methylene group, and an increase in absorbance in the peak observed at
1377 cm\textsuperscript{-1} that correspond to symmetrical bending
vibration of methyl groups (Guillen \& Cabo, 1997).

As seen in Figure 6, there are no additional peaks that provide
information about structure modifications rather than changes in single
and double bonds. It is proposed that the formation of this
oligomer/polymer may occur through the Diels Alder (Smith, 2017)
reaction mechanism, as seen in Fig. 4. As a result, the triglyceride
fatty acid chains are linked by cyclic structures. A rearrangement of
the double bonds from cis- to trans-, as a consequence of the reaction,
is also reported (Muik, Lendl, Molina-Diaz, \& Ayora-Canada, 2005). This
effect can be observed in results from FTIR and fatty acid composition,
by an increase of the trans isomer of 18:1 and 18:2.

HVACP treatment has not only been used to modify chemical structures but
has been studied mainly as a processing technique to reduce the
microbial load of food (Misra et al., 2019). The mechanisms of microbial
inactivation may include the production of UV light, oxidation of
membrane lipids, or protein oxidation (Liao et al., 2017). Therefore,
this technology is known as an oxidizer tool. In this study, the goal is
under the HVACP treatment to use hydrogen gas as a source of hydrogen
species and their effect on unsaturated fatty acids. Consequently, the
presence of oxygen was avoided because any trace could form reactive
oxygen species that may initiate oxidation reactions. Hydrogen gas was
flushed for 5 minutes, to reduce oxygen content below 0.01\%. However,
treated samples of soybean oil (1.9-5.0 meq/kg) showed a higher peroxide
value (PV) than untreated (0.19 meq/kg). These values were below 10
meq/kg, which is the limit of peroxide content requirement for fats and
oils (Pegg, 2005). PV is a critical parameter that should be monitored.

Further precautions can be adopted to remove oxygen from the plasma
chamber, or it would be required the use of antioxidants as additives.
Interestingly, results showed that longer treatment times reduce the PV.
Solid fractions of the samples treated for 6h have a PV value of
2.71+-0.6 meq/kg, being lower than 5.0+-0.9 meq/kg for the 2h-solid
treatment. Samples treated for 6h have less amount of double bonds due
to the possible dimerization via Diels-Alder (Ionescu \& Petrovic, 2009;
Mihail \& S., 2012; Smith, 2017) reaction as presented in Fig. 3
available to react with oxygen, which means that that samples treated
for longer treatment times are less susceptible to lipid oxidation.

\textbf{CONCLUSIONS}

In this study, we investigated the modifications of soybean oil using
HVACP hydrogen plasma. Soybean oil was treated with
H\textsubscript{2}species formed by HVACP treatment for 2, 4, and 6.
Samples were characterized by techniques such as gas chromatography
(GC), FTIR,\textsuperscript{1}H-NMR, \textsuperscript{13}C-NMR, 2D-NMR,
oxidation, and thermal properties.

HVACP treatment of soybean oil using hydrogen gas can transform the
liquid oil into a solid product. This study found that HVACP can
catalyze hydrogenation of double bonds of the oil and polymerize
triglycerides. Hydrogen species formed by HVACP treatment can convert
isolated double bonds which contain bis-allylic hydrogens into
conjugated double bonds. Besides, those triglycerides can be
hydrogenated or polymerized (via Diels Alder) by hydrogen species form
under cold plasma conditions. HVACP is an environmentally friendly
technique to hydrogenate plant oils without the generation of trans
fatty acids. The use of cold plasma to modify chemical structures can
contribute to the production of sustainable products obtained from green
processes.

\textbf{ACKNOWLEDGEMENTS}

This work was funded by the Department of Food Science of Purdue
University. Ximena Yepez gratefully acknowledges the financial support
of the office of the Secretary of Higher Education, Science, Technology,
and Innovation of the government of Ecuador for her graduate studies.
This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.

\textbf{REFERENCES}

AOAC. (2005). \emph{Official Methods of Analysis of AOAC International}
.

\emph{Association of Official Analysis Chemists International.}

Aragones, A. C., Haworth, N. L., Darwish, N., Ciampi, S., Bloomfield, N.
J., Wallace, G. G., \ldots{} Coote, M. L. (2016). Electrostatic
catalysis of a Diels--Alder reaction. \emph{Nature} , \emph{531} (7592),
88--91. https://doi.org/10.1038/nature16989

Behr, A., Witte, H., \& Bayrak, Z. (2013). Homogeneous metal complex
catalyzed conjugation of methyl linoleate. \emph{European Journal of
Lipid Science and Technology} , \emph{115} (7), 721--728.
https://doi.org/10.1002/ejlt.201200429

Biswas, A., Adhvaryu, A., Stevenson, D. G., Sharma, B. K., Willet, J.
L., \& Erhan, S. Z. (2007). Microwave irradiation effects on the
structure, viscosity, thermal properties and lubricity of soybean
oil.\emph{Industrial Crops and Products} , \emph{25} (1), 1--7.
https://doi.org/10.1016/j.indcrop.2006.04.001

Burgess, J. S., Matis, B. R., Robinson, J. T., Bulat, F. A., Keith
Perkins, F., Houston, B. H., \& Baldwin, J. W. (2011). Tuning the
electronic properties of graphene by hydrogenation in a plasma enhanced
chemical vapor deposition reactor. \emph{Carbon} , \emph{49} (13),
4420--4426. https://doi.org/10.1016/j.carbon.2011.06.034

Chew, S. C., \& Nyam, K. L. (2016). Oxidative Stability of
Microencapsulated Kenaf Seed Oil Using Co-extrusion
Technology.\emph{JAOCS, Journal of the American Oil Chemists' Society}
,\emph{93} (4), 607--615. https://doi.org/10.1007/s11746-016-2794-9

Crompton, R. (2012). Carbon hydrogen polymers. \emph{Thermal Stability
of Polymers} , 1--44.

Dijkstra, A. J. (2006). Revisiting the formation of trans isomers during
partial hydrogenation of triacylglycerol oils. \emph{European Journal of
Lipid Science and Technology} , \emph{108} (3), 249--264.
https://doi.org/10.1002/ejlt.200500335

El-Zeer, D. M., Samir, A., Elakshar, F., \& Garamoon, A. A. (2013).
Decaying of Nitrogen Second Positive System by Addition of
H\&lt;sub\&gt;2\&lt;/sub\&gt; Gas in Air DB Discharge. \emph{Journal of
Modern Physics} , \emph{04} (02), 160--167.
https://doi.org/10.4236/jmp.2013.42022

FDA. (2015). Final Determination Regarding Partially Hydrogenated
Oils.\emph{The Daily Journal of the United States Goverment} , \emph{80}
(116), 34650--34670.

Firestone, D. (2009). Official Methods and Recommended Practices of the
AOCS. In \emph{Iodine value of fats and oils cyclohexane method}(Sixth).

Guillen, M. D., \& Cabo, N. (1997). Characterization of edible oils and
lard by fourier transform infrared spectroscopy. Relationships between
composition and frequency of concrete bands in the fingerprint
region.\emph{Journal of the American Oil Chemists' Society} , \emph{74}
(10), 1281--1286. https://doi.org/10.1007/s11746-997-0058-4

Gunstone, F. D., Harwood, J. L., \& Harwood, J. L. (2007). \emph{The
Lipid Handbook with CD-ROM} . \emph{The Lipid Handbook with CD-ROM} .
https://doi.org/10.1201/9781420009675

Ionescu, M., \& Petrovic, Z. (2009). Cationic polymerization of
biological oils with superacid catalysts.

Jovanovic, D. (1998). Nickel hydrogenation catalyst for tallow
hydrogenation and for the selective hydrogenation of sunflower seed oil
and soybean oil. \emph{Catalysis Today} , \emph{43} (1--2), 21--28.
https://doi.org/10.1016/S0920-5861(98)00133-3

Kiefer, K. (1997). Derivatization of Corn Oil for Analysis by
GC.\emph{Supelco Reporter} , \emph{16} (3), 6.

Koehler, K. C., Anseth, K. S., \& Bowman, C. N. (2013). Diels-alder
mediated controlled release from a poly(ethylene glycol) based
hydrogel.\emph{Biomacromolecules} , \emph{14} (2), 538--547.
https://doi.org/10.1021/bm301789d

Liao, X., Liu, D., Xiang, Q., Ahn, J., Chen, S., Ye, X., \& Ding, T.
(2017). Inactivation mechanisms of non-thermal plasma on microbes: A
review. \emph{Food Control} , \emph{75} , 83--91.
https://doi.org/10.1016/j.foodcont.2016.12.021

Miao, S., Wang, P., Su, Z., \& Zhang, S. (2014). Vegetable-oil-based
polymers as future polymeric biomaterials. \emph{Acta Biomaterialia}
,\emph{10} (4), 1692--1704. https://doi.org/10.1016/j.actbio.2013.08.040

Mihail, I., \& S., P. (2012). Polymerization of Soybean Oil with
Superacids. \emph{Soybean - Applications and Technology} .
https://doi.org/10.5772/10547

Misra, N. N., Yepez, X., Xu, L., \& Keener, K. (2019). In-package cold
plasma technologies. \emph{Journal of Food Engineering} , \emph{244} ,
21--31. https://doi.org/10.1016/J.JFOODENG.2018.09.019

Muik, B., Lendl, B., Molina-Diaz, A., \& Ayora-Canada, M. J. (2005).
Direct monitoring of lipid oxidation in edible oils by Fourier transform
Raman spectroscopy. \emph{Chemistry and Physics of Lipids} ,\emph{134}
(2), 173--182. https://doi.org/10.1016/j.chemphyslip.2005.01.003

Ostrikov, K., Neyts, E. C., \& Meyyappan, M. (2013). Plasma nanoscience:
From nano-solids in plasmas to nano-plasmas in solids. \emph{Advances in
Physics} , \emph{62} (2), 113--224.
https://doi.org/10.1080/00018732.2013.808047

Pankaj, S. K., Wan, Z., De Leon, J. E., Mosher, C., Colonna, W., \&
Keener, K. M. (2017). High-voltage atmospheric cold plasma treatment of
different types of starch films. \emph{Starch - Starke} , \emph{1700009}
, 1700009. https://doi.org/10.1002/star.201700009

Pegg, R. B. (2005). Measurement Of Primary Lipid Oxidation
Products.\emph{Handbook of Food Analytical Chemistry} , \emph{1}
--\emph{2} , 515--529. https://doi.org/10.1002/0471709085.ch13

Schneider, C. (2008). An update on products and mechanisms of lipid
peroxidation. \emph{Molecular Nutrition and Food Research} ,\emph{23}
(1), 1--7. https://doi.org/10.1038/jid.2014.371

Smith, M. B. (2017). Pericyclic Reactions: The Diels-Alder Reaction.
In\emph{Organic Synthesis} (pp. 743--800). Elsevier.
https://doi.org/10.1016/B978-0-12-800720-4.00014-3

Wang, H., Maiyalagan, T., \& Wang, X. (2012). Review on recent progress
in nitrogen-doped graphene: Synthesis, characterization, and its
potential applications. \emph{ACS Catalysis} , \emph{2} (5), 781--794.
https://doi.org/10.1021/cs200652y

Yao, W., Mu, Y., Gao, A., Su, Q., Liu, Y., \& Zhang, Y. (2008).
Efficient ring-opening polymerization of epsilon-caprolactone using
anilido-imine-aluminum complexes in the presence of benzyl
alcohol.\emph{Polymer} , \emph{49} (10), 2486--2491.
https://doi.org/10.1016/j.polymer.2008.03.035

Yepez, X. V., \& Keener, K. M. (2016). High-voltage Atmospheric Cold
Plasma (HVACP) hydrogenation of soybean oil without trans-fatty
acids.\emph{Innovative Food Science \& Emerging Technologies} ,
\emph{38} , 169--174. https://doi.org/10.1016/j.ifset.2016.09.001

Zhao, X., Yang, J., Tao, D., \& Xu, X. (2014). Synthesis and
Tribological Properties of Air Plasma Polymerized Soybean Oil with
N-Containing Structures. \emph{Journal of the American Oil Chemists'
Society} , \emph{91} (5), 827--837.
https://doi.org/10.1007/s11746-014-2424-3\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig. 1. High voltage atmospheric cold plasma system for soybean oil
treatment (a). Schematic diagram (b): transformer, high voltage
electrode, ground electrode, dielectric barrier, plasma chamber, packing
film(cryovac), the sample of soybean oil. Treated samples (c): liquid
(x), gel (y), and solid (z).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig. 2. Proposed mechanism for the formation of conjugated double bonds
with of HVACP treatment (from left to right: 9,11,15-Octadecatrienoic
acid and 10,12,15-Octadecatrienoic)

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/331204/articles/457896-atmospheric-cold-plasma-treatment-of-soybean-oil-with-hydrogen-gas}

Fig. 3. NMR spectra of soybean oil untreated and treated for 6h with
hydrogen gas (a) \textsuperscript{1}H-NMR spectra, bottom to top:
untreated, treated-liquid, treated-gel, treated-solid, and PHO;
(b)\textsuperscript{13}C-NMR spectra, bottom to top: untreated, and
treated-gel; (c) 2D-NMR-HSQC spectrum of soybean oil-treated (gel
fraction) for 6h.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig. 4. Proposed Diels-Alder reaction between triglycerides.

\textbf{Hosted file}

\verb`image5.(null)`  available at \url{https://authorea.com/users/331204/articles/457896-atmospheric-cold-plasma-treatment-of-soybean-oil-with-hydrogen-gas}

Fig. 5. Gel permeation spectra of untreated soybean oil (black), and
treated samples for 2, 4, and 6h with hydrogen gas, at 80 kV.

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/331204/articles/457896-atmospheric-cold-plasma-treatment-of-soybean-oil-with-hydrogen-gas}

Fig. 6. FTIR spectra of soybean oil untreated and treated for 6h with
hydrogen gas.

\selectlanguage{english}
\FloatBarrier
\end{document}

