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\begin{document}

\title{Enzymatic hydrolysis and detection of 3-Monochloropropane-1,2-diol
esters}



\author[1]{ming fang}%
\affil[1]{Wuhan Polytechnic University}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
There are more and more studies on the detection method of
3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) at present.
By comparing these methods for the determination of 3-MCPD esters.
Indirect methods, which determine total amount of 3-MCPD after
hydrolysis of the esters, have an advantage over direct methods. The
existing indirect methods, however, may yield unreliable results or
require long hours of alkaline methanolysis. In contrast, the Indirect
enzymatic hydrolysis method has mild conditions and more accurate
results. In this study, we developed a reliable and rapid indirect
method for determinations of 3-MCPD esters. 3-MCPD esters was
enzymatized to 3-MCPD by indirect enzymatic hydrolysis method, and the
conditions of enzymatic hydrolysis were optimized, the content of 3-MCPD
after enzymatic hydrolysis was detected by gas chromatography-mass
spectrometry (GC-MS) and the yield was calculated. Finally, the optimum
conditions for enzymatic hydrolysis of 3-MCPD esters were determined.
According to the optimal enzymatic hydrolysis condition, the contents of
3-MCPD esters in four food oils were determined. The method is simple
and sensitive, and can meet the requirement of 3-MCPD esters detection
in general oils.%
\end{abstract}%



\sloppy


Enzymatic hydrolysis and detection of 3-Monochloropropane-1,2-diol
esters

Ting Xu \textsuperscript{1}, Zeng Qingru\textsuperscript{1}, Xin
Liu\textsuperscript{1,2}, Shensheng Xiao\textsuperscript{1,2}, Xiaoming
Jiang\textsuperscript{3}, Yuepeng Lu\textsuperscript{3}, Zhiyong
Gong\textsuperscript{1,2}, Yonggui Gao\textsuperscript{1,3,4}, Min
Fang\textsuperscript{1,2*}

\textsuperscript{1} Institute of Food Science and Engineering, Wuhan
Polytechnic University, Wuhan 430023, China

\textsuperscript{2} Hubei Collaborative Innovation Center for Processing
of Agricultural Products, Wuhan Polytechnic University, Wuhan 430023,
China

\textsuperscript{3} Key Laboratory of Edible Oil Quality and Safety for
State Market Regulation, Wuhan, 430040, China

\textsuperscript{4} Key Laboratory for Deep Processing of Major Grain
and Oil, Ministry of Education, College of Food Science and Engineering,
Wuhan Polytechnic University, Wuhan 430023, PR China

* Corresponding author:

Min Fang

E-mail:\emph{fangmin0227@126.com}

Address: Institute of Food Science and Engineering, Wuhan Polytechnic
University, Wuhan 430023, China.

Tel:+86-027-83924790

Fax:+86-027-83924790

\textbf{Abstract}

There are more and more studies on the detection method
of~3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) at
present. By comparing these methods for the determination of 3-MCPD
esters. Indirect methods, which determine total amount of 3-MCPD after
hydrolysis of the esters, have an advantage over direct methods. The
existing indirect methods, however, may yield unreliable results or
require long hours of alkaline methanolysis. In contrast, the Indirect
enzymatic hydrolysis method has mild conditions and more accurate
results. In this study, we developed a reliable and rapid indirect
method for determinations of 3-MCPD esters. 3-MCPD esters was
enzymatized to 3-MCPD by indirect enzymatic hydrolysis method, and the
conditions of enzymatic hydrolysis were optimized, the content of 3-MCPD
after enzymatic hydrolysis was detected by gas chromatography-mass
spectrometry (GC-MS) and the yield was calculated. Finally, the optimum
conditions for enzymatic hydrolysis of 3-MCPD esters were determined.
According to the optimal enzymatic hydrolysis condition, the contents of
3-MCPD esters in four food oils were determined. The method is simple
and sensitive, and can meet the requirement of 3-MCPD esters detection
in general oils.

\textbf{Keywords} : 3-MCPD esters; 3-MCPD; Indirect enzymatic hydrolysis
method; GC-MS

\textbf{Introduction}

The contamination of 3-MCPD esters is one of the hot issues in the field
of food safety that have been disputed internationally in recent
years\textsuperscript{{[}1{]}}.The formation of 3-MCPD esters is
directly related to the chemical refining of synthetic oil products, It
has been confirmed that 3-MCPD esters are found in hot processed foods
derived from grains, fish and meat products, coffee, nuts, potatoes and
edible oils\textsuperscript{{[}2-4{]}}. 3-MCPD esters are hydrolyzed in
vivo by intestinal pancreatic lipase to produce free 3-MCPD, 3-MCPD has
extremely obvious renal toxicity and high genotoxicity, which has been
strictly classified as 2B carcinogen by the International Cancer
Organization Working Group (IARC) of the United
Nations\textsuperscript{{[}5-6{]}}.

3-MCPD esters detection mainly has the direct determination method and
the indirect determination method. By comparing these methods for the
determination of 3-MCPD esters. Direct methods based on
LC-MS/MS\textsuperscript{{[}7--9{]}}are available in literature,
however, the great complexity of 3-MCPD esters may lead to the
determination of up to several tens of compounds in one sample for which
a respective number of internal standards are
required\textsuperscript{{[}10{]}}. This contributes to make the
procedure generally expensive, but the advantages of direct methods are
the lack of derivatization step and the possibility to get direct
information on the content of individual esters and for these reasons
they found wide application in the field\textsuperscript{{[}11{]}}.
Indirect methods for the determination of 3-MCPD esters in samples
requires a complex pretreatment process with many steps in advance, and
the change of conditions in each experimental step will easily affect
the accuracy of the final detection results, the methods are based on
derivatization, transforming 3-MCPD esters in free 3-MCPD, and

they include several stages such as transesterification, neutralization
and derivatization steps before quantification, using NaOH for
transesterification, sodium bromide for neutralization and phenylboronic
acid for derivatization\textsuperscript{{[}12{]}}. But in the alkaline
catalyzed indirect method, if the alkaline hydrolysis reaction time
conditions and salting-out conditions or temperature are not well
controlled, a part of 3-MCPD may be eliminated or extra produced, thus
making the determination result small or large. Acid catalyzed indirect
method is more stable and durable, but relatively time-consuming. In
contrast, the indirect enzymatic hydrolysis method has mild conditions
and more accurate results.

At present, there are more and more studies on the determination of
3-MCPD esters by indirect enzymatic hydrolysis method. However, there
are no study have reported the optimal conditions for enzymatic
hydrolysis of 3-MCPD esters. In this work, 3-MCPD esters was enzymolized
to 3-MCPD by indirect enzymatic hydrolysis method, and the conditions of
enzymatic hydrolysis were optimized, GC-MS was used for detection and
calculation of yield, then the optimum enzymatic conditions were
determined. Finally, the actual samples were detected by indirect
enzymatic hydrolysis method. The method is simple, reliable, and rapid,
and is suitable for a routine quality control of fats and
oils\textsuperscript{{[}13{]}}.

\textbf{Experimental Procedures}

\emph{2.1. Reagents}

As the ester-form standard reagent, 3-MCPD dioleate (purity 99.8\%) was
purchased from Wako Pure Chemical (Osaka, Japan). As the free-form
standard reagents, 3-MCPD (purity 98\%) was purchased from Macklin
Biochemical Co. Ltd (Shanghai, China,
\emph{www.macklin.cn),}3-MCPD-d\textsubscript{5} (purity 98.0\%) from
Larodan AB (Sweden,\emph{www.larodan.com)}. Phenylboronic acid (PBA)
(purity 95\%) was purchased from Sigma-Aldrich
(USA,\emph{www.sigmaaldrich.cn).}Lipase from Candida rugosa was
purchased from Sigma-Aldrich. Lipase from Candida sp. and lipase from
Thermophila sparsiformis was purchased from Shanghai Aladdin Biochemical
Technology Co. Ltd (Shanghai, China,\emph{www.aladdin.cn).}

A 30\% (w/v) sodium bromide aqueous solution was prepared and adjusted
pH with citric acid and disodium hydrogen phosphate aqueous solutions.
The lipase was dissolved with this solution to prepare an aqueous
solution of sodium bromide containing 150 U/mL lipase. A PBA solution
was prepared by dissolving 0.25 g PBA in 10 mL water/acetone mixed
solution (1:19, v/v).

1 mg of 3-MCPD esters standard was weighed, dissolved in ethyl acetate,
transferred to a 10 mL volumetric flask, volumetric to calibration with
ethyl acetatel. For the standard stock solutions, 300 \selectlanguage{greek}μ\selectlanguage{english}g/mL ethanol
solutions were prepared for 3-MCPD and 200 \selectlanguage{greek}μ\selectlanguage{english}g/mL ethanol solutions were
prepared for 3-MCPD-d\textsubscript{5} as internal standard stock
solutions. These stock solutions could be stored at [?] 20 for at least
three months. On the day of the study, the standard stock solution was
diluted with the 30\% sodium bromide aqueous solution to prepare 1.5
\selectlanguage{greek}μ\selectlanguage{english}g/mL standard solutions. Additionally, 2.0 \selectlanguage{greek}μ\selectlanguage{english}g/mL internal standard
solutions was prepared by combining the internal standard stock
solutions and diluting it with the 30\% sodium bromide aqueous solution.

\emph{2.2. Oil samples}

As oil samples, Palm oil, rapeseed oil and soy sauce were purchased in
supermarkets. Samples were spiked with ester standards and dispensed
into glass vials, and stored in the dark at room temperature.

\emph{2.3. Equipment}

Electronic analytical balance (Shanghai Minqiao Precision Scientific
Instrument Co. Ltd), a centrifuge (Model SC-3614; Anhui, China),
laboratory pH Meter (Ohaus Instruments Co. Ltd), a vortex mixer (SCI-VS;
SCILOGEX, USA), a GC-MS (Agilent 7890/5975C-GC/MSD; Agilent
Technologies, USA), a constant-temperature shaker (SHA-B; Guohua
company, China)

\emph{2.4. Enzymatic hydrolysis of 3-MCPD esters standard}

1 mg of 3-MCPD esters standard was weighed and dissolved in a 10 mL test
tube with ethyl acetate. After mixing, 20 uL of the above solution was
put into a 5 mL test tube. After adding 3 mL of the 30\% sodium bromide
aqueous solution containing 150 U/mL lipase, the mixture was shaken by a
high-speed shaker for 30 min at room temperature to hydrolyze the
esters. The mixture was then heated in a water bath at 80 for 10 min.
The mixture was cooled to room temperature, and 50 \selectlanguage{greek}μ\selectlanguage{english}L of 2.0 \selectlanguage{greek}μ\selectlanguage{english}g/mL
internal standard mix was added. Next, 3 mL of hexane was added and the
tube was vortexed for 10 s. The aqueous layer was transferred to a new
test tube, to which another 3 mL of hexane was added and the tube was
vortexed again for 10 sec. After the hexane layer was removed, 3-MCPD in
the aqueous layer and the deuterated free form internal standards were
derivatized by adding 100 \selectlanguage{greek}μ\selectlanguage{english}L of PBA solution and 3 mL of hexane, and
shaking at room temperature for 5-10 min. The organic layer was then
transferred to a new test tube containing sodium sulfate and
concentrated to approximately 0.5-0.8 mL under a stream of nitrogen.
After filtration through a 0.2 \selectlanguage{greek}μ\selectlanguage{english}m membrane filter, the sample was
subjected to GC-MS analysis.

\emph{2.5. Procedures for analysis of3-MCPD esters}

In a 10 mL test tube, 0.1 g of oil sample was dissolved in 200--500 \selectlanguage{greek}μ\selectlanguage{english}L
of isooctane. After adding 3 mL of 30\% NaBr aqueous solution containing
150 U/mL lipase, the mixture was shaken by a high-speed shaker for 30
min at room temperature. The test tube was heated for 10 min in an 80
water bath. The test tube was cooled to room temperature, and 50 \selectlanguage{greek}μ\selectlanguage{english}L of
2.0 \selectlanguage{greek}μ\selectlanguage{english}g/mL internal standard mixed solution was added. Next, 3 mL of
hexane was added and shaken for 10 min. The resulting aqueous layer was
transferred to a new test tube. Another 3 mL of hexane was added and the
tube was shaken for 10 sec; the resulting hexane layer was subsequently
removed. A 100 \selectlanguage{greek}μ\selectlanguage{english}L PBA solution was added to the aqueous layer, and the
mixture was agitated with a vortex mixer for 10 sec. Next, 3 mL of
hexane was added and shaken for 10 sec. The resulting organic layer was
transferred to a new test tube and concentrated to approximately
0.5--0.8 mL using a stream of nitrogen. After filtration through a 0.2
\selectlanguage{greek}μ\selectlanguage{english}m membrane filter, the samples were subjected to GC-MS analysis.

\emph{2.6.} \emph{GC-MS Analysis}

A GC capillary column was used with a (5\%-phenyl)-methylpolysiloxane
liquid phase, 30 m length, 0.25 mm internal diameter, and 0.25 \selectlanguage{greek}μ\selectlanguage{english}m film
thickness. The carrier gas was helium at a fixed flow rate of 1.2
mL/min. The sample was injected in splitless mode at 250. The column
oven temperature was held at 60 for 1 min, then raised to 150 at
10/min, 180 at 3/min, and finally 300 at 30/min, before being held
at 300 for 8 min (total run=32 min). MS was performed in positive
electron ionization mode with an ion source temperature of 230.
Quantitative and qualitative analyses were performed by selective ion
monitoring (SIM) using ions at m/z 147 and 196 for the 3-MCPD
derivative, 150 and 201 for the 3-MCPD-d5 derivative.

\textbf{Results and Discussions}

\emph{3.1. Validation of lipase hydrolysis}

To verify that the lipase acted only on 3-MCPD esters, three controls
were used. The comparison of lipase action on 3-MCPD and lipase action
on 3-MCPD esters, the comparison of lipase action on 3-MCPD esters and
lipase no action on 3-MCPD esters, the comparison of pure solution of
3-MCPD, lipase action on 3-MCPD and pure solution of lipase. As seen in
the chromatogram, comparison between the effect of lipase on 3-MCPD and
the effect of lipase on 3-MCPD esters (Fig. 1), the peak time, mass
spectrogram information and ratio in qualitative ion map of the two were
similar, indicating that the same substance was finally obtained, that
is, the enzymatic hydrolysis was successful.

Comparison between the effect of lipase action on 3-MCPD esters and
lipase no action on 3-MCPD esters (Fig. 2). 3-MCPD is produced by the
action of lipase on 3-MCPD ester, but the lipase does not act on the
3-MCPD ester does not produce the 3-MCPD, and 10.705 min is peak time of
3-MCPD esters. This indicated that the enzymatic hydrolysis was
successful.

Comparison among pure solution of 3-MCPD, lipase action on 3-MCPD and
pure solution of lipase (Fig. 3). The comparison of the three results
shows that, the response of pure solution of 3-MCPD and lipase action on
3-MCPD is almost the same. However, the pure solution of lipase do not
produce 3-MCPD peak, indicating that the lipase can not act on 3-MCPD.
Therefore, 3-MCPD produced by lipase action on 3-MCPD esters is
successful, and has nothing to do with the addition of 3-MCPD internal
standard during the experiment.

\emph{3.2 Selection of}

\emph{lipases}

To select the lipase suitable for hydrolysis of 3-MCPD esters, the
recovery of 3-MCPD esters was also analyzed using lipase from Candida
rugosa , lipase from Candida sp. and lipase from Thermophila
sparsiformis at 150 U/mL. All lipases were prepared at 150 U/mL in 30\%
NaBr buffer, and a 0.5 h lipolysis was performed at the optimal pH and
temperature proposed by the manufacturer in triplicate. As shown in
Table 1, 3-MCPD esters could not be enzymatically decomposed by lipase
from Thermophila sparsiformis. Among the lipases tested, lipase from
Candida sp. gave the highest recovery rate for 3-MCPD. These results
suggested that lipase from Candida sp. had higher substrate specificity
for hydrolysis of 3-MCPD esters than other lipases tested.

\emph{3.3 Optimization of hydrolysis condition for the lipase from
Candida sp.}

Firstly, pH were studied in the optimal range proposed by the
manufacturer (5 [?] pH [?]9). we studied the effect of different pH of the
3-MCPD on the yield. Hydrolysis was performed by adding 3 mL of 30\%
(w/v) sodium bromide aqueous solution (pH 5.0, pH 6.0, pH 7.0, pH 8.0,
pH 9.0) containing 150 U/mL of lipase. As shown in Fig.4, among the
tested pH, the yield of 3-MCPD was the highest when pH was 5.0,
achieving acceptable results at all three levels.It has been shown that
the change of pH value will cause the change of enzyme conformation,
leading to the change of enzyme catalytic
activity\textsuperscript{{[}14{]}}. The results show that the optimal pH
of lipase from Candida sp. is 5. What is more, the lipase from Candida
sp. is inactivated under alkaline conditions.

Secondly, to determine optimal lipase concentration in the hydrolysis
buffer, we analyzed 3-MCPD esters standard solution at 0.1 mg/mL by
using the hydrolysis buffer containing a concentration of lipase in the
range between 90 and 210 U/mL were studied in triplicate spanning, and
by adding 3 mL of 30\% (w/v) sodium bromide aqueous solution (pH 5.0)
after completion of the hydrolysis step of 30 min. Good yield for 3-MCPD
(95\%) were obtained when the lipase concentration was 150 U/mL at all
concentrations tested (Fig.5). From the result, it was decided to use
150 U/mL of lipase from Candida sp. in the hydrolysis buffer.

Thirdly, the effects of enzymatic hydrolysis time were studied by
varying from 10 min to 60 min. As shown in Fig.6, the yield for 3-MCPD
increased and then decreased with the increase of enzymatic hydrolysis
time. It was suggested that optimum time for enzymatic hydrolysis is 30
minutes. And with the increase of time, the lipase activity decreased.

Lastly, in order to find appropriate temperature for enzymatic
hydrolysis of 3-MCPD esters, different temperatures such as 15, 20,
25, 30 and 35 were studied in triplicate spanning. As shown in Fig.7,
as the temperature rises, the yield for 3-MCPD increased first, then
decreased, When the hydrolysis temperature reached 25, the lipase
activity reached the highest value, After that, the higher the
temperature, the smaller the yield for 3-MCPD. It can be seen that the
lipase is sensitive to temperature, and the activity of the lipase can
be increased by increasing the hydrolysis temperature appropriately. On
the other hand, the high hydrolysis temperature will reduce the lipase
activity and reduce the service life of the lipase.

\emph{3.4 Feasibility Study}

3.4.1 Standard curve, regression equation and detection limit

The 3-MCPD standard solution was used for derivatization to make the
standard curve, and then the sample was spiked with the standard
solution to determine the content of 3-MCPD ester in edible oil. Due to
the esterification reaction of 3-MCPD to free 3-MCPD, the structure
characteristics of 3-MCPD itself lead to the shortcomings of the
chromatographic peak width and low sensitivity in the direct
determination of 3-MCPD, so the test will be further derivatization of
3-MCPD\textsuperscript{{[}15{]}}.

The 3-MCPD standard solution of 10, 50, 100, 200, 400 ug was diluted
with the prepared standard stock solution,operate according to the steps
of 2.4 adding derivatization reagent (PBA), and test according to the
GC-MS Analysis conditions of 2.6. The standard curve was drawn with the
amount of 3-MCPD (X) as abscissa and the peak area (Y) as ordinate. At
the same time, 3 times SNR was used to determine the minimum detection
limit. Standard curve of 3-MCPD is showed in Fig.8. The linear range,
regression equation, correlation coefficient and detection limit of this
method are shown in Table 2.

3.4.2 Precision and recovery rate

Three levels of 3-MCPD ester recovery experiments were carried out with
vegetable oil as substrate, Repeat six times for each spiked level.
Under the optimal enzymatic hydrolysis conditions, the recovery of
3-MCPD was 92.1\%-101.4\%, the relative standard deviation (RSD) of
3-MCPD ester was 3.8\%-4.3\%, the results are shown in Table 3.
According to the regression equation, detection limit, recovery rate and
other methodological parameters, The method has the characteristics of
high sensitivity, good specificity and high precision for the
determination of 3-MCPD esters in vegetable oil. It can satisfy the
purpose of determining the total 3-MCPD esters in vegetable oil.

\emph{3.5 Determination of 3-MCPD esters in oil samples}

Soybean oil, rapeseed oil, palm oil and peanut oil were detected by gas
GC-MS, which was established by enzyme-hydrolyzed sample pretreatment
method. According to the experimental method of 2.5, each sample was
measured six times in parallel to take the average value, the content of
3-MCPD ester was calculated according to the peak area and linear
regression equation obtained by GC-MC detection. The contents of 3-MCPD
ester in palm oil, peanut oil, rapeseed and soybean oil were as follows:
5.52, 3.02, 2.87 and 2.73 mg/kg.

\textbf{Conclusion}

In this study, the optimal conditions for enzymatic hydrolysis of 3-MCPD
esters were obtained by single factor test as follows: lipase from
Candida sp. was selected, enzyme concentration was 150 U/mL, the
enzymatic hydrolysis temperature was 25, the enzymatic hydrolysis time
was 30 min, and the pH was 5. Secondly, the standard curve of the 3-MCPD
standard solution was drawn, and then the edible oil sample was
processed before enzymatic hydrolysis according to the optimal enzymatic
hydrolysis conditions, derived with PBA, and finally detected by GC-MC.
The content of 3-MCPD ester in four kinds of vegetable oil was obtained
by combining the standard curve, and the content of 3-MCPD ester in food
oil could be detected simply and quickly.

\textbf{Acknowledgements}

This study was supported by National Natural Science Foundation of China
(Grant No. 32001772), and Hubei Key Laboratory for processing and
Transformation of Agricultural Products (Wuhan Polytechnic University)
(Grant No. 2018HBSQGDKFA02).

\textbf{Authorship:~}

\textbf{Ting Xu:} Project administration, Resources, Writing- review \&
editing. \textbf{Min Fang:} Methodology, Software, Writing-original
draft. \textbf{Qingru Zeng:} Software, Investigation. \textbf{Xin
Liu:}Supervision and Data curation. \textbf{Shensheng Xiao:} Validation
and Investigation. \textbf{Xiaoming Jiang:} Resources, Writing- review
\& editing. \textbf{Yuepeng Lu:} Validation and
Investigation.\textbf{Zhiyong Gong:} Investigation and Funding
acquisition. All authors contributed to and approved the final draft of
the manuscript.\textbf{Yonggui Gao:} Reviewing and editing.

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\textbf{Hosted file}

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