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\begin{document}

\title{Enzymatic Conversion of Camellia Seed Oil into Glycerol Esters:
Synthesis, Characterization, and Mechanistic Analysis}



\author[1]{Jingzhi Wu}%
\author[1]{Jingjing Xiao}%
\author[1]{Suli Nie}%
\author[1]{Yan Chao}%
\author[1]{Peiwang Li}%
\author[1]{Changzhu Li}%
\author[1]{Zhihong Xiao}%
\author[2]{Hongmei Wu}%
\affil[1]{Hunan Academy of Forestry}%
\affil[2]{University of South China}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The conversion of triglycerides in edible oils into diacylglycerols
(DAGs) is of great significance for obtaining products with health
benefits. Camellia seed oil (C-oil), which is rich in oleic acid and
linoleic acid, is an excellent raw material for the production of DAGs.
In this study, the hydrolysis rate reached 87.14\% under optimal
hydrolysis conditions (reaction temperature of 60 \selectlanguage{ngerman}°C, reaction time 24
h, 30\% water content and 4\% enzyme addition) with RMIM as catalyst,
and camellia seed oil diacylglycerol (C-DAG) with a content of 62.49\%
was also given under optimal esterification conditions (vacuum system,
3\% enzyme addition, 2\% water addition, reaction temperature of 50 oC,
and substrate molar ratio of free fatty acid to glycerol of 1:1). The
high content of DAG was obtained by a coupled method, which eliminated
the purification steps and reduced production costs. C-oil, and C-DAG
have been characterized by means of GC, TG, DSC, and GC-IMS. Our results
showed that the enzymatic coupling method did not affect the structural
composition of the substances themselves, but did affect the
crystallization and melting properties of the oils. Moreover, the taste
of C-DAG was more delicate flavor than that of C-oil. Finally, the
reaction mechanism has been analyzed by means of infrared spectroscopy,
which showed that C-oil was mostly hydrolyzed to free fatty acids. C-DAG
exhibited ester C-O stretching vibrations in the range 1280--1030 cm-1,
indicating successful esterification reaction between camellia seed oil
free fatty acids (C-FFAs) and glycerol under catalysis by the enzyme.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


October 20, 2023

Dear reviewer,

Thank you very much for handling our manuscript and giving us an
opportunity for revision. Now we are pleased to submit our revised
article entitled ``\textbf{Enzymatic Conversion of Camellia Seed Oil
into Glycerol Esters: Synthesis, Characterization, and Mechanistic
Analysis} '' (Manuscript ID: JAOCS-23-0156).

A point-by-point response to reviewers' comments has been attached and
all the modifications were used the ''Track Changes'' function in the
revised manuscript. We appreciate the critical comments from reviewers
and we hope that all the questions have been satisfactorily addressed.
we believe that our novelty and scientific merits are presented in a
clearer manner in the revised manuscript for potential publication in
processes.

Use Track Changes to show where the manuscript has revised the
manuscript. Changes in the article are marked in blue font with yellow
markers.

Kindest regards,

Dr. Changzhu Li, representative of all of the authors

State Key Laboratory of Utilization of Woody Oil Resource,

Hunan Academy of Forestry,

658\#, Shaoshan South Street,

Changsha 410004, China

Email address: Lichangzhu2013@aliyun.com

\textbf{Response to Reviewer 1}

We are very grateful to your comments for the manuscript. According to
your advice, we amended the relevant part in manuscript. All of your
questions were answered one by one. We will make the necessary markings
in the manuscript with red markers.

\textbf{Question 1: The article contains a large number of language and
grammar errors.}

Thank you for your valuable comments. This article has undergone
extensive grammatical and linguistic changes and has been touched up by
native English speakers. And we hope the revised manuscript could be
acceptable for you.

\textbf{Question 2: The novelty is not found in this paper. There have
been many reports on the optimization of conditions for DAG, and the
comparison of C-oil, C-FFA, and C-DAG has no research significance.}

Thanks for your helpful comments. The novelty for this paper lies in the
fact that the higher content of diglycerides is obtained by a two-step
enzymatic coupling, which simplifies the purification process that needs
to be carried out in order to obtain a high content of diglycerides, and
achieves the diglyceride content that most of the current reports need
to be purified in order to achieve, and reduces the cost problems
associated with the purification step. The abstract of the article has
been revised to highlight the significance of the methodology by
replacing the abstract on page 2, lines 16-19 in the original manuscript
with the following:

The results showed that under the optimal conditions of hydrolysis and
esterification, the hydrolysis rate was 87.14\% and the DAG content was
62.49\%. The high content of DAG was obtained by the coupling method,
which reduced the cost of purification step in the production process.

\textbf{Question 3: Moreover, this paper is not written according to the
submission style of JAOCS. Unfortunately, it is not acceptable.}

Thanks for your thoughtful comments. The revised version of this article
was revised and rewritten in accordance with the JAOCS guidelines for
authors and the latest published articles.

\textbf{Response to Reviewer 2}

We are very grateful to your comments for the manuscript. According to
your advice and comments, we amended the relevant part in manuscript.
All of your questions were answered one by one.

\textbf{Question 1: This work could be of interest, but in its current
form it is difficult to assess. The text is poorly written, and it is
difficult to understand the meaning of many phrases. Furthermore, the
references are inappropriately placed in the text, which is riddled with
inconsistencies. For example: ''At present, there are few types of
healthy oils and fats on the market, and the quality is uneven.'' Are
you sure that there are few healthy oils on the market? Please provide a
reference. ''Compared with TAG, DAG has similar energy value,
digestibility and antiobesity characteristics. Their calorific value is
low, which has a positive effect on lipid metabolism, decreasing total
serum cholesterol, low-density lipoprotein cholesterol, and fat
accumulation, thereby reducing liver and abdominal fat levels.'' If you
affirm that TAG and DAG have the same properties, it is not worth
carrying out the study that you have carried out\ldots{}This is a sample
of the poverty of expression throughout the entire manuscript.}

We sincerely thank the reviewers for their careful reading. We have
rewritten lines 49-130 on pages 3-5 of the original manuscript as
follows:

Therefore, it is of great significance to further increase the content
of DAG in human diet for health. The preparation methods of DAGs are
principally chemical and biological enzyme methods. The chemical method
has advantages of high reaction rate and simple operation (Zhong et al.,
2010). However, it increases the cost incurred in removing organic
reagents from the product. The bioenzymatic method using lipase as a
catalyst can effectively avoid contamination with organic reagents. At
present, the main methods for the preparation of DAGs by bioenzymatic
methods are esterification, hydrolysis, glycerolysis, and
transesterification. The esterification method uses lipase as a catalyst
to esterify free fatty acids (FFAs) and glycerol in water to obtain
DAGs. This method requires the use of vacuum, silica gel, molecular
sieves, and nitrogen stream to separate the water produced in the
reaction (Arcos et al., 1998; Weber \& Mukherjee, 2004; Lo et al.,
2004). In the hydrolysis method, the fatty acids in TAG are substituted
with alcohol hydroxyl groups in the presence of water and lipase to form
FFAs, DAGs, monoacylglycerols (MAGs), and glycerol. The disadvantage of
this method is that the degree of hydrolysis is difficult to control and
often occurs at the oil-water interface (Li et al., 2018). The DAG
content obtained by a single method is not high, and the product has to
be purified by molecular distillation, solvent crystallization,
supercritical CO2 extraction, and silica column chromatography (Huang et
al., 2023; Li et al., 2018). Combining multiple methods may be an
effective way to prepare samples with high contents of DAG. Li et al.
(2021) partially hydrolyzed soybean oil using Lipase DF ``Amano'' 15 and
obtained DAG content of 55.2\% by molecular distillation. Other products
were combined with PGL AOL-V269 D esterification reaction and molecular
distillation to obtain high purity DAG (99.3\%). Liu et al. (2020)
prepared DAGs rich in \selectlanguage{greek}α-\selectlanguage{english}linolenic acid by a two-step enzymatic method.
The combination of Lipase TL100L and Lipase PCL hydrolyzed silkworm
pupae oil to obtain 96.51\% FFA, and then silkworm pupae oil diglyceride
oil with a purity of 97.01\% was obtained by molecular distillation
after esterification catalyzed by lipase PCL. Based on the above
analysis, it is indeed feasible to prepare a high content of DAG by
coupling two or more methods.

\textbf{Question 2: On the other hand, it is necessary to provide
evidence of the oxidative stability of the obtained DAG. For example,
diene index, peroxide value, etc.}

Thanks for the reviewer's valuable comments. The peroxide value of
glycerol diesters was in Table S3 in Supporting Materials in the
original manuscript, and is now supplemented and modified on page 17,
lines 890-891 (i.e., the last sentence of the second paragraph of 3.5
Determination of flavor) in the revised manuscript, with the results of
its peroxide determination added in the revised manuscript ''TABLE 1
Fatty acid composition of oil samples, as well as hydrolysis or
esterification rates and physicochemical indicators''. We examined the
literature and other avenues where the diene index is one of the most
important indicators of the properties and stability of FCC gasoline
during production and storage and found that the diene index is
infrequently used in edible oils. However, it is common in petroleum
characterization.

\textbf{Question 3: Furthermore, no information is provided on the
standards used to characterize the fatty acids. Please show
chromatograms. What quality protocol has been followed in the
determination of fatty acids? Thus, I recommend a severe editing and
restructuring of the entire manuscript, and that it be resubmitted.}

Thank you for your constructive suggestions. For the standards for the
determination of fatty acids, the specific method was added to the text
as National Standard of the People's Republic of China (GB 5009). 168:
2016), and the fatty acid gas chromatograms of the three oil samples are
shown in ''FIGURE S1 GC plots of the major fatty acids of three oil
samples of fatty acids'' in the Supporting Information.

\textbf{Response to Associate Editor}

We are very grateful to your comments for the manuscript. According to
your advice and comments, we amended the relevant part in manuscript.
All of your questions were answered one by one.

\textbf{Question 1 According to the issues raised by the reviewers, the
manuscript should be revised.}

Thanks to the advice of the editor, the revised draft has been modified
in accordance with the comments of the reviewing experts, and a separate
one-by-one response is provided.

\textbf{Question 2: The Introduction contains many content that not
relevant to this study. The authors are suggested to condensed to as
much as 3 pages.}

Thanks for your thoughtful comments. We have revised and rewritten the
abstract section of the original manuscript, deleted Reviewer 2
suggesting tedious sentences, examined similar sentences and the
introduction with many irrelevant to this study, revised and deleted it,
and finally have compressed it to 3 pages. Delete lines 46-48, 51-57,
58-63 on page 3, lines 80-81, 85-94, 100-103 on page 4, and lines
111-115, 118-121, 124-126 on page 5 of the original manuscript. And
rewrite lines 58-71 on page 3 and 73-76 on page 4 of the original
manuscript.

\textbf{Question 3: In Materials and methods, the city, province, and
country of the materials/equipment should be provided.}

Thanks for your constructive suggestion. In Materials and Methods,
revisions are made according to ''city, province, country where
material/equipment is located''.

\textbf{Question 4: There are issues with the English (syntax, grammar,
etc.) throughout the manuscript. These must be addressed with the help
of a native speaker or English language editing service, such as Wiley
Editing Services.}

Thanks for your thoughtful comments. English (syntax, grammar, etc.) in
the revised manuscript was touched up in a native speaker or English
editing service and marked in gray.

\textbf{Question 5: At the end of the paragraph, some sentences have two
period while other have no period, which need carefully revised.}

Thanks for your constructive suggestion. The original manuscript was
examined and an inaccurate expression of periods was indeed found to be
a problem, and the format of the article has been checked and corrected
in the revised manuscript.

\textbf{Question 6: The format of the references should be revised
according to the JAOCS author guideline. Please provide the whole
references in the reference list not in the text.}

Thanks for your thoughtful comments. The format of references has been
revised according to the JAOCS author guidelines and the latest
articles, and references are provided and referenced as folders.

\textbf{Response to Senior Associate Editor}

We are very grateful to your comments for the manuscript. According to
your advice and comments, we amended the relevant part in manuscript.
All of your questions were answered one by one.

\textbf{Question 1: Very confusing paper. A few varying items are
discussed and not fully articulated.}

Thanks to the advice given by the editor, we have read it carefully and
indeed found that the article appears to be unclear in its expression,
weak in its analytical purpose, and repetitively expressed in sentences,
resulting in a lack of clarity in the focus of the article. Various
parts have been revised or rewritten in the revised draft.

\textbf{Question 2: No flavor analysis was done! Only headspace
analysis. They are very different.}

Thanks for your helpful comments. In the revised version of page 16,
''3.5 Determination of flavor'', the flavor analysis was carried out by
headspace sampling using Flavour Spec\selectlanguage{ngerman}®, Gesellschaft für Analytische
Sensor systemembH, Dortmund, Germany. The flavor analysis was performed
by headspace injection using Flavour Spec®, Gesellschaft für Analytische
Sensor systemembH, Dortmund, Germany. To avoid misunderstanding, the
term ''headspace vial'' has been replaced with ''headspace sample vial''
throughout the text.

\textbf{Question 3: Is the paper about hydrolysis or esterification.}

Thanks for your thoughtful comments. The article is about the enzymatic
hydrolysis of tea seed oil to obtain fatty acids, and then
esterification of fatty acids with glycerol at the enzyme to obtain
diglycerides, which do not need to be purified to achieve 62.49\%
diglyceride content.

\textbf{Question 4: No purification of the C-FFA nor C-DAG were
disclosed. What was the final composition of the ''oil'' produced. Was
it a DAG oil or not? In most of the world, organic oils are only
purified. If any modification occurs (including enzymatic) it is not
considered as an organic oil but a modified oil only!}

Thanks for your helpful comments. We did not purify C-FFA and C-DAG.
Through testing, we found that the hydrolysis rate of the hydrolysis
reaction using the enzyme as a catalyst was 87.14\%, and after the
esterification reaction, a diglyceride content of 62.49\% was obtained,
and the rest were mainly TAG, MAG, FFA and trace elements. The resulting
DAG is modified oil. Additionally, ''DAG oil'' in the supporting
information has been changed to ''C-DAG''.

\textbf{Question 5: References are not correct. Please look at the
authors pages for requirements.}

Thanks for your valuable comments. The format of the references should
be revised according to the JAOCS author guideline.

\textbf{Question 6: English must be greatly improved or the paper will
be rejected without re-submission possible.}

Thanks for your constructive suggestion. We have touched up the
revisions and rewritten some of them.

\selectlanguage{english}
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\end{document}

