Accepted Manuscript

Title: Supercritical fluid extraction of free amino acids from

sugar beet and sugar cane molasses

Authors: Mona Varaee, Masoud Honarvar, Mohammad H.

Eikani, Mohammad R. Omidkhah, Narges Moraki

PII: S0896-8446(18)30450-9
DOI: https://doi.org/10.1016/j.supflu.2018.10.007
Reference: SUPFLU 4384

To appear in: J. of Supercritical Fluids

Received date: 5-7-2018

Revised date: 6-9-2018
Accepted date: 6-10-2018

Please cite this article as: Varaee M, Honarvar M, Eikani MH, Omidkhah
MR, Moraki N, Supercritical fluid extraction of free amino acids from sugar
beet and sugar cane molasses, The Journal of Supercritical Fluids (2018),
https://doi.org/10.1016/j.supflu.2018.10.007

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Supercritical fluid extraction of free amino acids from sugar beet and sugar
cane molasses
Mona Varaeea, Masoud Honarvara,*, Mohammad H. Eikanib, Mohammad R. Omidkhahc,
Narges Morakid

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a
Department of Food Science and Technology, College of Agriculture and Food Science, Science and Research

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Branch, Islamic Azad University (IAU), Tehran, Iran
b
Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST),

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Tehran, Iran
c
Department of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
d
Department of Fisheries Sciences, College of Marine Science and Technology, North Tehran Branch, Islamic Azad

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University (IAU) , Tehran, Iran

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
Corresponding author at: Department of Food Science and Technology, College of Agriculture and Food Science,
Science and Research Branch, Islamic Azad University (IAU), Tehran, Iran, P.O. Box 1477893855. Tel:
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+982144868536 ; Fax: +982144868538.

E-mail address: m.honarvar@srbiau.ac.ir (M. Honarvar).
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Graphical abstract
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Research Highlights:
 The SFE was carried out for the extraction of AAs for SGB and SGC molasses.

 The effects of pressure, temperature and time were evaluated to optimize the extraction.

 Extraction recovery of SGB molasses were higher than the ERs of SGC molasses.

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The application of SFE for SGB and SGC molasses improves the value added in sugar

industry and reduces the environmental pollution.

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Abstract
In this work supercritical fluid extraction (SFE) was used to extract free amino acids (AAs)

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from sugar beet (SGB) and sugar cane (SGC) molasses. The effect of different variables such as,
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pressure (150-350 bar), temperature (40-60 ˚C) and extraction time (10-90 min) was evaluated to
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optimize the extraction using response surface methodology (RSM).The results of SGB and SGC
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molasses extraction showed that the optimal condition were 184 and 316 bar; 43 and 50 ˚C and
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76 and 76 min, respectively. Under the optimum condition, the extraction recoveries of AAs for
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SGB and SGC molasses were 42% and 31% for aspartic acid, 63% and 37%, for glutamic acid,

46% and 48% for alanine and 31% and 20% for lysine sequentially. This study indicated that
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SFE might be employed to extract of AAs from SGB and SGC molasses with acceptable

selectivity and extraction efficiency.

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Keywords: Supercritical fluid extraction; Sugar beet molasses; Sugar cane molasses; Amino

acids extraction.
1. Introduction

Molasses is a viscous and dark liquid by-product of sugar beet (Beta vulgaris var.

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saccharifera) or sugar cane (Saccharum L.) obtained as the final effluent of sugar refinement.

Molasses contains profitable components such as fermentable carbohydrates (sucrose, glucose,

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fructose) and considerable nonsugar organic materials (betaine, other amino acids; minerals and

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trace elements; vitamins, etc.). Molasses has been mainly employed as a supplement for animal

feed and ethanol production [1-7].

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Amino acids (AAs) have central roles as building blocks of proteins and as intermediates in
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metabolism [8, 9]. Twenty AAs are common in make up the body; and eight of them are named
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as essential AAs, since the body cannot synthesis them from other components. Therefore, they
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must be obtained from foods or nutritional supplements. AAs are required in pharmaceutical and
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food domains thus high value-added AAs can be recovered from by-product and utilized in
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medical, cosmetic, animal feed, and other industrial applications [9, 10].

Techniques to extract these valuable components from raw materials are quite essential to
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obtain purified compounds. The most common method for the extraction of free AAs from plants

is solvent extraction using water or boiling mixtures of methanol–water [11, 12]. Recently,
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supercritical fluid extraction (SFE) using carbon dioxide, as a solvent-free method, has been

employed for extraction of vitamins, antioxidants, and AAs from food industry by-products [13-
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16]. Usually, supercritical carbon dioxide (SC-CO2) extraction has been considered as an

efficient method to extract low-polarity components. But, for the extraction of AAs which are
polar substances, polar modifiers or co-solvents, such as methanol and ethanol, should be used to

enhance the solute solubility.

SC-CO2 extraction displays several prevalence for instance; it does not need adding

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explosive or toxic solvents and leaves no toxic residue [17-21]. Previous works demonstrated

that SC-CO2 extraction has been utilized to extract different components, e.g. essential oils [22-

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25], lycopene [26], carotenoids [27, 28], glycosides [29] oleoresins [30], anthocyanins [31]

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astaxanthin [32].

Indeed, few investigation have been released about the SFE extraction of free AAs and

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neither of them was employed on liquid and viscous substances such as sugar beet (SGB) and

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sugar cane (SGC) molasses. In this view, the addition of a polar organic modifier, such as
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methanol, is necessary to raise the solute solubility. The significance of the extraction of AAs
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matter from SGB and SGC molasses is the recovering wastes as the raw material, and adequately

producing AAs from the sugar industry’s residues. The main goal of the present work is to obtain
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AAs from SGB and SGC molasses using SFE extraction and optimize the extraction method for
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molasses samples.
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The response surface methodology (RSM) as the experimental design procedure was

applied. It was based on the orthogonal central composite design (OCCD) employing
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temperature, pressure and extraction time as influential factors.

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2. Experimental

2.1. Raw materials, Chemicals and reagents

 The SGB and SGC molasses with 18-25 wt% water content were obtained from Hegmatan

Co., Ltd. (Hamedan, Iran) and Developed Sugar Cane Co., Ltd. (Ahvaz, Iran) respectively.

Hydrochloride acid, sodium hydroxide, sodium acetate, sodium borate, HPLC-grade acetonitrile,

carbonate buffers, methanol and anhydrous sodium sulphate were obtained from Merck

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(Darmstadt, Germany), FMOC-Cl, ADAM and amino acid standards (Aspartic acid, Glutamic

acid, Lysine, Alanine) were purchased from Sigma(Milano, Italy). All standard solutions were

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stored at 4℃ and protected from light. Carbon dioxide with 99.99% purity was obtained from

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Sabalan Co. (Tehran, Iran) and utilized in all of the extraction experiments.

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2.2. Apparatus and extraction procedure

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A Separex (Champigneulles, France) system in SFE mode was employed for all
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experiments. The extractions were accomplished using a 100 mL volume stainless steel
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extraction vessel. An adjustable separator (240 mL) from Separex Co. (Champigneulles, France)

was applied in the SFE system to collect the extracted amino acids and control. The required
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pressure was maintained by a back pressure regulator and checked by an automatic manometer.
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The temperature was checked by an automatic thermometer. The heating of the system was
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accomplished by an oven. The Separex pump (LGP-50) works with a maximum CO2 flow rate of

80 mL/min of liquid or 50 g/min up to 1000 bar. In addition, co-solvent pump works with a
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maximum 10 mL/min up to 400 bars. Filter absorbs bed cartridge in the recycling loop to trap

lightest and volatile particles. Stirrer supplies of a magnetic stirrer installed on the extractor at
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350 or 700 bar. SFE equipment is schematically represented in Fig. 1.

Fig. 1
 The suitability of the method was investigated for the extraction of AAs from SGB and

SGC molasses. To achieve to the lowest water content, 5 g of molasses was poured in a porcelain

mortar containing 70 g of anhydrous sodium sulphate, and the mixture was blended for a few

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minutes until an apparently dry material was obtained.75 g dried powder with 2-3 wt% water

content were mixed with glass beads (2 mm diameter; 1:2 weight ratio) to prevent agglomeration

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and act as a carrier to increase the surface area. The final powder was poured in 100 mL

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extraction cell. Then, another filter was placed on the top of the vessel and the vessel was

closed. Injection of methanol was occurred before pressurizing the cell with CO2. Therefore,

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using 30 mL methanol or nearly 6 g methanol/g molasses (dried basis) was added through the

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six-port valve into the CO2 stream at the flow rate of 1 mL/min. The system was equipped with
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an air-driven pump to deliver the CO2 to the extraction cell, which was placed in a temperature-
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controlled oven. Finally, SFE was carried out using a static extraction to enhance the sample-

solvent contact. In this work, after the pre-set static time, by opening the separator valve, CO2
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was transferred to the extract collector and CO2 gas is separated from the extract.
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The precision in pressure and temperature measurements were ±1 bar and ±1˚C,
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respectively. 5 min time was given to stabilize temperature and pressure. The separator

operating temperature was fixed at 25˚C. The extracts were collected in a 5 mL volumetric flask
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for further HPLC analysis.

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2.3. Derivatization procedure

AAs were derivatized (FMOC-AA) at room temperature using a pre-column procedure.

Under these conditions a volume to 300 μL of molasses or extracted AAs molasses (or a standard
solution of AAs) was added with 600 μL of a 200mM borate buffer (pH 10.0). Then, 600 μL of

15mM FMOCCl (in acetonitrile) was added to the extracted molasses and derivatization

occurred. The reaction was stopped after 5 minutes by the addition of 600 μL of 300mM ADAM

(water-acetonitrile, 1:1, v/v), and the reaction lasted for 1 min to form the FMOC-ADAM

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complex, Fig. 2. The sample was then filtered through a 0.45-µm polytetrafluorethylene (PTFE)

and analyzed by HPLC-UV in the wavelength of 263 nm. The total time required for the

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derivatization procedure was 6 min [33].

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Fig. 2

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2.4. HPLC analysis N
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The analysis of the AAs in extracts was performed by high performance liquid
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chromatography. The HPLC system consisted of a Spectra Physics (San Jose, CA) was equipped

with a 8700 XR ternary pump, a 20-µL Rheodyne (Cotati, CA) injection loop, an SP8792
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column heater, a 8440 XR UV-Vis detector that was set at 263 nm and a 4290 integrator linked
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via Labent to a computer. Chromatographic data were analyzed using ChromanaCH software,
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version 3.6.4 (Tehran, Iran). For separation, a 250- × 4.6 mm column packed with 5-µm particle

size C18 (Sugelabor, Madrid, Spain) was employed at 25°C. A mixture of sodium acetate 50 Mm
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(pH = 4.2) and acetonitrile (60:40) at a flow rate of 1.0 mL min−1 was implemented as the mobile

phase where the former and latter were used as eluent A and B respectively. All the
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chromatographic measurements were carried out in the linear range [33].

2.5. Response surface methodology

 RSM, based on implicating OCCD, was used to separately assess the role of three variables

on AAs extraction from SGB and SGC molasses. In principle it not only led to estimate the main

effects separately, but also by use of a fitted second-order mode, finding the optimum conditions

was applicable. The first-order two-level design with center runs is properly augmented to allow

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estimation of second-order terms. As the second-order response surface model is widely used for

process optimization [34]. The total number of experiments (N) to be attained by accomplishing

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OCCD which is equal to 20 by using Eq. (1):

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N = 2f + 2f + N0 (1)

where, f is the number of variables [35]. The three independent analyses variables and their

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ranges were according to pressure (X1) from 150 to 350 bar, temperature (X2) from 40 to 60 ˚C,
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extraction time from 10 to 90 min with five levels selected for each variables: -α, -1, 0, +1 and
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+α. The axial points are set at +α and -α from the center of the experimental area that was
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computed equal to ±1.5.The coded, ranges and levels of the independent variables employed in

the RSM design are listed in Table 1.For both raw materials, the experimental design based on
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OCCD containing three variables, needed 20 experimental runs with six at the central point was
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accomplished which presented in Table 2. All experiments were implemented in a randomized

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pattern to reduce the effect of unrecognized variability. All experiments except the center point

(0, 0, 0) were carried out in three replications and the central point was replicated six times.
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Table 1
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Table 2

The experimental data were fitted in Eq. (2) as a second-order polynomial equation

consisting of the linear and the interaction effects of each variable to predict Y variable [35]:
 (2)

k k k

Y= β0 + ∑ βjZj + ∑ βjj Zj2 + ∑ ∑ βij ZiZj

j=1 j=1 1<𝑗

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where, Y is the response or output (yield as total peak area), k is the number of the patterns, i and

j are the index numbers for patterns, β0, βi,βii, βij are the offset, linear, quadratic and interaction

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terms, respectively. Zi and Zj are independent variables (pressure, temperature and time).

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Response surfaces were depicted by the fitted model. The software package Design-Expert 8.0.3

(Stat-Ease, Minneapolis, MN, USA) was applied for experimental design, data analysis and

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obtaining the response surface plots.
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3. Results and discussion
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In the present study, SFE technique was used to determine the extraction of AAs from SGB
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and SGC molasses. There are considerable variables that can affect the extraction efficiency of
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AAs. As mentioned before, these include pressure, temperature and time of the extraction.

Optimization of theses parameters has been considered by using the RSM. The results have been
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discussed in the following sections.

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3.1. HPLC analysis of raw materials

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SGB and SGC molasses contain approximately eighteen AAs [36-38]. However, only four

of them, incl. aspartic acid, glutamic acid, alanine and lysine have been selected for this research

study. Aspartic and glutamic acids were selected due to the fact that they are the predominant

amino acids in the SGB and SGC molasses [37] and both are the most abundant neurotransmitter
in the central nervous system [39, 40]. Although, alanine and lysine are the trivial AAs in the

SGB and SGC molasses [37], alanine has a considerable role in transferring nitrogen from

tissues to the liver and cooperates in the metabolization of glucose for energy that leads to the

balance of glucose and nitrogen in the body. Lysine is an essential AA and cannot be synthesized

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by mammals [41, 42]. Samples consisting of 20 μL of the different SGB and SGC molasses were

derivatized using the FMOC procedure and analyzed by HPLC as control samples to determine

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the four aforementioned AAs. The identification of AAs in the samples was based on the

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comparison between the relative retention times of the AAs extracts with standards. The HPLC

results are shown in Table 3.

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Table 3

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3.2. SFE and statistical analysis
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The experimental data of the total peak area obtained from the OCCD for SGB and SGC
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molasses are presented in Table 2. Responses (RSGB and RSGC) were represented the total peak
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area.

Table 4 and 5 show analysis of variance (ANOVA) for SGB and SGC molasses,
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respectively. ANOVA was performed to confirm the suitability of the response surface model

and to evaluate the effect of the principal parameters and their interactions on the response.
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ANOVA was carried out with an F-test (lack of fit), for validation. The “lack of fit” was not
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significant (p = 0.05) for both SGB and SGC molasses data. F-values of SGB and SGC molasses

were 87.92 and 41.72 respectively, and they are both significant. F-value in ANOVA test

determines p-value which demonstrate the factors that were significant (p<0.05). As it can be

seen in the Tables 4 and 5, only the significant parameters have been kept into account to make
the model. The response equation fitted to the experimental data consisting of R2-value of the

SGB and SGC molasses are 0.9846, 0.9681, respectively. In this study, the adjusted R2 for both

SGB and SGC molasses were 0.9734 and 0.9449, obviously within acceptable limits of R2≥ 0.9.

Total peak area in both cases was selected as the optimization criteria.

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Table 4

Table 5

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For an experimental design with three factors, the mathematical model was expressed as

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presented in Eq. (3-4) for SGB and SGC molasses, respectively.

R SGB: -2.290E+003 -7.937× X1 – 8.413E+001× X2 + 2.639E+002× X3– 3.307E-001 × X1X3 –

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2.881 × X2X3 + 5.355E-002×𝑋12 + 2.150×𝑋22 - 3.334E-001×𝑋32 (3)

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R SGC : -1.234E+004 – 2.721E+ 001× X1 + 6.098E+002× X2 + 2.711E+001× X3 + 1.811E-001 ×
X1X2 + 4.158E-002 × X1X3 – 6.444E-001× X2X3 + 3.467E-002×𝑋12 - 6.152𝑋22
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(4)
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where X1, X2 and X3 are extraction pressure, temperature and time respectively. The response

surface plots were generated through a statistical process that describes the design and OCCD
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data. The goodness-of-fit of the empirical model for SGB (a) and SGC molasses (b), is described
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in Fig. 3. Actually the horizontal axis presents predicted peak area which the peak areas that will

expect to obtain and the vertical axis demonstrates experimental peak area which the peak areas
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that are attained during examinations. The Fig. 3 shows the points of predicted peak area and
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experimental peak area are nearly coinciding of each other’s.

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Fig. 3

3.4. Effects of extraction factors on the SFE

 In Figs. 4a-d and 5a-dthe relationship between the perceptively and response variables are

have been presented in a three-dimensional representation of the response surface and two-

dimensional contour plots. Desirability function was measured to detect the optimum conditions.

Figs. 4a-b and 5a-b demonstrate the interaction between extraction time and temperature in SGB

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and SGC molasses. As can be seen, in Figs. 4a-b by raising the extraction temperature in SGB

molasses samples from 40 to 43 ˚C, the extraction efficiency is enhanced impressively. By

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increasing the extraction temperature above 44˚C thermal denaturation and decomposition of

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AAs was occurred and the efficiency of extraction was decreased. It is in accordance with other

published works [43]. In addition; the effects indicate that the amount of extracted AAs was

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increased by intensifying the extraction time from 50 to 76 min. Figs. 5a and 5b indicate that by

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increasing the extraction temperature in the SGC molasses patterns from 50 to 57 ˚C, the
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extraction performance is increased considerably. The application of higher temperature above
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57 ˚C reduced the extraction efficiency. The results indicate that higher temperatures lead to AAs

decomposition. It is in accordance with other published works [43].

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Fig. 4a, b, c, d

Fig. 5a, b, c, d
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Compared to SGB molasses, the SGC molasses samples needs higher temperature since it

has a complex matrix comprising more tannin, starch, fibers, lignin, pectin, and minerals [44-47].
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Moreover, the amounts of extracted AAs in SGC were extremely grown from 76 to 90 min, but

in lower time it had not acceptable extraction efficiency.

 The effects of interaction pressure with extraction time are shown in Figs.4c-d and 5c-dfor

SGB and SGC molasses, respectively. Figs. 4c-d show lower pressures (184 bar) had important

role for recovery of AAs fromSGB molasses while as presented in Figs. 5c-d for SGC molasses,

higher pressures (316 bar) is required to increase the extraction efficiency. As it is obvious there

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is an inverse relation between the two independent factors of pressure and time. In other words in

lower pressure and longer duration the efficiency of extraction is higher; which may be due to

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the nature and structure of SGB molasses with less sticky compounds (i.e. tannin and fibers) [48,

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49].Moreover as if seen through the plot the contour (Fig. 4C) lines of the first and second

optimized points (184 bar, 76 min), (316, 20 min) have a significant distance which means there

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is a larger step to increase the amount of the extraction by increment of pressure and decrement

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of duration; which as it explains before it may be due to the chemical structure of SGB molasses.
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On the other hand, suitable extraction time of SGB is evaluated same as SGC molasses. The
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experiments were performed at various extraction times in the range of 10-90 min. The

extraction of AAs in both SGB and SGC molasses might increase by increasing the time from50
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to 76 min as presented in Figs. 4c-d and 5c-d. By increasing extraction time from 76 to 90 min,
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extraction performance has remained relatively constant. It can be concluded the best range of
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pressure and time for future researches on SGB molasses are 180 up to 320 bar, and 20 to 80

minutes.
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3.5. Determination of optimal SFE

On these experiments, the optimized SFE conditions for SGB were obtained as 184 bar, 43˚C

and 76 min and the result for total peak area was 2777, Table 6. Pucarevic et al. [2013] studied
the supercritical fluid extraction of Tebupirimphos residues (a pesticide) in SGB 240 bar and 45

˚C that expressed the influence of temperature and pressure.

In addition, the optimum conditions of SGC molasses were obtained at 316 bar, 50 ˚C and

76 min and 1104 was the maximum total peak area, Table 6.

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Table 6

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In comparison with the SGB molasses, SGC molasses needed higher temperatures and

pressures. It can be noticed that more viscous SGC molasses has more tannin, fibers, starch and

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minerals that make the AAs extraction more difficult [42-47]. Guan et al. [2014] investigated the

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antioxidants from SGC molasses in the same SFE extraction condition and reported nearly the

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similar results. Gracia et al. [2007] evaluated the isolation of aroma compounds from SGC pulps
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and Pasquini et al. [2005] investigated SGC bagasse by SFE. Their results are partly in
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agreement with our study where the effects of temperature and pressure were distinctly

represented.
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The HPLC chromatograms of the optimum SFE extract for SGB and SGC molasses are

presented in Figs. 6a-b.

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Fig. 6a
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Fig. 6b
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Extraction recovery (ER) was calculated as the ratio of the final concentration of the AAs

after extraction (Cn) to its initial concentration (C0) according to the following Eq. (5). The
amounts of initial AAs of SGB and SGC molasses and the extraction recovery of AAs are

presented in Table 3.

𝐶
ER (%) = 𝐶𝑛 ×100 (5)
0

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As the results of extraction recovery indicated, the ERs of SGB molasses were higher than

the ERs of SGC molasses. The mean value of extraction recovery for four AAs concerned with

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SGB molasses was 46% while this was 34% for SGC molasses. It can be perceived that the

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extraction yield of SGB molasses, containing higher concentration of AAs, was the results of less

sticky components, e.g., tannin, starch and fiber. Therefore, AAs extraction can be carried out

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more easily [44-46, 48-49]. The current results are better than those which have been reported
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elsewhere. AAs extraction from soft-shell fish egg performed by Shen et al. [2008] resulted to as
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low as 29% extraction recovery.
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Regarding selectivity of the SFE, it should be stated that SGB and SGC molasses are
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mixtures of sugars (53 and 64% w/w), non-sugar materials incl. AAs (19 and 10% w/w), water
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(16.5 and 20% w/w) and ash (11.5 and 8% w/w), respectively [44]. Obviously, it could be

anticipated to obtain a mixture containing AAs and also sugars in the separator. Actually, SFE of
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sugar types, its determination and its concentrations were not aim of this study but some of

distinct researches on carbohydrates has been studied [53-56] at the higher temperatures (60 to
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100 oC), and using ethanol as co-solvent. Carbohydrates have higher molecular weights and it is
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clear that ethanol is a better solvent for them. Because of probable AAs decomposition, in the

present study, intentionally lower temperatures were selected (40 to 60oC). In addition, methanol

as a more polar solvent and recognized solvent for extraction of AAs was applied. AAs are
generally more polar than the sugars of molasses and it could be expected that methanol

dissolves AAs more selectively [7].

4. Conclusions

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In this study, for the first time the SFE extraction was carried out successfully for the

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extraction of AAs for SGB and SGC molasses. The evaluation of the results demonstrated that

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SFE extraction is an effective method in order to extraction of AAs from SGB and SGC

molasses. Furthermore, the optimum conditions to extract the AAs with the highest total peak

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area were found. SGC molasses required higher temperature and pressure for extraction due to

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higher content of tannin, fiber, minerals and complicated matrix. Finally, it is worth to mention
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that the application of SFE for SGB and SGC molasses not only improves the value added in
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sugar industry and reduces the environmental pollution but also the finding in this study might be

employed for other industrial wastes with similar structure to extract of valuable AAs. The
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optimum operating conditions reported here correspond to a laboratory scale free AAs extraction
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from two types of molasses, helping development of the concept experimentally. Extractions at
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pilot or industrial scale may have a different set of optimum operating conditions, which would

depend on material pretreatment and configuration of the extraction plant.

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Acknowledgements
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The authors are gratified to Science and Research Branch, Islamic Azad University, Iranian

Research Organization for Science and Technology (IROST), Farogh laboratory, Tarbiat

Modares University, Hegmatan Sugar Co. and professors Mehrdad Ghavami and Hamid Asiabi

for their help and assistance.

References

[1] V. Valli, A. M. Gomez-Caravaca, M. F. Caboni, A. Bordoni, M. D. Nunzio, F. Danesi, Sugar

cane and Sugar beet molasses, antioxidant-rich alternatives to refined sugar. J. Agric. Food

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Chem. 60 (2012) 12508–12515.

[2] A. Baiano, Recovery of biomolecules from food wastes-a review. Molecules 19 (2014)

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14821-42.

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[3] C. Sguarezi, C. Longo, G. Ceni, G. Boni, M. F. Silva, M. D. Luccio, M. A. Mazutti, Inulinase

production by agro-industrial residues: optimisation of pretreatment of substrates and production

U
medium. Food and Bioprocess Technology 2 (2009) 409–414.
N
[4] J. B. Marcus, Carbohydrate Basics: Sugars, Starches and Fibers in Foods and Health: Healthy
A
Carbohydrate Choices, Roles and Applications in Nutrition, Food Science and the Culinary Arts ,
M

in: J. B. Marcus (Eds.), Culinary Nutrition the Science and Practice of Healthy Cooking,
D

Academic press, Cambridge Massachusetts, 2013, pp. 149-187.

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[5] M. A. Clarke, Syrups, in: B. Caballero, L. Trugo, P. M. Finglas (Eds.), Encyclopedia of

Food Sciences and Nutrition (Second Edition), Academic press, Cambridge Massachusetts,
EP

2003, pp. 5711–5717.

CC

[6] O. F. Sánchez, A. M. Rodriguez, E. Silva, L. A. Caicedo, Sucrose biotransformation to

fructooligosaccharides by Aspergillus sp. N74 free cells. Food and Bioprocess Technology 3
A

(2010) 662-673.

[7] L. C. Saric, B. V. Filipcev, O. D. Simurina, D. V. Plavsic, B. M. Saric, J. M. Lazarevic, I. L.

Milovanovic, Sugar beet molasses: properties and applications in osmotic dehydration of fruits

and vegetables, Food and Feed Research. 43 (2016) 135-144.

[8] B. Klejdus, L. Lojková, E. Kula, I. Buchta, P. Hrdlička, V. Kubáň, Supercritical fluid

extraction of amino acids from birch (Betula pendula Roth) leaves and their liquid

chromatographic determination with fluorometric detection. J. Sep. Sci. 31 (2008)1363–1373.

[9] G. Zhu, X. Zhu, Z. Xiao, R. Zhou, N. Feng, Y. Niu, A review of amino acids extraction from

PT
animal waste biomass and reducing sugars extraction from plant waste biomass by a clean

method. Biomass Conversion and Biorefinery. 5 (2015) 309–320.

RI
[10] W. Andlauer, P. Furst, Nutraceuticals: a piece of history, present status and outlook. Food

SC
Research International 35 (2002) 171–176.

[11] E. Arnáiz, J. Bernal, M.T. Martín, M.J. Nozal, J.L. Bernal, L. Toribio, Supercritical fluid

U
extraction of free amino acids from broccoli leaves, Journal of Chromatography A. 1250 (2012)

49-53. N
A
[12] M. Herrero, J. A. Mendiola, A. Cifuentes, E. Ibánez, Supercritical fluid extraction: Recent
M

advances and applications. Journal of Chromatography A. 1217 (2010) 2495–2511.

[13] S. Samadi, B. Mahmoodzadeh Vaziri, Two-structured solid particle model for predicting
D

and analyzing supercritical extraction performance, Journal of Chromatography A. 1506 (2017)

TE

101-108.
EP

[14] J. L. Bernal ,M.J. Nozal, L. Toribio, C. Diego, R. Mayo, R. Maestre, Use of supercritical

fluid extraction and gas chromatography–mass spectrometry to obtain amino acid profiles from
CC

several genetically modified varieties of maize and soybean. Journal of Chromatography A. 1192

(2008) 266-272.
A

[15] L. Lojková, B. Klejdus, J. Moravcová , V. Kubán, Supercritical fluid extraction (SFE) of

4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI)

from ground-coffee with high-performance liquid chromatographic-electrospray mass

spectrometric quantification (HPLC/ESI-MS). Food Addit Contam. 23 (2006) 963-73.

[16] C. T. Shen, S. L. Hsu, C. M. J. Chang, Co-solvent-modified supercritical carbon dioxide

extractions of cholesterol and free amino acids from soft-shell turtle fish egg. Separation and

PT
Purification Technology 60 (2008) 215–222.

[17] M. Herrero, A. Cifuentes, E. Ibanez, Sub- and supercritical fluid extraction of functional

RI
ingredients from different natural sources: Plants, food-by-products, algae and microalgae: A

SC
review. Food Chemistry 98 (2006) 136-148.

[18] S. M. Pourmortazavi, S. S. Hajimirsadeghi, Supercritical fluid extraction in plant essential

U
and volatile oil analysis. Journal of Chromatography A. 1163 (2007) 2–24.

N
[19] H. Wang, Y. Liu, S. Wei, Z. Yan, Application of response surface methodology to optimize
A
supercritical carbon dioxide extraction of essential oil from Cyperus rotundus Linn. Food
M

Chemistry 132 (2012) 582-587.

[20] I. H. Adil, H. I. Çetin, M. E. Yener, A. Bayındırl, Subcritical (carbon dioxide + ethanol)

D

extraction of polyphenols from apple and peach pomaces, and determination of the antioxidant
TE

activities of the extracts. The Journal of Supercritical Fluids 43 (2007) 55-63.

EP

[21] G. Kashif, P. Jiyong, C. Yong-Hee, Optimization of supercritical fluid extraction of

bioactive compounds from grape (Vitis labrusca B.) peel by using response surface
CC

methodology. Innovative Food Science & Emerging Technologies 11 (2010) 485-490.

[22] Z. J. Wei, M. Liao, H. X. Zhang, J. Liu, S. T. Jiang, Optimization of supercritical carbon

A

dioxide extraction of silkworm pupal oil applying the response surface methodology.

Bioresource Technology. 100 (2009) 4214–4219.

[23] Y. Yamini, M. Khajeh, E. Ghasemi, M. Mirza, K. Javidnia, Comparison of essential oil

compositions of Salvia mirzayanii obtained by supercritical carbon dioxide extraction and

hydrodistillation methods. Food Chemistry.108 (2008) 341–346.

[24] H. Kamali, N. Aminimoghadamfarouj, E. Golmakani, A. Nematollahi, The optimization of

PT
essential oils supercritical CO2 extraction from Lavandula hybrid through static-dynamic steps

procedure and semi-continuous technique using response surface method. Pharmacognosy

RI
Research 7 (2015) 57-65.

SC
[25] X. Xu, Y. Gao, G. Liu, Q. Wang, J. Zhao, Optimization of supercritical carbon dioxide

extraction of sea buckthorn (Hippophae thamnoides L.) oil using response surface methodology.

U
LWT 41 (2008) 1223–1231.

N
[26] J. A. Egydio, A. M. Moraes, A. M. Moraes, P. T.V. Rosa, Supercritical fluid extraction of
A
lycopene from tomato juice and characterization of its antioxidation activity. J. of Supercritical
M

Fluids 54 (2010) 159-164.

[27] M. Sun, F. Temelli, Supercritical carbon dioxide extraction of carotenoids from carrot using
D

canola oil as a continuous co-solvent. J. of Supercritical Fluids 37 (2006) 397–408.

TE

[28] M. D. A. Lima, D. Charalampopoulos, A. Chatzifragkou, Optimisation and modeling of

EP

supercritical CO2 extraction process of carotenoids from carrot peels. J. of Supercritical Fluids

133 (2018) 94-102.

CC

[29] A. Erkucuk, I. H. Akgun, O. Yesil-Celiktas, Supercritical CO2 extraction of glycosides from

Stevia rebaudiana leaves: identification and optimization. J. of Supercritical Fluids 51 (2009) 29-
A

35.
[30] M. P. Fernandez-Ronco, C. Ortega-Noblejas, I. Gracia, A. De Lucas, M. T. Garcia, M. T., J.

F. Rodriguez, Supercritical fluid fractionation of liquid oleoresin capsicum: Statistical analysis

and solubility parameters. The Journal of Supercritical Fluids, 54 (2010) 22–29.

[31] C. S. Ku, S. P. Mun, Optimization of the extraction of anthocyanin from Bokbunja (Rubus

PT
coreanus Miq.) marc produced during traditional wine processing and characterization of the

extracts. Bioresource Technology. 99 (2008) 8325-8330.

RI
[32] P. Thana, S. Machmudah, M. Goto, M. Sasaki, P. Pavasant, A. Shotipruk, Response surface

SC
methodology to supercritical carbon dioxide extraction of astaxanthin from Haematococcus

pluvialis. Bioresource Technology. 99 (2008) 3110–3115.

U
[33] A. Fabiani, A. Versari, G.P. Parpinello, M. Castellari, S. Galassi, High-Performance Liquid

N
Chromatographic Analysis of Free Amino Acids in Fruit Juices Using Derivatization with 9-
A
Fluorenylmethyl-Chloroformate. Journal of Chromatographic Science 40 (2002) 14-18.
M

[34] Y. Guan, Q. Tang, X. Fu, S. Yu, S. Wu, M. Chen, Preparation of antioxidants from

sugarcane molasses. Food Chemistry 152 (2014) 552-557.

D

[35] B. Muir, S. Quick, B.J. Slater, D.B. Cooper, M.C. Moran, C.M. Timperley, W.A. Carrick,
TE

C.K. Burnell, Analysis of chemical warfare agents: II. Use of thiols and statistical experimental
EP

design for the trace level determination of vesicant compounds in air samples, J.

Chromatography A. 1068 (2005) 315–326.

CC

[36] J. M. L. Mee, C. C. Brooks, R.W. Stanley, Amino Acid and Fatty Acid Composition of

Cane Molasses, J. Sci. Food Agric. 30 (1979) 429-432.

A

[37] D. E. Rearik, C. Mckey, The behavior of amino acids in chromatographic molasses

desugarization. Conference on sugar process research New Orleans, LA 1996,

http://www.arifractal.com/images/files/The-behavior-of-amino-acids-in-chromatographic-

molasses-desugarization-systems.pdf (accessed 15june 2018).

[38] S.H. Khan, G. Rasool and S. Nadeemm, Bioconversion of Cane Molasses into Amino

Acids. Pak. J. Agri. Sci. 43 (2006) 157-161.

PT
[39] S. P. H. Alexander, Glutamate, in: L. R. Squire (Eds.), Encyclopedia of Neuroscience,

Academic press, Cambridge Massachusetts, 2009, pp. 885-894.

RI
[40] A. J. Hubbard, D. K. Binder, Glutamate metabolism, in: A. J. Hubbard, D. K. Binder

SC
(Eds.), Astrocytes and Epilepsy, Academic press, Cambridge Massachusetts, 2016, pp. 197-224.

[41] M. J. York, Clinical Pathology, in: A. S. Faqi (Eds.), A Comprehensive Guide to

U
Toxicology in Nonclinical Drug Development (Second Edition), Academic press, Cambridge

Massachusetts, 2017, pp. 325-374. N

A
[42] N. V. Bhagavan, C. E. Ha, Amino acids, in: N. V. Bhagavan, C. E. Ha (Eds) Essentials of
M

Medical Biochemistry: with clinical cases, 2011, pp. 19-27.

[43] F. Tanaka, A. Tanaka, T. Uchino, Effect of high temperature drying on amino acids
D

decomposition in feed rice. Engineering in Agriculture, Environment and Food 10 (2017) 1-3.
TE

[44] Molasses-General Consideration, 1983 National Feed Ingredients Association West Des
EP

Moines, Iowa, http://rcrec-ona.ifas.ufl.edu/pdf/publications/molasses-general-considerations..pdf

(accessed 15 June 2018)

CC

[45] J. L. Dividich, R. Christon , J. Peiniau, A. Aumaitre, Proximate Chemical Analysis of Final

Cane Molasses and Effect of Feeding 30% Molasses on Intestinal Sucrose and Maltase Activities
A

in the rat. Animal Feed Science and Technology 3 (1978) 15-22.

[46] A. Steg, J. M. Van Der Meer, Differences in Chemical Composition and Digestibility of

Beet and Cane Molasses. Animal Feed Science and Technology 13 (1985) 83-91.
[47] L. Wong Sak Hoi, B. Martincigh, Sugar Cane plant fibers: Separation and Characterization.

Industrial Crops and Products 47 (2013) 1-12.

[48] J. Tjebbes, Utilization of Fiber and Other Non-Sugar Products from Sugar beet, in: M. A.

Clarke, M. A. Godshall (Eds.), Sugar Series, Chemistry and Processing of Sugar beet and

PT
Sugarcane, Volume 9, Elsevier Science, New Orleans, Louisiana, 9 (1988), pp. 139-145.

[49] R. A. Kitchen, Polysaccharides of Sugarcane and their Effects on Sugar Manufacture, in: M.

RI
A. Clarke, M. A. Godshall (Eds.), Sugar Series, Chemistry and Processing of Sugar beet and

SC
Sugarcane, Volume 9, Elsevier Science, New Orleans, Louisiana, 9 (1988), pp. 208-235.

[50] M. Pucarevic, V. Bursic, D. Pankovic, R. M. Nebojsa, M. Cara, I. Kecojevic, Supercritical

U
Fluid Extraction of Tebupirimphos Residues Sugar beet. The Journal of Animal & Plant Sciences

23 (2013) 277-280. N
A
[51] L. Garcia, J. F. Rodriguez, M. T. Garcia, A. Alvarez, A. Garcia, Isolation of aroma
M

compounds from sugar cane spirits by supercritical CO2 , J. of Supercritical Fluids 43 (2007) 37-

42.
D

[52] D. Pasquini, M. T. B. Pimenta, L. H. Ferreira, A. A. S. Curvelo, Sugar cane bagasse pulping

TE

using supercritical CO2 associated with co-solvent 1-butanol/water. J of Supercritical Fluids 34

EP

(2005) 125-131.

[53] F. Montanes, T. Fornari, R.P. Stateva, A. Olano, E. Ibanez, Solubility of carbohydrates in

CC

supercritical carbon dioxide with (ethanol + water) cosolvent. J. of Supercritical Fluids. 49

(2009) 16–22.
A

[54] F. Montanes, T. Fornari, A. Olano, E. Ibanez, Supercritical fluid purification of complex

carbohydrate mixtures produced by enzymatic transglycosylation and isomerized with

complexating reagents. J. of Supercritical Fluids. 53 (2010) 25–33.

[55] F. Montanes, A. Corzo, A. Olano, G. Reglero, E. Ibanez, Selective fractionation of

carbohydrate complex mixtures by supercritical extraction with CO2 and different co-solvents. J.

of Supercritical Fluids. 45 (2008) 189–194.

[56] E. Ibanez, F. Montanes, T. Fornari, A. Olano, Selective Fraction of Prebiotic Carbohydrates

PT
From Complex Mixtures by Supercritical CO2 with different cosolvents. 2007

https://pdfs.semanticscholar.org/bd18/7062dac497fec5c935a7050ed16c0ad44249.pdf (accessed

RI
15 June 2018).

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Figure Legends

Fig.1 Diagram of supercritical fluid extraction equipment; 1. CO2 Cylinder, 2. CO2 Pump, 3.

Chiller, 4. Solvent Pump, 5. Solvent reservoir, 6. Extraction cell, 7. Oven, 8. Back pressure

regulator, 9. Extract collector, 10. Separator, 11. Flow meter, 12. CO2 vent.

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Fig.2 Chemical reaction between FMOC-CL and ADAM.

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Fig.3 Goodness-of-fit of the empirical model with predicted for SGB (a) and SGC molasses (b).

Fig.4 Response surfaces and contour plots for: (a, b) Extraction time (min) vs. Temperature (˚C)

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in 184 bars; (c, d) Extraction time (min) vs. Pressure (bar) at 43 ˚C for SGB molasses.

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Fig.5 Response surfaces and contour plots for: (a, b) Extraction time (min) vs. Temperature (˚C)
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in 316 bars; (c, d) Extraction time (min) vs. Pressure (bar) at 50 ˚C for SGC molasses.
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Fig.6 The HPLC-UV chromatograms of optimized SFE at 184 bar, 76 min and 43˚C for SGB (a)
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and 316 bar, 76 min and 50 ˚C for SGC (b) molasses.

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Fig. 1
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FMOC-Cl + ADAM = FMOC-ADAM + HCl

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Fig. 3a

Fig. 3b
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Fig. 4a

Fig. 4b
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Fig. 4c
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Fig. 4d
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Fig 5a

Fig 5b
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Fig 5c

Fig 5d
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Fig 6a

Fig 6b
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 Table 1 Independent factors, their symbols and levels for the OCCDused for SGB and SGC
molasses
Factor Symbol Levels

-α -1 0 +1 +α

Pressure (bar) X1 150 184 250 316 350

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Temperature (˚C) X2 40 43 50 57 60

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Time (min) X3 10 24 50 76 90

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Table 2 Experimental values of the total peak area obtained for SGB and SGC molasses

No. X1 X2 X3 Total peak area

SGB SGC

1 184 57 76 1546 422

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2 316 57 24 2509 692

3 184 43 24 161 331

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4 184 57 24 1046 471

5 250 50 10 641 412

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6 250 50 50 1517 748

7 250 60 50 1718 106

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8 316 43 76 2226 804

9 316 57 76
N 1023 812
A
10 250 40 50 1847 34
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11 250 50 50 1455 635

12 250 50 50 1623 605

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13 250 50 90 1425 888

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14 250 50 50 1725 737

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15 350 50 50 2360 1178

16 184 43 76 2775 608

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17 150 50 50 1846 886

18 250 50 50 1459 724

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19 250 50 50 1544 561

20 316 43 24 1859 123

Table 3 AAs content of raw SGB and SGC molasses, optimized extracts of SGB and SGC, and
extraction recoveries at the optimum conditions (%)

Aspartic acid Glutamic acid Alanine Lysine

Raw material (mg/kg)

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SGB 152 141 157 29

SGC 141 155 132 16

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Extract (mg/kg)

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SGB 64 89 73 9

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SGC 44 58 63 3
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Extraction recovery (%)
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SGB 42 63 46 31
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SGC 31 37 48 20
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 Table4 ANOVA for SGB molasses
Source Sum of dfa Mean square F-Ratio p- Value Effect
square
Model 6.995E+006 8 8.744E+005 87.92 <0.0001 Significant

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X1-Pressure 6.525E+005 1 6.525E+005 65.61 <0.0001
X2-Tempreture 94351.58 1 94351.58 9.49 <0.0001

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X3-Extraction time 8.047E+006 1 8.047E+005 80.91 0.0105
X1X2 5505.15 1 5505.15 0.53 0.4834

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X2X3 1.966E+006 1 6.857E-006 197.72 <0.0001
X1X3 2.240E+006 1 2.701E-006 225.26 <0.0001

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𝑿𝟐𝟏 5.736E+005 1 1.080E-004 57.67 <0.0001

𝑿𝟐𝟐 92475.34 1 92475.34N 9.30 0.0111

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𝑿𝟐𝟑 5.736E+005 1 5.706E+005 57.37 <0.0001
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Residual 1.094E+005 11 9945.66

Lack of fit 54977.05 6 9162.84 0.84 0.5867 Not significant
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Pure Error 5425.26 5 10885.05

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Cor Total 7.104E+006 19

R2 0.9846
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R2 (adj) 0.9734
adegrees of freedom.
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Table5 ANOVA for SGC molasses
Source Sum of dfa Mean square F-Ratio p- Value Effect
square
Model 1.548E+006 8 1.934E+005 41.72 <0.0001 significant

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X1-Pressure 86306.08 1 86306.08 18.62 <0.0012
X2-Tempreture 32384.29 1 32384.29 6.99 0.0229

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X3-Extraction time 2.436E+005 1 2.436E+005 52.55 <0.0001
X1X2 48576.73 1 48576.73 10.48 0.0079

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X2X3 98349.45 1 98349.45 21.21 0.0008
X1X3 40947.64 1 40947.64 8.83 0.0127

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𝑿𝟐𝟏 2.404E+005 1 2.404E+005 51.86 <0.0001

𝑿𝟐𝟐 7.569E+005 1 N
7.569E+005 163.26 <0.0001
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𝑿𝟐𝟑 2486.76 1 2486.76 0.51 0.4904
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Residual 50997.93 11 4636.18

Lack of fit 20109.82 6 3351.64 0.54 0.7612 Not significant
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Pure Error 30888.11 5 6177.62

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Cor Total 1.598E+006 19

R2 0.9681
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R2 (adj) 0.9449
adegrees of freedom.
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Table 6 Optimized SFE conditions for the SGB and SGC molasses

Pressure (bar) Temperature (˚C) Extraction Time (min) Total peak area

Sugar beet 184 43 76 2777

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Sugar cane 316 50 76 1104

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39