Supercritical fluid extraction of free amino acids from sugar beet and sugar cane molasses

The Journal of Supercritical Fluids 144 (2019) 48–55

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Supercritical fluid extraction of free amino acids from sugar beet and sugar cane molasses

T

Mona Varaeea, Masoud Honarvara, , Mohammad H. Eikanib, Mohammad R. Omidkhahc, Narges Morakid ⁎

a

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

GRAPHICAL ABSTRACT

ARTICLE INFO

ABSTRACT

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

In this work supercritical fluid extraction (SFE) was used to extract free amino acids (AAs) from sugar beet (SGB) and sugar cane (SGC) molasses. The effect of different variables such as, pressure (150–350 bar), temperature (40–60 °C) and extraction time (10–90 min) was evaluated to optimize the extraction using response surface methodology (RSM).The results of SGB and SGC molasses extraction showed that the optimal condition were 184 and 316 bar; 43 and 50 °C and 76 and 76 min, respectively. Under the optimum condition, the extraction recoveries of AAs for 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 SFE might be employed to extract of AAs from SGB and SGC molasses with acceptable selectivity and extraction efficiency.

1. Introduction Molasses is a viscous and dark liquid by-product of sugar beet (Beta vulgaris var. saccharifera) or sugar cane (Saccharum L.) obtained as the

final effluent of sugar refinement. Molasses contains profitable components such as fermentable carbohydrates (sucrose, glucose, fructose) and considerable nonsugar organic materials (betaine, other amino acids; minerals and trace elements; vitamins, etc.). Molasses has been

⁎ Corresponding author at: Department of Food Science and Technology, College of Agriculture and Food Science, Science and Research Branch, Islamic Azad University (IAU), P.O. Box 1477893855, Tehran, Iran. E-mail address: [email protected] (M. Honarvar).

https://doi.org/10.1016/j.supflu.2018.10.007 Received 5 July 2018; Received in revised form 6 September 2018; Accepted 6 October 2018 Available online 11 October 2018 0896-8446/ © 2018 Published by Elsevier B.V.

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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.

mainly employed as a supplement for animal feed and ethanol production [1–7]. Amino acids (AAs) have central roles as building blocks of proteins and as intermediates in metabolism [8,9]. Twenty AAs are common in make up the body; and eight of them are named as essential AAs, since the body cannot synthesis them from other components. Therefore, they must be obtained from foods or nutritional supplements. AAs are required in pharmaceutical and food domains thus high value-added AAs can be recovered from by-product and utilized in medical, cosmetic, animal feed, and other industrial applications [9,10]. Techniques to extract these valuable components from raw materials are quite essential to 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, 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 [13–16]. 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 cosolvents, 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 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 [22–25], lycopene [26], carotenoids [27,28], glycosides [29] oleoresins [30], anthocyanins [31] astaxanthin [32]. Indeed, few investigation have been released about the SFE extraction of free AAs and neither of them was employed on liquid and viscous substances such as sugar beet (SGB) and sugar cane (SGC) molasses. In this view, the addition of a polar organic modifier, such as methanol, is necessary to raise the solute solubility. The significance of the extraction of AAs 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 AAs from SGB and SGC molasses using SFE extraction and optimize the extraction method for molasses samples. The response surface methodology (RSM) as the experimental

design procedure was applied. It was based on the orthogonal central composite design (OCCD) employing temperature, pressure and extraction time as influential factors. 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 (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 stored at 4℃ and protected from light. Carbon dioxide with 99.99% purity was obtained from Sabalan Co. (Tehran, Iran) and utilized in all of the extraction experiments. 2.2. Apparatus and extraction procedure A Separex (Champigneulles, France) system in SFE mode was employed for all experiments. The extractions were accomplished using a 100 mL volume stainless steel 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 pressure was maintained by a back pressure regulator and checked by an automatic manometer. The temperature was checked by an automatic thermometer. The heating of the system was 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 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 350 or 700 bar. SFE equipment is schematically represented in 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 49

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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 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 wt ratio) to prevent agglomeration and act as a carrier to increase the surface area. The final powder was poured in 100 mL 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, using 30 mL methanol or nearly 6 g methanol/g molasses (dried basis) was added through the six-port valve into the CO2 stream at the flow rate of 1 mL/min. The system was equipped with an air-driven pump to deliver the CO2 to the extraction cell, which was placed in a temperature-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 was transferred to the extract collector and CO2 gas is separated from the extract. The precision in pressure and temperature measurements were ± 1 bar and ± 1 °C, 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 for further HPLC analysis.

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

N = 2 f + 2f + N0

where, f is the number of variables [35]. The three independent analyses variables and their ranges were according to pressure (X1) from 150 to 350 bar, temperature (X2) from 40 to 60 °C, extraction time from 10 to 90 min with five levels selected for each variables: -α, -1, 0, +1 and +α. The axial points are set at +α and -α from the center of the experimental area that was 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 OCCD containing three variables, needed 20 experimental runs with six at the central point was accomplished which presented in Table 2. All experiments were implemented in a randomized 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. 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]:

2.3. Derivatization procedure AAs were derivatized (FMOC-AA) at room temperature using a precolumn 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 200 mM borate buffer (pH 10.0). Then, 600 μL of 15 mM FMOCCl (in acetonitrile) was added to the extracted molasses and derivatization occurred. The reaction was stopped after 5 min by the addition of 600 μL of 300 mM ADAM (water-acetonitrile, 1:1, v/v), and the reaction lasted for 1 min to form the FMOC-ADAM 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 derivatization procedure was 6 min [33].

k

k 2 jj Zj

2.4. HPLC analysis The analysis of the AAs in extracts was performed by high performance liquid 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 column heater, a 8440 XR UV–vis detector that was set at 263 nm and a 4290 integrator linked via Labent to a computer. Chromatographic data were analyzed using ChromanaCH software, version 3.6.4 (Tehran, Iran). For

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

0

+

k

Y=

jZj + j=1

j=1

Fig. 2. Chemical reaction between FMOC-CL and ADAM. 50

+

ij 1
ZiZj

(2)

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Table 1 Independent factors, their symbols and levels for the OCCDused for SGB and SGC molasses. Factor

Symbol

Pressure (bar) Temperature (˚C) Time (min)

X1 X2 X3

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

Levels -α

−1

0

+1

+α

150 40 10

184 43 24

250 50 50

316 57 76

350 60 90

Raw material (mg/kg) SGB SGC Extract (mg/kg) SGB SGC Extraction recovery (%) SGB SGC

Table 2 Experimental values of the total peak area obtained for SGB and SGC molasses. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

X1

184 316 184 184 250 250 250 316 316 250 250 250 250 250 350 184 150 250 250 316

X2

57 57 43 57 50 50 60 43 57 40 50 50 50 50 50 43 50 50 50 43

X3

76 24 24 24 10 50 50 76 76 50 50 50 90 50 50 76 50 50 50 24

Total peak area SGB

SGC

1546 2509 161 1046 641 1517 1718 2226 1023 1847 1455 1623 1425 1725 2360 2775 1846 1459 1544 1859

422 692 331 471 412 748 106 804 812 34 635 605 888 737 1178 608 886 724 561 123

Aspartic acid

Glutamic acid

Alanine

Lysine

152 141

141 155

157 132

29 16

64 44

89 58

73 63

9 3

42 31

63 37

46 48

31 20

3.2. SFE and statistical analysis The experimental data of the total peak area obtained from the OCCD for SGB and SGC molasses are presented in Table 2. Responses (RSGB and RSGC) were represented the total peak area. Table 4 and 5 show analysis of variance (ANOVA) for SGB and SGC molasses, 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. ANOVA was carried out with an F-test (lack of fit), for validation. The “lack of fit” was not 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. For an experimental design with three factors, the mathematical model was expressed as presented in Eqs. (3) and (4) for SGB and SGC molasses, respectively.

the response surface plots. 3. Results and discussion

R SGB: -2.290E + 003 -7.937× X1 – 8.413E+001× X2 + 2.639E +002× X3– 3.307E-001 × X1X3 – 2.881 × X2X3 + 5.355E-002× X12 + 2.150× X22 - 3.334E-001× X32 (3)

In the present study, SFE technique was used to determine the extraction of AAs from SGB and SGC molasses. There are considerable variables that can affect the extraction efficiency of 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 discussed in the following sections.

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 – (4) 6.444E-001× X2X3 + 3.467E-002× X12 - 6.152 X22 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 data. The goodnessof-fit of the empirical model for SGB (a) and SGC molasses (b), is described 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 that are attained during examinations. The Fig. 3 shows the points of predicted peak area and experimental peak area are nearly coinciding of each other’s.

3.1. HPLC analysis of raw materials 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 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 the four aforementioned AAs. The identification of AAs in the samples was based on the comparison between the relative retention times of the AAs extracts with standards. The HPLC results are shown in Table 3.

3.3. 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 and SGC molasses. As can be seen, in Figs. 4a-b by raising the extraction temperature in SGB molasses 51

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Table 4 ANOVA for SGB molasses. Source

Sum of square

dfa

Mean square

F-Ratio

p- Value

Effect

Model X1-Pressure X2-Tempreture X3-Extraction time X1X2 X2X3 X1X3

6.995E+006 6.525E+005 94351.58 8.047E+006 5505.15 1.966E+006 2.240E+006 5.736E+005

8 1 1 1 1 1 1 1

8.744E+005 6.525E+005 94351.58 8.047E+005 5505.15 6.857E-006 2.701E-006 1.080E-004

87.92 65.61 9.49 80.91 0.53 197.72 225.26 57.67

< 0.0001 < 0.0001 < 0.0001 0.0105 0.4834 < 0.0001 < 0.0001 < 0.0001

Significant

X22

92475.34

1

92475.34

9.30

0.0111

5.736E+005

1

5.706E+005

57.37

< 0.0001

1.094E+005 54977.05 5425.26 7.104E+006

11 6 5 19

9945.66 9162.84 10885.05

0.84

0.5867

Not significant

Source

Sum of square

dfa

Mean square

F-Ratio

p- Value

Effect

Model X1-Pressure X2-Tempreture X3-Extraction time X1X2 X2X3 X1X3

1.548E+006 86306.08 32384.29 2.436E+005 48576.73 98349.45 40947.64 2.404E+005

8 1 1 1 1 1 1 1

1.934E+005 86306.08 32384.29 2.436E+005 48576.73 98349.45 40947.64 2.404E+005

41.72 18.62 6.99 52.55 10.48 21.21 8.83 51.86

< 0.0001 < 0.0012 0.0229 < 0.0001 0.0079 0.0008 0.0127 < 0.0001

significant

X22

7.569E+005

1

7.569E+005

163.26

< 0.0001

2486.76

1

2486.76

0.51

0.4904

50997.93 20109.82 30888.11 1.598E+006

11 6 5 19

4636.18 3351.64 6177.62

0.54

0.7612

X12

X32 Residual Lack of fit Pure Error Cor Total R2 R2 (adj) a

0.9846 0.9734

degrees of freedom.

Table 5 ANOVA for SGC molasses.

X12

X32 Residual Lack of fit Pure Error Cor Total R2 R2 (adj) a

Not significant

0.9681 0.9449

degrees of freedom.

samples from 40 to 43 °C, the extraction efficiency is enhanced impressively. By increasing the extraction temperature above 44 °C thermal denaturation and decomposition of 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 increased by intensifying the extraction time from 50 to 76 min. Figs. 5a and 5b indicate that by increasing the extraction temperature in the SGC molasses patterns from 50 to 57 °C, the extraction performance is increased considerably. The application of higher temperature above 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]. 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]. 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 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 the nature and structure of SGB molasses with less sticky compounds (i.e. tannin and fibers) [48,49].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 is a larger step to increase the amount of the extraction by increment of pressure and decrement of duration; which as it explains before it may be due to the chemical structure of SGB molasses. On the other hand, suitable extraction time of SGB is evaluated same as SGC molasses. The 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 to 76 min as presented in Figs. 4c-d and 5 c-d. By increasing extraction time from 76 to 90 min, extraction performance has remained relatively constant. It can be concluded the best range of pressure and time for future researches on SGB molasses are 180 up to 320 bar, and 20 to 80 min. 3.4. 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. [50] 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 52

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investigated SGC bagasse by SFE. Their results are partly in agreement with our study where the effects of temperature and pressure were distinctly represented. The HPLC chromatograms of the optimum SFE extract for SGB and SGC molasses are presented in Figs. 6a-b. 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.

ER (%) =

Cn × 100 Co

(5)

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 SGB molasses was 46% while this was 34% for SGC molasses. It can be perceived that the 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 more easily [44–46,48,49]. The current results are better than those which have been reported elsewhere. AAs extraction from soft-shell fish egg performed by Shen et al. [2008] resulted to as low as 29% extraction recovery. Regarding selectivity of the SFE, it should be stated that SGB and SGC molasses are mixtures of sugars (53 and 64% w/w), non-sugar materials incl. AAs (19 and 10% w/w), water (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 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 (60–100 °C), and using ethanol as co-solvent. Carbohydrates have higher molecular weights and it is clear that ethanol is a better solvent for them. Because of probable AAs decomposition, in the present study, intentionally lower temperatures were selected (40–60 °C). 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].

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

at 316 bar, 50 °C and 76 min and 1104 was the maximum total peak area, Table 6. 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 minerals that make the AAs extraction more difficult [42–47]. Guan et al. [34] investigated the antioxidants from SGC molasses in the same SFE extraction condition and reported nearly the similar results. Gracia et al. [51] evaluated the isolation of aroma compounds from SGC pulps and Pasquini et al. [52]

Fig. 4. Response surfaces and contour plots for: (a, b) Extraction time (min) vs. Temperature (˚C) in 184 bars; (c, d) Extraction time (min) vs. Pressure (bar) at 43 °C for SGB molasses.

53

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Fig. 5. Response surfaces and contour plots for: (a, b) Extraction time (min) vs. Temperature (˚C) in 316 bars; (c, d) Extraction time (min) vs. Pressure (bar) at 50 °C for SGC molasses.

4. Conclusions

Table 6 Optimized SFE conditions for the SGB and SGC molasses.

Sugar beet Sugar cane

Pressure (bar)

Temperature (˚C)

Extraction Time (min)

Total peak area

184 316

43 50

76 76

2777 1104

In this study, for the first time the SFE extraction was carried out successfully for the extraction of AAs for SGB and SGC molasses. The evaluation of the results demonstrated that 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 area were found. SGC molasses required higher temperature and pressure for extraction due to higher content of tannin, fiber, minerals and complicated matrix. Finally, it is worth to mention that the application of SFE for SGB and SGC molasses not only improves the value added in 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 optimum operating conditions reported here correspond to a laboratory scale free AAs extraction from two types of molasses, helping development of the concept experimentally. Extractions at 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. Acknowledgements 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 Chem. 60 (2012) 12508–12515. [2] A. Baiano, Recovery of biomolecules from food wastes-a review, Molecules 19 (2014) 14821–14842. [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 medium, Food Bioproc. Tech. 2 (2009) 409–414. [4] J.B. Marcus, carbohydrate basics: sugars, starches and fibers in foods and health: healthy carbohydrate choices, roles and applications in nutrition, food science and the culinary arts, in: J.B. Marcus (Ed.), Culinary Nutrition the Science and Practice of Healthy Cooking, eds., Academic press, Cambridge Massachusetts, 2013, pp.

Fig. 6. The HPLC-UV chromatograms of optimized SFE at 184 bar, 76 min and 43 °C for SGB (a) and 316 bar, 76 min and 50 °C for SGC (b) molasses.

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