Effect of the extraction by thermosonication on castor oil quality and the microstructure of its residual cake

Industrial Crops & Products 141 (2019) 111760

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Effect of the extraction by thermosonication on castor oil quality and the microstructure of its residual cake

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P. López-Ordaza, J.J. Chanona-Péreza, , M.J. Perea-Floresb, C.E. Sánchez-Fuentesc, J.A. Mendoza-Pérezc, I. Arzate-Vázquezb, J. Yáñez-Fernándezd, H.H. Torres-Venturaa a

Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Av. Wilfrido Massieu Esq, Cda, Miguel Stampa s/n, C.P. 07738, Ciudad de México, Mexico Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, Luis Enrique Erro s/n, Zacatenco, C.P. 07738, Gustavo A. Madero, Ciudad de México, Mexico c Departamento Ingeniería en Sistemas Ambientales, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Wilfrido Massieu s/n, Gustavo A. Madero, C.P. 07738, Ciudad de México, Mexico d Departamento de Biotecnología Alimentaria, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, Av. Acueducto S/N Col. Barrio la Laguna, Ticomán, CP 07340, Ciudad de México, Mexico b

A R T I C LE I N FO

A B S T R A C T

Keywords: Microscopy techniques Physicochemical properties Ricinus oil Response surface methodology X-ray diffraction

The seed of Ricinus communis L. is a source of oil that can be used to produce biodiesel. In this study, the thermosonication (TS) extraction of castor oil is proposed and is compared with Soxhlet (S) extraction as the conventional method. The optimum conditions for the TS extraction of castor oil were determined by response surface methodology (RSM). The time at 25, 35, and 45 min and amplitudes of 25, 50, and 75% were independent variables, while the dependent variables were the oil yield, iodine index, peroxide index, saponification index, unsaponifiable matter, acidity index, and refraction index. The optimal conditions for the oil yield were an amplitude of 50%, 35 min, and a solid/liquid ratio of 1/10 g/mL; a yield of 61.12% was reached under these conditions; by S extraction, the yield was 57.3% after 8 h of extraction. The microstructure of the residual cake before and after extraction was evaluated by scanning electron microscopy and the cellulosic compounds, proteins, and lipids in the residual cake were identified and their distribution observed by means of confocal laser scanning microscopy. Microscopy techniques and image analysis were helpful in evaluating the changes in the microstructure that occur on the residual cake during the TS and S extraction and to understand the extraction mechanisms. The crystallinity index was calculated from X-ray diffraction spectra to interpret changes in the structure of the residual cake before and after use of the extraction methods. Therefore, the TS extraction improved the oil yield in shorter extraction time. Characterization of the residual cake opens the way to study a potentially usable material as a source of cellulosic compounds.

1. Introduction The castor bean (Ricinus communis L.) is a shrub that originates from Africa. It grows in the wild in large quantities (Ogunniyi, 2006). Its seed is commonly known as “ricin” or “higuerilla” (Ali et al., 2008; Scholza and da Silva, 2008). Ricinus communis L or castor bean is widely distributed in temperate and tropical regions. Castor seeds are rich in oil and content about 35 to 60% oil by weight sample (Danlami et al., 2015; Perdomo et al., 2013). In this way, R. communis L. is among the plants with the highest oil production, which makes it a profitable crop, with the advantage of being inedible and not competing with traditionally used field crops for food (Scholza and da Silva, 2008). The oil consists of approximately 80–90 % ricinoleic acid, 3–6 % linoleic acid,

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2–4 % oleic acid, and 1–5 % saturated fatty acids (Ogunniyi, 2006). The presence of ricinoleic acid, containing both a double bond and hydroxyl group, impart increased lubricity to the castor oil and its derivatives as compared to other vegetable oils, which can be used like a fuel additive (Goodrum and Geller, 2005). Unlike first-generation oils, reused cooking oils and animal fat oils, castor oil has no problems in storage due to cold flow properties (point of cold filter clogging and turbidity and flow points) and oxidative stability (Perdomo et al., 2013). The chemical properties and thermal stability of castor oil make it suitable to obtain several products such as pharmaceuticals, organic fertilizers, biological pest control, the manufacture of polymers, paint thinner, emulsifier, varnishes, and dyes (Kilic et al., 2013; Lorestani et al., 2012).

Corresponding author. E-mail address: [email protected] (J.J. Chanona-Pérez).

https://doi.org/10.1016/j.indcrop.2019.111760 Received 6 June 2019; Received in revised form 24 August 2019; Accepted 3 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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high reproducibility, quicker response to extraction process control, expand the use of inedible plants for the production of renewable energy (e.g. oil extraction for biodiesel production), easy implementation at industrial level, rapid return of investment a higher production rate with less process stages, and as well as a reduce consumption of fossil energy normally required in conventional extraction methods such as Soxhlet extraction, maceration or Clevenger distillation. By means of UAE full extractions can now be completed in minutes with high reproducibility and low energy consumption (Chemat et al., 2011, 2017; Chanioti and Tzia, 2017; Gaëlle-Sicaire et al., 2016). Overall, research related to UAE has been dedicated to studying of different issues, the most abundant reports are focused to evaluation of the effect of TS on the extraction yield, rate of the kinetics, optimization of extraction conditions, characterization and evaluation of the quality of the compounds obtained (Anaya-Esparza et al., 2017; Chanioti and Tzia, 2017; Samaram et al., 2015), without delving into the extractive mechanisms due to the use of ultrasonic radiation. The works focused on studying the mechanisms, microstructural changes and the effects that induce a better separation when the UAE is applied are less abundant (Cruz et al., 2011; Xiaoming et al., 2018). The mechanisms more frequently reported in the extraction by sonication are fragmentation, erosion, sonocapillarity, detexturation (disruption), local shear stress, and sonoporation, but usually, combine mechanisms could act during TS extraction (Chemat et al., 2017). As this technology has been successful in recovering different chemical compounds from different biological materials, some research has focused on reporting applications at industrial level (Juliano et al., 2017; Petigny et al., 2013; Pingret et al., 2012). In the case of the oils some studies on the optimization of the oil extraction assisted with ultrasound have been developed (Chemat et al., 2011; Hernandez-Santos et al., 2016; Juliano et al., 2017). There is a report of the oil separation by UAE from hemp seeds, it was found a short processing time and less solvent consumption than the Soxhlet method (Lin et al., 2012). After the extraction process, the residual cake suffers modifications in the composition and structure of the cell wall materials (Cruz et al., 2011). There are few studies that have evaluated the effect of TS on the microstructure of the residual cake obtained after oil extraction. Zhang et al. (2009) studied the effect of autoclaving pretreatment and ultrasonic treatment on the microstructure of almond powder by means of scanning electron microscopy (SEM), the micrographs evidenced that the almond powder became porous due to the structural breakage by the ultrasonic cavitation energy. Guimaraes et al. (2016) recovered castor oil cake (COC) to reinforced with banana fibers and sugarcane bagasse fibers and its morphology was observed by SEM, as can be noted, the microstructure studies on COC have only been carried out by SEM, and a feasibility study of the use of the residual cake as a cellulose source has not been carried out. As discussed above, the UAE has been widely used to separate natural compounds, bioactive, and, various types of oils from different plant material. However, as far as we are aware, the studies with TS for castor oil extraction are still scarce. There is a knowledge gap in the optimization of process conditions to extract castor oil by UAE, there are few microstructural studies that allow visualizing the effects and mechanisms caused by TS in the endosperm cells of the castor seeds. Moreover, could be novel to evaluate the structural damage caused by TS in the residual cake, since this information is valuable for recovery of the cellulose after the castor oil extraction. Therefore, the present investigation aimed to compare the performance of oil extraction from the castor seeds through two methods; Soxhlet (S) and thermosonication (TS). Response surface methodology (RSM) was used, the physicochemical parameters evaluated were: the oil yield (%), II, PI, SI, USM, AI, and RI. Moreover, the residual cake was studied by confocal laser scanning microscopy (CLSM), SEM, and X-ray diffraction (XRD) to evaluate the structural changes after the oil extraction, which can be relevant issue for recovery of cellulosic compounds such as hemicellulose and cellulose from residual cake, with

Furthermore, quality control during oil extraction is very significant for the success of its use in biodiesel production. The synthesis of biodiesel from castor oil is carried out by an alkaline transesterification, where triglycerides in the presence of alcohol are converted into a mixture of fatty acid esters and glycerol (Dias et al., 2013), but the presence of impurities could produce saponification reactions and reduce the quality of biodiesel. For this reason, is important preserve the castor oil quality during its extraction and storage, because high levels of oxidation, acidity, saponifiable matter, and water could diminish the yields of the transesterification to obtain biodiesel and also of the reactions used for the synthesis of other chemical products. Some of the parameters frequently used to evaluated the castor oil quality are iodine index (II), peroxide index (PI), saponification index (SI), unsaponifiable matter (USM), acidity index (AI), these indicators must be evaluated to known and control the effects of castor oil extraction with organic solvents and heat (Danlami et al., 2015; Ogunniyi, 2006; Pradhan et al., 2012). An important issue in biodiesel production from castor bean is the oil extraction, which can be carried out by different methods such as pressing, pre-pressing, and subsequent extraction with solvent by the Soxhlet method, which is the most common procedure for extracting oil or determining lipids at the laboratory (Chemat et al., 2017; GaëlleSicaire et al., 2016; Hernández-Hernández et al., 2016a, 2016b; Ogunniyi, 2006). However, this method has some drawbacks, such as high solvent and energy consumption, long time for extraction and that it still does not offer a good extraction performance of oil or good quality in the extract. (Da Porto et al., 2013). An alternative to improve extractive processes is the use of ultrasound technology, this is divided into two types, high intensity ultrasound (HIU), and low intensity ultrasound (LIU). LIU is frequently used in biomedical applications, while HIU generates high energy power, this is taken advantage for extraction of intracellular components in plant materials with low thermal damage and short processing time, HIU is characterized by low amplitude and high intensity, which allows the extraction of biological compounds from plant tissues. HIU can be of two types, ultrasonic bath or probe-type, both systems use a transducer as a source of ultrasound power. Commercial HIU devices commonly are designed at frequencies between 20 kHz and 100 kHz, but probes around 20 kHz are the most frequently used due to that during growth of bubbles and cycles of compression-rarefaction produce cavitation bubbles of higher sizes compared with baths at 40 kHz, for this reason, ultrasonic probes are preferred for extraction processes, additionally the major disadvantages of ultrasonic baths are a low reproducibility, low power of ultrasound delivered directly to the sample and the attenuation of the ultrasound waves by the water contained in the bath and the glassware used (Vinatoru, 2001; Anaya-Esparza et al., 2017; Chemat et al., 2017). Thermosonication (TS) or ultrasound-assisted extraction (UAE) is a combined process, that for example can linking an ultrasound probe with the typical Soxhlet apparatus at the dissolvent boiling temperature (Chemat et al., 2017). The use of ultrasonic waves and heat allows the extraction from several plant tissues in shorter periods of time, decreasing the energy cost. To counter the problems of the conventional extraction method, the UAE offers several advantages to improve extractive processes. UAE is an emerging technology that has proven highly efficient for the extraction of bioactive and aromatic compounds, antioxidants, pigments, phytochemicals, essential, edible and inedible oils from leaves (Chemat et al., 2017; Petigny et al., 2013), fruits (Pan et al., 2012), oilseeds (Zhang et al., 2008), different plant tissues (Anaya-Esparza et al., 2017; Samaram et al., 2015) and microorganisms such as algae (Adam et al., 2012) and yeasts (Meullemiestre et al., 2016). UAE has revolutionized the conventional extraction by maceration with solvents and heating, due to its diverse advantages compared with conventional extraction methods, these include a higher mass and energy transfer capability, reduction of extraction time and process temperature, low solvents and energy consumption, selective extraction, higher purity of the final product, smaller equipment size, 2

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Fig. 1. Extraction systems of castor oil: (a) Soxhlet extraction, (b) thermosonication extraction, and (c) ultrasonic processor power supply and sonicator probe features.

time (T) and the amplitude (A), while the percentage oil yield, II, PI, SI, USM, AI, and RI were the response variables. These parameters were performed according to the methods given in the following references: II (Nielsen, 2010), PI (Crowe and White, 2001), SI (AOAC, 2005), USM by the (AOAC, 1990), AI (Kirk and R y Egan H., 1996), and RI (Aurand et al., 1987). The central composite design allowed established the dependence between the response and the independent variables, and optimal conditions of the process were obtained. The independent variables were coded according to Eq. (1):

potential uses as wall materials in the encapsulation of bioactive compounds and in obtaining cellulose nanoparticles for mechanical reinforcement. Thus, with this approach, it may be possible to increase yields and reduce the operating costs of oil extraction from the castor bean at laboratory level and with potential applications to address the industrial production of biodiesel. 2. Materials and methods 2.1. Oil extractions methods

Xi =

Castor beans were obtained of Oaxaca Central Valleys, Mexico. The manually peeled seeds were ground in a blender (Hamilton Beach, Virginia, USA) for approximately 30 s, the crushed seed endosperm was sieved. The size particles used to set up for the extraction experiments were those retained between 12 and 14 meshes (ASTM). Additionally, the average particle size of the sieved was measured in a light microscope (Eclipse Ti-U, Nikon, Japan) and by means of image analysis of at least 200 particles, it was found that the average particle size was in the range of 1.5 ± 0.12 mm for all experiments. Oil extraction was carried out in a Soxhlet apparatus (Fig. 1(a)) as the comparison method, where 10 g of crushed castor beans were placed inside a cellulose cartridge of 33 mm × 80 mm (Whatman, 2800-338, GE Healthcare, UK). Hexane (06471, Fermont, Mexico) was used in the extraction in a sample-dissolvent ratio of 1:15 (g/mL) at 70 °C for 8 h. Then, thermosonication extraction (TS) was carried out in a modified Soxhlet system (Fig. 1(b)) coupled with a sonicator (VCX130, SONICS Vibra cell™, Newton CT, USA) at 20 kHz and 130 W with a 120 V generator (CV13), using a standard probe of the sonicator (length of 113 mm and diameter of 6 mm) (Fig. 1(c)). The probe was submerged into the mixture placed in the cellulose cartridge. In TS, the sample–dissolvent ratio and the temperature were equal to those applied in the Soxhlet extraction (S). With both methods, after extraction was completed, oil–hexane mixture was collected and the dissolvent was evaporated in a vacuum evaporator (R-3000, Büchi, Switzerland). To remove the remaining hexane, the ball flasks were placed in an oven (Dynamica BIO-performance, Meyzieu, France) at 70 °C for one hour. The oil obtained was weighed on an analytical balance (accuracy of 0.1 mg, Explorer OHAUS, USA) and the oil yield was calculated. All determinations were done in triplicate.

xi − x i0 Δxi

(1)

where Xi , xi, and xi0 are the dimensionless, actual, and initial values, respectively, of the variable i at the central point, Δxi is the change of xi with regard to unit variation of the dimensionless value. The process requires a square model, expressed in Eq. (2):

Yi = β0 + β1 X1 + β2 X2 + β11 X12 + β22 X22 + β12 X1 X2

(2)

where Yᵢ is the dependent variable, β0 is a constant, β1and β2 are linear terms, β11 and β22 are squared coefficients, and β12 is the term that represents the interaction. The experimental design and RSM were conducted using Design Expert 7.0. A well-fitting regression model was evaluated using ANOVA, R2, the probability (P), F-test. 3D plots obtained from RSM have used for analyzing the behavior of the independent variables on the dependent variables, and the optimal conditions were determined from model coefficients and statistical parameters. 2.3. Structural characterization of residual cake The residual cake was dried in a desiccator at room temperature overnight, thereby, solvent-free powders were obtained. The microstructural changes of the powders by both extraction methods were studied by means of SEM. Powders were placed in aluminum holders adhered with carbon tape and coated with carbon by cathodic sputtering (SPI Supplies, USA). The samples were observed by field emission SEM (JEOL JSM-7800 F, Japan) at 500 and 1000×, using a backscattered electron detector (BED). The SEM images were used to estimate the particle size of dry powders by image analysis methodology with ImageJ v.1.47 (National Institutes of Health, USA). In order to obtain the particle size, the major length of particles was obtained manually, following the methodology mentioned by Suarez-Najera et al. (2018). The lignocellulosic compounds, proteins, and lipids in the residual cake were identified and their distribution obtained by confocal laser

2.2. Experimental design In the experimental design, the effects of time (25, 35, and 45 min) and output amplitude of the transducer (25, 50, and 75%) on the castor oil extraction the RSM was used. The independent variables were the 3

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relationship between the parameters chosen, except for RI, which did not show a significant effect of the extraction variables. F and P values were used to determine the significant difference terms of each coefficient. Large F-value and a small P-value depict a more significant effect on the corresponding response variable (Chanioti and Tzia, 2017). Table 3 presents the values of the physicochemical parameters obtained experimentally by TS at 35 min, where better extraction performance was obtained at amplitudes of 25, 50, and 75%. In addition, the optimal points derived from RSM of the physicochemical parameters at 35 min are presented. It can be noticed that the experimental values of evaluated parameters closest to the optimal points obtained by RSM were obtained at an amplitude of 50% because no found significant differences between them. Additionally, a structural study was carried out using the experimental conditions to understand the changes that occurred in the residual cake under the extraction conditions (Table 3). Overall, the influence of the TS on the percentage oil yield can be observed; nearly 62% of the oil is extracted in 35 min at an amplitude of 50%, whereas S took 8 h to reach an oil yield of 57% (Table 1). The model has an R-squared of 0.98 (Table 2). The time and amplitude showed an effect on the linear terms (p < 0.05), while, the time had significance for the quadratic term (p < 0.05), which demonstrate that the percentage oil yield extraction depended on these operation variables. As has been by Hernandez-Santos et al. (2016) in their study of the effects of amplitude and time in ultrasound-assisted extraction on the physicochemical properties of pumpkin seed oil. In other study of the oil extraction from Kolkhoung (Pistacia khinjuk) kernel with pulsed ultrasound probe (30 kHz), evaluated the effect of conditions process (amplitude of 0, 25, and 50% and at different temperatures, 30, 40, and 50 °C) on yield and quality of extracted oil (Hashemi et al., 2015), where the highest yield (77.5% w/w), an increase in the oil quality, and improvement of the extraction process were obtained under similar conditions (50% and 50 °C) herein studied. Fig. 2a shows the relationship between oil extraction and amplitude, where it can be observed that from 25% to 50% of amplitude, oil extraction increases. The increase in amplitude can be attributed to the cavitation, in which the propagation of the sound waves during contraction and expansion of the liquid and solid produce some bubbles; subsequently, those bubbles grow and break, which causes soluble substances from the solid phase to dissolvent to flow with greater velocity. However, at 75% amplitude, the oil yield begins to decrease slightly, which may be due to the fact that the larger amplitude causes wave instability due to high pressure generated in the thermosonication system (Suslick, 1995). The high pressure generated in the TS is associated with the larger amplitude causes wave instability in the liquid phase which induces faster bubbles collapse, and eventually, the rupture of cell tissue, facilitating the extraction of compounds that are only soluble in hexane. The effect of amplitude in the UAE has been reported

scanning microscope (CLSM, LSM NLO 710, Carl Zeiss, Germany). Fluorescent brightener (Calcofluor white M2R) was used to stain the cellulose and hemicellulose, fluorescein 5 (6) isothiocyanate (FITC) at 0.01% (w/v) was used for the proteins, and oil red O at 0.01% (v/v) was used for the lipids. For each staining, the wavelengths used were 405, 488, and 633 nm respectively. All dyes were obtained from SigmaAldrich. The images were acquired by the software ZEN 2010 (Carl Zeiss, Germany). Oil objective lens at 1024 × 1024 pixels and stored in TIFF format. The XRD of residual cake powders was carried out by means of a diffractometer (Rikagu MiniFlex 600, Tokyo, Japan), CuKα radiation source at 40 kV, scans from 2θ = 3 to 60°, at 3 s/step. The crystallinity index (CI) was calculated using the XRD deconvolution method. Crystalline peaks were separate of the amorphous contributions, from pattern diffraction with a curve-fitting, using Origin Pro v.8.0 (OriginLab Corporation, USA). To evaluate the CI, cotton cellulose (435236, Sigma-Aldrich, USA) was used as the reference cellulose (RC); in this case, five crystalline peaks, 101 at 15°, 10 1¯ at 16.4°, 021 at 20.7°, 002 at 22.6°, and 040 at 34.7° were considered (Park et al., 2010), while for the residual cake powders only the crystalline peak 002 shifted to the left at 19° was observed. The CI was calculated as given in Eq. (3):

CI (%) =

Crystalline peak area × 100 Total area

(3)

2.4. Statistical analysis Significant differences (p < 0.05) were verified by ANOVA with a Tukey test (SigmaPlot software v. 12.0, Systat Software, Inc., USA). 3. Results and discussion 3.1. Oil extraction Table 1 shows the responses of the physicochemical parameters (II, PI, SI, USM, AI, and RI) to the castor oil extraction by S and TS. For the oil yield and the results from the experimental design of two controlled variables, namely time and amplitude as a percentage in the ranges of 25 to 45 min and 25 to 75%, respectively, were chosen to fit the limits of the TS. This was carried out with 13 experiments by multivariate study. Overall, for all physicochemical parameters, the standard deviation ranged from 0 to 0.82 with a maximum variation of the coefficient of 1% (data not shown). This confirmed that the reproducibility of experiments was accurate. The results of the fitting model are shown in the (Table 2), R2 values were used as a measure of the model goodness. The determination coefficients (R2) were 0.99, 0.93, 0.93, 0.93, 0.88, 0.96, and 0.50 for the percentage oil yield, II, PI, SI, USM, AI, and RI, respectively, which means that the models represent a real

Table 1 Responses of dependent variables of the castor oil extraction by thermosonication and average experimental values of physicochemical parameters. Exp.

Time (min)

Amplitude (%)

Oil yield (%)

II (g I2/100 g Oil)

PI (meq O2/kg Oil)

SI (mg KOH/g Oil)

USM (%)

AI (mg KOH/g Oil)

RI20 °C

1 2 3 4 5 6 7 8 9 10 11 12 13

25 45 35 35 35 35 35 25 35 25 35 45 45

75 75 50 50 75 50 25 50 50 25 50 50 25

55.57 60.96 60.93 61.50 57.71 60.90 47.97 59.53 61.12 44.84 61.11 61.10 47.59

85.14 85.49 85.70 85.80 85.23 85.82 85.70 85.00 85.80 84.85 85.80 85.90 85.67

4.75 4.90 4.68 4.69 4.76 4.70 4.63 4.68 4.70 4.65 4.70 4.85 4.70

180.30 180.92 180.83 180.81 180.79 181.00 180.49 180.51 180.84 180.34 180.86 180.63 180.35

0.54 0.56 0.55 0.56 0.55 0.55 0.55 0.54 0.55 0.54 0.55 0.56 0.56

0.65 0.70 0.70 0.68 0.68 0.69 0.65 0.67 0.69 0.65 0.69 0.70 0.65

1.4760 1.4760 1.4760 1.4760 1.4760 1.4760 1.4750 1.4760 1.4760 1.4759 1.4760 1.4760 1.4760

II: Iodine index; PI: Peroxide index; SI: Saponification index; USM: Unsaponifiable Matter; AI: Acidity index; RI: Refraction index. 4

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Table 2 Analysis of variance for the response surface of the oil extraction by thermosonication and physicochemical parameters. Coefficient

Oil yield (%)

p < 0.05

II

p < 0.05

PI

p < 0.05

SI

p < 0.05

USM

p < 0.05

AI

p < 0.05

RI

p < 0.05

β0 β1 β2 β12 β11 β22 F–value R2 Adj R2 CV Lack of fit (p–value)

61.08 1.62 5.64 0.66 –0.70 –8.18 149.62 0.99 0.98 1.35 0.00

< 0.0001 0.0013 < 0.0001 0.1289 0.1721 < 0.0001 – – – – –

85.77 0.04 –0.08 –0.08 –0.28 –0.26 19.69 0.93 0.88 0.15 0.01

0.0005 0.0002 0.1448 0.2263 0.0079 0.0103 – – – – –

4.70 0.07 0.07 0.03 0.07 –0.01 19.02 0.93 0.88 0.60 0.01

0.0006 0.0005 0.0004 0.1192 0.0060 0.6066 – – – – –

180.85 0.13 0.14 0.15 –0.23 –0.16 18.78 0.93 0.88 0.05 0.35

0.3216 0.9780 0.0851 0.9461 0.4010 0.3206 – – – – –

0.55 0.01 0.00 0.00 –0.00 –0.00 10.31 0.88 0.80 0.62 0.99

0.0040 0.0002 1.0000 1.0000 0.6324 0.6324 – – – – –

0.69 0.01 0.01 0.01 –0.00 –0.02 30.76 0.96 0.93 0.83 0.93

0.0001 0.0007 0.0007 0.0030 0.2989 0.0002 – – – – –

1.48 0.00 0.00 –0.00 0.00 –0.00 1.41 0.50 0.14 0.02 –

0.3280 0.8776 0.1223 0.8505 0.1796 0.1218 – – – – –

II: Iodine index; PI: Peroxide index; SI: Saponification index; USM: Unsaponifiable Matter; AI: Acidity index and RI: Refraction index. The differences are considered significant at p < 0.05. Table 3 Experimental results of Soxhlet extraction and thermosonication extraction at different amplitudes for 35 min and optimal points obtained by response surface methodology. Physicochemical parameters Yield Oil (%) II (g I2/100 g Oil) PI (meq O2/kg Oil) SI (mg KOH/g Oil) USM (%) AI (mg KOH/g Oil) RI (–)

Soxhlet

25% Amplitude ac

57.30 ± 1.01 85.66 ± 0.55 5.03 ± 0.15 180.60 ± 0.79 0.71 ± 0.50 0.75 ± 0.30 1.476 ± 0.00

50% Amplitude

ab

abc

47.97 ± 0.16 85.70 ± 0.12 4.63 ± 0.14 180.49 ± 0.27 0.55 ± 0.01 0.65 ± 0.01 1.475 ± 0.00

61.12 ± 0.33 85.80 ± 0.40 4.70 ± 0.06 180.87 ± 0.54 0.55 ± 0.01 0.69 ± 0.01 1.476 ± 0.00

Optimal points by RSM

75% Amplitude

60.88 85.66 4.69 180.81 0.55 0.69 1.475

57.71 ± 0.04bc 85.23 ± 0.46 4.76 ± 0.22 180.79 ± 0.68 0.55 ± 0.01 0.67 ± 0.01 1.476 ± 0.00

II: Iodine index; PI: Peroxide index; SI: Saponification index; USM: Unsaponifiable matter; AI: Acidity index; RI: Refraction index. Values (mean ± standard deviation) in the same row with the same letters are significantly different (p < 0.05).

2013), although it could be used as a lubricant or hydraulic brake fluid (Yusuf et al., 2015). At higher values of the amplitude, PI increases (Fig. 2c), a significant effect (p < 0.05) of the amplitude and time on the linear terms and quadratic was presented in Table 2. This behavior may be due to prolonged extraction times and high temperatures during TS, forming in the initial stage of oxidation, compounds that contain hydroperoxide as functional group which does not change to secondary oxidation compounds as peroxyl, alkoxyl radicals, aliphatic aldehydes, alcohols and ketones (Samaram et al., 2015). The PI is related to oil oxidation as a result of hydroperoxide formation at double bond positions. In castor oil, the double bond on C-9 of ricinoleic acid is believed to be protected against hydroperoxide formation by the hydroxyl group on C-12 (Yusuf et al., 2015). Overall, the results obtained (Table 1) demonstrate that the PI is low and does not change during the extraction experiments, which indicates that the oils are more stable to oxidation. The stability of castor oil against oxidative rancidity is presented due to low iodine and peroxide values observed for the castor oil (Yusuf et al., 2015). For the SI, the effects of amplitude and time on the linear, interaction and quadratic terms not had a significant difference. In Fig. 2d, the effects of time and amplitude on the SI are shown; the SI highest value was around of 180, at amplitudes and times intermediate. High values of saponification (∼175) indicate that the oil has the potential to be used in the soap and cosmetics industries. In USM, the time showed a significant effect (p < 0.05) on the linear term, as shown in Table 2. The USM of the castor seed oil extracted by TS extraction presented an average value of 0.55% and the S extraction method gave a slightly higher value of 0.71%; it can be seen that the USM obtained with both methods were found to be low and can be comparable to the USM value of olive pomace oil given by IOCC Trade Standards (Council, 2003), which is about 3%. Different values of USM have been reported for vegetable oils such as walnut oil (3.73%), rice bran oil (4%), olive oil (1.5%), and sesame oil (1.5%) (Chanioti and Tzia, 2017). Pradhan et al. (2012) obtained a USM of 0.94% for castor oil. Values of USM less than 2% are

in previous reports (Chemat et al., 2011, 2017, Anaya-Esparza et al., 2017; Hashemi et al., 2015), the increase of amplitude is directly linked to ultrasonic intensity or energy transmitted to system and subsequently to pressure of the shockwaves, theoretically, this produces a rapid and intense bubbles collapse, that increase cavitation phenomenon and extraction efficiency. However in batch systems at higher amplitudes, the transfer phenomena are transients and, the medium viscosity and operation temperature change with process time proceeds, in special liquid agitation, could be a predominant event instead of cavitation, this could cause a low energy transmission in the liquid media and a slight decrease in yield extraction could be expected, such as was observed in the current study. Nevertheless, Chemat et al. (2017) recommend that amplitude should be increased when liquids (oils) with high viscosity are processed with TS, but the energy to reach the cavitation threshold must be considered. For these reasons, to choose an optimum range of operating conditions of the TS, equipment scale-up and for each specific solid matrix extracted, previous optimization studies are required. Besides, the use of power ultrasound, when combined with heat treatment, reduces the time taken to rupture the endosperm cells, allowing the castor oil to be extracted in a shorter time, after which the oil yield is not changed by the effect of TS. The highest value of II was presented at 50% and 45 min, as can be seen in Table 1. Time showed a significant effect on the linear and quadratic terms (p < 0.05). However, the amplitude and interaction terms were non-significant. (Fig. 2b) shows that II increases slightly with increases in the time. The II determines the amount of unsaturation of fatty acids from castor seed oil (g I2/100 g oil); this index is an indicator of total unsaturation of oil, as a measure of their susceptibility to oxidation. The higher value of 85.90 obtained for the II (Table 1, Exp. 12) when is compared with the ASTM standard and general specifications for industrial grade castor oil (WHC, 2012), the II level is low (with II values of 50 to 100). The low iodine value observed for the castor oil is characteristic of non-drying oils with a low unsaturation level. This means that the oil may not be suitable as alkyd resin for paint formulation or varnishes (Yang et al., 5

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Fig. 2. Response surfaces of a) percentage oil yield; b to g) physicochemical parameters of castor oil in function of sonication conditions (amplitude and time). II: Iodine index, PI: Peroxide index, SI: Saponification index, USM: Unsaponifiable matter, AI: Acidity index, RI: Refraction index at 25 °C.

with subcellular organelles (protein and lipid bodies) and the cell wall can be observed; a similar microstructure has been described by PereaFlores et al. (2011). Image analysis showed that the average size of endosperm cells of native castor seeds was 50 ± 9 μm (Fig. 4a), while the lipid bodies had an average size of 12 ± 1 μm. Fig. 4b and Fig. 3b shows the residual cake after oil extraction by S, where it can be observed the destroyed the structure of cell walls and dispersed lipid bodies with severe damage to their membranes, with some voids and noticeable shrinkage due to oil diffusion. By SEM it can be observed that there are two main particle types: extracted lipid bodies and cell wall fragments. Both types of particles were measured by image analysis; the average particle size of the cell wall fragments was 27 ± 9 μm, while the lipid bodies had an average particle size of 10 ± 2 μm. After Soxhlet (S) extraction, the lipid bodies were reduced in size in comparison with those observed in the endosperm cells of the native seeds, since these lost their membranes and shrunk; also the cell wall of endosperm cells was broken into large fragments due to the extraction process. In the case of protein bodies, their number decreased and structural damage could be seen. All microstructural changes were caused by high temperature and the long duration of the extraction process with S. SEM images of flaked caraway seeds extracted by S (Hexane, 60 min at 69 °C) showed cell rupture (detexturation) and diffusion of the extracts (Chemat et al., 2004), however, the structural damage in cellular tissue of caraway seeds was less drastic than endosperm tissue of castor seeds studied herein, since the processing time was longer (8 h). Fig. 5 shows SEM images of the microstructural changes of the

a potential feedstock, which can be used in biodiesel production (Chanioti and Tzia, 2017). With regard to the AI, the time and amplitude had an effect on the linear and interaction terms (p < 0.05), and the quadratic term was significant for amplitude variable. Fig. 2(f) presents the effect of time and amplitude on the AI (mg KOH/g oil), which was evaluated as a measure of the potassium hydroxide required to neutralize the oil. In amplitudes and intermediate times, values of 0.67 for the TS and 0.71 for S were obtained. Both extraction methods gave a low acid value compared with other studies (10 mg/g) (Canakci, 2007). The difference in acid value in comparison with those obtained by other studies could be due to factors such as maturity stage and storage time of the seeds. It is well known that the acid value could oscillate about 10% if the oil is stored approximately 90 days, thus the acid value can be reduced by the reaction between hydroxyl and carboxyl groups to form estolides from oil molecule (Canakci, 2007). A similar RI was obtained at 20 °C (Table 1) by both extraction methods in all experiments. The RI value was 1.476 and was in almost the same range as the value reported by Dias et al. (2013) for castor oil, which was 1.47 at 25 °C.

3.2. Structural study of residual cakes A study of the effect on the residual cake microstructure before and after extraction (S and TS) was performed by means of SEM, CLSM, and XRD. Fig. 3a and b show the native castor seed and residual cake after oil extraction by S, respectively. Fig. 3a shows SEM images of the endosperm cells of the native castor seed, where cells of isodiametric form 6

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Fig. 3. Scanning electron microscopy images at different magnifications: a) native castor seed and b) residual cake after oil extraction by the Soxhlet method. cw: cell wall; cwf: cell wall fragments; lp: lipid bodies; pb: protein bodies and v: voids. Scale bars correspond to 10 μm.

lipid bodies Fig. 4b obtained by TS at amplitudes of 50% (8 ± 2 μm) and 75% (9 ± 2 μm) are smaller than those obtained at 25% (11 ± 2 μm) and by S (10 ± 2 μm) and native lipid bodies (12 ± 1 μm). In all cases, the size of the lipid bodies had a significant difference between amplitudes. Specifically, at amplitudes of 50 and 75%, the shrinkage was more severe and was linked with high oil yield under these TS conditions. These microstructural changes were compared to reported in other biological materials, for instance, fragmentation mechanism has been observed in chlorophyll extraction from spinach leaves, and this effect is more remarkable in ultrasound extraction than maceration process (Chemat et al., 2017). Detexturation effect is linked with severe destruction of cell wall material, this effect has been observed in flaked caraway seeds extracted by sonication (Chemat et al., 2004). In boldo leaves erosion effect was studied by scanning electron microscopy (SEM), where surface and trichomes microstructure were affected by ultrasound waves, erosion phenomenon was attributed to the improvement of extraction (Petigny et al., 2013). While sonoporation by ultrasound technology has been used to cell permeabilization

residual cake from thermosonication (TS) extraction of oil at different amplitudes. Thus, smaller fragments of cell walls (fragmentation and detexturation effects) and lipid bodies with more damage (erosion effect) and voids (sonoporation) as well as noticeable shrinkage (oil diffusion) were observed at amplitudes of 50% (Fig. 5b and 75% Fig. 5c) compared to 25% Fig. 5a. This showed that the TS caused remarkable damage to the cell walls and yielded fragments with lower sizes Fig. 4a at amplitudes of 50% (18 ± 5 μm) and 75% (19 ± 5 μm) than at 25% (26 ± 6 μm) while in the S method (27 ± 9 μm, see Fig. 3b). Statistical analysis indicated that the size of cell wall fragments induced by S and TS at 25% no had significant differences (p > 0.05); in the same way, the treatments at 50% and 75% showed no significant differences between amplitudes Fig. 4a. In general, an increase in sonoporation (voids) and erosion to the lipid bodies membranes were observed when TS was used (Figs. 5a-c) in comparison with the residual cake obtained by S extraction (Fig. 3b), which can be attributed to the fact that these voids are preferential spaces for oil diffusion during extraction. Consequently, the sizes of

Fig. 4. a) Particles sizes of the native cells (N) and cell wall fragments (cwf), b) particle sizes of the lipid bodies (lb) in the native cells at different amplitudes of the thermosonication extraction. S: Soxhlet extraction. Bars marked by the same letters have no significant differences between them (p > 0.05). 7

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Fig. 5. Scanning electron microscopy images at different magnifications of residual cakes after extraction of castor oil by the thermosonication process during 35 min and at different amplitudes: a) 25%, b) 50%, and c) 75%. cwf: cell wall fragments; lp: lipid bodies; pb: protein bodies, v: voids. Scale bars correspond to 10 μm.

by S, the channels of cellulose-hemicellulose show breaking of the cell wall into several fragments, as was also observed in SEM images, where fragmentation and detexturation effects were evident. The proteins seem more dispersed than in the native sample due to the thermal treatment by S, perhaps because some protein and lipid components of the lipid bodies membranes were disassembled by the action of the dissolvent. Moreover, the typical spherical shape of lipid bodies in native samples was affected by the extraction process and in the images of S, the spherical shape of lipid bodies became irregular; these changes are linked with the shrinkage observed in SEM images and the results provided by the image analysis. In the case of the lipids channel, in native samples the lipids can be observed in abundance but are distributed in the whole of the cellular structure of endosperm cells; this effect is caused by the cutting done during sample preparation, while after treatment by S, the presence of lipids decreased due to the separation of the lipids by diffusion towards the dissolvent and only some lipid agglomerates could be seen. Integrated images (Fig. 6a and b) provide a comparative snapshot of the effect of S on the microstructure of the endosperm cells of native castor seeds, where rupturing of cell walls, deformation of lipid bodies, and removal of lipids were evidenced. Fig. 7 shows CLSM images of the residual cakes obtained under different TS conditions. Cellulose-hemicellulose images at different amplitudes evidenced that drastic rupturing of the cell walls of endosperm cells was caused by TS, and this promoted more efficient oil extraction compared to S (Fig. 6), whereas in the protein channels, the

and inactivation of pathogen microorganisms. In the case of lipids bodies content in endosperm cell of castor seeds, its size is close to the diameter of yeats (depending on the species and growth stage) and it is frequently observed sonoporation phenomenon in biological systems of comparable sizes as bacteria, yeasts and oil glands treated with ultrasound, as has been reported by Ugarte-Romero et al. (2006); Meullemiestre et al. (2016); Veillet et al. (2010), respectively. Kerr (2004) found that heat treatment breaks the cell wall structures. Also, the effects involved in ultrasound can accelerate the eddies in the dissolvent and internal molecular diffusion (Yusuf et al., 2015; Yang et al., 2013) and facilitate the diffusion of solvent into the sample matrix (Chanioti and Tzia, 2017), which could be generated by the rupture of the cell walls in the induction of ultrasonic cavitation and local shear stress, which homogenizes the TS system, releasing more oil in comparison with the S method. CLSM requires fluorochromes to evidence the presence of specific compounds into biological materials. Taking advantage of this capability, the main components in castor beans were stained to describe the microstructural changes caused by the extraction processes. Fig. 6 shows the presence of cellulose-hemicellulose, lipids, and proteins in castor seeds before and after oil extraction by the S method. CLSM (Fig. 6a) evidenced the abundance of cellulose and hemicellulose (blue color) in the cell wall of endosperm cells, while proteins (green color) and lipids (red color) are notorious in lipid bodies and the cytoplasmic matrix, as shown in the channels of the proteins and the lipids as well as in the integrated image. In Fig. 6b, showing the residual cake obtained 8

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Fig. 6. Confocal laser scanning microscopy images stained with calcofluor for identification of cellulose-hemicellulose (blue), fluorescein 5(6)-isothiocyanate (FITC) for proteins (green), and oil red O for lipids (red) and the integrated image. a) Native castor seed; b) residual cake after extraction of oil by the Soxhlet method. Scale bars correspond to 10 μm.

Fig. 7. Confocal laser scanning microscopy images of residual cakes after extraction of castor oil by thermosonication during 35 min and at different amplitudes (a, b, and c). Stained with calcofluor for identification of cellulose-hemicellulose (blue color), fluorescein 5(6)-isothiocyanate (FITC) for proteins (green color), and oil red O for lipids (red color) and integrated images. Scale bars correspond to 10 μm. 9

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expansion of lipid bodies was observed at the amplitude of 25% (Fig. 7a), although at amplitudes of 50 and 75% (Fig. 7b and c) the lipid bodies showed drastic deformation of their spherical shape and more severe shrinkage, probably caused by sonoporation, erosion and oil diffusion mechanisms (Chemat et al., 2017), similarly to what was observed by SEM and image analysis. Also, the dispersion of proteins was wider with TS than with the S method, which demonstrated that TS caused more damage to the microstructure of the cell wall and the lipid bodies membranes in comparison with S extraction. This may be associated with the better yield achieved in the oil extraction when TS is used. The lipid channel confirms this fact: at an amplitude of 50%, the fluorescence of lipids was lower than at other amplitudes of TS, and this coincided with the best yield of oil obtained at the amplitude of 50%. The integrated images confirmed the low presence of oil at amplitudes of 50 and 70%, caused by greater damage to the cellular structure of endosperm cells when TS was applied compared to the S method. CLSM provides relevant information to evaluate changes in the structural components of the seed during the extraction and as a visual indication of the extraction performance, as it provides information on the damage occurred in the parenchymatic tissue. To our knowledge, there are no studies by CLSM that demonstrate the distribution of compounds in the residual cake during the oil extraction process. CLSM has been demonstrated to be useful in providing information about the distribution of structural compounds and their release during extraction, which could be of interest as sources of lignin and cellulose (Hernández-Hernández et al., 2016a, 2016b). To evaluate the effect of the extraction on the cellulose structure, XRD of the residual cake powders was performed. Fig. 8 shows the diffractogram of the reference cellulose (RC) and of the residual cake from TS at different amplitudes and from S. In the RC pattern, five peaks are showed at 2θ = 15°, 16.4°, 20.7°, 22.6°, and 34.7°, that correspond to the planes: 101, 10 1¯, 021, 002, and 040, respectively. This pattern is characteristic of cellulose type I which is the main constituent of wood and plant tissues (Park et al., 2010). The diffraction patterns and CI obtained for each treatment are shown in Fig. 8. The diffractograms show that for all the powders extracted there is a peak at around 20°, which may be the displacement of the cellulose peak assigned to 23°. However, the peaks showed different intensities between extraction treatments. The peak heights decreased as the extraction treatment became more drastic, namely at 50% amplitude in the TS treatment and with a longer extraction time, the latter, in the case of the Soxhlet treatment. In this way, a wider and lower-altitude peak is observed for the powders of the residual cake treated by S. This lower intensity may be due to the treatment conditions of high temperature and long extraction time could cause that the peak to become less intense but not disappear. The highest peak was found in the sample extracted at 25%

Fig. 9. Diagram of the microstructural changes and extraction combined mechanisms that the seed undergoes when the oil is extracted by Soxhlet (S) and thermosonication (TS) methods from castor seeds.

amplitude, followed by the sample extracted at 75% amplitude, which did not show a great difference in the decrease with respect to the one extracted at 50% amplitude. This behavior can be explained in relation to the microstructure of the residual cake, where, as described above, the presence of voids is due to the greater oil output, leading to the higher extraction yield which was obtained at 50%. Another explanation for the decrease of the peaks is the crystallinity, which may be due to the presence of oily residues with non-crystalline polymers as pectin, lignin, and hemicelluloses, which caused a decrease in the CI of XRD patterns (Hernández-Hernández et al., 2016a, 2016b). Taking as reference the highest CI, which was obtained by the RC, the CI of the residual cake decreases as the extraction treatment becomes more drastic in terms of longer extraction time and greater amplitude (Fig. 8), which could lead to cellulose less ordered in surface layers. This analysis confirms that the extraction by TS can be an option to extract oil, with the possibility of taking advantage of the cellulose content, for which, it is suggested to extract the proteins contained in the material to improve the crystallinity and cellulose quality. Finally, Fig. 9 presents a diagram that schematizes the microstructural changes that the seed undergoes when the oil is extracted by S and TS. Four stages of extraction are proposed: The first stage represents the changes undergone by the seed through the first contact with the ultrasonic waves and heat (TS) and heat alone (S). In this stage, the seeds experience intercellular disruption (fragmentation mechanism), which may be due to the spread of sound waves (TS) and the long duration of the extraction process (S), promoting cell separation in both cases. In the second stage, the rupture of the cell walls (detexturation effect) causes the release of the soluble substances from the solid phase to the dissolvent. As the extraction time proceeds, in TS,

Fig. 8. X-ray diffraction pattern of the residual cake obtained by different methods and under different conditions in comparison with the reference cellulose (RC) and the crystallinity index (CI) of RC and castor residual cakes. 10

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contribute to the development of technologies for the extraction of oils by TS at the industrial level. This work provides an overview of the most important aspects to extract by TS castor oil from castor seeds, and integrated the optimization of process conditions, the evaluation of the quality of the extracted oil, while another novelty was the use of microscopy and X-ray diffraction techniques to study the extraction mechanisms that occur during extraction of the castor oil using TS.

the ultrasonic waves traveling through the dissolvent cause bubbles to collapse more drastically, leading to disruption of the cellular tissue and thus the release of the lipid bodies by local shear stress mechanism. In the case of S, the long duration of the thermal process causes microstructural damage similar to treatment by TS. The third stage refers to the analysis of the three pathways that lipid bodies take as the course of extraction continues. One proposed pathway occurs at low amplitudes (25%), with the internal distortion of the organelles by erosion effect, which begins to leak their contents, and the internal molecular diffusion gives rise to an increase in oil diffusion and erosion, which leads to slight shrinkage of the lipids bodies. The other path that lipid bodies could take happens at high amplitudes (50% and 75%), where the waves travel at a higher velocity due to ultrasound, resulting in an increase in the voids (sonoporation) and damage (erosion) to the lipid bodies membranes such that the oil comes out more easily, and consequently the lipid bodies shrink and the oil yield is increased. The last path refers to the lipid bodies obtained by S, which were extracted from cell wall fragments; these lipid bodies have fewer voids and less shrinkage than those obtained at high amplitudes. The previous stages were proposed according to the microstructural changes observed by means of SEM and CLSM in the native seed and after the extraction by S and TS. A remarkable aspect of the microstructural study of the residual cake was to find that the extraction of castor oil by TS occurred by different mechanisms, due to the compartmentalized structural inside the endosperm cells (cell wall, lipids and protein bodies), due to this hierarchical organization the oil extraction by conventional methods required to be exhaustive (about 8 h), while the extraction TS showed to be efficient in a short process time and with less energy consumption, since that different extraction mechanisms occurred in combination, (fragmentation, local shear stress, diffusion, erosion, sonoporation, detexturation), this also turned out to be adequate to minimize damage to the cell wall material (cellulose) compared to S extraction.

Acknowledgments Pedro López Ordaz wishes to thank CONACyT and BEIFI, COFAA, Instituto Politécnico Nacional (IPN) in Mexico for the scholarship provided during his PhD studies, and the financial support provided by CONACyT projects (239899,268660) and SIP-IPN (20170517, 20171797, 20180455, 20195158) References Ali, R.M., Elfeky, S.S., Abbas, H., 2008. Response of salt stressed Ricinus commnunis L. To exogenous application of glycerol and/or aspartic acid. J. Biol. Sci. 171–175. https:// doi.org/10.3923/jbs.2008.171.175. AOAC, 2005. Oils and fats. In: Firestone, D., Yurawecz, M.P. (Eds.), AOAC Official Methods of Analysis, 18th ed. AOAC International, Gaithersburg, MD (USA) Chapter 41. AOAC, 1990. Official Methods of Analysis, 15 ed. USA. Anaya-Esparza, L.M., Velazquez-Estrada, Rita M., Roig, Artur X., García-Galindo, H.S., Sayago-Ayerdi, S.G., Montalvo-Gonzalez, E., 2017. Thermosonication: an alternative processing for fruit and vegetable juices. Trends Food Sci. Technol. 61, 26–37. https://doi.org/10.1016/j.tifs.2016.11.020. Aurand, L.W., Woods, A.E., Wells, M.R., 1987. Food Composition and Analysis: AVI. Van Nostrand Reinhold Co., New York. Canakci, M., 2007. The potential of restaurant waste lipids as biodiesel feedstocks. Bioresour. Technol. 98, 183–190. https://doi.org/10.1016/j.biortech.2005.11.022. Chanioti, S., Tzia, C., 2017. Optimization of ultrasound-assisted extraction of oil from olive pomace using response surface technology: oil recovery, unsaponifiable matter, total phenol content and antioxidant activity. LWT - Food Sci. Technol. 79, 178–189. https://doi.org/10.1016/j.lwt.2017.01.029. Chemat, S., Lagha, A., AitAmar, H., Bartels, P.V., Chemat, F., 2004. Comparison of conventional and ultrasound-assisted extraction of carvone and limonene from caraway seeds. Flav. Frag. J. 19, 188–195. https://doi.org/10.1002/ffj.1339. Chemat, F., Zille, H., Khan, M.K., 2011. Applications of ultrasound in food technology: processing, preservation and extraction. Ultrason. Sonochem. 18, 813–835. https:// doi.org/10.1016/j.ultsonch.2010.11.023. Chemat, F., Rombaut, N., Gaëlle Sicaire, A., Meullemiestre, A., Fabiano-Tixier, A.S., Abert-Vian, M., 2017. Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrason. Sonochem. 34, 540–560. https://doi.org/10.1016/j.ultsonch.2016.06.035. Council, I.O.O., 2003. Trade Standard Applying to Olive Oil and Olive Pomace Oil. Crowe, T.D., White, P.J., 2001. Adaptation of the AOCS official method for measuring hydroperoxides from small-scale oil samples. J. Am. Oil Chem. Soc. 78 (12), 1267–1269. Cruz, R.M.S., Vieira, M.C., Fonseca, S.C., Silva, C.L.M., 2011. Impact of Thermal Blanching and Thermosonication Treatments on Watercress (Nasturtium officinale) Quality: Thermosonication Process Optimisation and Microstructure Evaluation. Food Bioprocess. Tech. 4 (7), 1197–1204. https://doi.org/10.1007/s11947-0090220-0. Da Porto, C., Porretto, E., Decorti, D., 2013. Comparison of ultrasound-assisted extraction with conventional extraction methods of oil and polyphenols from grape (Vitis vinifera L.) seeds. Ultrason. Sonochem. 20 (4), 1076–1080. https://doi.org/10.1016/j. ultsonch.2012.12.002. Danlami, J.M., Arsad, A., Zaini, M.A.A., 2015. Characterization and process optimization of castor oil (Ricinus communis L.) extracted by the soxhlet method using polar and non-polar solvents. J. Taiwan Inst. Chem. Eng. 47, 99–104. https://doi.org/10.1016/ j.jtice.2014.10.012. Dias, J.M., Araujo, J.M., Costa, J.F., Alvim-Ferraz, M.C.M., Almeida, M.F., 2013. Biodiesel production from raw castor oil. Energy 53, 58–66. https://doi.org/10.1016/j.energy. 2013.02.018. Adam, F., Abert-Vian, M., Peltier, G., Chemat, F., 2012. “Solvent-free” ultrasound assisted extraction of lipids from fresh microalgae cells: a green, clean and scalable process. Bioresour. Technol. Rep. 114 (2012), 457–465. https://doi.org/10.1016/j.biortech. 2012.02.096. Gaëlle-Sicaire, A., Abert Vian, M., Fine, F., Carré, P., Tostain, S., Chemat, F., 2016. Ultrasound induced green solvent extraction of oil from oleaginous seeds. Ultrason. Sonochem. 31, 319–329. https://doi.org/10.1016/j.ultsonch.2016.01.011. Goodrum, J.W., Geller, D.P., 2005. Influence of fatty acid mehyl esters from hydroxylated vegetable oils in diesel fuel lubricity. Bioresour. Technol. 96 (7), 851–855. https:// doi.org/10.1016/j.biortech.2004.07.006. Guimaraes, J.L., Cursino, A.C.T., Ketzer Saul, C., Sierrakowski, M.R., Ramos, L.P., Satyanarayana, K.G., 2016. Evaluation of Castor oil cake starch and recovered glycerol and development of "Green" composites based on those with plant fibers.

4. Conclusions The most relevant contribution of this work was to use an emerging methodology to extract oil in a shorter extraction time without compromising the oil quality. The optimal conditions for the extraction of castor oil by TS were determined by means of RSM, with a good oil extraction yield in comparison with the S and greater stability against oxidation due to its low peroxide index. Based on the RSM and the structural studies, the best conditions for extraction of the oil were 35 min and 50% amplitude. Thus, the current work provides important information about physicochemical properties of castor oil obtained by thermosonication, to our knowledge, there are no studies related with this issue, and another contribution was improving the extraction yield using this technology. This is a relevant case of study, due to the several applications in the industrial sector of castor oil, such as hydraulic brake fluid, cosmetics industry and as an alternative biofuel source, among others uses. In addition, the residual cake was evaluated microstructurally after the extraction. The samples presented lignocellulosic compounds which underwent some microstructural changes due to the increase in the amplitude during the extraction by TS, which was reflected in structural changes in cellulose-hemicellulose, in the presence of lipid bodies in the residual cake observed by CLSM, and in the decrease of the CI from the diffraction pattern of XRD. The use of CLSM, SEM, XRD and image analysis allowed contribute to the elucidation of the extraction mechanisms and the microstructural changes that occur during the extraction by TS and S. Moreoever, the characteristics of the cellulose composition of the residual cake open the possibility of conducting research on the lignocellulosic compounds after the oil extraction, which can be separated and used as wall material (hemicellulose) and as mechanical reinforcement in the form of cellulose nanoparticles. This study provides important information for the extraction by TS of oils from biological materials at the laboratory level, which could 11

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