Supercritical Anti-solvent Process as an Alternative Technology for Vitamin Complex Encapsulation Using Zein as Wall Material: Technical-economic Evaluation

Accepted Manuscript Title: Supercritical Anti-solvent Process as an Alternative Technology for Vitamin Complex Encapsulation Using Zein as Wall Material: Technical-economic Evaluation Authors: M. Thereza M.G. Rosa, V´ıctor H. Alvarez, Juliana Q. Albarelli, Diego T. Santos, M. Angela A. Meireles, Marleny D.A. Salda˜na PII: DOI: Reference:

S0896-8446(19)30071-3 https://doi.org/10.1016/j.supflu.2019.03.011 SUPFLU 4499

To appear in:

J. of Supercritical Fluids

Received date: Revised date: Accepted date:

31 January 2019 19 March 2019 20 March 2019

Please cite this article as: Rosa MTMG, Alvarez VH, Albarelli JQ, Santos DT, Meireles MAA, Salda˜na MDA, Supercritical Anti-solvent Process as an Alternative Technology for Vitamin Complex Encapsulation Using Zein as Wall Material: Technical-economic Evaluation, The Journal of Supercritical Fluids (2019), https://doi.org/10.1016/j.supflu.2019.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Supercritical Anti-solvent Process as an Alternative Technology for Vitamin Complex Encapsulation Using Zein

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as Wall Material: Technical-economic Evaluation

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M. Thereza M. G. Rosaa,b*, Víctor H. Alvarezb,c, Juliana Q. Albarellia,

LASEFI/DEA/FEA (School of Food Engineering)/UNICAMP (University of

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a

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Diego T.Santosa, M. Angela A. Meirelesa, Marleny D. A. Saldañab

Department of Agricultural, Food and Nutritional Science, University of Alberta,

Camber Technology Corporation, Edmonton, AB, Canada T6G 2M9

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Edmonton, AB, Canada T6G 2P5

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Campinas), R. Monteiro Lobato, 80; 13083-862, Campinas, SP, Brazil

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Author for correspondence*: [email protected] / Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5.

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Phone: +1 780 492 8018 Fax: +1 780 492 8914 (M.T.M.G. Rosa).

Graphical abstract

Supercritical AntiSolvent Process

Filter

Thermostatic bath Heating bath

CO2 Pump

Blocking Valve Temperature Controller

Line Filter

Manometer

HPLC Pump

Flow totalizer

Back Pressure Regulator

Temperature Controller

Cylinder CO2 Solution Reservoir

Precipitated Vitamins-Zein Particles

Filter Precipitation Vessel

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Precipitated Zein Particles

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Flask

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Highlights

δ-Tocopherol, riboflavin and β-carotenewere encapsulated.

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Microcapsules showed particle size from 8 to 18 µm and spherical

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morphology.

The operational conditions presented a significant influence on precipitation

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yield.

The best result was obtained with a pressure of 16 MPa.

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CO2 flow is the key economic factor of SAS process for vitamin formulation.

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

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Abstract

The objective of this study was to investigate the technical-economic feasibility of

supercritical antisolvent technique for the precipitation of a vitamin complex containing riboflavin, δ-tocopherol and β-carotenein zein microcapsules. First, the following parameters were investigated for the precipitation of pure zein: pressure (7.0–16.0 MPa),

anti-solvent flow rate (20–60 g/min), solution flow ratio (0.5–1.5 mL/min) and concentration of zein in an aqueous ethanol solution (0.02–0.04 g/mL). At optimized SAS condition for zein precipitation (pressure of 16 MPa, temperature of 313 K, zein concentration of 0.02 mg/mL, solution flow rate of 1 mL/min and anti-solvent flow rate of 60 g/min) it was then performed the co-precipitation of the vitamins with zein. The

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results showed that the mean particle size of the microcapsules varied from8 to 18 µm,

depending on the vitamins encapsulated, being its morphology spherical, meanwhile the

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precipitation yield was within the range of 41–82g/100g.

Keywords

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Supercritical fluids; δ-tocopherol; riboflavin;β-carotene; Co-precipitation

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1

Introduction

Vitamins are essential for life. These essential constituents of food are required for

the normal growth, self-maintenance and functioning of human and animal bodies. The food industry seeks to incorporate vitamins into food systems due to the increasing interest for products with high nutritional value [1].Vitamins are a class of organic

compounds classified in two main groups: fat-soluble and water-soluble vitamins. Among water-soluble vitamins, the B group including B1, B2, B6 and B12 are the most important [2]. Fat-soluble vitamins include A, E, D, and K vitamins, among which vitamin E isthe main

dietary

lipid-soluble

antioxidant

[3].In

contrast,

β-carotene

(pro-

vitamin A carotenoid) has important and well-established biologic effects, such as control

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of cell differentiation [4].

Vitamin deficiency is usually associated with some severe diseases. Therefore, the

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development of foods fortified with vitamins is likely to have played an important role in

the current nutritional health and well-being of populations in industrialized countries [5]. In this context, the development of products enriched with vitamins would be particularly

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useful in combating the vitamins deficiency in certain population groups.

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Vitamins are sensitive to environmental stresses such as temperature, light, and

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oxygen which may result in reduction or loss of bioactivity [6]. Development of delivery

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systems may improve the stability of vitamins against environmental stresses.

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Encapsulation is an important application to develop functional food products via delivery of vitamins, which provides enhanced product functionality and stability

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including controlled release of the vitamin during storage [7]. Methods traditionally used for particle formation and encapsulation can suffer from

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some drawbacks such as poor control of particle size and morphology, degradation of thermo sensitive compounds, low encapsulation efficiency, and low yield [8-10]. The use

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of supercritical CO2 (scCO2) has become a more controlled method to producing delivery systems. Some of the advantages of the several supercritical fluid-based techniques are: very small particles may be produced; particle sizes are easily controlled; applicability shown for a wide variety of substances; efficient separation of the solvent and anti-solvent

of the particles after precipitation; operation at moderate temperatures and in an inert atmosphere that avoids degradation of thermolabile and oxidable substances [11-13]. The supercritical anti-solvent (SAS) technique uses the scCO2 as an anti-solvent for the precipitation and encapsulation of the solute already dissolved in organic solvents[14, 15]. In this work, vitamins and the wall material are dissolved using ethanol and the

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ethanolic solution obtained is sprayed into a precipitation vessel. The scCO2is also injected into the system causing a rapid contact between the two media. This generates

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higher supersaturation ratios of the solution, resulting in fast nucleation and growth, and

consequently creates microcapsules. Zein, a major storage corn protein, was chosen as wall material because it is one of the few categories of food biopolymers that are soluble

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in organic solvents miscible with CO2 [16]. SAS process was successfully proposed to

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coprecipitate zein with a model drug in some works [17, 18].

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In this context, the objective of this work was to explore the possibility of using

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SAS process to manufacture food grade vitamins delivery systems. Zein was used as wall

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material for the formation of the microcapsules. Riboflavin, δ-tocopherol and β-carotene were used as vitamin complex, which can be incorporated into milk beverages. A GRAS

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(Generally Recognized as Safe) solvent, 94g/100g aqueous ethanol was used as the solvent. The process was studied experimentally and the data obtained were used to

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perform the mass and energy balance simulation using a commercial flow sheeting

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software in order to verify the bottlenecks of the technology through economic evaluation.

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2.1

Materials and Methods Materials

Corn Zein, with a mean particle size of 167.5 μm, and the vitamins, riboflavin (>90g/100 g), δ-tocopherol (>90g/100 g), and β-carotene (>93g/100 g) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ethyl alcohol (Anhydrous), used to prepare the vitamins solutions, was purchased from Commercial Alcohols (Brampton,ON, Canada). Carbon dioxide, having purity of 99.9g/100 g, was supplied by Praxair

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(Edmonton, AB, Canada) and used as antisolvent in the SAS process. All chemicals were

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used directly without further purification.

Experimental apparatus and procedure

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Fig. 1 presents a schematic diagram of the experimental apparatus for SAS

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precipitation on a laboratory scale. For this process, the solution and the antisolvent fluid

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(scCO2) were injected continuously into the precipitation vessel. The zein solution was

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prepared by dissolving zein (0.02 g/mL) in a solution of anhydrous ethanol:deionized

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water (0.94:0.06g/g) using an ultrasonic bath (Aquasonic, 75T, Portland, New York) for 10 min followed by centrifugation (Fisher Scientific, AccuSpinTM 400, Osterode,

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Germany) at 1,400 g for 10 min. The δ-tocopherol was mixed directly with the zein solution. Solutions containing riboflavin and β-carotene (0.025 g/mL) were prepared in

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the same way than zein solution. The portions of the riboflavin and β-carotene solution supernatant were added in the δ-tocopherol plus zein solution for the encapsulation of

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both vitamins. The vitamin complex, with δ-tocopherol, riboflavin and β-carotene, was obtained using a supersaturated vitamins solution. In this study, the runs were performed on a modified SAS apparatus provided by Separex (Champigneulles, France). Briefly, the unit consisted of a cylindrical precipitation vessel, with an internal volume of 251 mL and internal diameter of 4 cm,

one pump for CO2 displacement (Separex, P200, Champigneulles, France), and an HPLC liquid pump (Gilson, 307,Villiers-le-Bel, France) used for solution delivery. The liquid CO2was cooled through a heat exchanger (Separex, C1000, Champigneulles, France) and then flowed through a heater. Once the desired conditions of pressure, temperature and antisolvent flow rate was achieved,30 mL of the vitamin solution was pumped into the

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precipitation vessel. Afterwards, the flow of the vitamins solution was stopped and only pure CO2 continued to be run through the precipitation vessel for 60 min in order to dry

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and remove the residual solvent from the microcapsules. After the precipitation vessel

was depressurized and the microcapsules were carefully collected with a spatula and stored in a refrigerator (Coldstream,Winnipeg, Manitoba, Canada) at 2 °C under

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protection from light until subsequent analysis.

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For riboflavin encapsulation, a zein plus riboflavin solution was prepared as

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follows: riboflavin solution was made with 94g/100g aqueous ethanol at concentration of

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0.025 g/mL and after ultrasonic treatment the solution was centrifuged. Around 0.01 g of

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riboflavin was solubilized and a portion of 5, 10 or 15 mL riboflavin solution supernatant was mixed with the 50 mL zein solution.

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At the end of the process, the particles were collected and loaded in a glass vial. The glass vial was kept in an oven at 45 °C for one week. The initial and final weight of

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the particles was obtained. The weight loss of the particles was lower than 0.5%.

Particle analysis and characterization

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2.3

The mean particle size and the particle size distribution of the samples were measured using a laser diffraction particle size analyzer (LS 13320, Beckman Coulter, Fullerton, Cal, USA) after the microcapsules were well dispersed in ultrapure Milli-Q

water containing Tween 20 (0.02g/g), which stabilize the suspension. The morphologies of the microcapsules were observed using a scanning electron microscope (SEM,S-450, Hitachi, Japan).The microcapsules were mounted onto brass stubs and sputter-coated with gold in an argon atmosphere prior to examination under SEM. The precipitation yield was determined by weighting the total amount of particles

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collected in the precipitator chamber. The percentage of precipitation yield was calculated by the ratio between the mass of microcapsules collected in the precipitation chamber

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after each assay and the mass of vitamins and zein present in the organic solution added to the precipitation chamber at each experiment.

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Vitamin encapsulation efficiency

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The vitamins inside the core of the particles were calculated as the total in the bulk

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particles (non-encapsulated and encapsulated) minus the content on the surface (non-

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encapsulated). Total and surface ß-carotene, δ-tocopherol or riboflavin were analyzed

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using HPLC (UV or fluorescence detector) method. About 0.05 g of microcapsule was dissolved in 1mL (96% ethanol) solution. The sample was placed in a sonicator bath at

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room temperature. After 10 min, the sample was extracted using 1 mL of hexane (ßcarotene, δ-tocopherol) or water (riboflavin). The sample was centrifuged at 5000 rpm

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for 15 min at room temperature. The upper phase was collected and the total vitamins were analyzed by HPLC method. The surface vitamins were determined by the use of

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0.05 g of microcapsule washed using 1 mL of hexane (ß-carotene, δ-tocopherol) or of water (riboflavin). The sample was centrifuged at 5000 rpm for 15 min at room temperature. The supernatant was collected and vitamins were analyzed using HPLC method. Zein particles were not soluble in hexane or water and did not interfere with the HPLC method.

Analysis of vitamins was achieved on a Varian 9010 unit (Walnut Creek, Calif., U.S.A.) equipped with a Supelco LC-Diol column (25 cm × 4.6 cm × 5 μm, Supelco Inc., Bellefonte, Pa., U.S.A.). Shimadzu RF535 fluorescence detector (Shimadzu Scientific Instruments Inc., Columbia, Md., U.S.A.) and a Shimadzu UV detector (Shimadzu Scientific Instruments Inc., Columbia, Md., U.S.A.) were used. Sample was injected with

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a Hewlett Packard 1050 autosampler with a 25-μL sample loop. The mobile phase for fat

soluble vitamins consisted of 0.6% (v/v) iso-propanol in hexane (both HPLC grade from

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Fisher Scientific, Fair Lawn, N.J., U.S.A.) flowing at 1.0 mL/min. The mobile phase for water soluble vitamins consisted of water HPLC grade from Fisher Scientific (Fair Lawn, N.J., U.S.A.) flowing at 1.0 mL/min. For tocopherol, the detector was set at 265 nm for

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the excitation and 330 nm for the emission. For b-carotene, the UV detector was set at

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265 nm. For rivoflavine, the UV detector was set at 450 nm Total run time for each sample

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was 60 min.

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2.4 Process modeling and simulation description

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A flowsheet model of the co-precipitation of vitamins and zein by SAS process was developed using the commercial software Aspen Plus®. Matlab simulation software

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was used to perform thermal integration and economic analysis of the evaluated process. In this study, the problem resolution was carried out following the steps:

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Process data was gathered from the performed experiments Aspen Plus® flow sheeting software was used to model mass and energy flows of

the process. The model was used to calculate the associated heat and power balances. 3. Pinch analysis methodology [19,20] was used to perform the thermal integration of the process aiming at the reduction of process steam requirements.

4.

An economic model was developed using data obtained from the flowsheeting

software Aspen Plus® and the results obtained by the thermal integration model. OSMOSE simulation tool was used in its basic level to perform thermal integration and economic analysis. OSMOSE (OptimiSation Multi-Objectifs de Systemes Energetiques integres, which means “Multi-Objective OptimiZation of integrated Energy

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Systems”) is a computation platform that was built in MATLAB (MATrix LABoratory,

MathWorks, Natick, MA, USA), developed and continuously improved at École

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Polytechnique Fédérale de Lausanne in Switzerland for the design and analysis of

integrated energy systems. The platform allows one to link Aspen Plus® software for a

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2.4.1 Mass and energy flows using Aspen Plus

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complete suite of computation and result analysis tools.

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The thermodynamic model used to represent the SAS process was the RK-ASPEN

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model. The solution preparation step was simulated in Aspen Plus® considering a mixing

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tank. The composition of the final solution was set according with experimental data using the design specification tool of Aspen Plus®. SAS unit was simulated considering

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that the CO2 sent to the process is initially cooled to -10°C and compressed to the desired pressure. It is then heated to the process temperature, reaching the supercritical

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conditions. Later, the precipitation vessel of 500 L is filled with the vitamin solution and the supercritical fluid is passed through it. As the process was studied in a stationary

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regime, it was considered that two SAS unities working in parallel were used to achieve a continuous inlet and outlet material flow. After the precipitation process, the organic solvent diluted in supercritical CO2 is sent to a depressurization tank to separation. At this stage, the pressure is reduced to 50 bar and temperature is set at 25°C. Considering that

the initial 5% of scCO2 is removed from the process, as it contains the higher amount of solvent, and the remaining is recycled to the process.

2.4.2 Thermal integration using the Pinch Method Once the heat and power requirements of the transformations are defined in the energy

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and mass flow model, a model is developed by the user inside MatLab that extracts from the commercial software (Aspen Plus®) the values of temperature and heat from the

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process streams. The thermal effects of each sequence of operations are grouped and

constitute the units whose flow rates are to be computed in the integration problem. The hot and cold utility needs to be considered. In order to supply the energy requirement

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above the pinch, combustion of fuels available on-site is considered. A model of steam

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network is used to consider a cogeneration process. Based on the pinch analysis

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methodology [11-13], the optimal thermal process integration is computed in the MatLab

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platform after the maximum heat recovery potential between hot and cold streams is

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defined and a minimum approach temperature ΔTmin, which represents the energy capital trade-off between the energy savings obtained by heat exchange and the required heat

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exchangers investment, is considered. The optimal utility integration is obtained when the combined production of fuel, power and heat are maximized, which minimizes the

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operating cost by solving the heat cascade problem using a mixed integer linear programming technique. All heat streams generated by the energy and mass flow model

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were considered in the thermal integration analysis. At this stage of the analysis, it was not carried out the initial design and optimization of the heat exchanger network. 2.4.3 Economic modeling and economic performance indicators

The economic model was built in MatLab using information extracted from the Aspen Plus model. In order to accomplish an economic evaluation of the process viability at industrial scale, lab results were scaled-up considering that the same performance would be obtained. This criterion, which has been used by other authors for supercritical fluid-based processes [21,22], assumes that the process will have identical performance

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with respect to yield at the laboratory and industrial scales if the same process conditions

are used (temperature, pressure, extraction time, etc.). As the process was studied in a

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stationary regime, it was considered that two SAS unities working in parallel were used

to achieve a continuous inlet and outlet material flow. To calculate the total investment cost, the major process equipment were roughly sized and their purchase cost were

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calculated and adjusted to account for specific process pressures and materials using

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correlations from literature and manufacturers [23, 24]. The equipment cost estimated

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were: the SAS extractor and pumps, the CO2 separation tank, two mixing tanks, a

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centrifuge and the ultrasonic homogenizer. The total investment cost was then calculated

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using multiplication factors to take into account indirect expenses like installation costs, contingencies and auxiliary facilities. All costs were updated by using the Chemical

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Engineering Plant Cost Index [23,24]. Cost of manufacturing (COM) estimation for the evaluated process was

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accomplished based on the methodology of [23,24]; in which variable cost (operational costs which are dependent on the production rate and consist in raw material costs,

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operational labor, utilities, among others), fixed costs (do not dependent on production rate and include territorial taxes, insurance, depreciation, etc.) and general expenses (cover business maintenance and include management, administrative sales, research and development costs) are calculated. These three components are estimated in terms of five main costs: fixed capital investment (FCI), cost of utilities (CUT), cost of operational

labour (COL), cost of waste treatment (CWT) and cost of raw materials (CRM). Utility costs considered the electricity and the cooling requirements under 293 K. COM was calculated as presented in Equation 1. COM = (VC + FC + GE)*(1 + 0.03COM + 0.11COM + 0.05COM),

(Eq.1)

In which 0.03COM represents the royalties; 0.11COM the distribution and selling and

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0.05COM the research and development investments.

It was also evaluated the COM divided by the amount of product produced in a year as

COM

prod



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presented in Equation 2 . COM

(Eq. 2)

. prod

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m

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Where m is the mass flow calculated per year, the sub-index susp is the particle produced

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at the SAS process.

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The yearly total costs are calculated according to Eq. (1), where Cop is the operating cost of the plant expressed in US$/y,  is defined as the annualization factor

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(Eq. (2)), i is the interest rate and n is the lifetime assumed for the investment. Ctot = Cop + (total investment)

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 = (i * (i + 1)n)/((i + 1)n – 1)

Eq. (1) Eq. (2)

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Table 6 shows the list of economic assumptions.

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3.1

Results and Discussion Precipitation of zein A preliminary study of zein precipitation was done in order to select the best

operational conditions to encapsulate the vitamins. Supercritical conditions were tested, however not presented good results for particle formation, and then subcritical conditions were used. The variables studied included the zein concentration (0.02 - 0.04 g/mL),

antisolvent (20 - 60 g/min) and solution (0.5 - 1.5 mL/min) flow rates and precipitation pressure (7.0 - 16.0 MPa). Table 1 presents the summary of working conditions and the results of mean particle size and precipitation yield obtained in all experimental precipitation runs of pure zein. First, the precipitation was carried out at typical conditions for SAS processes of

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10MPa and 313 K. In this study, a temperature of 313 K was fixed for all runs to minimize thermal degradation of vitamins associated with high temperature exposure. Higher

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temperatures may contribute to faster degradation of vitamins. Coronel-Aguilera and San Martín-González [25] evaluated the effect of temperature on carotenoid encapsulation and observed that the highest temperature (353 K) showed less than half carotenoid

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content of the lowest temperature (333 K) treatment. The pressure of 10 MPa was kept

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constant for the study of the influence of zein concentration, antisolvent and solution flow

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rates.

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Runs 1, 2 and 4were undertaken to study the effect of antisolvent flow rate at 3

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levels, namely, 20, 40 and 60 g/min. These experiments were carried out with an operating solution flow rate of 1 mL/min. In the SAS process, a CO2 molar fraction in the

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precipitator greater than or equal to 0.7 is preferable to avoid the partial extraction of the product [26]. Therefore, a CO2 molar fraction above 0.90 was used in this work. As can

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be observed from runs 1, 2 and 4, only at 60 g/min antisolvent flow rate it was possible to successfully obtain dry particles of zein. Literature shows that a raise in the CO2 flow

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rate led to a decrease in the solute solubility, increasing the maximum supersaturation, decreasing the amount of solute that remains dissolved in the solvent. This phenomenon can be observed due the differences of solubility between zein and the solvents used in the SAS process. The solubility of any substance on a solvent it is a matter of state and phase equilibria. In this study, the solvent power of the CO2 only depends on its pressure

and temperature. The increase of the CO2 flow rate changed the ratio between ethanol and CO2, and probably mixtures with higher CO2 proportion has a smaller solvent power for zein [27, 28]. In the range of experimental conditions studied, the higher antisolvent flow rate promoted a more intense mixing between the solution and antisolvent, enhancing mass

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transfer between CO2 and the solvent (94 g/100g ethanol) in the atomized droplets so that

the solvent could be extracted. Using this antisolvent concentration, it was possible to

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completely dry the particles during the time of 1 hour. These findings are in agreement with those obtained by Zhong et al. [29] on precipitation of zein by aerosol solvent

extraction system (ASES) using scCO2 as anti-solvent and 90 g/100g aqueous ethanol as

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solvent where no particles were produced at 20 g/min antisolvent flow rate and some

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particles were produced at 50 g/min but not completely dry.

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The effect of solution flow rate was also evaluated. On the basis of some previous

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works, an efficient atomization of the liquid jet is observed at the solution flow rate of 1

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mL/min [30,31]. From Table 1,runs 3, 4 and 5 produced larger particles compared to the unprocessed zein particles, which present a mean particle size of 167.5 μm. Lower particle

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size and a higher precipitation yield were obtained at 1 mL/min solution flow rate (run 4). Franceschi et al. [33] examined the effect of solution flow rate (1 – 4 mL/min) and

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observed that the solution flow did not present a significant effect on particle size and on yield of the precipitated particles. The authors concluded that solution flow rate of 1

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mL/min is suitable for encapsulation. Runs 4 and 6 were performed to study the influence of zein concentration.

However, the experiment at zein concentration of 0.04 g/mL did not yield particles, but a film of zein at the bottom and the walls of the precipitation vessel. Increasing the zein concentration there was a change in the equilibrium properties of the system zein +

ethanol + CO2. For instance the critical point of mixture was modified well as the equilibria conditions between zein and ethanol. In this sense, the CO2 flow employed was not able to dislocate the equilibrium and thus to act as a powerful antisolvent for the mixture, since was maintained the others experimental conditions of SAS process. Therefore, this result can be attributed to mass transfer limitations, which led to the

the best value of zein concentration was estimated to be 0.02 g/mL.

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formation of a continuous layer when the main aim was to obtain fine particles. However,

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With runs 4, 7, 8 and 9, the influence of pressure in the range of 7 – 16 MPa was

studied while maintaining the rest of the process conditions constant. At 7 MPa, a film was produced instead of particle formation. Reverchon and Della Porta [30] performed a

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study on the antibiotics precipitation, finding that at 9 and 9.5 MPa, a continuous film of

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tetracycline on the surface ofthe precipitator was obtained. This behavior was attributed

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to a low solubility of solvent in supercritical CO2 at this pressure, with the consequent

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formation of a liquid solution at the bottom of the precipitator and solute precipitation

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when this liquid solution was progressively dried. Pressure ranges from 7 to 16 MPa were studied. High pressure treatment is

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expected to be less detrimental than thermal processes to low molecular weight food compounds such as flavouring agents, pigments, vitamins, etc., as covalent bondings are

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not affected by pressure. Also, the structure of macromolecules like, for example, proteins may change under the influence of pressure [34]. This change was visually observed at

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pressures above 13 MPa, in which zein color changed from yellow to white after processing. This change was not expected to affect the zein encapsulation capacity. According to Franceschi et al. [33], pressure is the variable that more strongly influence the particle size. This occurs due the large influence that pressure have on behavior of system in the phase equilibria. The formation of particles is obtained when

the mixture (zein + ethanol + CO2) is above the critical point, in other words, when the system presents a single phase [35]. However, a complete evaluation of effects of this operational parameter on morphology and particle size distribution must be made using phase equilibrium data of the system zein + ethanol + CO2. In this study, an increase in pressure led to a decrease in the mean particle size, while in the precipitation yield had an

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opposite effect, with an increase in the precipitation yield due to a raise in the pressure. However, a variation in pressure in the range 13 and 16 MPa had a very small effect on

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both the size of the particles and the precipitation yield.

He et al. [36] also found that particle sizes decreased with increases in pressure, which it can be explained in terms of the volumetric expansion of the liquid phase.

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Because of the high solubility of CO2 in the solvent, the volumetric expansion of solvent

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in scCO2 increases rapidly with increasing operating pressure at a given temperature,

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lowering its solvent power for the solid solute and leading to a higher supersaturation

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ratio of the expanded liquid solution, which subsequently results in the formation of

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smaller particles.

Regarding process yield, Martın and Cocero [36] and Franceschi et al. [33] found

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that the precipitation yield decreased with pressure, due to a density increase of the solution (anti-solvent plus organic solvent), thus increasing the solubility of solute in this

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solution, with a consequent stream fraction carried in the effluent solution. However, in this study an increase on pressure increased considerably the precipitation yield. This

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result can be attributed to better conditions of zein precipitation due the system to be possibly above your critical point conform discussed previously. Runs 9 and 9R show the reproducibility of zein precipitation from aqueous ethanol solutions. The particle size and precipitation yield attested the very good reproducibility of the experiments with small differences attributed to random experimental errors. A

more detailed study about the SAS process reproducibility is presented at the vitamins encapsulation runs. Based on these runs, the best operational condition which may be suitable for vitamins encapsulation corresponds to run 9, obtained with a pressure of16 MPa, temperature of 313 K, zein concentration of 0.02 g/mL, antisolvent flow rate of 60 g/min

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and solution flow rate of 1 mL/min. The drying time was 1 hour for 30 mL of solution

3.2

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injected into the precipitation vessel.

Co-precipitation of vitamins and zein

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The vitamins encapsulation was carried out using the best operational conditions

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as determined from the previous zein precipitation runs. The main idea for co-

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precipitation is that the particles of the material to be encapsulated (core) are smaller than

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those of the encapsulating material (shell). The co-precipitation of δ-tocopherol and zein

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was studied in runs 10 – 13 of Table 2. Run 13 showed that at 0.25 g δ-tocopherol resulted in very low precipitation yield of 1.68 g/100g suggesting that the δ-tocopherol

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concentration has reached its limit, because in this concentration it is a viscous liquid, and thus was not possible to completely dry by SAS. The SAS technology is usually applied

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to precipitate substances and form particles from solutions, however a viscous material also can be processed but at a lower concentration [13]. The δ-tocopherol amounts

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between 0.05 and 0.15 g had relatively little effect on precipitation yield and particle size. The higher yield (67.39 g/100g) and lower particle size (10.80 µm) was obtained at run 11, being selected to the co-precipitation with all vitamins studied in this work. In regard to the yield of riboflavin microcapsules, runs 14-16 showed that no relevant differences between the studied concentrations were detected. In fact, the solute

concentration has no effect on precipitation yield. Franceschi et al. [33] concluded that the precipitation yield is statistically influenced by system temperature and pressure, and anti-solvent flow rate. The final microcapsules obtained with the riboflavin and δ-tocopherol vitamins presented precipitation yields in the range of 57–78g/100g. Only δ-tocopherol amount

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was varied in these runs. Higher yields were achieved by Franceschi et al. [33] in the precipitation of β-carotene microparticles using dichloromethane as solvent. The authors

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obtained precipitation yield of 71–94g/100g depending on the process parameter values.

On the other hand, Kikic et al. [38] obtained even lower yields compared to this work with the antisolvent precipitation of vitamin B6 from ethanol solution, between 19 and

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55g/100g.The precipitation from non-chlorinated solvents, such as ethanol, resulted in

N

low yield in the Majerik et al. [39] study, suggesting that these solvents act as co-solvent

A

and increase the solubility of the solute in the supercritical phase.

M

Runs 10, 11, 12, 17, 18 and 19 were repeated under identical operating conditions

ED

to demonstrate the reproducibility of experimental results in the SAS process. The data in Table 2 showed that an acceptable reproducibility has been found in most of the cases.

PT

The highest standard deviation for a precipitation yield was obtained in run 12, of 11.4g/100g. The difference between the precipitation yields may arise from the difficulty

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to collect every particle in the precipitation vessel, since some of them were stuck to the vessel wall and to the filter. This difficulty produces a large error relative to the amount

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of processed material. For particle size, a highest standard deviation of 4.2 µm was detected in run 10. Table 3 presents the runs performed using supersaturated solutions. The amount chosen forβ-carotene was the highest concentration where no presence of precipitate was observed in 50 mL of 94g/100g aqueous ethanol plus zein solution. The final solution had

the same concentration of riboflavin and β-carotene. An ultrasonic treatment during all the solution injection was applied to avoid the vitamins precipitation. The highest precipitation yield was 82g/100g for the encapsulation of δ-tocopherol plus β-carotene, and the complex of vitamins, including the riboflavin, δ-tocopherol and β-carotene, yielded around 68g/100g.

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Zein was selected by Patel et al. [40] as a wall material for synthesizing polymeric colloidal particles containing curcumin. These particles were synthesized using an

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antisolvent precipitation method, in which the removal of the organic solvent was

performed under reduced pressure using a rotatory evaporator. However, the zeincurcumin composite particle formation had a low yield of about 60g/100g.According to

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the authors, the incorporation of curcumin in the zein made the dispersion more unstable

N

and led to aggregate formation which in turn accounted for a lower yield.

A

Therefore, in an attempt to increase the precipitation yield, the zein plus vitamins

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solution was stabilized using surfactants like Tween 20 or sodium lauryl sulfate (LSL).

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However, the obtained results were not satisfactory because was formed a film covering the filter and the walls of the precipitator for both surfactants. These results suggested

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solution.

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that maybe the surfactants used were not suitable for stabilizing the zein plus vitamins

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3.2.1

Particle morphology and size

The effectiveness of the SAS technique in producing very small particles is based

on the very fast expansion process of the liquid droplets when they come in contact with scCO2, which rapidly diffuses into the liquid droplets produced by the nozzle, expands

them in the form of balloons and finally the explosion of balloons produces the required micro or nanoparticles [30]. The microcapsules containing vitamins and zein, obtained in this study, presented particle size ranging from about 8 to about 18μm. Franceschi et al. [33] obtained particles with relatively larger size for the precipitation of β-carotene. The precipitated powders

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obtained by authors presented mean particle size varying from 3.2 to 96.8 µm.

Fig. 2 shows the SEM micrographs of the microcapsules obtained at Run 23 (δ-

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tocopherol, 0.15g; β-carotene 0.02 g) at pressure of 16 MPa, temperature of 313 K, zein

concentration of 0.02 mg/mL, solution flow rate of 1 mL/min and anti-solvent flow rate of 60 g/min. The SEM pictures obtained in this work reveal that the use of different

U

vitamins does not influence the spherical morphology of the precipitated microcapsules.

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On the other hand, it also suggest that the vitamins were not completely encapsulated by

A

the zein due the presence of vitamin in the wall material. Fig. 3 shows a photograph of

M

the microcapsules (right side) containing a vitamin complex (Run 24, δ-tocopherol,

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0.15g; Riboflavin, 0.01 g; β-carotene, 0.02 g) obtained via SAS, which will be incorporated into milk beverages in future studies, meanwhile in the left side is presented

PT

the Zein particles precipitated by SAS (Run 9). SEM micrographs showed that from run 9 to 24 the size of particles decrease. Run 9 showed more amount of particles higher than

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10 µm with cracks.

The results of efficiency of encapsulation are shown in Table 4. The results showed

A

that the Riboflavin vitamin had higher encapsulation capacity in the core particle, related to the physicochemical characteristics of both the vitamin and zein as wall material.

3.3 Process evaluation using simulation tools

The effectiveness of the SAS technique in producing very small particles is based on the very fast expansion process of the liquid droplets when they come in contact with scCO2, which rapidly diffuses into the liquid droplets produced by the nozzle, expands them in the form of balloons and finally the explosion of balloons produces the required micro or nanoparticles [30]. The amount of sc CO2 used usually is much higher compared

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with Supercritical Fluid Extraction (SFE) processes. Thus, the total amount of particles

producedestimated in our similation was 10.99 t/year. Table 6 shows the main economic

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results for this analysis.

The amount of CO2 used in the SAS process strongly influences the economical variables. The replacement of 5% of CO2 lost per precipitation cycle represents 56.4% of

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the raw material costs. The use of excess CO2 increases not only the raw material price

N

but also the amount of electricity used in the process by the CO2 pump and, consequently,

A

the total investment. CO2 is usually used in a large excess in SAS process, and a more

M

accurate study of the necessary amount of this solvent to obtain the particles is still

ED

necessary experimentally. Therefore a sensitivity analysis was undertaken in order to evaluate possible scenarios that considered a lower solvent mass to feed mass ratio (S/F)

PT

ratio, maintaining the same performance of the process (Table 7). The market value for multivitamins varies greatly depending on the type of

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encapsulated vitamin, vitamin source, and so on. Among the three vitamins studied for encapsulation, the most expensive is vitamin B2, riboflavin. A market value search for

A

riboflavin vitamins (400 mg) revealed values ranging from 0.10 to 0.6 USD/capsule (each capsule containing approximately 0.4 g). The estimated value for the cost of manufacturing (COM) of riboflavin vitamin by SAS process proposed in this study with the initial CO2 flow would be USD 0.2 / capsule, however, presenting a lower riboflavin concentration than the market, demonstrating the potentiality of supercritical anti-solvent

process as an alternative technology for vitamin complex encapsulation. On the other hand, with the reduction of 50% of the CO2 flow, it was possible to reduce the COM of this multivitamin by 23.7% (from a COMprod from 502.95 to 389.69 USD/kg, Table 7). In a recent previous studywe also observed that the main economic bottle neck of the scCO2 antisolvent-based processes is the CO2 flow. The decrease of CO2 flow

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demonstrated to be the main parameter to decrease the energy demand and cost of

manufacturing of the antisolvent-based process developed by [41] for quercetin particle

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production. Thus, further experimental studies should be done towards optimization of this parameter by the engineers interested in this technology. This study is part of abroad project that aims more collaboration between the experimental groups and the groups that

U

work with process simulation tools in order to find technology bottle necks to help the

N

successful implementation of novel sub/supercritical fluid-based technologies by the

M

Conclusions

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4

A

industry.

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This work explored the possibility of using the SAS to manufacture food grade vitamins delivery systems, and the technical-economic results obtained here may be

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relevant as a supportfor the successful design of controlled delivery systems applied to food products.

A

In this work the encapsulation of a vitamin complex, containing riboflavin, δ-

tocopherol and β-carotene in zein by SAS technique with scCO2 as antisolvent was studied. According to the experimental results microcapsules with a mean particle size varying from 8 to 18 µm and spherical morphology were formed. The operational conditions presented a significant influence on particle size and precipitation yield. The

best result was obtained with a pressure of 16 MPa, temperature of 313 K, zein concentration of 0.02 g/mL, antisolvent flow rate of 60 g/min and solution flow rate of 1 mL/min (run 9), which may be suitable for encapsulation. Regarding economic aspects the main bottle neck of the scCO2 antisolvent-based process evaluated was the CO2 flow.

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ACKNOWLEDGMENTS

We are grateful to the Natural SciencesandEngineeringResearchCouncilof Canada

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(NSERC) and to theFaculty of Agricultural, Life and Environmental Sciencesfor

fundingthisproject. M. Thereza M. G. Rosa is grateful to the National Council of Technological and Scientific Development (CNPq, processes 140641/2011-4) for a

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doctoral fellowship and the Coordination for the Improvement of Higher Education

N

Personnel (CAPES, process 99999.002445/2014-00) for the receipt of PDSE scholarship.

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Juliana Q. Albarelli thanks FAPESP (process 2013/18114-2) for the post-doctoral

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fellowship. Diego T. Santos thanks CNPq (processes 401109/2017-8; 150745/2017-6) for

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the productivity grant.

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the post-doctoral fellowship. M. Angela A. Meireles thanks CNPq (302423/2015-0) for

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References

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[1] M. Marsanasco, A.L. Márquez, J.R. Wagner, S. del V Alonso, N.S. Chiaramoni, Liposomes as vehicles for vitamins E and C: An alternative to fortify orange juice and offer vitamin C protection after heat treatment, Food Research International, 44 (2011) 3039-3046. [2] P. Moreno, V. Salvado, Determination of eight water-and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography, Journal of chromatography A, 870 (2000) 207-215. [3] Y. Luo, B. Zhang, M. Whent, L.L. Yu, Q. Wang, Preparation and characterization of zein/chitosan complex for encapsulation of α-tocopherol, and its in vitro controlled release study, Colloids and Surfaces B: Biointerfaces, 85 (2011) 145-152. [4] W.A. Pryor, W. Stahl, C.L. Rock, Beta carotene: from biochemistry to clinical trials, Nutrition reviews, 58 (2000) 39-53.

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[5] I. Darnton-Hill, R. Nalubola, Fortification strategies to meet micronutrient needs: sucesses and failures, Proceedings of the Nutrition Society, 61 (2002) 231-241. [6] M.D. Saldaña, J.S. dos Reis Coimbra, L. Cardozo-Filho, Recovery, encapsulation and stabilization of bioactives from food residues using high pressure techniques, Current Opinion in Food Science, 5 (2015) 76-85. [7] D.J. McClements, E.A. Decker, Y. Park, J. Weiss, Structural design principles for delivery of bioactive components in nutraceuticals and functional foods, Critical reviews in food science and nutrition, 49 (2009) 577-606. [8] E.K. Silva, V.M. Azevedo, R.L. Cunha, M.D. Hubinger, M.A.A. Meireles, Ultrasoundassisted encapsulation of annatto seed oil: Whey protein isolate versus modified starch, Food Hydrocolloids, 56 (2016) 71-83. [9] E.K. Silva, M.A.A. Meireles, Influence of the degree of inulin polymerization on the ultrasound-assisted encapsulation of annatto seed oil, Carbohydrate Polymers, 133 (2015) 578-586. [10] J.M.G. Costa, E.K. Silva, A.A.C. Toledo Hijo, V.M. Azevedo, M.R. Malta, J.G.L. Ferreira Alves, S.V. Borges, Microencapsulation of Swiss cheese bioaroma by spray-drying: Process optimization and characterization of particles, Powder Technology, 274 (2015) 296-304. [11] Z. Knez, E. Weidner, Particles formation and particle design using supercritical fluids, Current Opinion in Solid State and Materials Science, 7 (2003) 353-361. [12] M.J. Cocero, Á. Martín, F. Mattea, S. Varona, Encapsulation and co-precipitation processes with supercritical fluids: fundamentals and applications, The Journal of Supercritical Fluids, 47 (2009) 546-555. [13] M.T.M. Gomes, D.T. Santos, M.A.A. Meireles, Trends in particle formation of bioactive compounds using supercritical fluids and nanoemulsions, Food and Public Health, 2 (2012) 142-152. [14] S.-D. Yeo, E. Kiran, Formation of polymer particles with supercritical fluids: A review, The Journal of Supercritical Fluids, 34 (2005) 287-308. [15] E.K. Silva, M.A.A. Meireles, Encapsulation of Food Compounds Using Supercritical Technologies: Applications of Supercritical Carbon Dioxide as an Antisolvent, Food and Public Health, 4 (2014) 247-258. [16] Q. Zhong, M. Jin, D. Xiao, H. Tian, W. Zhang, Application of supercritical anti-solvent technologies for the synthesis of delivery systems of bioactive food components, Food Biophysics, 3 (2008) 186-190. [17] P. Franco, E. Reverchon, I. Marco, Production of zein/antibiotic microparticles by supercritical antisolvent coprecipitation, Journal of supercritical fluids, 145 (2019), 3138. [18] P. Franco, E. Reverchon, I. Marco, Zein/diclofenac sodium coprecipitation at micrometric and nanometric range by supercritical antisolvent processing, Journal of CO2 utilization, 27 (2018), 366-373. [19] F. Maréchal, B. Kalitventzeff, Targeting the minimum cost of energy requirements: a new graphical technique for evaluating the integration of utility systems, Computers & Chemical Engineering, 20 (1996), 225-230. [20] B. Linnhoff, D.W. Townsend, D. Boland, G.F. Hewitt, B.E.A. Thomas, A.R. Guy, R.H. Marsland, User Guide on Process Integration for the Efficient Use of Energy, first ed., IChemE, Rugby, United Kingdom, 1982.

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[21]C.G. Pereira, M.A.A. Meireles, Supercritical fluid extraction of bioactive compounds: Fundamentals, applications and economic perspectives. Food Bioprocess Technology, 3 (2010), 340–372. [22] M.M.R. Melo, H.M.A. Barbosa, C.P. Passos, C.M. Silva, Supercritical fluid extraction of spent coffee grounds: Measurement ofextraction curves, oil characterization and economic analysis. Journal of Supercritical Fluids, 86 (2014), 150–159. [23] R.B. Turton, B. Wallace, J.S. Whiting, D. Bhattacharyya, Analysis, synthesis and design of chemical processes, third ed., Prentice Hall, Upper Saddle River, NJ, USA, 2009. [24] G. Ulrich, P. Vasudevan, A guide to chemical engineering process design and economics a practical guide, second ed., CRC, Boca Raton, FL, USA, 2003. [25] C.P. Coronel-Aguilera, M.F. San Martín-González, Encapsulation of spray dried βcarotene emulsion by fluidized bed coating technology, LWT-Food Science and Technology, 62 (2015) 187-193. [26] E. Reverchon, I. De Marco, Supercritical fluid extraction and fractionation of natural matter, The Journal of Supercritical Fluids, 38 (2006) 146-166. [27] F. Miguel, A. Martin, T. Gamse, M. Cocero, Supercritical anti solvent precipitation of lycopene: Effect of the operating parameters, The Journal of supercritical fluids, 36 (2006) 225-235. [28] E. Franceschi, A. De Cesaro, S. Ferreira, J. Vladimir Oliveira, Precipitation of βcarotene microparticles from SEDS technique using supercritical CO2, Journal of food engineering, 95 (2009) 656-663. [29] Q. Zhong, M. Jin, D. Xiao, H. Tian, W. Zhang, W., Application of supercritical antisolvent technologies for the synthesis of delivery systems of bioactive food components. Food Biophysics, 3 (2008), 186-190. [30] E. Reverchon, G. Della Porta, D. Sannino, P. Ciambelli, Supercritical antisolvent precipitation of nanoparticles of a zinc oxide precursor, Powder technology, 102 (1999) 127-134. [31] E. Reverchon, G. Della Porta, M. Falivene, Process parameters and morphology in amoxicillin micro and submicro particles generation by supercritical antisolvent precipitation, The journal of supercritical fluids, 17 (2000) 239-248. [32] E. Reverchon, G. Della Porta, Production of antibiotic micro-and nano-particles by supercritical antisolvent precipitation, Powder Technology, 106 (1999) 23-29. [33] E. Franceschi, A.M. De Cesaro, M. Feiten, S.R. Ferreira, C. Dariva, M.H. Kunita, A.F. Rubira, E.C. Muniz, M.L. Corazza, J.V. Oliveira, Precipitation of β-carotene and PHBV and co-precipitation from SEDS technique using supercritical CO< sub> 2, The Journal of Supercritical Fluids, 47 (2008) 259-269. [34] P. Butz, R. Edenharder, A.F. Garcı ́a, H. Fister, C. Merkel, B. Tauscher, Changes in functional properties of vegetables induced by high pressure treatment, Food Research International, 35 (2002) 295-300. [35] P. Benelli, S.R. Rosso Comim, J. Vladimir Oliveira, R.C. Pedrosa, S.R.S. Ferreira, Phase equilibrium data of guaçatonga (Casearia sylvestris) extract + ethanol + CO2 system and encapsulation using a supercritical anti-solvent process, The Journal of Supercritical Fluids, 93 (2014) 103-111.

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[36] W.Z. He, Q.L. Suo, H.L. Hong, G.M. Li, X.H. Zhao, C.P. Li, Supercritical antisolvent micronization of natural carotene by the SEDS process through prefilming atomization, Industrial & engineering chemistry research, 45 (2006) 2108-2115. [37] A. Martın, M. Cocero, Numerical modeling of jet hydrodynamics, mass transfer, and crystallization kinetics in the supercritical antisolvent (SAS) process, The Journal of supercritical fluids, 32 (2004) 203-219. [38] I. Kikic, N.D. Zordi, M. Moneghini, D. Solinas, Antisolvent precipitation of vitamin B6: a thermodynamic study, Journal of Chemical & Engineering Data, 56 (2011) 49784983. [39] V. Majerik, G. Charbit, E. Badens, G. Horváth, L. Szokonya, N. Bosc, E. Teillaud, Bioavailability enhancement of an active substance by supercritical antisolvent precipitation, The Journal of Supercritical Fluids, 40 (2007) 101-110. [40] A. Patel, Y. Hu, J.K. Tiwari, K.P. Velikov, Synthesis and characterisation of zein– curcumin colloidal particles, Soft Matter, 6 (2010) 6192-6199. [41] G. Lévai, A. Martín, A. Moro, A.A. Matias, V.S.S. Gonçalves, M.R. Bronze, C.M.M. Duarte, S. Rodríguez-Rojo, M.J. Cocero, Production of encapsulated quercetin particles using supercritical fluid technologies, Powder Technology, 317 (2017), 142-153.

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Figure Captions

A

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M

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Fig. 1:Schematic diagram of the SAS precipitation apparatus.

I N U SC R

Filter

Thermostatic bath Blocking Valve

Heating bath

HPLC Pump

Manometer

Temperature Controller

Line Filter

Back Pressure Regulator

Temperature Controller

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PT

Solution Reservoir

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Cylinder CO2

A

M

A

CO2 Pump

Flask

Filter Precipitation Vessel

Flow totalizer

Run 9

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PT

ED

M

A

N

Run 8

A

Run 18

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Fig. 2: SEM micrograph of the precipitated vitamins in zein.

Run 19

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Run 23

A

N

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Run 22

A

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ED

M

Fig. 3: Zein particles (Run 9) and vitamin complex in zein microcapsules (Run 24).

Table Captions Table 1: SAS precipitation of zein at 313 K: summary of experiments, mean particle size and precipitation yield.

Table 2: SAS co-precipitation of δ-tocopherol and riboflavinin zeinat optimized SAS

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condition for zein precipitation.

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Table 3: SAS co-precipitation of δ-tocopherol, riboflavin andβ-carotene in zeinat optimized SAS condition for zein precipitation.

N

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Table 4: Efficiency of encapsulation of vitamins in zein particles.

M

A

Table 5: List of assumptions of the economic analysis of the evaluated process

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Table 6: Results for the economic analysis

PT

Table 7: Sensitivity analysis results for different solvent mass to feed mass ratio (S/F)

A

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ratios maintaining the same performance of the process

Table 1: SAS precipitation of zein at 313 K: summary of experiments, mean particle size and precipitation yield. C0zein (g/mL)

FCO2 (g/min)

FSOL

P (MPa)

(mL/min)

Yield (g/100g)

0.02

20

1

10

-

2

0.02

40

1

10

-

3

0.02

60

0.5

10

4

0.02

60

1

10

5

0.02

60

1.5

10

6

0.04

60

1

7

0.02

60

1

8

0.02

60

9

0.02

60

9R

0.02

60

No particles

41.11

122.7

8.14

564.6

-

No particles

7

-

No particles

1

13

54.67

9.69

1

16

57.53

8.51

1

16

51.98

10.56

N

U

445.7

A

10

M

ED

PT CC E

No particles

18.58

* R = replicate.

A

(µm)

SC R

1

Particlesize

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Run

Table 2: SAS co-precipitation of δ-tocopherol and riboflavin in zein at optimized SAS condition for zein precipitation. Run

δ-

Riboflavin

Tocopherol(g)

Yield (g/100g)

(mg)

Particle (µm)

0.05

-

52.98

14.36

10R

0.05

-

49.93

8.40

11

0.10

-

63.09

8.97

11R

0.10

-

71.68

12.64

12

0.15

-

41.54

10.38

12R

0.15

-

57.65

10.67

13

0.25

-

1.68

SC R

IP T

10

Not

-

2.5

66.62

15

-

5.0

71.96

14.21

16

-

7.5

67.17

11.24

17

0.05

5.0

56.88

15.84

17R

0.05

5.0

69.98

16.67

18

0.10

5.0

56.51

13.51

18R

0.10

5.0

59.24

9.78

19

0.15

5.0

74.15

12.12

19R

0.15

5.0

77.58

8.71

A

M

ED

PT CC E

U

14

N

particles

A

size

* R = replicate.

12.51

sufficient

Table 3: SAS co-precipitation of δ-tocopherol, riboflavin and β-carotene in zein at optimized SAS condition for zein precipitation. δ-

Riboflavin

tocopherol (g)

(g)

β-

Yield

carotene (g)

(g/100g)

-

-

0.02

72.00

21

-

0.01

-

69.40

22

-

0.01

0.02

73.74

23

0.15

-

0.02

81.75

24

0.15

0.01

0.02

24R

0.15

0.01

0.02

A

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PT

ED

11.79 14.05 11.96 14.63

65.61

17.80

68.95

17.52

U

M

A

N

* R = replicate.

size

(µm)

SC R

20

Particle

IP T

Run

Table 4: Efficiency of encapsulation of vitamins in zein particles.

PT CC E A

Riboflavin (mg/g particle) Total -

Core -

4.461 -

1.876 -

2.387 2.877 -

4.766 5.048 -

2.379 2.171 -

4.885 -

10.266 -

5.381 -

4.290 6.902 8.624

8.765 11.207 13.744

4.475 4.305 5.121

2.585 -

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Surface -

N

U

SC R

Core Surface Total Core 0.1290 0.2894 0.1603 0.1535 0.2673 0.1138 0.1861 0.3819 0.1958 0.2225 0.3893 0.1669 0.2095 0.4627 0.2532 0.2293 0.4718 0.2425 0.0582 0.1112 0.0530 0.0709 0.1234 0.0525 0.1124 0.1529 0.0405 0.0968 0.1591 0.0623 0.1283 0.2097 0.0814 0.1279 0.2131 0.0852 0.6929 0.5384 0.0182 1.3718 0.1444 0.1936 0.0492 0.0833 0.1367 0.2041 0.0674 0.0728 0.1557 0.2059 0.0502 -

A

Total 9.4994 11.4916 12.4056 12.5215 13.4555 14.5708 -

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9 9R 10 10R 11 11R 14R 17 17R1 18 18R 19 19R 20 20R 22 21 23 23R 24

Surface 8.8065 10.9532 12.3874 11.1497 13.3722 14.4980 -

δ-tocopherol (mg/g particle)

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Run

β-carotene (mg/g particle)

Table 5: List of assumptions of the economic analysis of the evaluated process Value

Unit

Days worked in a year

330

(days/year)

Zein

40.001

(USD/kg)

Ethanol

0.721

(USD/kg)

Tocopherol

40.001

CO2

0.302

Electricity

0.053

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Economic data

U

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Raw materials prices

M

A

N

(USD/kg) (USD/kg)

(USD/kWh)

ED

Cold demand under

0.0284

PT

293K

(USD/kWh)

Hot utility cost (steam

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low pressure)

1

0.0524

(USD/kWh)

calculated based on a medium value from different manufacturers; 2data from (Santos

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et al., 2014); 3data from (Albarelli et al., 2014); 4data from (Pereira and Meireles, 2010).

Table 6: Results for the economic analysis Value

Unit

Total investment

2.33

MUSD

Utility cost

77,914.19

USD/year

Variable cost

4.12

MUSD/year

Fixed cost

0.50

MUSD/year

General production cost

0.02

MUSD/year

COM

5.53

COMprod

502.95

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Economic data

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PT

ED

M

A

N

U

MUSD/year USD/kg

Table 7: Sensitivity analysis results for different solvent mass to feed mass ratio (S/F) ratios maintaining the same performance of the process Economic data

Value

Unit

process

kg/h 15000

13500

10500

7500

0

10

30

50

Total investment

2.33

2.29

2.18

2.06

Utility cost

77.9

72.2

60.6

48.7

TUSD/year

Variable cost

4.12

3.91

3.49

3.07

MUSD/year

Fixed cost

0.50

0.50

0.48

0.46

MUSD/year

0.02

0.02

0.02

0.02

MUSD/year

COM

5.53

A

CO2 flow to the

4.75

4.22

MUSD/year

COMprod

502.95

479.32

431.78

383.69

USD/kg

Reduction on the

cost

5.27

M

ED PT CC E

IP T

%

MUSD

U N

General production

A

SC R

scCO2 flow