Analysis of different protocols for the cleaning of corn starch adhering to stainless steel

Accepted Manuscript Analysis of different protocols for the cleaning of corn starch adhering to stainless steel José M. Vicaria, Encarnación Jurado-Alameda, Otilia Herrera-Márquez, Vanessa Olivares-Arias, Alejandro Ávila-Sierra PII:

S0959-6526(17)31933-9

DOI:

10.1016/j.jclepro.2017.08.232

Reference:

JCLP 10491

To appear in:

Journal of Cleaner Production

Received Date: 22 May 2017 Revised Date:

23 August 2017

Accepted Date: 25 August 2017

Please cite this article as: Vicaria JoséM, Jurado-Alameda Encarnació, Herrera-Márquez O, OlivaresArias V, Ávila-Sierra A, Analysis of different protocols for the cleaning of corn starch adhering to stainless steel, Journal of Cleaner Production (2017), doi: 10.1016/j.jclepro.2017.08.232. 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.

ACCEPTED MANUSCRIPT

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Starch detergency

temperature microparticles ozonation enzyme recovery flow time

Ozone generator O2

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pH

O2+O3

Gas washing flasks

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Surfactants

Ozone analyser

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α-amylase

O2+O3

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Variables:

O2

Starch adhered to stainless steel Washing bath circuit

ACCEPTED MANUSCRIPT Wordcount: 7708

Title Analysis of different protocols for the cleaning of corn starch adhering to stainless steel

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Authors José M. Vicaria, Encarnación Jurado-Alameda, Otilia Herrera-Márquez, Vanessa Olivares-Arias, Alejandro Ávila-Sierra

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Address

Chemical Engineering Department, Faculty of Sciences, University of Granada, Avda Fuentenueva,

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s/n, 18071 Granada, Spain

Corresponding author José M. Vicaria Abstract

Cleaning-in-place is a process used in food industries to maintain the hygiene in the process.

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This work analyses the effect that different cleaning protocols exert on the cleaning of starch adhering to stainless-steel surfaces. The objective is to optimize the cleaning process reducing its environmental impact by diminishing the consumption of reagents and the energy consumption.

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These protocols include different processes (ozonation, enzyme recovery), experimental conditions (pH, temperatures, time, recirculation flow, ozone gas concentration) and composition of the washing solutions (enzyme, surfactant, silica microparticles). In the absence of enzyme, the maximum

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detersive effectiveness (61.9%) was obtained using a protocol that includes ozone and fatty ethoxylated alcohol at 45ºC. In the presence of the enzyme, the maximum detergency (97.0±4.7%) was obtained using the highest enzyme concentrations (1.00 g/L), 60ºC and 45 min. Finally, the viability of reusing the enzyme was confirmed after different washing cycles that incorporated intermediate centrifugation stages.

Keywords Starch, cleaning protocol, amylase, ozone, microparticles, enzyme reutilization

1. Introduction

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ACCEPTED MANUSCRIPT The world starch market of corn is predicted to grow more than 4.8% until the year 2024. In the food industry, starch is used as a thickener in gelification processes and as a water-retention agent in a great variety of products such as soups, sauces, ice creams, beer, pastry, etc. (Singh et al., 2007). Starches or their derivatives usually have problems of adhesion to the inner surfaces of pipes and accessories, these build-ups being difficult to eliminate.

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The cleaning process in the food industry is a critical operation. Therefore, the cleaning and disinfecting need to be repeated regularly (Liu et al., 2002). The removal of starch and amylaceous products in the food industry presents difficulties due to the type of bonds formed with hard surfaces (Singh et al., 2007), depending on the detersive efficiency of several factors such as the properties

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and concentration of the soiling agent, substrate properties, or time (von Rybinski, 2007). Generally, these procedures do not take into account the specific soiling agent that must be eliminated. Often it is necessary to develop new detergent formulations or methods that improve the cleaning and reduce

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the cost of the process (Khalid et al., 2016; Mauermann et al., 2009). Different works have examined the adhesion of starch in the textile sector (St. Laurent et al., 2007), but the behaviour of starch on hard surfaces in the food industry has not been as thoroughly studied. Starchy solutions are often high-viscosity liquids and therefore the cleaning requires high temperatures that depend on the concentration and physical structure of the gel (Fryer and Asteriadou, 2009) .

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The aqueous starchy solutions and the stainless steel are negatively charged to pH alkalines and therefore the forces of electrostatic repulsion are strong while bonding forces are weak. In an acidic medium the starch particles cause the repulsion forces to diminish and thus result in lower cleaning efficiency (Otto et al., 2016). The use of aqueous alkaline solutions to clean corn starch is very

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frequent and effective (Nor Nadiha et al., 2010). Lai et al. (2004) suggested that the ions in the alkaline solution in the rich amorphous regions in amylose break the intermolecule bonds. Amylopectin is more difficult to dissolve in an alkaline solution, the total solubility being favoured

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by stirring (Han and Lim, 2004). Experimental results show that the amylose is more expanded in an alkaline solution (Hanselmann et al., 1995). Alternatives to the use of high-temperature cleaning with alkaline media include aqueous solutions containing surfactants or ozone, which provide good cleaning and disinfection results (Pascual et al., 2007) while increasing the biodegradability of the washing solutions (Guzel-Seydim et al., 2004; Vicaria et al., 2016). The incorporation of enzymes (α-amylase) enables adequate cleaning of starch under mild operation conditions (neutral pH, low temperatures) (Mitidieri et al., 2006). These hydrolyse the starch, facilitating its cleaning (Pongsawasdi and Murakami, 2010) and avoiding its redeposition. Other studies have used nanoparticles or enzymes and nanoparticles to clean different soiling agents (Chengara et al., 2004; Lee et al., 2009). However, the process of 2

ACCEPTED MANUSCRIPT cleaning hard surfaces is complex, as different interactions occur between surfactants, enzymes, soils, and surfaces that exert diverse effects on the effectiveness of the washing with detergent formulations (Roy and Mukherjee, 2013). The present work offers an analysis of the way in which the cleaning of corn starch adhering on stainless steel is affected by different variables (pH, time, surfactants, ozone, flow recirculation,

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microparticles, enzymes, and enzyme reutilization). The aim is also to analyse whether the use of enzymes and microparticles increases detergency due to the adsorption of enzymes onto the microparticles and subsequent adhesion of these to starch, thereby creating a differential concentration of enzyme near the starch. Also, the possibility of recovering enzyme by centrifugation

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of the washing solutions that contain the enzyme and microparticles is analysed. This would enable the reuse of the enzyme in several cleaning cycles, reducing the cost and environmental impact of the

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cleaning process.

2. Materials and methods

2.1. Materials

Commercial cornstarch (Maizena®, 11.50% moisture, 0.29% fat, and 0.3% protein) was used

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as the soiling agent (Jurado-Alameda et al., 2015a). Different commercial surfactants have been studied (weight was in dry form). The structure of surfactants are shown in Figure 1. •

A fatty ethoxylated alcohol (FAE): non-ionic surfactant called Findet 1214N-23 (Kao Corporation S.A), molecular weight 629 g/mol, and water content 0.3% in weight (Bravo

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Rodriguez et al., 2005), HLB=14.4 and CMC 0.021 g/L (37°C) (Martínez-Gallegos et al., 2011), and readily biodegradable by aerobic degradation (Jurado-Alameda et al., 2013). An alkylpolygycoside (APG): non-ionic surfactant called Glucopon 650 (Henkel KgaA,

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Düsseldorf, Germany), HLB=11.9, water content 50.4% in weight , CMC 0.073 g/L (37ºC), molecular weight 397 g/mol (Bravo Rodriguez et al., 2005), readily biodegradable by aerobic degradation (Jurado-Alameda et al., 2011), and biodegradable by anaerobic degradation. •

A linear alkyl benzene sulfonate (LAS): ionic surfactant (Petresa, Cádiz, Spain), water content 54.6% in weight and molecular weight 342 g/mol, CMC 1.018 g/L (37ºC) (Martínez-Gallegos, 2005), readily biodegradable by aerobic degradation (Lechuga et al., 2014), and not biodegradable by anaerobic degradation.

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An amine oxide (AO): non-ionic/cationic surfactant - pH dependent called Oxidet DM-20 (Kao Corporation S.A), a lauramine oxide, water content 67.7% in weight.

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ACCEPTED MANUSCRIPT The choice of these surfactants is because they are surfactants usually used in cleaning processes and environmental friendly. FAE is considered as readily biodegradable by aerobic degradation (JuradoAlameda et al., 2013). APG is considered as readily biodegradable by aerobic degradation (JuradoAlameda et al., 2011), and biodegradable by anaerobic degradation. Besides FAE, APG and AO are considered as “eco-friendly”, being able to be used to formulate, as example, detergent with eco-

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label. These surfactants also have a good wettability. LAS is also considered as readily biodegradable by aerobic degradation (Lechuga et al., 2014), and not biodegradable by anaerobic degradation. However, it is considered suitable to incorporate it into the study as it is one of the most widely used surfactants worldwide.

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A commercial thermostable amylase (EC 3.2.1.1) from B. licheniformis supplied by Sigma (A3403-500KU) was used. Its optimal pH range is 7–9 and optimal temperature 40-60ºC. The washing solutions that contained α-amylase were performed in buffer pH 7.0 [3.63 g/L KH2PO4 (PA,

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Panreac) and 8.93 g/L Na2HPO4·2H2O (PRS, Panreac)] (Sørensen, 1909). The enzymatic activity was measured regularly to assess the α-amylase stability during the testing period. The activity was constant for all experimental times.

Hydrophilic silica microparticles Sipernat 50 (MP) (specific surface area 500

m2/g,

mean

diameter 50 µm, density 180 g/L, SiO2 content ≥ 97%, sulfate content ≤ 1.0%) were incorporated into

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the washing solutions. These particles were supplied by Evonik Industries AG (Hanau–Wolfgang, Germany). When the silica microparticles are included in the washing solution, they are previously dispersed in water by sonication for 15 min (Sonorex RK 106 S, Bandelin). If the washing solution contains enzyme, the enzyme is added after sonication and before the washing process begins.

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The aqueous solutions used were:

2N H2SO4 prepared with H2SO4 95% (PA AnalaRNormapur, VWR).

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Phenol 5% (w/v) (PA, Panreac). For its dissolution, the phenol was liquefied by heating in a

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thermostatically controlled bath. The solution had to be preserved in a sealed container and protected from light. •

5% KI (PRS, Panreac).

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0.2 g/L Na2SO3 (PRS, Panreac). The detergency assays were made in NaOH aqueous solutions (pH 13) or buffer pH 7.

Different components were incorporated (enzymes, microparticles, surfactants) in order to complete the formulation of the washing solution. The enzymatic solutions were prepared in buffer pH 7.

2.2 Soiling agent and substrate preparation. Soiling procedure

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ACCEPTED MANUSCRIPT The surface that is soiled is called the substrate. The substrate used consisted of spherical wads of stainless-steel fibres AISI 410 (Figure 2.a), with a diameter of approx. 2.0-2.1 cm, a weight of 0.80-0.82 g, a fibre width of 0.51 mm approx., free volume fraction of wads of approx. 93% (Jurado et al., 2015a). The soiling agent was gelatinized corn starch. The aqueous solution of gelatinized cornstarch (8% w/w) was prepared in the following way: the aqueous suspension of

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starch was heated at 70ºC with constant stirring for an hour (Souza and Andrade, 2002). For this soiling agent, the temperature should not exceed 73ºC. The gel was cooled at room temperature for at least 12 h. The composition of the dried gelatinized corn starch was protein 0.37 g/100g, fat 0.42 g/100g, carbohydrates 90.37 g/100g, moisture 7.84 g/100g, ash 0.99 g/100g, Na 46.55 mg/100g, Ca

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38.96 mg/100g, K 290.36 mg/100g, and Mg 32.55 mg/100g (Jurado-Alameda et al., 2015b). Eight spherical wads were soiled with gelatinized starch gel in the following way: (a) each wad was submerged in starch gel, impregnating the surface uniformly; (b) the soiled wads were placed in

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an oven (Hotcold-S, Selecta) at 60ºC to be dried for 12 h; (c) the eight dried wads were removed and weighed. The starch retained was evaluated by the difference in the weight of the clean and soiled wads. This quantity should be similar in the different assays, so that the eight wads used in each washing assay contained a mass of dry starch of 2.0±0.2 g. The spherical wads soiled with corn starch are shown in Figure 2.b [weight 1.04-1.07 g, with a free volume fraction of wads of approx.

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82% (Jurado-Alameda et al., 2015b)]. The use of flat surfaces allows to evaluate the effect of some variables, but the amount of dirt that can be adhered per unit of element is smaller than when rough surfaces are used. In addition, the use of rough surfaces in the BSF device also allows taking into account in detergency evaluation some stages that occur in the real washing such as redeposition. The

process.

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use of a rough substrate and dry starch in the washing trials hampers wetting and thus the cleaning

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2.3. Method of analysis of the total soluble carbohydrates (phenol-sulphuric method) The total soluble carbohydrates in the washing solution were analysed by the phenol– sulphuric colorimetric method (DuBois et al., 1956). This method enables the quantification of the starch remaining on the substrate and in the washing solution. For this, washing samples were added to test tubes containing 1 mL of 2 N sulphuric acid and then placed in a digester (Spectroquant TR320, Merck) at 100ºC for 30 min to hydrolyse the starch that the solution contains. Later, the tubes were cooled with ice. In this way, the starch is chemically hydrolysed. Afterwards, 0.5 mL of this solution was added to 0.5 mL of an aqueous solution of phenol 5% (w/v) and 2.5 mL of H2SO4 (95%). This was stirred and left to cool at room temperature for 15 min. The absorbance of the samples was measured at 490 nm 15 min later, using a spectrophotometer Cary 100 Bio UV–Visible (Varian). The 5

ACCEPTED MANUSCRIPT concentration of starch in the samples was calculated using a calibration curve obtained with Dglucose anhydrous (PA, Panreac) multiplying by a correction factor of 0.9 which considered the stoichiometric relation between starch and glucose in the acid hydrolysis of the starch (Jaiswal and Prakash, 2011; Lampitt et al., 1947).

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2.4. Evaluation of starch detergency in a BSF device The washing of the starch soil adhering to the stainless-steel substrate was studied in a modified Bath-Substrate-Flow system (BSF) (Jurado-Alameda et al., 2016) that includes an ozonation system. This device enables an evaluation of the detersive efficiency reached on applying

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different methods and detergent formulations on hard surfaces. The BSF device simulates a “cleaning-in-place” (CIP) system of industrial cleaning. Its usefulness is to evaluate the effectiveness of detergent formulas against different soils and substrates. This BSF device allows the modification

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of the elements acting in the detersive process, such as the substrate, soiling agent, wash-bath composition, temperature, and recirculation flow of the system.

Figure 3 shows a simplified scheme of the device. It has a tank (1) that contains the washing solution (1 L of capacity); a peristaltic pump (model 5006, Heidolph) (2) that supplies a 60 L/h flow; a packed column (3) of 50 mL of capacity, 2.5 cm in diameter and 8.5 cm in height, where the

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substrate with the soiling agent is placed; a thermostatically controlled bath (4) (model Ultraterm, casa P-Selecta) which recirculates the water through a jacket of the tank and column while maintaining the temperature constant in their interior; and the gas diffuser (5) through which a current of oxygen with ozone passes towards the solution in the tank. The ozone generator

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(AnserosPeripherals COM-AD, Germany) produces the ozone in situ from a stream of oxygen. The concentration of ozone in the ozone-oxygen mixture is determined by an ozone analyser (Ozomat GM-6000-PRO, Anseros, Germany). The volumetric flow of the ozone-oxygen gas flow is 40 NL/h,

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and the concentration of the inflow of ozone is 42.3 g/m3. The gas leaving the BSF passes through washing flasks containing 5% KI aqueous solution that eliminates the ozone in the gas stream before being released into the atmosphere. The washing solution flows from the tank upwards towards the column and afterwards returns to the tank. The recirculation maintains the agitation of the washing solution. The variables tested in the BSF are indicated in Tables 1-5. The washing procedure is the following: 1.2 L of washing solution is added to the device and the pump is turned on. After the solution is thermostatically controlled, the recirculation flow and the substrate with the soiling agent are placed in the column. In each of the washing trials made in the BSF, eight wads are previously soiled with the starch (Figure 2.b). Then the oxygen-ozone flow begins (when ozone is used for the trial) and the pump is turned on, beginning the washing process. 6

ACCEPTED MANUSCRIPT Periodically a sample is taken from the interior of the tank and the starch concentration in the solution is determined by the phenol-sulphuric method (Section 2.3). After the test, the pump is stopped and the wads are removed from the column. Finally, the wads are dried in the oven at 60ºC for 24 h and weighed. All the experiments are made in triplicate. After the end of each assay, the BSF device is cleaned. For this, the washing solution is emptied and the device is rinsed with 3 cycles of

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water (50ºC) for the entire device for 5 min. In the samples extracted from the washing trials where ozone was used, the residual ozone had to be eliminated as it could be present before evaluating the detergency. For this, 0.2 mL of 0.2 g/L Na2SO3 aqueous solution is added to 1 mL of washing sample. The Na2SO3 reacts with the excess

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ozone. No interference at all was detected using Na2SO3. The experiments were performed in triplicate in most cases.

The effectiveness of the cleaning or detergency (De, %) was calculated from the samples

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extracted from the tank using Eq.(1), where mbath is the mass of the soil in the wash bath (this is determined considering the concentration of starch suspended in the wash bath, evaluated by the phenol-sulphuric method (Section 2.3) and the volume of washing solution used) and mstarch is the total quantity of starch adhering to the substrate at the outset of the experiment. De (%) = mbatch · 100 / mstarch

Eq. (1)

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With the application of Eq.(1), the composition of the washing solution in the BSF was considered uniform in the device (perfect-mixture hypothesis). Also, it was considered that the volume of the sampling was negligible.

Also, the detergency found at the end of the washing process was evaluated from the difference

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in the weight of the wads before and after the washing trial. This weight difference corresponds to the mbath. The detergency values at the final times evaluated by the phenol-sulphuric method and by the difference in weight were similar, with weight differences being lower than 5% in all cases.

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Each experimental series made was called “Tx-yy”, where x is the number of the table in which the experimental results are indicated and yy is the number of the assay shown in this table.

2.5. Evaluation of starch detergency by solutions with enzyme in the presence or absence of MP Washing assays were made in the BSF device to evaluate the detergency achieved when different washing solutions containing enzyme are used. The detergency was studied for the enzymatic solutions prepared in buffer pH 7 in the presence of MP and under different experimental conditions (Tables 4 and 5). Also, the effect of centrifugation on the enzymatic solutions containing MP was analysed.

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ACCEPTED MANUSCRIPT For this, the washing assays were carried out following the instructions indicated in Section 2.4. In this case, the washing solution (1.2 L) used in the BSF was an enzyme solution in the presence or absence of MP. In the case of studying the influence of MP (concentrations assayed 0.33 and 0.66 g/L), these suspensions were prepared with buffer pH 7 (Section 2.1) and, afterwards, they were placed in the BSF. Once the temperature of the assay was reached, the enzyme is added to reach the

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desired final concentration (0.01-0.02 g/L) in the washing solution. In the case of not using MP, after the buffer pH 7 was thermostatically controlled, the necessary enzyme was used. Periodically, a sample was extracted from the tank and the concentration of soluble carbohydrate in solution was determined by the phenol-sulphuric method (Section 2.3). After 50 min of assay, the recirculation

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was stopped and the wads were removed from the column. Finally, they were oven dried at 60ºC for 24 h and each wad was weighed. All the experiments were made in triplicate. The detergency found after 50 min by the phenol-sulphuric method by weighing did not differ by more than 5%. It was

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verified that the presence of MP did not affect the detergency evaluation. Afterwards a study was made of the possibility of recovering the enzyme from the washing solution by centrifugation. The aim was to evaluate the detersive capacity that can be recovered by reusing part of the washing solution. For this, after the washing, the solution was centrifuged (Universal Centrifuge 320R Hettich) for 3 min at 8000 rpm, removing 80% of the volume of the supernatant, keeping 20% of the

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remaining volume. Next, this remaining volume was reconstituted (in assays with MP, a small precipitate appeared in the bottom containing the MP precipitate that possibly retained part of the enzyme adsorbed) with buffer pH 7 until reaching the original volume (1.2 L). This was the solution used for the second wash cycle (Table 5). Afterwards, a new washing process was carried out using a

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new group of eight soiled wads. Beforehand, the BSF was cleaned as indicated in Section 2.4. Similarly, the third wash cycle was carried out (Table 5). All the assays were made in triplicate. These reconstituted washing solutions contained MP with adsorbed enzyme, sugars, and the remains

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of starch in suspension. The quantity of starch present at the beginning of the test was determined (following the method defined in Section 2.4) and this was subtracted from the detergency measured in the successive cycles. In all the experiments conducted, the detergency values determined by the weight difference at the finishing time were similar to those reached by the phenol-sulphuric method, this giving differences of less than 5%. These recovery processes were meant to determine the recovery of the detersive capacity that could be reached in an industrial process where the enzyme is removed from the washing solution by centrifugation, this implying considerable economic savings in the industrial washing process.

3. Results and discussion 8

ACCEPTED MANUSCRIPT 3.1. Cleaning of starch with aqueous solutions and surfactants (FAE and APG) For the cleaning of starch with aqueous solutions to be effective, alkaline aqueous solutions are usually used, since they give rise to diffusion of hydroxyl ions towards the starch structure, allowing a swelling process (Lai et al., 2004) and the degradation of the internal structure of the

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starch (Han and Lim, 2004). The washing of starch with buffer pH 7 at 45ºC has been assayed confirming that the detergency at pH 7 was negligible (Serie T1-01).

The cleaning of starch using an aqueous solution pH 13 at 40-45ºC has been assayed following the procedure and using the device described in Section 2.4 with a recirculation flow of 60 L/h

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(Series T1-02, 03, 04 and 05). In all cases, the detergency increased almost lineally for the first 25 min but slowly lost detersive velocity until reaching 25% detergency after about 30 min. The cleaning results under similar experimental conditions were slightly higher after 40 min, reaching

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nearly 30% detergency. Experiments made at (pH 13, 40ºC) (Serie T1-07) with a 30 L/h recirculation flow, registered detergency values of 29.3±2.7% after 45 min. These values are similar to those found using the double recirculation flow (60 L/h) (Serie T1-05). It appears that, under these working conditions (pH=13, 40-45ºC) greater detergency is not achieved by increasing the recirculation flow of the washing solution in the BSF device. However, after 30 min it was found that

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in the absence of MP the detergency reached with 30 L/h flow recirculation (22.1±0.8%) (Serie T201) was less than at 60 L/h (28.4±1.1%) (Serie T2-04), and hence the flow recirculation appears to exert a positive influence on detersive effectiveness. The highest detergency values are reached at the highest washing time (45 min). For shorter time periods (30 min) the flow used does have an

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influence, probably because at shorter times the drag phenomenon, which is more pronounced, predominates on increasing the flow.

The influence of temperature on the cleaning with aqueous solution at pH 13 was analyzed

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using a 30 L/h recirculation flow. Series T1-06 to 08 are results at 45 min and 30, 40, and 60ºC, respectively. The detergency was 23.3±2.0%, 29.3±2.7%, and 44.7±4.8%, respectively. The temperature increase of the wash strongly affected the detergency reached, both in the absence as well as the presence of surfactants, reaching greater detersive capacity at 60ºC. The detergency did not significantly improve on incorporating 1.00 g/L of the surfactant FAE in the cleaning solution (Series T1-09 to 11). Thus, at pH 13, 45 min and 30, 40, and 60ºC, the detergency was 28.7±2.0%, 21.3±4.0%, and 47.0±3.3%, respectively. On adding 1.00 g/L of the surfactant APG to the washing solution, working under similar experimental conditions (pH 13, 45 min, 40-60ºC) (Series T1-12 and 13), the detergency achieved was 25.7±2.2% and 51.6±4.5%, respectively. Maximum detergency was

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ACCEPTED MANUSCRIPT achieved with the surfactant APG and the maximum temperature, although the increase in detergency with respect to the aqueous solution was not significant.

3.1.1. Influence of adding microparticles to the washing system Also, the effect exerted by the addition of microparticles to the detergent formulations used to

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clean starch was analyzed. The variables analyzed were washing time (30-45 min), MP concentration (0.00-1.00 g/L), recirculation flow (30-60 L/h) and the use of aqueous solutions with 1.00 g/L of surfactants (FAE and APG) (Table 2). In almost all cases, the addition of MP (0.10-1.00 g/L) reduced the detergency at both flow rates tested (30-60 L/h). The results appear to indicate that the

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presence of microparticles and surfactants (FAE and APG) reduced the detergency regardless of the flow rate tested in most cases (or else had no effect at all), probably because both were adsorbed over the soil, hampering its elimination. Nevertheless, in the cases in which the washing solution included

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microparticles, it was found that there was an increase in detergency with a greater recirculation flow. Similar results were found when washing times of 30 min were used.

No significant improvement in detergency was appreciated on adding MP (at concentrations of up to 1.00 g/L) to the washing solutions under the test conditions. The joint incorporation of surfactant (FAE or APG) and MP, either did not significantly affect detergency or significantly

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reduced it, primarily at low flow rates (30 L/h) and with the surfactant FAE. Jurado-Alameda et al. (2015a) also studied the influence of MP concentration in the cleaning process, and no significant influence on detergency was found after such additions. 3.1.2. Influence of the use of ozone in the washing system

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Table 3 shows the cleaning of starch when the ozone was used in the cleaning protocol. It was found that the pH strongly influenced detergency (Series T3-16 to 20), reaching the highest

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values at pH 13. Also, greater detergency resulted at higher temperatures, although the concentration of ozone in the solutions was lower, as Henry's law indicates. It is also investigated how the cleaning of starch was affected by the use of aqueous solutions at pH 13 accompanied by ozone (oxygen-ozone flow 40 NL/h, ozone concentration 42.3 g/m3) and aqueous solutions containing different surfactants with a concentration of 1.00 g/L at different temperatures. The detergency found in the washing assays that used ozone (pH 13, 80 L/h recirculation flow, 45 min) (Series T3-01 to 03) had a mild rise in temperature, reaching a detergency of 42.2±2.6% a 45ºC, which shows that it is similar to the detergency achieved under the usual washing conditions (Serie T1-08) (60ºC, pH 13, 30 L/h recirculation flow, 45 min)

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ACCEPTED MANUSCRIPT (44.7±4.8%). Therefore, the presence of ozone in the washing medium did not appear to improve the detergency under these conditions. The addition of different surfactants diminished detergency at all the temperatures tested for the LAS anionic surfactant and for the AO surfactant (except at 45ºC). It did not affect the incorporation of the APG surfactant, and the use of the FAE surfactant had a positive effect. The total

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LAS concentration diminished with ozonation time, this effect being more pronounced at higher temperatures (Jurado-Alameda et al., 2012).This could explain why the detergency decreased when LAS was added compared with the results found with ozone alone. Tehrani-Bagha et al. (2012) also studied the effect of ozone on the degradation of LAS, reporting that in basic media the degradation

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accelerates. Lechuga et al. (2014) also demonstrated that ozone degrades LAS to a higher degree than did APG. That is, 50% of the LAS was eliminated after 7.13 min of reaction while the APG degraded by the same amount after 25.8 min of ozonation. Beltrán et al. (2000) also studying the effect of

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degradation of LAS by ozone, found that the degradation was faster at pH values higher than 10. The addition of ozone and surfactant APG or AO under the best washing conditions assayed (45ºC) gave rise to detergency values close to those reached and the usual washing conditions (Serie T1-08) (pH 13, 60ºC, 30 L/h recirculation flow, 45 min) (44.7±4.8 %). The joint use of ozone and FAE surfactant strengthened detergency in the experiments made in the absence of surfactant at all

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the temperatures tested. Thus, at 25ºC, detergency reached 39.7±1.1% (Serie T3-13) against that found in the absence of FAE (36.7±2.8%, Serie T3-01), this increase being greater at higher temperatures, where notable improvements in detergency efficiency were reached when ozone and FAE were used jointly. Thus, at 35ºC, detergency of 54.7±2.9% (Serie T3-14) was reached using

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ozone and FAE, while without the surfactant the detergency was only 36.0±1.8%, (Serie T3-02). At 45ºC the detergency rose to 61.9±3.2% (Serie T3-03) when ozone and FAE were used, while without the surfactant the detergency fell to only 42.2±2.6% (Serie T3-03). The use of ozone therefore

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significantly boosted detergency at temperatures lower than normally used in the food industry by the incorporation of the surfactant FAE. The favourable effect found with this surfactant may be related to the greater wettability of the solutions containing this surfactant. Hence, García Martín et al. (2014) found that FAE reached high wettability values, i.e. higher than for AO. This could be explained, at least qualitatively, by the fact that higher detergency values were reached using the surfactant FAE. However, Lechuga et al. (2013) investigated the ozonation of linear alkyl benzene sulfonates (LAS), ether carboxylic derivative surfactants (EC), fatty-alcohol ethoxylates (FAE), and alkylpolyglucosides (APG) and found that the surfactant with the greatest toxicity and lowest surface-tension value was FAE, while the one with the lowest toxicity and the highest surface-tension 11

ACCEPTED MANUSCRIPT value was LAS. Surfactants with aromatic rings, such as LAS, or surfactants with glycosidic groups, such as APG, show lower toxicity. Meanwhile, ether groups in the FAE and EC, and carboxylic acid derivates in the EC, increase toxicity after the ozonation. Lechuga et al. (2012) reported that the ozonation process only slightly improved the biodegradation of amide oxide (AO) based surfactants.

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3.2. Using enzymes to clean starch adhering to stainless steel When enzymes are used to clean starch, it is necessary to work at pH near neutrality, a condition under which α-amylase enzyme is stable. The present work studies the effect that different factors, such as the enzyme concentration (E), surfactants, or MP concentration, exert on the

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detergency of starch with the use of α-amylase in pH 7 buffer solution.

Table 4 shows the results in the washing assays that contained enzyme and under different experimental conditions, temperature (40-60ºC), and enzyme concentrations (0.03-1.00 g/L). A

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notable increase in detergency is found on raising the temperature. At a 0.06 g enzyme/L, a temperature rise of between 40 and 60ºC tripled the detergency after 30-45 min (Series T4-01 and T4-03, T4-07 and 09) working under the same experimental conditions. At 60ºC, an increase in the enzyme concentration of between 0.03 and 1.00 g/L augmented the detergency significantly, reaching values of 88.5±2.7% after 30 min (Series T4-02 and 05) and 97.0±4.7% after 45 min (Series T4-08

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and 11). The use of enzyme in the cleaning process gave better detergency results at neutral pH values in the cleaning of starch.

At 60ºC and 0.06 g enzyme/L the effect of the presence of MP on detergency was also analysed. It was found that with 1.00 g/L de MP, the detergency was not affected. Thus, at 30 min, the

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detersive effectiveness was 20.9±3.2% (Serie T4-06), i.e. practically equal to that reached without MP (23.3±2.4%) (Serie T4-03). At 45 min, the trend was similar, reaching 36.8±2.2% without MP (Serie T4-09) as opposed to 39.3±0.5% with MP (Serie T4-12).

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Table 4 also shows the detergency reached with aqueous solutions containing 0.06 g/L of enzyme and 1.00 g/L of surfactants (LAS, APG and FAE). The cleaning was assayed with solutions at pH 7 in a BSF device and 30 L/h of recirculation flow at 40 and 60ºC for 45 min. No significant differences were detected in experiments conducted at 40ºC and 60ºC, either at 30 min (Series T4-01, 13, 14 and T4-03, 17, 16) or at 45 min (Series T4-07, 19, 18 and T4-09, 21, 22). However, when 1.00 g/L of the anionic surfactant LAS was used, significant differences in detergency were found at 30 min of washing (Serie T4-15) and at 45 min (Serie T4-20) with respect to the detergency experiment without surfactant (Serie T4-03), where the values at 45 min were 40.4±1.6% with 1.00 g/L LAS as opposed to 36.8±2.2% without (Serie T4-09). Therefore, although the addition of 1.00

12

ACCEPTED MANUSCRIPT g/L of APG or FAE had no effect at all on detergency, the presence of LAS appeared to improve the results.

3. 3 Reutilization of enzyme using microparticles Considering the high detergency values reached in the washing processes that incorporated

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enzymes in the neutral washing solutions, the adsorption of enzymes onto microparticles and their subsequent reutilization in different wash cycles was tested in order to reduce the cleaning costs. To evaluate the effectiveness of this approach, the results were compared with those reutilizing the enzyme without microparticles.

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The incorporation of silica MP into the enzyme solutions had a double objective. On the one hand, it was to analyse whether the adsorption of the enzyme onto the particles and subsequent adhesion of the particles to the starch caused a differential increase in the enzyme concentration near

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the starchy soils and therefore a greater detergency in the washing process using the same concentration of enzyme. On the other hand, the possibility of removing the MP from the wash bath by centrifugation would enable the reuse of the enzyme.

Different studies have used nanoparticles to clean different soiling agents (Chengara et al., 2004; Wasan and Nikolov, 2003) as well as the combined use of enzymes and nanoparticles on the

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starch removal (Lee et al., 2009). The joint use of the two components in detergent formulations for the cleaning of starchy soils was found to improve detergency (Soleimani et al., 2012). Table 5 shows the experimental results. It was found that the detergency after 50 min in the first washing cycle reached 23.7±1.2% for an enzyme concentration of 0.01 g/L (Serie T5-01). After

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centrifugation of the washing solution, 80% of the supernatant was removed and the remaining 20% was reconstituted until reaching the original volume, and a new washing assay was conducted (as described in Section 2.5). The detergency in the second washing cycles was 8.3% (Serie T5-02)

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while in the first washing cycle a detergency of 23.7±1.2% was obtained, i.e. 34% of the detergency initially reached. This result appears to indicate that after the enzymatic solution was centrifuged, it recovered little of the enzyme and therefore, with the reuse of the solution, there was an appreciable loss of detergency (35% of the original detergency is recovered). For an enzyme concentration of 0.02 g/L (Serie T5-05), the detergency reached, on reconstituting 20% of the solution after the wash following the removal of 80% of the supernatant (second washing cycle), was 10.9% (Serie T5-06). This means a 33% recovery of the detergency of the first cycle (33.1%, Serie T5-05), a value similar to that reached with an enzyme concentration of 0.01 g/L. A new centrifugation process enabled a third washing cycle (Serie T5-07), reaching 3.7% detergency (34% of the detergency reached in the second washing cycle; Serie T5-06). 13

ACCEPTED MANUSCRIPT Table 5 shows the detergency reached with aqueous solutions with different MP concentration and containing different enzymatic concentrations (method described in Section 2.5). The detergency reached after 50 min was 22.9±0.1% for an enzyme concentration of 0.01 g/L and an MP concentration of 0.33 g/L (Serie T5-03; MP:E ratio equal to 33). After the centrifugation of the solution, the removal of 80% of the supernatant and the reconstitution of the remaining 20% to reach

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the original volume, the second washing cycle was conducted (following the method described in Section 2.5). The detergency reached was 11.5±0.1% (Serie T5-04), i.e. 49% of the initial detergency, a greater recovery than found without MP.

In the same way as for an enzyme concentration of 0.02 g/L and an MP concentration of 0.66

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g/L (Serie T5-08), which an MP:E ratio equal to 33, the detergency reached after 50 min was 31.5±2.2%, similar to that reached without MP (33.1%) (Serie T5-05). After a second washing cycle the detergency was 16.1±0.7% (Serie T5-09), and thus it was possible to recover 51% of the

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detergency of the first washing cycle (31.5±2.2%, Serie T5-08). A new centrifugation process enabled a third washing cycle (Serie T5-10), reaching 8.2% of detergency, for 51% of the detergency reached in the previous wash (Serie T5-09).

4. Conclusions

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A study has been made of the process of washing starch adhering to stainless-steel surfaces. At pH 13 and 40-60ºC, there were no notably significant differences in the detergency when the solution lacked surfactant, with FAE or with APG, although it appeared to be lower when these two surfactants were included. At 60ºC in the presence of 1.00 g/L of APG, higher detergency values

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were reached than without surfactants (51.6±4.5%). Also, it was found that the addition of MP to the washing solutions in concentrations of 0.00-1.00 g/L did not improve the detergency. The joint incorporation of the surfactant (FAE or APG) and the MP, either had no significant effect on

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detergency or significantly reduced it. The incorporation of ozone or ozone and the surfactants LAS, APG, AO, and FAE (1.00 g/L) in the pH 13 washing solutions showed greater detergency when a higher temperature was tested (45ºC). The greater detersive effectiveness (61.9±3.2 %) was achieved in the washing process that included ozone and the surfactant FAE at 45ºC. At pH 7 the maximum detergency values were achieved using the highest enzyme concentrations (1.00 g/L) at 60ºC. The presence of LAS together with the enzyme increased the detergency values (the highest detergency value reached). The use of enzyme and silica microparticles in the washing process at neutral pH enabled the reuse of the enzyme after each wash, recovering 50% of the enzymatic activity.

14

ACCEPTED MANUSCRIPT Acknowledgements This work was financed by the project CTQ2015-69658-R of the "Ministerio de Economía y Competitividad", Spain. We thank Evonik Industries AG (Hanau–Wolfgang, Germany) for providing

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the microparticles.

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ACCEPTED MANUSCRIPT Tables captions Table 1. Detergency of starch. Influence of time, temperature, pH, recirculation flow and surfactant concentration (SD = standard deviation) Table 2. Detergency of starch. Influence of time, recirculation flow, surfactant concentration and MP concentration (pH=13, 40ºC) (SD = standard deviation)

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Table 3. Detergency of starch. Influence of temperature, pH, surfactant concentration and ozone (45 min, 80 L/h recirculation flow, 40 NL/h oxygen-ozone flow, 42.3 g/m3 ozone concentration) (SD = standard deviation)

Table 4. Detergency of starch. Influence of time, temperature, surfactant concentration, enzyme

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concentration, and MP concentration (pH=7, 30 L/h recirculation flow) (SD = standard deviation) Table 5. Detergency of starch. Influence of enzyme concentration, MP concentration, and enzyme

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recycle (pH=7, 50 min, 60ºC, 60 L/h recirculation flow) (SD = standard deviation)

20

ACCEPTED MANUSCRIPT Figure captions Figure 1. Surfactants. a) Fatty ethoxylated alcohol (FAE), b) alkylpolygycoside (APG), c) linear alkyl benzene sulfonate (LAS), d) amine oxide (AO). Figure 2. Spherical wads. a) without soiling agent, b) with soiling agent. Figure 3. Scheme of the BSF device with ozonation system. (1) jacketed tank, (2) peristaltic pump,

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(3) packed column, (4) thermostatically controlled bath, (5) gas diffuser. Figure 4. Influence of time on detergency of starch with an aqueous solution at pH=13. 40ºC, 60 L/h

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recirculation flow.

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Table 1.

No No No No No No No No FAE / 1.00 FAE / 1.00 FAE / 1.00 APG / 1.00 APG / 1.00

De (%) SD (%) 0.6 25.0 28.9 25.4 29.9 23.3 29.3 44.7 28.7 21.3 47.0 25.7 51.6

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60 60 60 60 60 30 30 30 30 30 30 30 30

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7 13 13 13 13 13 13 13 13 13 13 13 13

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45 40 40 45 45 30 40 60 30 40 60 40 60

Surfactant / Concentration (g/L)

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60 30 40 30 40 45 45 45 45 45 45 45 45

Recirculation flow (L/h)

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T1-01 T1-02 T1-03 T1-04 T1-05 T1-06 T1-07 T1-08 T1-09 T1-10 T1-11 T1-12 T1-13

Time (min) T (ºC) pH

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0.0 1.2 2.7 0.0 0.0 2.0 2.7 4.8 2.0 4.0 3.3 2.2 4.5

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Table 2.

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MP concentration (g/L) De (%) SD (%) 0.00 22.1 0.8 0.10 16.2 3.0 1.00 16.3 2.7 0.00 28.4 1.1 0.10 26.6 0.5 1.00 24.7 0.0 0.00 28.5 2.3 0.10 24.6 1.8 1.00 26.2 1.9 0.00 33.7 4.3 0.10 34.1 3.3 1.00 32.1 4.0 0.00 10.5 1.5 0.10 12.1 2.1 1.00 11.1 2.0 0.00 22.8 1.0 0.10 22.7 2.9 1.00 16.8 0.3 0.00 22.2 3.2 0.10 21.1 1.7 1.00 16.8 1.1 0.00 31.1 4.7 0.10 31.0 2.8 1.00 22.0 1.0 0.00 17.8 1.6 0.10 17.8 1.5 1.00 17.5 1.3 0.00 23.2 0.6 0.10 29.6 1.6 1.00 30.6 2.0

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Surfactant / Concentration (g/L) No No No No No No No No No No No No FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 APG / 1.00 APG / 1.00 APG / 1.00 APG / 1.00 APG / 1.00 APG / 1.00

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Serie Time (min) Recirculation flow (L/h) T2-01 30 30 T2-02 30 30 T2-03 30 30 T2-04 30 60 T2-05 30 60 T2-06 30 60 T2-07 45 30 T2-08 45 30 T2-09 45 30 T2-10 45 60 T2-11 45 60 T2-12 45 60 T2-13 30 30 T2-14 30 30 T2-15 30 30 T2-16 30 60 T2-17 30 60 T2-18 30 60 T2-19 45 30 T2-20 45 30 T2-21 45 30 T2-22 45 60 T2-23 45 60 T2-24 45 60 T2-25 30 30 T2-26 30 30 T2-27 30 30 T2-28 30 60 T2-29 30 60 T2-30 30 60

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0.00 0.10 1.00 0.00 0.10 1.00

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APG / 1.00 APG / 1.00 APG / 1.00 APG / 1.00 APG / 1.00 APG / 1.00

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30 30 30 60 60 60

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45 45 45 45 45 45

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T2-31 T2-32 T2-33 T2-34 T2-35 T2-36

25.7 27.0 22.1 28.4 34.4 28.6

2.2 2.8 3.2 1.3 0.7 0.4

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Table 3.

36.7 36.0 42.2 26.6 30.5 32.8 33.9 40.3 42.8 31.9 27.8 48.7 39.7 54.7 61.9 3.4 9.4 2.6 4.4 45.0

2.8 1.8 2.6 4.7 3.3 2.5 1.9 1.9 3.9 5.1 2.6 2.8 1.1 2.9 3.2 0.0 0.0 0.0 0.0 5.5

RI PT

No No No LAS / 1.00 LAS / 1.00 LAS / 1.00 APG / 1.00 APG / 1.00 APG / 1.00 AO / 1.00 AO / 1.00 AO / 1.00 FAE / 1.00 FAE / 1.00 FAE / 1.00 No No No No No

SC

13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 3 3 9.6 9.6 13

M AN U

25 35 45 25 35 45 25 35 45 25 35 45 25 35 45 25 45 25 45 40

De (%) SD (%)

TE D

T3-01 T3-02 T3-03 T3-04 T3-05 T3-06 T3-07 T3-08 T3-09 T3-10 T3-11 T3-12 T3-13 T3-14 T3-15 T3-16 T3-17 T3-18 T3-19 T3-20

pH Surfactant / Concentration (g/L)

EP

T (ºC)

AC C

Serie

ACCEPTED MANUSCRIPT

Table 4. Enzyme concentration (g/L)

40

No

0.06

No

8.2

1.1

T4-02

30

60

No

0.03

No

15.3

1.3

T4-03

30

60

No

0.06

No

23.3

2.4

T4-04

30

60

No

0.15

No

31.4

2.2

T4-05

30

60

No

1.00

No

88.5

2.7

T4-06

30

60

No

0.06

1.00

20.9

3.2

T4-07

45

40

No

0.06

No

12.3

0.2

T4-08

45

60

No

0.03

No

27.1

1.9

T4-09

45

60

No

0.06

No

36.8

2.2

T4-10

45

60

No

0.15

No

49.9

0.2

T4-11

45

60

No

1.00

No

97.0

4.7

T4-12

45

60

No

0.06

1.00

39.3

0.5

T4-13

30

40

APG / 1.00

0.06

No

7.1

0.4

T4-14

30

40

FAE / 1.00

0.06

No

7.3

1.3

T4-15

30

60

LAS / 1.00

0.06

No

34.0

1.8

T4-16

30

60

APG / 1.00

0.06

No

23.6

0.8

T4-17

30

60

FAE / 1.00

0.06

No

19.5

1.8

T4-18

45

40

APG / 1.00

0.06

No

12.8

1.0

T4-19

45

40

FAE / 1.00

0.06

No

12.3

0.2

T4-20

45

60

LAS / 1.00

0.06

No

40.4

1.6

SC

30

AC C

EP

TE D

M AN U

T4-01

MP concentration (g/L) De (%) SD (%)

RI PT

Serie Time (min) T (ºC) Surfactant / Concentration (g/L)

ACCEPTED MANUSCRIPT

45

60

APG / 1.00

0.06

No

34.5

3.0

T4-22

45

60

FAE / 1.00

0.06

No

31.9

1.5

AC C

EP

TE D

M AN U

SC

RI PT

T4-21

ACCEPTED MANUSCRIPT

Table 5. Enzyme concentration (g/L)

Recycle

MP concentration (g/L) De (%) SD (%)

T5-01

0.01

First washing cycle

0.00

T5-02

0.01

Second washing cycle

0.00

T5-03

0.01

First washing cycle

0.33

T5-04

0.01

Second washing cycle

0.33

T5-05

0.02

First washing cycle

0.00

T5-06

0.02

Second washing cycle

0.00

T5-07

0.02

Third washing cycle

0.00

T5-08

0.02

First washing cycle

T5-09

0.02

Second washing cycle

T5-10

0.02

Third washing cycle

23.7

0.0

22.9

0.0

11.5

0.1

33.1

0.0

10.9

0.0

3.7

0.0

0.66

31.5

2.2

0.66

16.1

0.7

0.66

8.2

0.0

M AN U

SC

8.3

TE D EP AC C

1.2

RI PT

Serie

ACCEPTED MANUSCRIPT −( −

2−

2) − b)

R = carbon chain length (C12-C14) (70% C12, 30% C14) n=9.9 d)

R = carbon chain length C12

AC C

EP

TE D

M AN U

SC

c) R = carbon chain length (C10-C13) (16.6% C10, 32.6% C11, 28.9% C12, 21.9% C13)

R = carbon chain length (C8-C14) DP = degree of polymerization

RI PT

a)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

1

Highlights.

2

The cleaning of starch adhering to stainless-steel surfaces was analyzed

3

Influence of ozone, enzyme, temperature or microparticles on detergency was studied

4

In the absence of enzyme, maximum detergency is obtained with fatty alcohol and ozone

5

Highest detergency is reached with highest enzyme concentrations and temperatures

6

Enzymes and microparticles allows the recovery of detersive activity

AC C

EP

TE D

M AN U

SC

RI PT

7

1