Effect of drying temperature on the antioxidant capacity of a cathodic

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Effect of drying temperature on the antioxidant capacity of a cathodic electro-activated whey permeate Amrane Djouab, and Mohammed Aider ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05962 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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ACS Sustainable Chemistry & Engineering

Effect of drying temperature on the antioxidant capacity of a cathodic electro-activated whey permeate

Amrane Djouab a, Mohammed Aïder* a,b

a) Department of Soil Sciences and Agri-Food Engineering, Université Laval, Quebec, Qc, G1V 0A6, Canada b) Institute of Nutrition and Functional Foods (INAF), Université Laval, Quebec, Qc, G1V 0A6, Canada

* Corresponding author: Université Laval, Pavilion P. Comtois, Department of Soil Sciences and Agri-Food Engineering, 2255 Rue de l’Agriculture, Quebec, Qc, Canada, G1V0A6. Tel: (418) 656-2131 Ext: 409051 E-mail: [email protected]

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ABSTRACT The aim of the present work was to study the antioxidant capacity of spry dried whey permeate (WP) that was subjected to a cathodic electro-activation. In this context, electroactivation technology was applied to WP in view to in situ convert a part of lactose into lactulose (prebiotic) with a simultaneous inducing of Maillard reactions products (MRPs) which are known to have high antioxidant capacity. The antioxidant activity (AA) of the electro-activated and dried whey permeate (EAWP) was evaluated by the DPPH scavenging activity, reducing power, ABTS•+ Radical scavenging assay and iron chelating capacity. The effect of the drying temperature on the AA of the EAWP was also evaluated. The obtained data demonstrated that electro-activation significantly (p < 0.001) enhanced the AA of WP and that this AA was mainly due to the intermediate MRPs, as shown by the highest absorbance at 294 nm. Moreover, the results showed that the drying temperature significantly influenced the AA of EAWP.

Key words: Electro-activation, whey permeate, lactulose; antioxidant activity, Maillard reaction products.


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INTRODUCTION Sweet whey is obtained after curd is separated during the cheese or casein manufacturing, and the production of 1 kg of cheese generates approximately 9 kg of whey which is composed of approximately 94% water, 5% lactose, 0.6% protein, and 0.5 ash. Quantitatively, whey contains almost 50% of all solids present in the used milk with lactose as the major component. Minor components, such as hydrolyzed peptides of κ-casein, free amino acids, residual lipids and bacteria, citric and lactic acids (0.02–0.05%), non-proteinic nitrogen compounds (urea and uric acid), and vitamins of the B group are also found in the whey

1-3.

The global demand for whey

proteins is continuously increasing because of their broad range of functionality, and nutritional value. To recover whey proteins, liquid whey is subjected to membrane processes such as microfiltration and ultrafiltration. Whey proteins are retained by the membrane, whereas water and smaller molecules such as lactose, small peptides, free amino acids and salts cross the membrane. This fraction constitute the whey permeate (WP) which is mainly composed of water (93%), lactose (5%), and minerals (0.53%) with minimal fat (0.36%) and protein (0.85%) content 4. However, while the whole whey or whey protein concentrates/isolates can be easily used in the food industry, the WP has so far been of little economic value and causes serious environmental issues. Actually, WP is mainly commercialized as powder for animal feed 5. To reach the objective of zero waste in the dairy industry, all milk components must be effectively and economically used. In this context adding net value to whey permeate is a perspective way to follow. Indeed, whey permeate contains high amount of lactose that can be valorized by conversion into lactulose which is a prebiotic directly in situ without fractionating of whey permeate. This will allow to generate an ingredient with prebiotic effect that can compete with other prebiotics dominating the market such as inulin, and other non-digestible carbohydrates obtained from natural sources such as polysaccharides following enzymatic and chemical hydrolysis 6. Indeed, recently, Djouab and Aider (2018) demonstrated that lactose can be converted into lactulose directly in situ of whey permeate with a conversion level of 35-40% by using the 3 ACS Paragon Plus Environment


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electro-activation technology 7. The authors showed that the obtained product is rich on lactulose (prebiotic) and presented indices of Maillard reaction products (MRPs) which can exhibit high antioxidant activity. The Maillard reactions, also known as non-enzymatic browning reaction, occur between carbonyl groups of reducing sugars, aldehydes or ketones and amino groups of free amino acids, peptides and proteins. This reaction evolves resulting in a series of complex products depending on the stage of the reaction, usually known as MRPs. Three stages are commonly considered: early, intermediate and final stages. In the early stage, after the formation of glycosylamines, a subsequent rearrangement proceeds into the Amadori compounds. The intermediate stage involves changes that lead to the formation of different products as a result of reactions like cyclation, sugar dehydration, fragmentation, and amino acid degradation. At the final stage, browning color appears due to the formation of brownish polymers known as melanoidins. This reaction is affected by several parameters such as pH, water activity, composition, physical structure of the food system, type of reducing sugars, presence or absence of oxygen, temperature and heating time. Several studies reported that MRPs have many beneficial properties including antimutagenic effect

8-9;

antimicrobial, prebiotic, and antihypertensive activities

10;

antioxidant

capacity such as high reducing power, DPPH radical scavenging and ferrous chelating activities 11-13.

This range of active properties gives them the status of a unique multifunctional ingredient

for eventual used in functional foods. The application of electro-activation technology on defatted sweet whey demonstrated that it induces simultaneous in situ isomerization of lactose into lactulose and the formation of MRPs having high antioxidant activity

14-16.

These authors demonstrated that the induction of MRPs

directly in whey by electro-activation was possible even at 10 °C, which is very important to avoid the adverse effect of heating. Thus, in the present study we hypothesized that the application of the electro-activation technology in a tri-compartmental reactor to the WP will allow inducing the formation of the MRPs that are responsible for the AA of the electro-activated whey permeate (EAWP). In this context, this work is aimed to study the antioxidant activity of EAWP. 4 ACS Paragon Plus Environment

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Specifically, the objectives of the present investigation were: (i) to study the effect of electroactivation and drying temperature (140, 170 and 200 °C) on the antioxidant activity of electroactivated whey permeate (EAWP) and lactose solution, and (ii) to study spectrophotometrically the in situ formation of Maillard reaction products (MRPs) in cheese whey permeate following cathodic electro-activation.



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MATERIALS AND METHODS Chemicals and reagents Sodium sulphate (Na2SO4) and sodium phosphate dibasic (Na2HPO4) were purchased from Anachemia (Montreal, Canada). Potassium chloride (KCl) was from EMD Chemicals Inc. (Gibbstown, NJ, USA). Trichloroacetic acid was from Fisher Chemical (Geel, Belgium). Lactose (≥99% purity), sodium phosphate monobasic (NaHPO4), potassium ferricyanide [K3Fe(CN)6], iron(III) chloride (FeCl3), 2,2-diphenyl-1-picrylhydrazyl (DPPH; >95%), iron(II) chloride (FeCl2), potassium persulfate (K2S2O8; ≥99%), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt (ABTS; ≥99%) were purchased from Sigma-Aldrich (Ontario, Canada). The whey permeate (WP) powder was from Agropur Cooperative (Quebec, Canada). The CMI-7000S cation exchange membrane (CEM) and the AMI-7001S anion exchange membrane (AEM) (Membranes International Inc., Ringwood, NJ, USA) were used directly in the reactor without any pretreatment. In situ generation of MRPs in WP using electro-activation The in situ generation of MRPs in WP was conducted by using an electro-activation reactor as described in our previous work 7. Three configurations were studied by applying a fixed electric current intensity of 330 mA. Na2SO4 and KCl were used as electrolytes in the anodic and cathodic compartments, respectively. The electro-activation treatment was conducted at ambient temperature (21 ± 2 °C). These configurations were used according to our previous work and correspond to the higher level of lactose conversion into lactulose with a minimal side reaction (galactose formation). WP and lactose solutions were used and freshly prepared following overnight mixing for a complete hydration of lactose. The electro-activation reactor was filled with deionised water after each use in view to maintain a high membrane hydration. The detailed configurations are summarized in Figure 1 and are as follow:


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Configuration 1: Anodic (+) compartment filled with Na2SO4 (0.25 M)/Central compartment filled with KCl (0.1 M)/Cathodic (–) compartment filled WP (6%, w/v) + KCl (0.1 M). Cycle duration: 21 min. Configuration 2: Anodic (+) compartment filled with Na2SO4 (0.25M)/Central compartment filled with KCl (0.1 M)/Cathodic (–) compartment filled with lactose (5%, w/v) + KCl (0.1 M). Cycle duration: 14 min. Configuration 3: Anodic (+) compartment filled with Na2SO4 (0.25M)/Central compartment filled with WP (6%, w/v)/Cathodic (–) compartment filed with WP (6%, w/v). Cycle duration: 35 min. Once the solutions of EAWP under Configuration (1 and 3) and EALac under the Configuration 2 were obtained, they were stored at 4 °C in plastic bottles before drying. Spry and freeze drying procedure of the electro-activated whey permeate The spry drying of the solutions obtained from the Configuration 1, 2 and 3 were performed by using a BÜCHI mini spray dryer (model B-290, Flawil, Switzerland) with a 1.5 mm diameter spray nozzle orifice. The selected drying air temperatures were 140, 170 and 200 °C while the feed rate was set at 10 mL/min. These values were determined from preliminary tests, and the drying air flow rate was maintained according to the recommendation chart of the manufacturer. The powders coming out of the cyclone were recovered in a glass container connected to the extremity of the cyclone. After electro-activation, the solutions were immediately pre-frozen at -20 C to avoid any further modification. These samples were considered as control versus the spry dried samples. The freeze dried powders from the solutions of EAWP (Configuration 1 and 3), EALac (Configuration 2) were obtained by using a freeze-dryer Reep Vertis 2 freeze-dryer (Sp Scientific, Gardiner, NY, USA). To carry out freeze drying, the already pre-frozen electro-activated samples at -20 C were



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further frozen at -40 °C in a stainless steel trays during 48 h. After that, they were freeze dried until residual humidity of approximately 3%. Antioxidant capacity measurement For the determination of the antioxidant capacity, the freeze-dried powders and those obtained by spry drying at 140, 170 and 200 °C were reconstituted in a form of aqueous solutions according to their respective initial concentrations as follows: the EAWP powder (EAWPP) from Configuration 1 and 3 was reconstituted at a concentration of 6 % (w/v) and the EALac powder (EALacP) from Configuration 2 was reconstituted at a concentration of 5% (w/v). The obtained aqueous solutions from these powders were used for the antioxidant analyses. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity The DPPH radical scavenging activity of the EAWPP and EALacP was evaluated by using the method described by Kareb et al. (2017) as follows: aliquot of each sample (250 µL) was added to 1 mL of 0.1 mM DPPH solution prepared the same day in methanol. The reaction mixture was then vortexed and kept in the dark at ambient temperature (20 ± 2 °C) for 30 min. The absorbance was measured in a 96-well microplate by using Epoch-microplate spectrophotometer (BioTek Instruments, Inc. Winooski, VT, USA) at 517 nm. The control consisted of a mixture of methanol and DPPH solution without adding samples

17.

The percentage of DPPH radical scavenging

activity (RSA) in percentage (%) was calculated by using the following formula (Eq. 1): 𝑅𝑆𝐴(%) =

𝐴𝑏𝑠c ― 𝐴𝑏𝑠s 𝐴𝑏𝑠c

× 100

(Eq. 1)

Where Absc is the absorbance of control while Abss is the absorbance of sample. Reducing power The reducing power of the EAWPP and EALactP were determined according to the method used by Kareb et al. (2017). One mL of each sample was mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide in a screw cap tube. The mixture was incubated in an air convection oven at 50 °C for 20 min followed by cooling at ambient temperature (20 ± 2 °C). Afterward, 2.5 ml of 10% trichloroacetic acid (w/v) were added 8 ACS Paragon Plus Environment

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and the mixture was centrifuged (Beckman Coulter, Miami, FL, USA) at 5000 ×g for 10 min at 20 °C. The upper layer (2.5 mL) was mixed with 2.5 mL deionised water and 1 ml of 0.1% of ferric chloride (FeCl3). The control consists of all the reactants except that sample was substituted with deionised water. The absorbance was measured after 10 min of incubation at 700 nm in a 96-well microplate using the Epoch-microplate spectrophotometer (BioTek Instruments, Inc. Winooski, VT, USA). A higher absorbance indicates a higher reducing power. The assays were carried out in triplicate and the results are expressed as mean values ± standard deviations 14. ABTS•+ Radical scavenging effect The antioxidant activity of the EAWPP and EA-LacP reconstituted solutions samples in the reaction with stable ABTS•+ radical cation was determined according to the method of Re et al. (1999) as follows: The ABTS•+ radical cation was pregenerated by reacting 7 mM of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt (ABTS) with 2.45 mM potassium persulfate (K2S2O8) in equal quantities and incubating for 12–16 h in the dark at ambient temperature (20 ± 2 °C) until the reaction was complete and the absorbance was stable. The absorbance of the ABTS•+ solution was equilibrated to 0.700 (± 0.02) by diluting with ethanol at ambient temperature. Then 1 ml of ABTS•+ was mixed with 10 μl of the test samples and the absorbance was measured at 734 nm after 6 min of reaction time. 18. All experiments were repeated three times. The ABTS•+ radical scavenging effect was estimated as percentage of inhibition (Eq. 2). 𝐴𝐵𝑇𝑆• + 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) =

𝐴734 𝑛𝑚c ― 𝐴734 𝑛𝑚𝐴s 𝐴734 𝑛𝑚c

× 100

(Eq. 2)

Where A734 nmc is the absorbance of control while A734 nms is the absorbance of sample. Iron chelating capacity The chelating capacity of Fe2+ of the EAWP powders was evaluated using the procedure used by Kareb et al. (2017). Briefly, 250 L of the reconstituted powders solution of EA-WP configuration 1 and 3, and EA-Lac were beforehand diluted with 2500 L of H2O then 50 L of FeCl2 (2 mM) and 100 µL ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic 9 ACS Paragon Plus Environment


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acid monosodium salt hydrate, 5mM) were added and vortexed for 10 seconds. After 10 min of incubation at ambient temperature (20 °C), the mixture was centrifuged at 5000 ×g for 5 min. After that 300 L of each sample was transferred to a 96-well microplate and the absorbance (A) was read at 562 nm using an Epoch-microplate spectrophotometer (BioTek Instruments, Inc. Winooski, VT, USA). The control consists on the mixture of all the reagents with deionised water in place of the sample. 14. The results were expressed as chelating ability in (%) using the below equation (Eq. 3): Fe2+ chelating capacity (%) = [(A562 nmcontrol – A562nmsample)/ A562nmcontrol)] × 100

(Eq. 3)

Intermediate and final MRPs measurement The uncolored intermediate compounds which are the important precursors of Maillard reaction and absorb in the UV region were measured at 294 nm

19,

while the brown polymers

developed in the more advanced stages of the Maillard reaction were measured at a wavelength of 420 nm 20. The optical density was evaluated on a 300 µL of each sample by using the Epochmicroplate UV–Visible spectrophotometer (BioTek Instruments, Inc. Winooski, VT, USA). Statistical analysis Statistical significance of the compared data was tested by one-way analysis of variance (ANOVA) by using SAS software (V9.3, SAS Institute Inc., Cary, NC, USA) at p < 0.05 level significance.


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RESULTS AND DISCUSSION DPPH scavenging activity In the present study, electro-activation of the whey permeate (WP) was conducted in the cathodic compartment of the electro-activation reactor where reducing conditions were created following water molecules electrolysis at the solution/cathode interface. Thus, it has been expected that the electro-activated whey permeate (EAWP) will exhibit an antioxidant activity that can be confirmed by the DPPH test. Indeed, stable radical DPPH• is used for the determination of primary antioxidant activity, expressed as the free radical scavenging activities 21. DPPH• radical activity is used to evaluate the ability of a given compound to act as free radical scavenger or hydrogen donor, and to evaluate its overall antioxidant capacity 22. It is based on the reduction of alcoholic DPPH solutions measured at 517 nm in the presence of a hydrogen donating antioxidant (AH) due to the formation of the non-radical form DPPH–H by the following reaction (Eq. 4): DPPH• + AH

DPPH–H + A•

(Eq. 4)

The free radical A• reacts with another molecule, produced by a parallel reaction (Eq. 5): A• + A•

A–A

(Eq. 5)

The Figs. 2a-b represents the percentage of inhibition of the free radical DPPH• by the different EAWP and EALac reconstituted powders from freshly EAWP and after 48 h storage, respectively. The initial WP solution has a DPPH inhibition activity of 11.79 % while that of lactose solution showed a significantly (p < 0.001) lower activity of 3.82%. This difference can be explained by the free amino acids, peptides and reducing sugars present in WP that acted synergistically or cumulatively as hydrogen donors. As depicted in the Figure 2a, the DPPH radical scavenging effect of the EAWP-1-Solution, EALac-2-Solution and EAWP-3-Solution were improved by 4.63, 10.09 and 4.79% in comparison to their initial solutions, respectively. After drying (Figure 2a), it appears that drying temperature influenced positively and significantly (p ˂ 0.001) the antioxidant activity of the samples whatever the type of the used reactor configuration. The temperature of 200 °C gives the highest AA while 140 °C the lowest. 11 ACS Paragon Plus Environment


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Furthermore, in the Configuration-1 no significant difference between the drying temperatures of 140 and 170 °C was observed while in the two other configurations (2 and 3) the difference between the three applied temperatures was significant. In the Configuration-1 (Figure 2a) the AA of WP was enhanced by 22.63, 27.87 and 50.68% while in the Configuration-3 by 15.98, 34.04 and 60.06% for the EAWPP-140, 170 and 200 °C samples compared to the initial WP, respectively. As for the Configuration-2, the AA was improved by 30.49, 57.44 and 80.74 % for the EALacP-140, 170 and 200 °C samples, respectively. The effect of storage and drying temperature of EAWP and EALac samples is shown in Figure 2b. The DPPH radical scavenging effect of the 48 h stored EAWP-1-Solution, EALac-2Solution and EAWP-3-Solution were improved by 30.99, 50.08 and 45.12%, respectively. Furthermore, it can be seen that the impact of drying temperature is different depending on the reactor configuration. Indeed, drying temperature enhanced the AA of the obtained powders in the case of the Configuration-2, whereas in the Configuration-3 the AA was stable and no significant difference was observed between the three obtained powders (EAWPP-3 (140, 170 and 200°C)). However, in the Configuration-1 a significant decrease of AA was observed only in the case of EAWPP-140 °C then increased with EAWPP-1-170 and 200 °C. In the Configuration-1 and 48 h storage (Figure2b) the AA was enhanced in comparison to the freshly EA samples by 2.40, 8.89 and 0.11% while in the Configuration-3 by 33.72 and 14.62% for the EAWPP-140 and 170 samples, respectively. In the case of EAWPP-3-200 °C a significant decrease of AA was observed. As for the Configuration-2 the AA of the initial lactose solution was improved by 29.68, 17.46 and 1.36% for the EALac solution, EALacP-140, 170 and 200 °C, respectively. The DPPH radical activity can be due to the presence of the intermediate reductones of the MRPs which are known to break the radical chain by hydrogen atom donating 23. The results we obtained in this study indicated higher AA capacity than those found by Kareb et al. (2017) 14 who found that EA-whey-7% has DPPH scavenging activity ranging from 40 to 45 % and 40 to 50% when 400 and 500 mA current intensity was applied during 15 to 45 min, 12 ACS Paragon Plus Environment

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respectively. This difference can be explained by the high reactivity of low molecular weight peptides and free amino acids with the reducing sugars allowing the easiness of MRPs formation. Furthermore, the amine groups of the free amino acids easily accessible to the reducing sugars than that of whey proteins having complex structure. Regarding the impact of storage on the AA of EAWP and EALac solutions, a significant increase of the AA was observed whatever the type of the reactor configuration. This increase can be explained by the formed MRPs (Table 1) which were favoured by the high solution pH 11-12. The increased AA was a result of the reactivity between the reducing sugars and amino groups which is highly enhanced by the alkaline pH of the medium 19. Also, the open chain form of the sugars and the unprotonated form of the amino group, being the reactive forms, are favored at such pH 24. In contrast, in acidic pH, the highly protonated form of the amino groups is less reactive with the reducing sugars 24. This protonated form is pHdependent (pKa of the amino groups). It was also reported that the AA of peptides was dependent on their molecular weight distributions, and that the small molecular weight peptides have higher potential for MRPs formation

12.

Concerning the mode of drying, the lyophilisation does not

influence de AA of the EA solution whatever the type of configuration. Reducing power (RP) The results of the RP of the EAWP and EALac before and after 48h of storage are summarized in the Figs. 3a-b. WP initial solution has low RP, due to the presence of peptides and free amino acids, whereas lactose initial solution has no RP. Compared to the initial WP solution, the RP of the freshly electro-activated WP solution increased by 4.03 and 7.03 fold for the EAWP1 and EAWP-2 Solutions, respectively, while in the EALac-2-Solution the increase was of 243.75 fold. After drying at 140 and 170 °C (Figure3a), the RP decreased for the three reactor configurations whereas at 200 °C the RP significantly (p < 0.001) increased, except for EALacP200 °C which RP was stable. Furthermore, for each reactor configuration, the obtained powders at 200 °C have the highest RP and that of the EAWPP-configuration-3 was enhanced by 9.21 fold. After 48 h storage of the EAWP solution (Figure3b), no significant difference of the RP was 13 ACS Paragon Plus Environment


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observed between the EAWP-Solutions obtained under Configurations 1, 3 and EALac-Solution from Configuration-2 compared to the freshly EA solutions (Figure 3a). This result demonstrates that storage did not affect the RP of the EA-Solutions. The application of the drying temperature on the EAWP-1-Solution at 140 and 170 °C decreased the RP value while at 200 °C a significant increase of the RP was observed. In the Configuration-2, the drying enhanced significantly (p < 0.01) the RP whereas in the Configuration-3 the temperature of drying did not influence the RP. The results obtained suggest that the Configuration-3 yielded the highest RP values. This can be explained by the longer electro-activation duration and the corresponding higher Redox potential of the solutions. In the lyophilized samples no significant difference (p ˂ 0.05) was observed in the AA in comparison to the EA solutions whatever the type of configuration. The results we obtained in this study indicated higher RP values than those found by Kareb et al. (2017) who found that the RP of EA-whey have optical density ranging from 0.320 and 0.800 when 400 mA of current intensity was applied during 15 and 45 min, respectively 14. This indicates that EAWP possess higher RP (antioxidant activity) than EA-whey even if lower current intensity and short electro-activation time were applied. The observed high RP values indicate that high amount of MRPs was formed that could act as electron donors, a fact that is in good agreement with Yoshimura et al. (1997) who reported that hydroxyl groups of MRPs play an important role in the reducing activity 25. Indeed, RP is indicator of the overall AA potential by which antioxidant molecules are able to donate electrons to deactivate free radicals, reducing them into more stable and unreactive species 26. ABTS•+ radical inhibition The scavenging of ABTS•+ radical is assumed as an electron transfer process, as shown by Eq. 6 as follows 27: ABTS• + + 𝑒 ― →ABTS

(Eq.6)

The results of the ABTS•+ radical assay of the EAWPP and EALacP of freshly EA whey permeate and after 48 h storage are shown in Figs. 4a-b. The WP initial solution has an inhibition 14 ACS Paragon Plus Environment

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activity of 7.87% while lactose initial solution has no activity. This difference is due to the electron donator capacity of the peptides and free amino acids of WP. For freshly EA whey permeate (Figure 4a), the ABTS•+ radical inhibition activity was enhanced by 32.48 and 77.63% by using the reactor Configurations-1 and 3, respectively, while in the case of the Configuration-2 (EALacSolution) the inhibition was increased by 68.27%. When drying is applied to the EAWP-Solution (Configuration-1 and 3) and EALac-Solution (Configuration-2), a significant decrease of radical inhibition activity was observed although the elevation of the drying temperature significantly (p < 0.001) increased the level of the ABTS radical inhibition (Figure 4a). However, only in the case of the EAWPP-3-200°C where a significant increase of the ABTS•+ radical scavenging activity was observed reaching a value of 95.02%. After 48 h storage of the EA whey permeate (Figure 4b), the ABTS•+ radical inhibition activity of EAWP-Solution obtained by the reactor Configuration-1 decreased by 15%, while when drying at 140, 170 and 200°C was applied the difference was not significant between the powders. In contrast, in the Configurations 2 and 3, the ABTS•+ radical inhibition activity was increased. The percentage of inhibition was 11.41, 53.90, 32.99 and 24.67% for the EALac-2-Solution and EALacP-2- 140, 170 and 200 °C, respectively and 6.85, 23.24, 14.12 and 1.06 % for the EAWP3-Solution and EAWP-3-140, 170 and 200 °C, respectively. Globally, the samples from the Configuration-3 showed the best activity with the ABTS assay. The Configuration-2 showed intermediate AA capacity expressed by the ABTS assay, which could be due by the caramelization products generated at high pH. Indeed, during non-enzymatic browning of foods, various degradation products are formed via caramelization of carbohydrates without involving amine groups

19, 28,

and that caramelization reactions contribute to overall browning under high pH

conditions 29. Furthermore, the products from caramelization process are reported to exhibit high AA, such as acetone extract from glucose caramelization which prevents soybean oil oxidation 2930.

Regarding the mode of drying, lyophilized samples don’t indicate any significant difference

with the EA solutions whatever the type of configuration. 15 ACS Paragon Plus Environment


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Iron chelating capacity Ferrous ion chelating activity of the EAWPP and EALacP of freshly and after 48 h electroactivation is shown in Figs. 5a-b. The WP initial solution exhibited iron-chelating power of 39.82%, whereas lactose initial solution did not show activity. This difference is due to the presence of free amino acids and peptides in WP. Indeed, the ability of peptides and some amino acids such as tyrosine, methionine, histidine, lysine, and tryptophan to inhibit lipid oxidation by their chelating pro-oxidative ions was reported 31. Once electro-activation was applied to WP and lactose solutions (Figure 5a), the chelating power was significantly (p < 0.001) enhanced whatever the reactor configuration used, and for each solution type, no significant difference between the EA solutions was observed. This enhance rate is of 51.26, 91.44 and 49.78 % for the EAWP-1-Solution, EALac-2-Solution and EAWP-3Solution. When drying was applied, this iron chelating activity was less influenced by the drying temperature in the Configurations-1 and 3, while in the Configuration-2 this activity significantly (p < 0.001) decreased to disappear when drying temperature of 200 °C was applied. This can be explained by the fact that at this temperature, the high degree of caramelization induced the destruction of molecules responsible of the chelating effect (Configuration-2). In the Configuration-1, the chelating power of initial WP was enhanced by 51.04, 49.75 and 41.77% whereas in the Configuration-3 by 46.77, 39.19 and 38.11 for the EAWPP-140, 170 and 200 °C samples compared to the initial WP, respectively. As for the Configuration-2, the chelating power was improved by 68.09, and 14.89% for the EALac- 140, 170 samples, respectively. However, no change was observed for the EALac-200 °C. The storage at 4 °C (Figure 5b) significantly (p < 0.001) decreased the iron chelating power of the EALac from Configuration-2 and EAWP from Configuration-3 solutions with values of 69.88 and 14.26%, respectively, while it was stable in the case of the EAWP from Configuration-1. When drying was applied, only EALac from Configuration-2 was affected with a value of 51.27 and 10.30% for the EALacP-2-140 and EALacP-2-170 °C samples, respectively. The mode of drying, lyophilisation versus spry drying 16 ACS Paragon Plus Environment

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indicate that lyophilisation was the mode that preserve the entire AA significantly in comparison to the EA solutions whatever the type of configuration. MRPs have been found to be effective as iron-chelating compounds and the chelating activity can be attributed to the hydroxyl or pyrrole groups 25. Furthermore, Zhuang and Sun (2011) demonstrated that low molecular weight peptide fractions of MRPs have effective metal-chelating ability in lysine-glucose model system and this property may explain some of the antioxidant mechanisms of MRPs. Our results are in agreement with those found by Kareb et al. (2017) on the EA-Whey 14. By this chelation process, MRPs of EAWP can operate as a secondary or preventive antioxidants; effectively inhibiting oxidation without directly interacting with oxidative species. Indeed, iron ions can cause lipid peroxidation that can produce free radicals and peroxides. Hence, metal chelating activity indicates antioxidant and antiradical activity 26. Also, the ability to chelate ferrous ions is an indication whether MRPs existing in the EAWPPs contain potential secondary antioxidants. Intermediate and final MRPs measurement The absorbance at 294 and 420 nm corresponding to the intermediate and final MRPs are given in Table 1. The obtained results demonstrated that electro-activation of WP and lactose solutions induced the formation of the intermediate and final MRPs. For the freshly EA solutions, the absorbance of the EA-solutions at 294 nm are classified as follows: EAWP-SolutionsConfiguration-3˃ EAWP-Solutions-Configuration-1˃ EALac-Solution-Configuration-2. Once dried, a significant decrease of the absorbance was observed except in the case of EAWP-3-200°C with a stable absorbance. Although the drying temperature decreased the absorbance, the samples obtained at 200 °C revealed the presence of high amounts of intermediate MRPs. After 48 h storage, even though a small decrease of intermediate MRPs was observed in the EAWP-1 and EALac-2 solutions, the general tendency showed an increasing of MRPs with the EAWPConfiguration-3 which have the highest value. This result is in concordance with the AA in the test of reducing power, ABTS radical assay and ferrous chelating activity where EAWP17 ACS Paragon Plus Environment


Page 18 of 33

Configuration-3 gave the highest AA. In the Configuration-2 with lactose (absence of amino groups), caramelization phenomena occurred and was enhanced (catalyzed) by the alkaline conditions

32

with a formation of different products similar to those resulting from the Maillard

reaction. Indeed, caramelization of carbohydrates starts with the opening of the hemiacetal ring followed by enolization, which proceeds via acid and base-catalyzed mechanisms leading to the formation of isomeric carbohydrates. In acid media, low amounts of isomeric carbohydrates are formed, whereas dehydration is favored, leading to furaldehyde formation, such as 5hydroxymethyl-2-furaldehyde from hexoses and 2-furaldehyde from pentoses. In alkaline media, dehydration reactions are slower than in neutral or acid media, but fragmentation products such as acetol, acetoin, and diacetyl are formed. All of these compounds react to produce brown polymers and flavouring compounds (Olano and Martínez-Castro 2004). Thus, these particularities can be used to measure Maillard reaction intensity through the development of brown color detected at 420 nm 33. Just after electro-activation (Table 1), the brown color intensity of the EA-solutions at 420 nm is classified as follows: EAWP-Solution-Configuration-3˃ EAWP-SolutionConfiguration-1˃ EALac-Solution-Configuration-2. This classification is in concordance with those of the intermediate MRPs estimated at 294 nm indicating that the evolution of the Maillard reaction was in the same direction. After drying, significant decrease of the absorbance was observed in all cases. At the end of the storage, while a small diminution of the absorbance of the EA-solutions was observed, a significant increase of the absorbance at 420 nm corresponding to the final MRPs was noticed after spry drying. The Configuration-3 gave the highest results in terms of the colored products which were in concordance with the dark color of the product in comparison with the Configurations-1 and 2. Indeed, pH of the medium significantly affect the Maillard reaction. In alkaline medium, Schiff-base is easily formed and promote the Maillard reaction with a quick formation of brown compounds. The Maillard reaction produced in amino acid/sugars model system has been known to be associated with the formation of compounds with pronounced antioxidant activity

25.

Moreover, pH strongly influences amino acids protonation 18

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which is essential to initiate the condensation step of the Maillard reaction which is increased in alkaline pHs, and higher pH favours the reductone formation over furfural production from the Amadori products, leading to colour development positively correlated with the antioxidant activity

34. 20.

Generally, brown colour development is It is also interesting to underline that the

intermediate MRPs in the studied samples are more presents than that of final products which are subsequently the responsible of the AA. This finding is very interesting because the intermediate MRPs produced during electro-activation are harmless to human health. Finally, in the studied EAWP, the reducing sugars lactulose, lactose and galactose are present together with different low molecular weight peptides and free amino acids, and the Maillard reaction was thus favored at pH˃7.

CONCLUSION In this study, the antioxidant activity (AA) of electro-activated whey permeate (EAWP) was studied in freshly electro-activated and after 48 h storage of EAWP. The effect of drying temperature on the AA was also studied. The results showed that the antioxidant capacity was significantly improved after the application of electro-activation. Drying temperature also affected the AA capacity and was dependent of the reactor configuration. It can be also concluded that intermediate MRPs are the main formed molecules following the reaction between the reducing sugars and amino groups present in the WP. These MRPs are responsible of the enhanced AA of the EAWP which are known to be nontoxic for humans. Furthermore, the storage of the EA-Solution during 48 h enhanced the AA which was positively correlated with the formation of absorbing (uncoloured) products at 294 nm. Globally, EAWP-Configuration-3 showed the highest AA. Finally, the enriched by lactulose EAWP can be used as food ingredient with double function combining prebiotic effect and antioxidant activity. ACKNOWLEDGEMENTS 19 ACS Paragon Plus Environment


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The authors would like to thank Diane Gagnon for her valuable help in carrying out this research. The financial support of Fonds de recherche du Québec – Nature et Technologie (FRQNT) is acknowledged.


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(11) Kim, J.-S.; Lee, Y.-S., Antioxidant activity of Maillard reaction products derived from aqueous glucose/glycine, diglycine, and triglycine model systems as a function of heating time. Food Chem. 2009, 116 (1), 227-232, DOI 10.1016/j.foodchem.2009.02.038. (12) Yu, M.; He, S.; Tang, M.; Zhang, Z.; Zhu, Y.; Sun, H., Antioxidant activity and sensory characteristics of Maillard reaction products derived from different peptide fractions of soybean meal hydrolysate. Food Chem. 2018, 243, 249-257, DOI 10.1016/j.foodchem.2017.09.139. (13) Lertittikul, W.; Benjakul, S.; Tanaka, M., Characteristics and antioxidative activity of Maillard reaction products from a porcine plasma protein–glucose model system as influenced by pH. Food Chem. 2007, 100 (2), 669-677, DOI 10.1016/j.foodchem.2005.09.085. (14) Kareb, O.; Gomaa, A. I.; Champagne, C. P.; Jean, J.; Aïder, M., Impact of electro-activation on antioxidant properties of defatted whey. Int. Dairy J. 2017, 65, 28-37, DOI 10.1016/j.idairyj.2016.09.009. (15) Kareb, O.; Gomaa, A. I.; Champagne, C. P.; Jean, J.; Aïder, M., Electro-activation of sweet defatted whey: Impact on the induced Maillard reaction products and bioactive peptides. Food Chem. 2017, 221, 590-598, DOI 10.1016/j.foodchem.2016.11.134. (16) Kareb, O.; Champagne, C. P.; Aïder, M., Contribution to the production of lactulose-rich whey by in situ electro-isomerization of lactose and effect on whey proteins after electro-activation as confirmed by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Dairy Sci. 2016, 99 (4), 2552-2570, DOI 10.3168/jds.2015-10037. (17) Kareb, O.; Gomaa, A. I.; Champagne, C. P.; Jean, J.; Aïder, M., Impact of electro-activation on antioxidant properties of defatted whey. Int. Dairy J. 2017, 65, 28-37, DOI 10.1016/j.idairyj.2016.09.009. (18) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C., Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26 (9), 1231-1237, DOI 10.1016/S0891-5849(98)00315-3. 22 ACS Paragon Plus Environment

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(28) Ajandouz, E. H.; Puigserver, A., Nonenzymatic browning reaction of essential amino acids: effect of pH on caramelization and Maillard reaction kinetics. J Agric Food Chem 1999, 47 (5), 1786-93, DOI 10.1021/jf980928z. (29) Benjakul, S.; Visessanguan, W.; Phongkanpai, V.; Tanaka, M., Antioxidative activity of caramelisation products and their preventive effect on lipid oxidation in fish mince. Food Chem. 2005, 90 (1), 231-239, DOI 10.1016/j.foodchem.2004.03.045. (30) Rhee, C.; Kim, D. H., Antioxidant Activity of Acetone Extracts Obtained From a Caramelization-Type Browning Reaction. J. Food Sci. 1975, 40 (3), 460-462, DOI 10.1111/j.1365-2621.1975.tb12504.x. (31) Peñta-Ramos, E. A.; Xiong, Y. L., Antioxidant Activity of Soy Protein Hydrolysates in a Liposomal System. J. Food Sci. 2002, 67 (8), 2952-2956, DOI 10.1111/j.13652621.2002.tb08844.x. (32) Namiki, M., Chemistry of Maillard Reactions: Recent Studies on the Browning Reaction Mechanism and the Development of Antioxidants and Mutagens. In Adv. Food Res., Chichester, C. O.; Schweigert, B. S., Eds. Academic Press: 1988, 32, 115-184. (33) Kim, J.-S.; Lee, Y.-S., Study of Maillard reaction products derived from aqueous model systems with different peptide chain lengths. Food Chem. 2009, 116 (4), 846-853, DOI 10.1016/j.foodchem.2009.03.033. (34) Bates, L.; Ames, J. M.; MacDougall, D. B.; Taylor, P. C., Laboratory Reaction Cell to Model Maillard Color Development in a Starch-Glucose-Lysine System. J. Food Sci. 2006, 63 (6), 991996, DOI 10.1111/j.1365-2621.1998.tb15840.x.


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Table 1: Absorbance of the intermediate and final Maillard reaction products (MRPs) at before (420 nm) and after storage (294 nm). Before storage

After storage

Before storage After storage

294 nm

294 nm

420 nm

420 nm

WP-6%-Solution

0.001 ± 0.000

0.001 ± 0.000

0.001 ± 0.000

0.026 ± 0.000

Lac-5%- Solution

0.001 ± 0.001

0.001 ± 0.001

0.001 ± 0.001

0.006 ± 0.001

EAWP-1- Solution

0.293 ± 0.004

0.132 ± 0.001

3.398 ± 0.021

3.177 ± 0.032

EAWP-1-Lyophilised

0.290 ± 0.006

0.133 ± 0.003

3.400 ± 0.014

3.177 ± 0.020

EAWPP-1- 140 °C

0.090 ± 0.001

0.085 ± 0.002

1.854 ± 0.004

2.507 ± 0.028

EAWPP-1- 170 °C

0.072 ± 0.001

0.090 ± 0.001

1.666 ± 0.020

2.451 ± 0.030

EAWPP-1- 200 °C

0.118 ± 0.001

0.109 ± 0.001

2.075 ± 0.013

2.499 ± 0.096

EALac-2- Solution

0.196 ± 0.025

0.195 ± 0.003

2.869 ± 0.060

2.413 ± 0.015

EAWP-2-Lyophilised

0.194 ± 0.003

0.197 ± 0.004

2.861 ± 0.009

2.411 ± 0.005

EALacP-2- 140 °C

0.064 ± 0.001

0.212 ± 0.002

0.657 ± 0.004

2.283 ± 0.017

EALacP-2- 170 °C

0.128 ± 0.002

0.198 ± 0.002

0.963 ± 0.014

2.174 ± 0.016

EALacP-2- 200 °C

0.109 ± 0.002

0.169 ± 0.001

1.642 ± 0.010

2.323 ± 0.011

EAWP-3- Solution

0.544 ± 0.009

0.446 ± 0.006

3.732 ± 0.002

3.712 ± 0.001

EAWP-3-Lyophilised

0.542 ± 0.005

0.444 ± 0.004

3.729 ± 0.004

3.710 ± 0.002

EAWPP-3- 140 °C

0.295 ± 0.004

0.424 ± 0.008

3.369 ± 0.029

3.695 ± 0.008

EAWPP-3- 170 °C

0.281 ± 0.004

0.455 ± 0.022

3.211 ± 0.042

3.707 ± 0.039

EAWPP-3- 200 °C

0.338 ± 0.004

0.423 ± 0.001

3.686 ± 0.006

3.706 ± 0.002

Product type

*WP-6%-Solution and Lac-5%- Solution are used as control samples.



Page 26 of 33

Figures captions Figure 1. Conceptual schematic representation of the used three compartmental electro-activation reactor. AEM: anion-exchange membrane; CEM, cation exchange membrane. Figure 2. DPPH scavenging activity of EAWPP (configuration 2 and 3) and EALacP (configuration 2) as influenced by temperature during spry drying. WP: whey permeate, EAWPP: electro-activated whey permeate powder, EALacP: electro-activated lactose powder. Error bars correspond to the standard deviation (n=3): (a) freshly electro-activated samples (without storage), and (b) after 48 h storage. Figure 3. Reducing power of EAWPP (configuration 2 and 3) and EALacP (configuration 2) samples without storage as influenced by temperature during spry drying. WP: whey permeate, EAWPP: electro-activated whey permeate powder, EALacP: electro-activated lactose powder. Error bars correspond to the standard deviation (n=3): (a) freshly electro-activated samples (without storage), and (b) after 48 h storage. Figure 4. ABTS•+ radical assay of EAWPP (configuration 2 and 3) and EALacP (configuration 2)) as influenced by temperature during spry drying. WP: whey permeate, EAWPP: electroactivated whey permeate powder, EALacP: electro-activated lactose powder. Error bars correspond to the standard deviation (n=3): (a) freshly electro-activated samples (without storage), and (b) after 48 h storage. Figure 5. Chelating effect of EAWPP (configuration 2 and 3) and EALacP (configuration 2) as influenced by temperature during spry drying. WP: whey permeate, EAWPP: electro-activated whey permeate powder, EALacP: electro-activated lactose powder. Error bars correspond to the standard deviation (n=3): (a) freshly electro-activated samples (without storage), and (b) after 48 h storage. Figure 6. Schematic representation of the induced Maillard reaction products (MRPs) following a cathodic electro-activation of whey permeate.


Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

O2

AEM

CEM

Cl– Anode

H+ + H+ H

H2 2

OH-

OHOH-

K+

Cathode

Configuration I Configuration II Configuration III

Na2SO4 (0.25 M)

KCl (0.25 M)

WP (6 %) or Lactose (5%) in KCl (0.1 M)

WP (6 %) in KCl (0.1 M)


P-

W

ct 6% -S os eo 5% lut io EA EA W So n W P-1 luti on PP -1 Sol u EA Lyo tion p W P hil EA P-1 ized W -1 40 P EA P-1 °C W -1 70 P ° EA P-1 -2 C EA La 0 La c-2- 0 °C c2- solu tio EA Ly La oph n i c EA P-2 lize d La cP 140 EA -2 ° La - 1 C 70 cP °C EA -2EA W 20 P 0 W °C PP -3s -3 - L olut EA yo ion p W PP hiliz EA -3 ed W -1 40 P EA P-3 °C W -1 7 PP 0 ° -3 -2 C 00 °C

La

W

Pct 6%os So e5% lut io E EA AW So n W P-1 luti on PP -1 Sol u EA Lyo tion p W PP hiliz EA -1 ed W - 14 P 0 EA P-1 °C W 17 P 0 EA P-1 °C EA La - 2 La c-2 00 ° cP C -2 Sol -L ut EA io y La oph n i c EA P-2 lize La - 1 d 4 c EA P-2 0 °C La 17 c 0 EA P-2 °C EA W - 2 W P-3 00 ° PP C -3 Sol -L ut EA yo ion p W P hil EA P-3 ized W - 14 P 0 EA P-3 °C W 17 PP 0 ° -3 -2 C 00 °C La

DPPH scavenging effect (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DPPH scavenging effect (%)


100 90

90

80


Page 28 of 33

Figure 2

a

80

70

60

50

40

30

20

10

0

100

b

70

60

50

40

30

20

10

0

28

W P6

% ct os - S o eEA 5% lutio EA WP So n lu P W t PP 1- S ion -1 ol u EA Lyo tion p W P hil EA P-1 ized W - 14 P 0 °C EA P-1 W 17 P 0 °C EA P-1 EA La 20 0 c La °C cP -2so -2 l ut EA Ly io o La ph n i c EA P-2 lize La - 1 d 4 c EA P-2 0 °C La 17 c EA P-2 0 ° C EA WP - 2 0 P 0 W °C PP -3so -3 l u EA Lyo tion p W P hil EA P-3 ized W - 14 P 0 °C EA P-3 W 17 PP 0 ° -3 -2 C 00 °C

La

W

Pct 6% -S os eo 5% lut io EA EA W S o n W P-1 luti on PP -1 Sol u EA Lyo tion p W P hil EA P-1 ized W -1 40 P EA P-1 °C W -1 7 P EA P-1 0 °C EA La - 2 La c-2 00 °C cP -2 Sol ut EA Ly io La oph n i c EA P-2 lize d La cP 140 EA -2 ° La - 1 C 7 c EA P-2 0 °C EA W 2 W P-3 00 °C PP -3 Sol -L u EA yo tion p W P hil EA P-3 ized W -1 40 P EA P-3 °C W -1 7 PP 0 ° -3 -2 C 00 °C La

Absorbance at 700 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Absorbance at 700 nm

Page 29 of 33 ACS Sustainable Chemistry & Engineering

Figure 3 1.400

1.200

1.400

a

1.000

0.800

0.600

0.400

0.200

0.000

1.200

b

1.000

0.800

0.600

0.400

0.200

0.000

29


La

Pct 6% os -S eol 5 u EA %- tion S EA W o W P-1 luti on PP -1 Sol u EA Lyo tion p W PP hili EA -1 zed W -1 4 P EA P-1 0 °C W -1 70 P °C EA P-1 EA La - 20 c La 0 ° cP -2so C -2 l EA Ly utio La oph n c i EA P-2 lize La - 1 d 4 c EA P-2 0 ° C La cP 170 °C EA -2EA W 20 W P-3 0 ° C PP -3 solu EA Lyo tion p W PP hili EA -3 zed W -1 4 P EA P-3 0 °C W -1 PP 70 ° -3 -2 C 00 °C

W Pct 6% os -S eo 5% lut io EA EA W So n W P-1 luti on PP -1 Sol u EA Lyo tion p W P hil EA P-1 ized W -1 4 P EA P-1 0 °C W -1 7 P EA P-1 0 °C EA La 2 La c-2 00 cP - S °C -2 ol EA Ly utio La oph n c i EA P-2 lize d La cP 140 EA -2 °C EA Lac 17 W P-2 0 ° C PP 20 -3 EA Lyo 0 °C p W P- hili ze EA 3W So d lu P EA P-3 tion W 14 P EA P-3 0 °C W -1 PP 70 ° -3 -2 C 00 °C La

W

(%) of ABTS•+ inhibition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

% of ABTS•+ inhibition


90

100

90


Page 30 of 33

Figure 4

100

a

80

70

60

50

40

30

20

10

0

b

80

70

60

50

40

30

20

10

0

30

Chelating effect (%) Pct 6% os e -S EA - 5% olu EA W - S tion W PP- olu PP 1- tio -1 So n EA - Ly luti W oph on EA PP- iliz W 1- 1 ed EA PP- 40 W 1- °C EA PP 170 EA La -1- °C La cP- 20 cP 2- 0 ° C -2 S EA - L olut La yop ion EA cP- hili La 2- zed EA cP- 140 °C L 2EA acP 170 °C EA W -2W PP- 20 PP 3- 0 ° -3 So C EA - Ly luti W oph on EA PP- iliz W 3- 1 ed EA PP- 40 W 3- 1 °C PP 70 -3 - 2 °C 00 °C La

W

Chelating effect (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

W La Pct 6% os e- - So EA 5% luti EA W - S on W P- olu PP 1- tio -1 So n EA - Ly luti W op on h EA PP- ilis 1 W - 1 ed EA PP- 40 W 1- 1 °C EA PP- 70 EA L 1- °C La ac- 20 cP 2- 0 ° C -2 S EA - L olut y La op ion EA cP- hili La 2- sed EA cP- 140 ° La 21 C EA cP-2 70 °C EA W W P- 20 PP 3- 0 ° -3 So C EA - Ly luti W op on h EA PP- ilis 3 W - 1 ed EA PP- 40 W 3- 1 °C PP 70 -3 - 2 °C 00 °C

Page 31 of 33 ACS Sustainable Chemistry & Engineering

Figure 5

100 90 80 70 60 50 40 30 20 10 0

90

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100

b

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Figure 6


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For Table of Contents Use Only

Electro-activation technology supports sustainable development by offering a possibility to exclude or significantly reduce the use of chemical alkalis and acids.


Effect of drying temperature on the antioxidant capacity of a cathodic

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