4 Effects of ascorbic acid and light on reactions in fresh-cut apples

unibz Freie Universität Bozen Libera Università di Balzano Università Liedia de Bulsan Faculty of Science and Technology PhD in Mountain Environment and Agriculture (29th cycle)

PhD Dissertation REACTION CALORIMETRY AS A TOOL FOR STUDYING THE QUALITY OF FRESH-CUT FRUITS AND THE EFFICACY OF VARIOUS PRESERVATION TREATMENTS

PhD Coordinator: Prof. Tonon Giustino

Supervisor: Prof. Matteo Scampicchio

Co-supervisor: Prof. Aberto Schiraldi

Co-supervisor: Prof.ssa Tanja Mimmo

Candidate: Hasan S. M. Kamrul

Year 2017

unibz Freie Universität Bozen Libera Università di Balzano Università Liedia de Bulsan Faculty of Science and Technology PhD in Mountain Environment and Agriculture (29th cycle)

PhD Dissertation REACTION CALORIMETRY AS A TOOL FOR STUDYING THE QUALITY OF FRESH-CUT FRUITS AND THE EFFICACY OF VARIOUS PRESERVATION TREATMENTS

PhD Coordinator: Prof. Tonon Giustino

Supervisor: Prof. Matteo Scampicchio

Co-supervisor: Prof. Aberto Schiraldi

Co-supervisor: Prof.ssa Tanja Mimmo

Candidate: Hasan S. M. Kamrul

Year 2017

i

Table of Contents Page Table of contents............................................................................................................ i Summary ....................................................................................................................... iv Riassunto ...................................................................................................................... v Zusammenfassung ...................................................................................................... vi

Chapter I ......................................................................................................................... 1 1 State of the art ......................................................................................................... 1

1.1 Introduction ........................................................................................................ 2 1.2 Calorimetric process analyser ............................................................................ 4

1.2.1 A reaction calorimeter .................................................................................. 6 1.2.2 A microcalorimeter ....................................................................................... 8

1.3 Application of different innovative treatments ................................................... 10 1.3.1 UV-C light .................................................................................................. 10 1.3.2 Pulsed light ................................................................................................ 13 1.3.3 Emulsion technology ................................................................................. 15

1.4 Traditional dipping treatment ............................................................................ 17 1.5 Research objectives ......................................................................................... 18 1.6 Research hypotheses ...................................................................................... 19 1.7 References ....................................................................................................... 20

Chapter II ...................................................................................................................... 28 2 Nanoemulsion as nano-carrier for fresh-cut fruits: a review ............................ 28

2.1 Introduction ...................................................................................................... 30 2.2 Potential functional compounds to be carried by nanoemulsion systems ........ 34

2.2.1 Antimicrobial agents .................................................................................. 34 2.2.2 Antioxidant agents/Anti-browning agents................................................... 38 2.2.3 Texture enhancers ..................................................................................... 39

2.3 Nanoemulsion formulation ............................................................................... 41 2.4 Nanoemulsion characterization ........................................................................ 46 2.5 Conclusion ....................................................................................................... 52 2.6 References ....................................................................................................... 54

Chapter III ..................................................................................................................... 65 3 Food and Ascorbic Scavengers of Hydrogen Peroxide .................................... 65

3.1 Introduction ...................................................................................................... 67

ii

3.2 Materials and methods ..................................................................................... 68 3.2.1 Materials .................................................................................................... 68 3.2.2 Reaction calorimetry apparatus . ............................................................... 69 3.2.3 Optimized procedure ................................................................................. 70

3.3 Results ............................................................................................................. 70 3.3.1 Solutions of Ascorbic Acid ......................................................................... 70 3.3.2 Scavenging Properties of Some Food Products. ....................................... 72

3.4 Discussion ........................................................................................................ 74 3.5 Conclusions...................................................................................................... 84 3.6 References ....................................................................................................... 86

Chapter IV .................................................................................................................... 88 4 Effects of Ascorbic Acid and Light on Reactions in Fresh-cut Apples by Microcalorimetry ......................................................................................................... 88

4.1 Introduction ...................................................................................................... 90 4.2 Methods ........................................................................................................... 91

4.2.1 Fresh cut apple samples............................................................................ 91 4.2.2 Dipping treatments .................................................................................... 92 4.2.3 Pulsed light treatments .............................................................................. 92 4.2.4 UV-C treatments ........................................................................................ 92 4.2.5 Isothermal microcalorimetry ....................................................................... 93 4.2.6 Oxygen measurement ............................................................................... 93

4.3 Results and Discussions .................................................................................. 94 4.3.1 Calorimetric signal ..................................................................................... 94 4.3.2 Oxygen consumption ................................................................................. 96 4.3.3 Fitting of the experimental points ............................................................... 97 4.3.4 Effect of Ascorbic acid ............................................................................... 98 4.3.5 Application of light treatments .................................................................. 100

4.4 Conclusion ..................................................................................................... 102 4.5 Acknowledgments .......................................................................................... 102 4.6 References ..................................................................................................... 103

Chapter V ................................................................................................................... 106 5 Free-radical Scavenging Capacity using Fenton reaction by Reaction Calorimetry ................................................................................................................ 106

5.1 Introduction .................................................................................................... 108 5.2 Materials and methods ................................................................................... 109

iii

5.2.1 Materials .................................................................................................. 109 5.2.2 Reaction calorimetry apparatus ............................................................... 110 5.2.3 Optimized procedure ............................................................................... 111

5.3 Results and discussions ................................................................................. 111 5.3.1 Reaction calorimetry of the Fenton reaction ............................................ 111 5.3.2 pH effect on the rate of the Fenton Reaction ........................................... 117 5.3.3 Iron (Fe2+) effect on the rate of the Fenton Reaction .............................. 119 5.3.4 Hydrogen peroxide (H2O2) effect on the rate of the Fenton Reaction ...... 120 5.3.5 Free-radical scavenging activity of antioxidant compounds ..................... 121 5.3.6 Free-radical scavenging capacity of food products .................................. 123

5.4 Conclusion ..................................................................................................... 125 5.5 References ..................................................................................................... 127

Chapter VI .................................................................................................................. 131 6 Conclusions and Future Prospects .................................................................. 131

6.1 General conclusion ........................................................................................ 132 6.2 Future prospects ............................................................................................ 135

Acknowledgement..................................................................................................... 136

iv

Summary

This research was aimed to investigate the quality changes of fresh-cut fruits and the

efficacy of various preservation treatments by calorimetric process analyser. First, the

study was focus on the development of a novel method based on reaction calorimetry

for monitoring the oxidation reaction in foods. The instrument measures the heat flow

signal (W) released during the reaction. Such heat flow as well as its integral yields the

heat (J) of the reaction. The overall heat was used as index to express the antioxidant

capacity of the food samples. The oxidation reaction was investigated between food

containing antioxidants and an oxidant reagent (i.e. hydrogen peroxide). The results

suggested to use of reaction calorimetry to investigate the antioxidant capacity of fruit

juices, fruit puree, tea, coffee and alcoholic beverages, like wines without time

consuming sample pre-treatment protocol. The reliability of the approach is assessed

through the study of the reaction between hydrogen peroxide and ascorbic acid at

different concentrations and pH at 250C. The second aim was focused to evaluate the

efficiency of traditional and innovative preservative treatments on fresh-cut fruits using

novel calorimetric approach for monitoring the reaction. Fresh-cut apples (Malus

domestica cv. Golden Delicious) were subjected to different stabilization treatments,

such as dipping with ascorbic acid solutions (traditional treatment), exposure to UV-C

and pulsed light (innovative treatments). The rate of reaction of treated fresh-cut apples

was investigated with microcalorimetry. The apple slices treated with ascorbic acid,

pulsed light or UV-C treatments showed decrease in the heat flow than control, which

confirm the reduction of fruit reaction. The heat flow signal was proportional to the

concentration of ascorbic acid or pulsed light dose used, but was not linearly

proportional to the fluence of the UV-C treatment. The findings of this study suggest

that innovative treatments based on the irradiance of light were able to preserve and

enhance the stability of fresh-cut apples, and also suggest the suitability of calorimetry

to determine the stability of fresh-cut fruits.

Moreover, to accelerate the oxidation reaction in foods, reaction calorimetry method

was extended using Fenton type reaction for its industrial application. The method was

performed on same samples like previous application of this method, and the results

were promising, which made ten times faster of the oxidation of antioxidant compounds

(i.e. ascorbic acid) and food samples.

v

Riassunto Questa ricerca ha avuto lo scopo di indagare i cambiamenti qualitativi della frutta fresca tagliata e l'efficacia di vari trattamenti di conservazione mediante l’analisi calorimetrica. Il primo obiettivo della ricerca consisteva nello sviluppo di un nuovo metodo basato sulla reazione calorimetrica per il monitoraggio della reazione di ossidazione negli alimenti. Lo strumento misura il segnale del flusso di calore (W) prodotto durante la reazione, cosi come il suo integrale, ossia il calore (J) emesso durante la reazione. Il calore totale è stato usato come indice per esprimere la capacità antiossidante dei vari campioni di alimenti. La reazione di ossidazione è stata studiata negli alimenti contenenti antiossidanti a cui sono stati aggiunti agenti con azione ossidante (ad esempio l’acqua ossigenata). I risultati suggeriscono di utilizzare la reazione calorimetrica per indagare la capacità antiossidante dei succhi di frutta, purea di frutta, tè, caffè e bevande alcoliche come il vino, senza la necessità di avere un protocollo di pre-trattamento del campione. L’affidabilità di questo approccio è stata valutata mediante lo studio della reazione tra l’acqua ossigenata e l’acido ascorbico a varie concentrazioni e diversi pH alla temperatura di 25°C. Il secondo obiettivo è stato orientato a valutare l’efficienza dei trattamenti di conservazione tradizionali ed innovativi sulla frutta fresca tagliata usando un approccio nuovo come quello della calorimetria per il monitoraggio della reazione. Le mele tagliate (Malus domestica cv. Golden Delicious) sono state soggette a diversi trattamenti di stabilizzazione, quali l’immersione nell’acido ascorbico (trattamento tradizionale), e l’esposizione ai raggi UV-C e la luce pulsata (trattamento innovativo). La velocità di reazione della mela tagliata e trattata è stata misurata con la microcalorimentria. Le fette di mela trattate con acido ascorbico, UV-C o luce pulsata hanno mostrato un flusso di calore minore rispetto al controllo, ciò conferma la riduzione della reazione della frutta. Il segnale di flusso di calore è stato proporzionale alla concentrazione dell’acido ascorbico e alla luce pulsata ma non in modo lineare rispetto al trattamento con UV-C. I risultati di questa ricerca suggeriscono che l’uso dei trattamenti innovativi basati sull’irradiazione con luce sono in grado di preservare la stabilità della mela fresca tagliata; inoltre suggeriscono l’adeguatezza nell’uso della calorimetria nella determinazione della stabilità della frutta fresca tagliata. Inoltre, per accelerare la reazione d’ossidazione negli alimenti, la reazione calorimetrica è stata applicata alla reazione di “Fenton”, usata nell’industria. Il metodo è stato eseguito applicando il metodo descritto precedentemente ed i risultati sono stati incoraggianti, in quanto la reazione di ossidazione dei composti antiossidanti (come l’acido ascorbico) e dei campioni alimentari è stata 10 volte più veloce.

vi

Zusammenfassung

Das Ziel dieser Studie war die kalorimetrische Untersuchung von

Qualitätsveränderungen von frischen geschnittenen Früchten und die Wirksamkeit von

verschiedener Konservierungsbehandlungen.

Die erste Zielsetzung dieser Studie war die Entwicklung einer neuartigen

reaktionskalorimetrischen Methode für die Überwachung die Oxidationsreaktion in

Lebensmitteln. Der Kalorimeter misst dabei das Signal des Wärmestroms (W), der

während der Reaktion befreit wird. Das Instrument nicht nur den Wärmestrom, aber

auch dessen Integral, die Reaktionswärme (J). Die Gesamtwärme wurde als Index für

das antioxidierende Fähigkeit der Lebensmittelproben herangezogen. Die

Oxidationsreaktion wurde zwischen Lebensmitteln, die Antioxidantien enthaltenen und

einem Oxidierungsmittel, wie z.B. Wasserstoffperoxid untergesucht. Die Ergebnisse

deuten auf die Anwendung der Reaktionskalorimetrie für die Untersuchung der

antioxidierenden Fähigkeit von Fruchtsäften, Fruchtmus, Tee, Kaffee und alkoholische

Getränke wie Wein, ohne zeitintensivem Vorbehandlungsprotokoll. Die Verlässlichkeit

dieser Herangehensweise wurde geprüft, indem die Reaktion von Wasserstoffperoxid

mit Ascorbinsäure in verschiedenen Konzentrationen und bei unterschiedlichem pH, bei

25°C.

Die zweite Zielsetzung war neuartige und weiterentwickelte

Konservierungsbehandlungen mit Kalorimetrie zu bewerten. Frisch geschnittene Äpfel

(Malus domestica cv. Golden Delicious) wurden an unterschiedlichen

Stabilisierungsbehandlungen untergezogen, wie Eintauchen in Ascorbinsäure-Lösung

(traditionelle Behandlung), sowie Belichtung mit UV-C und gepulstes Licht (innovative

Behandlungen). Die Reaktionsrate den Apfelzuschnitten wurde mit Mikrokalorimetrie

untergesucht. Apfelzuschnitte, behandelt mit UV-C, gepulstem Licht und Ascorbinsäure,

zeigen eine Abnahme an Wärmestrom im Unterschied zur Negativkontrolle. Dies

bestätigt die Reduktion der Obstes Reaktion. Der Wärmestrom war proportional zur

Konzentration der Ascorbinsäure und den Dosis des gepulsten Lichtes. Der

Wärmestrom war jedoch nicht linear proportional zur Einwirkung mit UV-C Licht. Die

Ergebnisse dieser Studie zeigen, dass innovative Obstbehandlungen auf der Grundlage

von Lichtbestrahlung die Konservierung und Stabilität von Schnittobst erhöhen.

Außerdem konnte die Eignung von kalorimetrischen Methoden zur Untersuchung der

Stabilität von frischem Schnittobst bestätigt werden.

vii

Um die Oxidierungsreaktion in Lebensmitteln zu beschleunigen, wurde die

reaktionskalorimetrische Methode um die Fenton-Reaktion für dessen industrielle

Anwendung erweitert. Die Methode wurde an dieselben Proben wie die vorherige

Methode angewandt. Die Ergebnisse waren vielversprechend, da die Oxidierung von

Antioxidantien (z.B. Ascorbinsäure) und Lebensmittelproben um das Zehnfache

beschleunigt wurde.

viii

Organization and Structure of the thesis

Chapter 1 discusses the general information of fresh-cut fruits, production and

significance together with the review of their quality evaluation methods and processing

treatments. It also focuses on the formulation of research objectives and hypotheses.

Chapter 2 presents a review on nanoemulsion as nano-carrier for fresh-cut fruits:

formulation, characterization and its application. Chapter 3 discusses about the

development and application of novel method based on reaction calorimetry to

investigate the scavenging capacity of foods. The method was validated with different

concentrations of ascorbic acid and pH. This method was also next applied in chapter 4

to investigate the effects of ascorbic acid and light on reaction in fresh-cut apples.

Chapter 5 discusses the extended application of reaction calorimetry method via Fenton

reaction to monitor the reaction of foods. Finally, chapter 6 summarized the general

conclusions and future prospects of this work.

1 State of the art

1

Chapter I

1 State of the art

1 State of the art

2

1.1 Introduction

International Fresh-cut Produce Association (IFPA) defines fresh-cut products as fruits

or vegetables that have been washed, peeled/trimmed, cut into a fully usable product,

which is subsequently packaged to offer the consumers with high nutritional value,

sensory and convenience, while preserving freshness. Contrarily to such healthy and

quality-oriented definition, in practice, fresh-cut fruits processing operations alter the

integrity of the product, damage the tissues and initiate enzymatic reactions, which

bring negative effects on the quality and shelf life.

Nowadays, consumers demand fresh, convenient, flavoured, and functional fresh-cut

fruits that claim health benefits. The benefits of fruits and vegetables on our diet are

well known. For instance, fruits and vegetables contain a number of functional

compounds, namely, antioxidants, caroteniods, flavonoids, phenols, dietary fibres,

vitamins, minerals and essential oils (Gil et al., 2006). These compounds control

different biological activities, such as improving the immune response, lowering the

incidence of certain type of cancer, heart diseases and degenerative diseases (Kaur

and Kapoor, 2001). Therefore, consumption patterns of fresh-cut fruits are changing

toward a healthy diet owing to an evident relationship to health. The demand of this

natural, fresh, convenient and high quality product has raised the production and

consumption.

The deteriorations reaction occurring in fresh-cut fruits are mainly associated with

cutting operations. Such processing results in surface discoloration, tissue softening,

water loss, aroma and flavour loss, off-flavour development, as well as microbial growth

and spoilage (Oms-Oliu et al., 2010; Cortez-Vega et al., 2008). Each step of processing

(Figure 1.1) may have an effect on the quality of the products. For instance, cutting

outcomes in a fruit that has at least one surface exposed to air. This enhances

oxidation reaction as well as the possible contamination of microbes. Oxidation reaction

leads to the appearance of pink, grey or brown colour within minutes if not treated with

inhibitors (Cacace et al., 2002). In addition, contaminated fresh-cut fruits may cause

safety problems as they are typically consumed without undergoing a thermal treatment

(Badosa et al., 2008). The cutting operations also lead to increase the respiration rate

and ethylene production in fruits (Rojas-Grau et al., 2009), which drive many other

metabolic and physicochemical changes (Cortez-Vega et al., 2008). All these reactions

1 State of the art

3

are responsible for deterioration the quality and shorten the shelf life of fresh-cut fruits

(Kader, 2002; Rojas-Grau et al., 2009).

Figure 1.1: Flow diagram of fresh-cut fruit production

There are number of preservation treatments that are applied on fresh-cut fruits for

improving their quality and shelf life, such as traditional dipping treatments with the

solution of antioxidant and antimicrobial agents, modified atmosphere packaging, and

wax coating. Some of these treatments have been proved effective in improving

particular quality parameters such as texture or colour but not in flavour and/or other

physiological aspects (Pal et. al., 2004; Singh and Pal, 2008). However, in some cases,

these treatments show considerable drawbacks. For instance, extensive use of

chemicals changes the typical fruit taste or irreversibly alters its texture. To overcome

these drawbacks, several innovative solutions have been recently proposed. These

include UV-C light, pulsed light and edible nano-emulsion coating incorporating with

functional compounds (e.g. antioxidants, antimicrobials) treatments. The application of

these preservation treatments on fresh-cut fruits may improve the quality during their

processing and preservation (Gómez-López et al. 2007; Aguiló-Aguayo et al. 2014;

Ignat et al. 2014; Zambrano-Zaragoza et al. 2014).

To evaluate those effects of traditional and innovative processing treatments on fresh-

cut fruits, the rate of fruit respiration, oxidation, and some quality attributes (i.e. colour,

Fruit Selection

1st step: washing and disinfection

Peeling, cutting or shreding

2nd step: washing

Treatment (i.e. dipping)

Packaging

Refrigeration

1 State of the art

4

acidity, pH, soluble solids, texture, water loss, vitamin C, or the sensorial profile) have

been generally assessed. These assessments make the use of destructive

measurements that may require time consuming sample pre-treatment and extraction

protocol, and the application of expensive analytical techniques (i.e. chromatographic

analysis of phenols, vitamins and carotenoids). Instead, method based on calorimetry to

measure oxidation reaction of fruits may overcome such drawbacks. This method

measures the rate of heat production (or heat-flow) of samples contained in the reaction

cell or ampoule, regardless of their physical state (i.e. liquid, solid, viscous, or gas),

non-destructively and without any pre-treatment protocol.

Thus, the overall goal of this research is to measure oxidation reaction in fresh-cut fruits

by a novel calorimetric approach. To achieve this goal, first task is to develop a simple

method based on reaction calorimetry to monitor the oxidation reaction in foods. The

second task is to evaluate the efficiency of traditional and innovative preservation

treatments on fresh-cut fruits by applying novel calorimetric approach.

1.2 Calorimetric process analyser

Calorimetry is a method to measure the heat effect of a process, which can be physical,

chemical or microbiological changes. Calorimeter is an instrument based on heat

sensors that is able to measure the heat flow generated during an exothermic or

endothermic process. Probably, the first calorimeter was developed and used in 1782-

1783 by Lavoisier and Laplace to determine the heat produced during chemical

changes. This was the ‘ice-calorimeter’, in which a significant amount of ice was used at

constant temperature to melt down (Lavoisier and Laplace, 1780). Nowadays, several

types of calorimeter have evolved such as adiabatic, constant-volume, constant-

pressure, differential scanning calorimeter, isothermal calorimeter, isothermal titration

calorimetry (van Herwaarden, 2000).

One of the main goal of food processing is to minimize those reactions that may cause

spoilage (oxidation, growth of microorganisms, enzymatic activities, concentration of the

reactive compounds) with the intention to extend the shelf-life of foods. Structural and

functional changes take place at the micro-and macromolecular level of food products

during processing, resulting in a modification of their physical, chemical, organoleptic,

1 State of the art

5

and nutritional properties. Food products may have a broad range of structures such as

solid, liquid, mixture of multi-liquid or solid, liquid-solid, liquid-gas and solid-gas. The

combination of complex structures making up complex food materials that make

challenging to characterise the food systems. Several thermal and non-thermal

methods are applied to address such characteristics of foods before and after

processing. Calorimetry method is promising and opens new opportunities in area of

food science to optimize the processing and storage condition by predicting the

physical, chemical and microbiological properties of foods. Probably, the first food-

related calorimetric measurement was studied by Dubrunfaut (1856). Although he did

not have access to a calorimeter of the type we use today, he studied the energy and

heat balance of wine fermentation vat.

Foods or biological samples undergo change even when they are kept at constant

temperature. All changes such as physical, chemical or microbiological in origin may

produce heat, which can be studied with the calorimeter. Furthermore, heat is involved

at each step of food preparation such as mixing, cooking and processing. During such

preparation step, food products undergo different transformation like melting,

crystallization, gelatinization, denaturation, oxidation, etc. All these transformations

occur in a certain range of temperature and are associated with heat changes.

Calorimetric data from these studies can be analysed to evaluate thermodynamic

stability of various phases for a rational design of food product formulations and

process conditions. However, detection and monitoring of small quantities of heat,

especially at the initial of the above-mentioned event, requires to use high sensitive

calorimeter.

For investigating thermal properties of foods, differential scanning calorimetry (DSC) is

being used as a main thermal analysis technique (Schiraldi et al., 1999; Harwalkar and

Ma, 1990; Raemy et al., 2000). However, the small size of the DSC cells make difficult

of in situ peripherals for dosing, sampling, mixing, and stirring. Therefore, fast reaction

following such step of food ingredients is difficult to monitor with this technique. Another

limit of DSC investigations comes with its open pans, the water contained in the sample

evaporates with a large endothermic effect that conceals any other thermal signal.

1 State of the art

6

Innovative instruments like reaction calorimeter CPA 202 and microcalorimeter (TAM

III) allow to overcome such drawbacks. In the following sections, a description of these

two types of high sensitive calorimeter is presented.

1.2.1 A reaction calorimeter

Reaction calorimeter is a calorimeter in which a chemical reaction is initiated within a

closed insulted chamber. Reaction heats are measured and the total heat is obtained

by integrating heatflow versus time curve. There are four main methods for measuring

the heat in reaction calorimeter such as heat flow, heat balance, power compensation

and true heat flow. The chemical process analyzer (CPA 202) is a true heat flow based

reaction calorimeter.

Figure 1.2: From left: Reaction calorimeter CPA 202, cross-section of CPA 202, and

scheme of CPA 202 reactor base.

The reaction calorimeter CPA 202 is originally developed by ChemiSense® (ChemiSens

AB, Lund, Sweden), suppresses uncontrolled temperature differences between the

content of the reactor and its surrounding jacket. This means that all the heat from the

reactor to the thermostat bath flows through the heat flow transducer, which is located

between the bottom of the reactor and a Peltier element. CPA 202 performs rather well

with either solid or liquid samples, without need of calibration, and without requiring any

1 State of the art

7

sample pre-treatment (Nilsson and Hess, 2008). The instrument measures the heat flow

signal (W) released during the reaction. Such heat flow as well as its integral yields the

heat of the reaction (J). Figure 1.2 reported the schematic illustration of reaction

calorimeter CPA 202.

The measuring chamber (reactor cell) of the instrument consists of a very sensitive heat

flow sensor and peltier element. The main sensor is installed between the reactor base

and the Peltier element. A Peltier element is used as pump to transfer heat from the

reactor to the surrounding thermostatting liquid. The heat flow that is forced through the

sensor will create a small temperature gradient, and is precisely measured by a True

Heat Flow transducer. The heat flow can be calculated according to following equation:

𝐻𝑒𝑎𝑡 𝑓𝑙𝑜𝑤 = 𝜆×𝐴

𝑑×(𝑇2 − 𝑇1) (1.1)

Where, λ is the specific heat conductivity (W/m.0C), A is the heat transfer area (m2), d is

the thickness of the heat transfer area (m), T2-T1 is the temperature difference (0C). The

values of λ, A and d are well known and constant.

The heat (enthalpy, ΔH) of the overall reaction can be also estimated based on the

integration of the heat flow (φ) during the experiment time t (Wadsö et al., 2004).

mole

dtH

t

t t 0

(1.2)

Finally, with the knowledge of the enthalpy of the process, the heat flow curve provides

a direct estimate of the rate of the process (r) (O’Neill, Beezer, Mitchell, Orchard, &

Connor, 2004):

Hr

(1.3)

The equation (2) and (3) are the connections between a calorimetric measurements

and the corresponding kinetic rate law. For processes of food stuffs and other complex

materials enthalpy change rates are not as easily defined as for simpler reactions, but

measured heat flow and heat can still be used to model kinetics of e.g., degradation

process (Hansen, 2000).

1 State of the art

8

The CPA 202 reaction calorimeter is being used in many different fields of food science.

For instance, monitoring of fermentation, crystallization, esterification, dissolution

processes of foods and ingredients have been successfully analysed by this instrument

(Landau, 1996; Chemisens, 2004; Widell and Karlsson, 2006; Nilsson and Hess, 2008;

Fransson et al., 2014; Varga et al., 2007). Recently, we have used this instrument to

investigate the antioxidant capacity of foods and ingredients (Kamrul et al., 2015).

1.2.2 A microcalorimeter

Microcalorimeter is not microsize instrument but is the measurement of heat in micro-

quantity. The instrument (TAM III) developed by TA Instrument company, New castle,

Delaware, USA. It has four channels consisting six independent minicalorimeters and

able to analyse simultaneously. It’s multichannel microcalorimeter (24 minicalorimeters)

operating in a wide range of temperature from 15 to 1500C.

Figure 1.3: Schematic illustration of a microcalorimeter and its sample holder

1 State of the art

9

In each calorimeter, heat is allowed to flow between the reaction cell or ampoule

containing sample and the heat sink. The instrument consists oil bath thermostat, which

keeps constant temperature through peltier element with the level of accuracy of 0.0001 0C. It is based on a similar approach as described in the previous part of this work and

can be operated in isothermal, step-isothermal and temperature scanning mode.

Briefly, the principle of this method is based on the measurement of the heat flow

generated inside an ampoule containing the sample, where the reaction takes place.

The ampoule was maintained at isothermal condition. Figure 1.3 shows the schematic

of microcalorimeter and its different parts.

Today, microcalorimetry is an emerging tool for the study of various phenomena such

as mixing, dilution, wetting, neutralization, oxidation, and enzyme reaction in the field of

food science and technology. It has been successfully used to monitor the kinetics of

microbial growth in packed fresh-cut carrots (Riva et al., 2001), carrot juice (Alklint,

2003), and meat (Gram and Sogaard, 1985). Furthermore, fermentation of dairy

products (Schaffer et al., 2004), oxidation in plant oils (Dridi et al., 2016), tissue

respiration of vegetables (Wadso et al., 2004; Gomez et al., 2004) among other

foodstuffs. These authors suggested that microcalorimetry could be particularly suitable

to assess the oxidation reaction or respiration of fresh-cut fruits. Fresh-cut fruits are

metabolic active tissues and show physiological responses to environment. As a result

of their metabolism they produce heat. This heat can be directly measured by

calorimeter and used to assess the level of biological activity. Calorimetric

measurements of the rate of heat production have been used to provide a direct

indication of the metabolic responses such as respiration and reaction to provoked

stress by processing or pre-treatments of this food product (Wadso et al., 2004, Wadso

et al., 2009; Rocculi et al., 2012). Nevertheless, non-destructive and non-invasive to the

sample, measures the rate of heat production (or heat-flow) of samples regardless of

their physical state (i.e. liquid, solid or gas) without any previous pre-treatments are the

important motivation of this technique for application on fresh-cut fruits.

1 State of the art

10

1.3 Application of different innovative treatments

1.3.1 UV-C light

Ultraviolet (UV) light is a light or an electromagnetic radiation that covers a broad range

spectrum from visible infrared with wavelength of a meter or more, down to X-rays with

wavelength of less than a billionth of a meter (Figure 1.4). Typically, the wavelength for

UV light process ranges from 100 to 400 nm. This range may be further subdivided into

315-400 nm (UV-A), 280-315 nm (UV-B) and 200 to 280 nm (UV-C). Ultraviolet light

treatment has been shown very effective for disinfection of drinking water and to

decontaminate equipments, devices, packaging materials, and many other surfaces in

the food industries (Bintsis, Litopoulou-Tzanetaki, and Robinson, 2000; Bolton and

Linden, 2003; Koutchma, keller, Chirtel, and Parisi, 2004). Among different types of UV

light (Figure 1.4), UV-C light recognised as powerful non-thermal germicidal method

(Guerrero-Beltrán and Barbosa-Cánovas, 2004). In most studies of the UV-C

destruction or inactivation of microorganisms and enzymes, a low-pressure UV lamp is

used, which emits nearly monochromatic light at 253.7 nm, exhibited highest germicidal

effectiveness (Bolton and Linden, 2003). To meet the growing demand of fresh-cut

fruits or minimally processed fruits and vegetables, such non-thermal food preservation

technique is promising to replace the conventional chemical and thermal sterilization for

microbial reduction processes. The goal of non-thermal food preservation technique is

to eliminate deteriorative factors in foods and food products without affecting the

nutritive value of the product itself. This technology is easy to use and characterised by

favourable costs, energy and maintenances (Barbosa-Canovas, Pothakamury, Palou,

and Swanson, 1998; Bintsis, Litopoulou-Tzanetaki, and Robinson, 2000). In addition, it

does not leave any residues; no formation of toxic or significant non-toxic products

during treatment (Keyser, Muller, Cilliers, Nel, and Gouws, 2008).

The degree to which the destruction or inactivation of microorganisms occurs by UV

radiation is directly related to the UV dose or fluence. UV-C intensity flux or radiation is

usually expressed in W/cm2, and the dose or fluence exposure is expressed as J/cm2

(Bintsis et al., 2000). The UV-C dose (D) is defined as:

D = I253.7*t

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Where, D is the dose (J/cm2), I253.7 is the intensity or dose rate (W/cm2), and t is the

retention time in second (Change et al., 1985; Morgan et al., 1989; Stevens et al.,

1999).

Figure 1.4: Electromagnetic spectrum (Guerrero-Beltrán and Barbosa-Cánovas, 2004)

Generally, UV-C germicidal effect is mainly observed at the nucleic acid level, where,

radiation absorbed by DNA can stop cell growth and lead to cell death (Liltved and

Landfald, 2000). In details, the mechanism for destruction/inactivation involves the

absorption of ultraviolet light by DNA or RNA pyrimidine bases causing a photochemical

reaction in which a chemical dimer is formed between the two bases (Figure 1.5). The

dimer inhibit the formation of new DNA (or RNA) chain in the process of cell replication

(mytosis), thus resulting cell death of microorganisms and enzymes by UV light

(Guerrero-Beltrán and Barbosa-Cánovas, 2004).

Figure 1.5: Left: Effect of UV light on DNA structure

(https://en.wikipedia.org/wiki/Ultraviolet); Right: UV-C chamber (inside photo)

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Food and Drug Administration has already approved UV-C light as a disinfectant

technology for surface treatment of foods (USDA-FDA, 2002). UV-C light is widely used

for eliminating surface microorganisms of meat, poultry and fish as well as sugar

syrups, apple ciders and fruit juices (Choi and Nielsen, 2005; Tran and Farid, 2004;

Stother, 1999; Wallner-Pendleton, Summer, Froning, and Stetson, 1994; Huang and

Toledo, 1982; Nakayama and Shinya, 1981). This technology is recognised as simple

and environmental friendly ways to destroy microorganisms, but not yet free from

drawbacks. For instance, UV-C light technology lies in poor penetration in the food

products, whether liquids or solids. This reason may particularly suitable of UV-C light

application for surface treatment of products where microbial and enzymatic activities

mainly occur. For that reason, the use of UV-C light to extend the shelf life of fresh-cut

fruits and vegetables has been raised interest. Other drawbacks may be oxidation of

lipids, vitamins and flavour compounds due to its extensive application which may result

in formation of unpleasant flavour, colour fading of foods and loss of nutritional value

(Manzocco, Kravina, Calligaris, and Nicoli, 2008; Ramirez, Hough, and Contarini, 2004;

Beckolet, 1990). Contrarily, recent experimental evidences show that under certain

processing conditions, UV-C treated foods tend to maintain their taste, colour and

vitamin profile (Manzocco et al., 2011; Tran and Farid, 2004; Keyser, Muller, Cilliers,

Nel, and Gouws, 2008). The decontamination effects on fresh-cut fruits and vegetables

are not improved by increasing intensity of the UV-C light treatment (Fonseca and

Rushing, 2006; Escalona et al., 2010). Prolong UV-C light exposure may result in

significant browning in fresh-cut apples (Gomez et al., 2010). However, it is well known

that the stability of these properties is often dependent on the inactivation of

microorganisms and enzymes present in fresh-cut fruits. The activity of enzymes and

microorganisms is strongly affected by UV-C light treatment. Therefore, UV-C treatment

is being used as novel non-thermal approach to inactivate or to destroy undesired

enzymes and microorganisms that limits the overall quality depletion compared to

conventional inactivation method (Manzocco et al., 2011; Yong-Gui and Zu, 2012;

Rodoni et al., 2012).

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1.3.2 Pulsed light

Pulsed light (PL) is also a non-thermal technique of food preservation in which intense

and short duration pulses of broad spectrum (white light) is used to inactivate

microorganism in foods (Gomez-Lopez et al., 2007). Dunn et al. (1995) noticed that the

wavelength of PL process ranges from 170 to 1200 nm including ultraviolet (UV), visible

and near infrared light region. The sample to be treated is exposed to at least 1 pulse of

light (typically 1 to 20 pulses per second (s)) with a duration range 1µs to 0.1s (Dunn et

al., 1991); each pulse having an energy density in the range of about 0.01 to 50 J/cm2

at the surface of the sample. In most cases, a few flashes applied in a fraction of a

second provide a high level of microbial inactivation. Pulsed light is an improved form of

UV-C and works with the help of Xenon lamps (Wekhof, 2000). This technology is

characterised using following units:

Fluence rate (W/cm2): It is the energy received from the lamp by the sample per unit

area per second.

Fluence / Dose (J/cm2): It is the energy received from the lamp by the sample per unit

area during treatment.

Peak power (W): It is measured as pulse energy divided by the pulse duration.

Exposure time (s): Necessary time in second during treatment

This method involves the generation of pulsed light with gradually increasing from low

to high energy and then releases the highly concentrated energy as broad spectrum

burst on the surface of foods to ensure microbial decontamination. The electromagnetic

energy gets stored in the capacitor and is then released in the form of light, which

results in power amplification and minimum energy consumption (Green et al., 2005).

The inactivation efficacy of pulsed light depends on fluence (J/cm2) and the number of

pulses delivered. Figure 1.6 shows the schematic pulsed light generation.

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Figure 1.6: Schematic representation of pulsed light treatment plant

The microbial inactivation is characterized by several mechanisms. For instance, pulsed

light induces photochemical, photothermal and photophysical reactions in foods

(Anderson et al., 2000; Krishnamurthy et al., 2007). The UV-rich light causes

photochemical changes, while visual and infrared light cause photothermal changes.

Photothermal effect may causes denaturation of DNA and proteins. Photophysical

effect (caused by high energy pulses) is the vibration of the molecules within the

material or bacteria cell. This effect can cause the rapture of the bacterial membrane.

The lethal effect of pulsed light can be due to photochemical or photothermal or

photophysical mechanism or all may exist simultaneously. However, it is suggested that

the photochemical effect is the main mechanism of the pulsed light treatment on

microbial inactivation. PL can cause structural changes of DNA of microorganisms and

enzymes. Thymine dimmers form within the DNA that can inhibit DNA transcription and

replication in the cell, which can lead to cell death (Miller et al., 1999; Wang et al.,

2005). Figure 1.7 represents the pulsed light treatment plant of fresh-cut fruits.

Figure 1.7: Pulsed light chamber (inside photo)

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Pulsed light is largely used to decontaminate packaging materials (plastic films, bottles,

etc) and appears as a novel food decontamination technology. This technology has

been approved by the FDA for decontamination of foods and food surfaces (FDA,

1996). Yet, food composition affects the efficacy of the treatment, proteins and oils

decreased the decontamination of PL (Charles et al., 2013). For this reason, fruits and

vegetables could be particularly suitable for pulsed light treatment (Elmnasser et al.,

2007). Low penetration on the food surfaces, lack of residual compounds, rapid

inactivation of microorganisms and great flexibility are some of the advantages of this

technology for food application (Elmnasser et al., 2007). Several authors have been

suggested for surface decontamination of fresh-cut fruits and vegetables, ready-to-eat

meat products, as well as for killing microorganisms in fruit juices and infant foods

(Marquenie et al., 2003; Romos-Villarroel et al., 2011; Hierro et al., 2011; Choi et al.,

2011; Caminiti et al., 2011). However, few studies have focused on the effects of pulsed

light on physiology and quality aspects of fresh-cut fruits and vegetables such as Ignat

et al. (2014) on fresh-cut apple, Aguilo-Aguayo et al. (2014) on fresh-cut avocado and

Charles et al. (2013) on fresh-cut mangoes. Dunn et al. (1989) have been

demonstrated the Polyphenol oxidase activity inhibition in treated potatoes by PL. This

can be linked to the reduction of browning of potatoes. Therefore, the use of pulsed

light as an emerging non-thermal technology on fresh-cut fruits could be an alternative

to thermal and chemical methods for rapid and effective inactivation of microorganisms

to extend their shelf life without compromising their quality and sensorial properties as

well as their nutritional value.

1.3.3 Emulsion technology

Emulsion is a colloidal dispersion system in which one liquid is being disperse into

others (Figure 1.8). There are wide application of emulsion technology in

pharmaceuticals and cosmetics industries to deliver nutraceuticals, colour, flavour,

antioxidant and antimicrobial agents (Jiahui Hu, Johnston, & Williams, 2004; Wissing,

Kayser, & Müller, 2004). In the recent years, this technology has attracted interest in

food science to encapsulate functional compounds in food preservation such as

carbonated beverage and salad (Monsalve-Gonzalez & Ochomogo, 2009), fresh fruits

(Kim, Oh, Lee, Song, & Min, 2014; Zambrano-Zaragoza et al., 2013), fresh-cut fruits

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(Zambrano-Zaragoza et al., 2014). Thanks to its high encapsulation efficiency, high

bioavailability, high physical stability and low turbidity advantages (Qian & McClements,

2011). Moreover, entrapping these functional compounds using nanotechnology

approaches can improve their functionality and efficacy. Immobilization of nano-droplets

on the surface of foods through nanoemulsion could be an interesting approach. Small

size droplet may not only enhance the transport of active compounds through biological

membrane but also increases the surface/ volume ratio, which may leads to improve

functionality.

Figure 1.8: Illustration of emulsion formation (http://slideplayer.com/slide/9069003/)

Nanoemulsion is a promising technology as it is able to encapsulate above mentioned

functional compounds as nano-carriers (McClements et al., 2007; McClements, 2010;

Sagalowicz & Leser, 2010; Velikov & Pelan, 2008). Nanoemulsion solution may

improve the quality of fruits and functional properties of coatings when using as coating

of fruits. Nanoemulsion (Figure 1.9) is an emulsion system (mixture of two immiscible

liquids) with the droplet size in ranges from 10 to 100 nm (McClements, 2005).

Nanoemulsion systems act as protective layer or as system for the control release of

functional compounds such as antioxidant, antimicrobial or other additives on fruit

surface (Silva, Cerqueira, & Vicente, 2011). Nanoemulsion may contribute to barrier

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properties to moisture loss, water hydration, gas exchange, suppression physiological

disorder since this system show an increased surface area (Rao & McClements, 2012).

This system results in delay ripening, reduce moisture loss, and to help to maintain

aroma and flavour (Olivas & Barbosa-Cánovas, 2005). This system also shows better

distribution and homogeneity on the fruit’s surface (Rao & McClements, 2012). A

review has been discussed about nanoemulsion in Chapter 2.

Figure 1.9: Schematic of O/W nanoemulsion (Tang and Sivakumar, 2010)

1.4 Traditional dipping treatment

The common way to control enzymatic browning reaction in fresh-cut fruits is dipping of

fruit pieces into an aqueous solution of antibrowning agents. Several types of

antibrowning agents (Table 1.1) are used to control browning of fresh-cut fruits. Some

act directly as inhibitor of polyphenol oxidase (PPO), other by rendering the medium

inadequate for the development of browning reaction. Probably, the most widely used

antibrowning agent is ascorbic acid, which is recognised as a GRAS substance by the

U.S. Food and Drug Administration (FDA) for its use to prevent browning of fresh-cut

fruits. Ascorbic acid reduces the o-quinones back to diphenols, and reduces the PPO

activity (Martinez and Whitaker, 1995; Whitaker, 1994; Golan-Goldhirsh et al., 1992).

Moreover, ascorbic acid may also applied in combination with other organic acids and

calcium salts to prevent enzymatic browning of fruits (Soliva-Fortuny et al., 2001, 2002).

Toivonen and Brummell (2008) studied the effects of ascorbic acid in combination with

calcium on PPO activity and to prevent cell and membrane breakdown in fresh-cut

fruits. Now-a-days, edible coating as carrier of antibrowning agents is being used to

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increase their effectiveness in controlling browning of fresh-cut fruits (Baldwin et al.,

1996; Rojas-Graü et al., 2008; Oms-Oliu et al., 2008). Baldwin et al. (1996)

demonstrated that ascorbic acid was very effective to inhibit browning of fresh-cut

apples when incorporated into edible coating rather than using into an aqueous solution

to treat fruit pieces by dipping.

Table 1.1: Dipping treatment on fresh-cut fruits using different stabilizing agents

Fruit Stabilizing treatment Reference Apple 1% AA Jang and Moon (2011) 2% AA Gil et al. (1998) 7% CaA Fan et al. (2005) 1%AA+0.2%CA or 0.5% NaCl Raybaudi-Massilia et al. (2007) 0.75% AA+0.75%CaCl2 Rocha et al. (1998) 1% AA+0.5%CaCl2 Soliva-Fortuny et al. (2001) 0.5% AA+1%CaCl2+0.1%PA Varela et al. (2007) 0.001M HR+0.5M IAA+0.05

CaP+0.0025M cys Buta et al. (1999)

0.5% AA+0.5%CA in edible coating McHugh and Senesi (2000) 1% AA+1%CaCl2 in edible coating Lee et al. (2003) 1% AA+0.5%CA+0.25% CaCl2 in

edible coating Wong et al. (1994)

1% AA or 0.5% cys Perez-Gago et al. (2006) Melon 2.5 mM AA Lamikanra and Watson (2001) 1% AA+0.5%CaCl2 Oms-Oliu et al. (2007) 2.5% CAL Luna-Guzman and Barrett (2000) Pear 2% AA+0.01% HR+1%CaCl2 Arias et al. (2008) 1% AA+0.5%CaCl2 Soliva-Fortuny et al. (2002c) 2% AA+1%CaCl2+0.5% cys Gorny et al. (2002) 0.5% AA+0.01% HR+1%CaL Dong et al. (2000) AA: Ascorbic acid, CaA: Calcium Ascorbate, CA: Citric acid, NaCl: Sodium chloride,

CaCl2: Calcium chloride, PA: Propionic acid, HR:4-hexylresorcinol, IAA: Isoascorbic

acid, cys: cysteine, CAL: Calcium lactate, CaP: Calcium propionate

1.5 Research objectives

The research objectives of this thesis are:

To develop a novel method based on calorimetric process analyzer for

measuring oxidation reaction in foods.

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To evaluate the efficiency of traditional and innovative preservation treatments

on fresh-cut fruits reaction by novel approach.

1.6 Research hypotheses

The primary question regarding the research work is centred on the development of a

novel method based on reaction calorimetry and its application on foods to assess

oxidation reaction. Secondly, to explore the advantages of traditional and innovative

preservation treatments on fresh-cut fruits to improve their quality and shelf life. Theses

central ideas have the following hypothesis:

Calorimetric performance based on heat released can be applied for the

assessment of oxidation reaction in foods.

How effectively the traditional and innovative treatments influence on the

metabolic activity of fresh-cut fruits?

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2 Nanoemulsion as nano-carrier for fresh-cut fruits: a review

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CHAPTER II



29

Abstract

The increasing consumer’s demand regarding the healthy diet has promoted research

on novel approach for preserving fresh-cut fruits without the necessity of using

preservative. Thanks to emulsion based coating technology for their ability to improve

food quality that has been considered in food preservation. The quality of fresh-cut fruits

is pertinent and determines the consumer acceptance based on the combination of

parameters including appearance, texture, flavour and nutritional value. This review

discusses some recent advances for improving of the quality and safety of fresh-cut

fruits with respect to the use of nanoemulsion as carrier of functional compounds such

as antimicrobial agents, antioxidants and texture enhancers. It focuses especially on

the use of natural functional compounds in food preservation as alternative to chemical

additives. The controlled release of functional compounds from nanoemulsion coating

on fruit surface may overcome their strong flavour and potential toxicity drawbacks in

comparison with conventional coating. The formation and characterization of

nanoemulsion are also reviewed.

Keywords: nanoemulsion, fresh-cut fruit preservation, antioxidants, antimicrobials,

texture enhancers.

This work is under submission to a scientific journal


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2.1 Introduction

The increasing consumer’s demand of fresh-cut fruits is driving the researchers to

pursue for using natural preservative and for rejecting the synthetic or chemical

additives. Thus, food preservation can be enhanced. Moreover, consumption patterns

are changing toward a healthy diet owing to an evident relationship between fresh-cut

fruits and health. Fresh-cut fruits are rich sources of biological active compounds,

micronutrients and nutraceuticals (Vasco, Ruales, & Kamal-Eldin, 2008). These

compounds show a number of biological properties such as antioxidant, anti-genotoxic,

anti-inflammatory, anti-allergic, anticancer and anti-diabetic. As a result, there is a

global trend towards the intake of fresh-cut fruits containing health promoting properties

and their nutritional values. However, the quality of fresh-cut fruits is unwavering based

on physical appearance and freshness characteristics at the time of purchasing by

consumers (Kader, 2002). Fresh-cut fruits processing operations including peeling,

cutting, slicing/shredding alter the integrity of the product, damage the tissues and

initiate enzymatic reactions. These induce adverse effects on the quality such as

surface browning, unexpected flavour, water loss, dehydration and texture breakdown

(Oms-Oliu, Rojas-Graü, González, Varela, Soliva-Fortuny, Hernando, Munuera,

Fiszman & Martín-Belloso, 2010). Furthermore, the existence of microbes on the

surface of fruits may affect the safety of the product as typically fruits are consumed

without undergoing any sanitizing treatments (Badosa, Trias, Parés, Pla, & Montesinos,

2008). Consequently, these issues shorten shelf life of fresh-cut fruits, increasing the

production costs and reducing consumer acceptance.

A number of antimicrobials, antioxidants and texture enhancers from natural sources

may allow reducing or retarding the above mentioned negative effects, improving the

quality with their great performance, and eventually replacing the use of their synthetic

counterparts in fresh-cut fruits (de Oliveira, Ramos, Ramos, Piccoli, & Cristianini, 2015;

Irkin & Esmer, 2015). However, the effectiveness of these functional compounds may

be restricted by their physicochemical properties, stability under certain conditions or

solubility when incorporated to foods. Therefore, there is a need of encapsulating them

into a delivery system, understood as those in which a functional compound is

entrapped into a carrier (Fathi, Mozafari, & Mohebbi, 2012), that allow overcoming the

mentioned issues.


31

Recent scientific findings demonstrate that the emulsion technology (mixture of two

immiscible liquids) can serve as innovative alternative to formulate food products; able

to deliver antioxidants antimicrobial, nutraceuticals, colour, and flavour (Jiahui Hu,

Johnston, & Williams, 2004; Wissing, Kayser, & Müller, 2004). Emulsion may

formulates using vegetable oil (i.e. corn oil, olive oil, sesame oil, rapeseed oil, sunflower

oil, and soybean oil), animal oil (i.e. fish oil), vegetable waxes (i.e. carnauba, candelilla,

and sugar cane wax), animal waxes (i.e. beewax, lanolin, and wool grease), various

essential oils together with emulsifier (lecithin, whey protein isolate, tween 20, 40, 60,

and 80) and water (Acevedo-Fani, Soliva-Fortuny, & Martín-Belloso, 2017; Galus &

Kadzińska, 2015).

Figure 2.1: Schematic representation of coating process of foods

The application of emulsion based coating has been known for centuries in improving

and extending shelf life of fresh-like foods. A thin layer of coating material is directly

layered by dipping or drenching on foods (Figure 2.1); and used as a food wrap without

varying the main constituents or the processing means. Particularly, Coatings have

been considered in food preservation (Chillo, Flores, Mastromatteo, Conte,

Gerschenson, & Del Nobile, 2008; Perez-Gago, Serra, & Río, 2006), for their capability


32

to improve quality and to extend shelf life by controlling and /or preserving physic-

chemical properties (i.e. colour, firmness, respiration, water loss, vapour) of various

food products (Krochta, 2002; Rojas-Graü, Tapia, & Martín-Belloso, 2008). The

formulation of emulsion for coating approach creates a new window to link the

characteristics of lipophilic and hydrophillic active compounds. This enhances

functionality of active compounds of coating on the products. A number of studies on

the incorporation of functional compounds into emulsion based coating as appeared in

recently published journals (Cerqueira, Souza, Teixeira, & Vicente, 2012; Perdones,

Vargas, Atarés, & Chiralt, 2014; Pereda, Amica, & Marcovich, 2012; Perez‐Gago,

Serra, Alonso, Mateos, & Del Rio, 2003).

Among different types of emulsion, nanoemulsion is the most promising approach as it

is able to encapsulate functional compounds as nano-carriers (Chen, Weiss, & Shahidi,

2006; McClements, Decker, & Weiss, 2007; McClements, 2010; Sagalowicz & Leser,

2010; Velikov & Pelan, 2008; Yuan, Gao, Zhao, & Mao, 2008). Nanoemulsion is an

emulsion system in which droplet size ranges from 10 to 100 nm thus it is able to work

at molecular level in foods (Mason, Wilking, Meleson, Chang, & Graves, 2006;

McClements, 2005; Tadros, Izquierdo, Esquena, & Solans, 2004). Thanks to its high

encapsulation efficiency, high bioavailability and physical stability (Qian & McClements,

2011). Due to its fundamental properties, this technology may present several benefits

as carrier of useful compounds over unadventurous emulsions. For instance, reduced

particle size may increase the surface volume ratio which enhances the transport of

active compounds through membranes consequently leading to improve functionality.

On the other hand, nanoemulsions are kinetically stable and transparent colloidal

dispersion which are suitable for a wide range of food applications (Solans, Izquierdo,

Nolla, Azemar, & Garcia-Celma, 2005). Particularly, nanoemulsion may rising-approach

for fresh-cut fruits or fresh-like foods for improving their quality and shelf life when

coated by dipping. Because this technology has ability of serving as reservoirs of active

compounds, protecting them and modulating their controlled release in response to

certain triggers (Acevedo-Fani, Soliva-Fortuny, & Martín-Belloso, 2017; Kuan, Yee-

Fung, Yuen, & Liong, 2012; Rojas-Graü, MA., Soliva-Fortuny, R., and, Martίn-Belloso,

2009).


33

As an example, Kim (Kim et al., 2014) used nanoemulsion as coating formulating with

the addition of an antimicrobial agent (i.e. lemongrass oil) and carnauba wax on

grapeberry to inhibit foodborne pathogens that contaminate grape berry. The result

revealed that nanoemulsion coating on berries inhibited Salmonella typhimurium and

Escherichia coli O175: H7, when inoculated them on the berries by more than 3.2 and

2.6 log cfu/g, respectively. This coating was also effective on reducing the losses of

weight, firmness, phenolic content, antioxidant activity during storage at 4 and 250C for

28 days in the berries. Zambrano-Zaragoza et al., (2013) noticed the reduction of

weight loss and firmness on guava when coated by nanoemulsion based on carnauba

wax and xanthan gum. The inclusion of α-tocopherol in nanoemulsion system exhibited

lowest browning index and preserved firmness of fresh-cut apple during 18 days of

storage than control (Zambrano-Zaragoza, Mercado-Silva, Del Real, Gutiérrez-Cortez,

Cornejo-Villegas, & Quintanar-Guerrero, 2014).

Figure 2.2: The distribution of publication related to “nanoemulsion on food application”

(2000-2016: Science Direct-Elsevier SD Freedom Collection).

However, although these promising results, more intense investigation are needed to

assess the potential of nanoemulsion applied to fresh-cut fruits. Figure 2.2 shows the

distribution of scientific manuscripts published over the past few years on nanoemulsion

0 50 100 150 200 250 300 350 400 450 500

2000

2002

2004

2006

2008

2010

2012

2014

2016

Number of publications

Year


34

in the field of food processing. Among these, just 10 % of the studies are found to be

reported on this technology to improve the quality and prolong the shelf life of fresh-cut

products. Due to these limited studies, more researches are essential for improving the

knowledge showing the potential of this technology for future industrial implementation.

Based on these considerations, the main goal of this review is to update the information

available on the use of nanoemulsion as carrier of functional compounds (antioxidant/

anti-browning, antimicrobial, and texture enhancer) for coating of fresh-cut fruits to

improve their quality and shelf life. In addition, an overview of different methods to

formulate, and characterise the nanoemulsion is presented.

2.2 Potential functional compounds to be carried in nanoemulsion application

In the following paragraphs, the application of nanoemulsion coating as carrier of

functional compounds (antioxidant/ anti-browning, antimicrobial, and texture enhancer)

to improve the quality of fresh-cut fruits are summarised in Table 1 and discussed in.

2.2.1 Antimicrobial agents

Fresh-cut fruits are more susceptible to microbial contamination and proliferation than

intact fruits due to slicing and cutting operations needed to prepare them to be

consumed. Moreover, they are consumed without a lethal microbiological treatment,

thus severe safety problems can be encountered (Badosa, Trias, Parés, Pla, &

Montesinos, 2008). Studies demonstrated that the incidence of foodborne diseases

from fruit sources increased in the last few years (Berger, Sodha, Shaw, Griffin, Pink,

Hand, & Frankel, 2010). Therefore, seeking for innovative techniques to retard

microbial growth and spoilage of fresh-cut fruits is of great interest. Traditionally,

spraying or dipping treatments with the solution of antimicrobial agents is mostly used

to prevent microbial growth and to extend the shelf life of fresh-cut fruits. Food

antimicrobials are chemical compounds that may delay microbial growth or cause

microbial death when they are incorporated into the food matrix (Davidson, Critzer, &

Taylor, 2013). Antimicrobial agents from chemical sources including organic acids

(acetic, lactic, citric, malic, propionic, tartaric, sorbic and their salts), fatty acid esters

(glyceryl monolaurate), polypeptide (lysozyme, peroxidase, lactoferrin, nisin), nitrites

and sulphites are normally used for this purpose (Oms-Oliu, Rojas-Graü, González,


35

Varela, Soliva-Fortuny, Hernando, Munuera, Fiszman & Martín-Belloso, 2010).

However, in the last few years there has been a considerable interest from the

consumers to eat fresh-cut fruits not prepared with chemically synthesised additives.

Therefore, a complex mixture of non-volatile and volatile compounds from plants

sources as essential oils (lemongrass, cinnamon, oregano) has been discovered and

studied as potential alternative to chemical synthetic food additives (Franssen, Krochta,

& Roller, 2003). Researches demonstrate that they can be widely used in foods,

pharmaceuticals and cosmetics industries, thanks to their strong antimicrobials,

antioxidants and flavouring properties (Burt, 2004). However, some issue needed to be

solved for the application of antimicrobial agents on fresh-cut fruits as their diffusion and

adsorption from the surface into the product, which may limit the action as antimicrobial

agents (Min & Krochta, 2005). In this sense, edible coatings and films with the inclusion

of antimicrobial compounds are being used to the inhibitory effects against

microorganisms (Rojas-Graü, Soliva-Fortuny, & Martίn-Belloso, 2009). Many

publications have reviewed about the effectiveness of edible coatings containing

antimicrobial agents. They have been proved as carrier of functional compounds such

as antimicrobials and antioxidants to preserve minimally processed fruits products as

an alternative (Oms-Oliu, Soliva-Fortuny, & Martín-Belloso, 2008; Rojas-Graü, Soliva-

Fortuny, and, Martίn-Belloso, 2009). The antimicrobial action of essential oils may be

attributed to their phenolic compounds and their interaction with microbial cell

membrane. The lipophilic compounds of essential oils may induce the disturbance of

cell membrane, disrupting the proton motive force, electron flow, active transport and

coagulation of cell content, which may cause the cell breakdown (Burt, 2004). However,

the hydrophobicity nature of these compounds make their use problematic in food

formulations, due to their some degree of solubility, lose of activity, toxicity, and impact

on organoleptic food properties when used at high dose as well as economic

considerations (Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer,

2011). Therefore, entrapping these compounds using nanotechnology approaches may

overcome these issues and improve their antimicrobial efficacy. Thus, nanoemulsion

could be an innovative emerging approach to encapsulate, protect and controlled

release of antimicrobial agents, which may increase inhibitory effects against spoilage

and pathogenic microbial growth on the fruit surface (Weiss, Takhistov, & McClements,

2006). Moreover, small droplet size may not only enhance the transport of active

compounds through biological membrane but also increase the surface/ volume ratio,


36

which may lead to improve functionality. The major advantage of nanoemulsion is that

they can be used as carrier of both lipophilic and hydrophilic antimicrobial compounds

(Jiahui, Johnston, & Williams, 2004; McClements, Decker, & Weiss, 2007; Qian &

McClements, 2011).

Several factors need to be taken into account for formulating nanoemulsion as coating

material with the incorporation of antimicrobial agents such as nature of foods, emulsion

components and concentrations, emulsion stability, droplets size, solubility of

antimicrobial compounds, and effectiveness of the antimicrobial compounds into the

coatings (Qian & McClements, 2011). For this reason, fundamental studies must be

carried out to evaluate the effect of an antimicrobial compound incorporated into

nanoemulsion before its application to the real food systems. A high shear rate is

required to generate interface area with nano-sized droplets (Delmas, Piraux, Couffin,

Texier, Vinet, Poulin, Cates, & Bibette, 2011). Microfluidization can produce

nanoemulsion with droplet size ranges from 60 to 600 nm (Hatanaka, Chikamori, Sato,

Uchida, Debari, Onoue, & Yamada, 2010; McClements & Rao, 2011; Qian &

McClements, 2011) and their stability can be improved by incorporating substances

known as stabilizer, emulsifiers or other functional compounds (McClements & Rao,

2011). Several researchers studied the effectiveness of natural antimicrobial

compounds encapsulated in nanoemulsion systems such as oregano oil (Bhargava,

Conti, da Rocha, & Zhang, 2015), essential oils containing lemongrass, clove, tea tree,

thyme, geranium, marjoram, palmarosa, rosewood and mint (Salvia-Trujillo, Rojas-

Graü, Soliva-Fortuny, & Martín-Belloso, 2015a; Ahmad, Benjakul, Prodpran, & Agustini,

2012), tropical fruit by-product extracts (Silva, Hill, Figueiredo, & Gomes, 2014), basil oil

(Ghosh, Mukherjee, & Chandrasekaran, 2013), mandarin essential oils (Severino, Vu,

Donsì, Salmieri, Ferrari, & Lacroix, 2014). In these studies, basil oil, oregano oil,

lemongrass, clove, thyme loaded nanoemulsion showed bactericidal action against

Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus

aureus. These studies proved that nanoemulsion made from natural extracts may

consider as potential carrier of antimicrobial agents for fresh-cut fruits. Salvia-Trujillo et

al. (2015b) studied the effects of nanoemuslion-based edible coatings with 0.5 % and

1% (v/v) concentration of lemongrass essential oil on fresh-cut Fuji apples and found

complete inhibition of the natural microflora during 2 weeks shelf life. Nano-sized

ranges droplets exhibited a faster and greater inactivation of Escherichia coli compared


37

to conventional emulsion (Salvia-Trujillo, Rojas-Graü, Soliva-Fortuny, & Martín-Belloso,

2015b). Nanoemulsion coating was formulated with incorporation of lemongrass oil by

Kim et al. (2014) for coating grape berries, and investigated antimicrobial effects for

improving the microbiological safety and shelf life. These authors observed the

reduction of Salmonella typhimurium and Escherichia coli O157:H7 more than 3.2 and

2.6 log cfu/g respectively during 28 days at 4 and 25°C, whereas no inhibition was

observed on the product coated without the inclusion of lemongrass oil in

nanoemulsion. Jo et al. (2014) reported that nanoemulsion coating containing

lemongrass oil inhibited the population of aerobic bacteria such as Escherichia coli

O157:H7 and Listeria monocytogenes compared to uncoated apples during 5 months of

storage. Furthermore, the population of yeast and molds on the uncoated apples was

2.2 log CFU/g, whereas yeast and molds were not detected on the coated apples. Such

results were also observed by Bhargava et al. (2015) when fresh lettuce was coated

with 0.1 % (v/v) oregano oil based nanoemulsion and by Zambrano-Zaragoza et al.

(2014) for fresh-cut apples coated by nanoemulsion incorporating α-tocopherol.

However, the application of nanoemulsion on fresh-cut fruits is not free from drawbacks.

Due to the reduction of particle size of materials in nanometer scale, may an alteration

of the biological behaviour within the human body can occur, thus changing its potential

for promoting toxicity (Hagens, Oomen, de Jong, Cassee, & Sips, 2007). The

nanoparticle in nanoemulsions can diffuse into mucous layer coating the enterocytes,

which may increase the transit time in the gastrointestinal tract compared to

conventional emulsion (Bouwmeester, Dekkers, Noordam, Hagens, Bulder, De Heer,

Ten Voorde, Wijnhoven, Marvin, and Sips, 2009). An increase of the transit time may

enable more lipid digestion, and greater fraction of the functional compound to be

solubilised by micelles and absorbed by enterocytes (Acosta, 2009). Therefore,

nanoemulsion increases the bioavailability of bioactive compound that may be toxic at

high doses. For many bioactive compounds, an increase in bioavailability may be either

desirable or may have no adverse effect on human health. Some of the components

e.g., solvents, surfactants, and emulsifiers used to prepare nanoemulsions are toxic

when consumed at high doses. Theses organic solvents are usually removed by

evaporation during preparation of the nanoemulsion, but some residual solvent may

remain in the final product. The potential toxicity of certain bioactive compounds,

emulsifiers, surfactants, and solvents that are suitable for utilization on fruits or within


38

foods have been published by different organization such as WHO (www.who.int), FDA

(www.fda.gov) and European Food Safety Authority (www.efsa.europa.eu). These

organisations carry out technical reviews of the toxicity data of different additives and

established safe usages levels and analytical protocols for testing toxicity. However,

further and important researches need to be addressed to assess this aspect.

2.2.2 Antioxidant agents/Anti-browning agents

The crucial quality parameters of fresh-cut fruits are colour and appearance. The

mechanical operations during processing induce undesirable changes of colour

promptly. It is evidenced that the main cause of these undesirable changes is oxidation

phenomenon due to polyphenol oxidase (PPO) enzyme, which, in presence of oxygen,

converts phenolic compounds into dark colour pigments (Zawistowski, Biliaderis, &

Eskin, 1991). The common way to control this undesirable colour or enzymatic

browning is the direct immersion of cut fruits in aqueous solution of antioxidant/ anti-

browning agents. The most extensively used antioxidant is ascorbic acid (McEvily,

Iyengar, & Otwell, 1992) to control enzymatic browning of cut-fruits. Furthermore, thiol-

containing compounds including cysteine, N-acetylcysteine, reduced glutathione

(Gorny, Hess-Pierce, Cifuentes, & Kader, 2002), carboxylic acids such as citric acid and

oxalic acid (Jiang, Pen, & Li, 2004), resorcinol derivatives (Oms‐Oliu, Aguiló‐Aguayo, &

Martín‐Belloso, 2006), phenolic acids (Chen, Wei, & Marshall, 1991) have been also

used as effective antioxidant/anti-browning agents on fresh-cut fruits. However, the

effectiveness of such surface treatment is not free from drawbacks. For instance,

limitations are caused by the loss of the functional compounds, their limited solubility,

limited adsorption and diffusion into the fruit surface, their loss due to oxidation reaction

with oxygen of the environment, etc. when they are directly applied on fruits surface.

Typically, edible coatings have been used as carriers of antioxidant/anti-browning

agents to control enzymatic browning of fresh-cut fruits/minimally processed fruits and

vegetables (Baldwin, Nisperos, Chen, & Hagenmaier, 1996; Oms-Oliu, Soliva-Fortuny,

& Martín-Belloso, 2008; Rojas-Graü, Soliva-Fortuny, and Martίn-Belloso, 2009).

However, the widespread use of edible coatings shows some drawback such as

objectionable flavour and potential toxicity at high dose of functional compounds

(Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer, 2011). Therefore,

the design of new systems in order to reduce their dose to be incorporated in foods is a


39

current challenge. Regards these aspects, immobilization of nano-droplets on the

surface of fresh foods through nanoemulsion could be a promising approach.

Nanoemulsion coating is an effective system for encapsulating of natural antioxidant or

antibrowning compounds such as carotenoids, α-tecopherol in the oil phase (Li, Zheng,

Xiao, & McClements, 2012; McClements et al., 2007; McClements, 2010), which

reduced browning index of fresh-cut fruits compared to the use of antioxidant applied

alone (Perez-Gago, Serra, & Del Rio, 2006). Functionality and bioaccessibility of

encapsulated active compounds in nanoemulsion can increase with decreasing the

droplet size of emulsion (Wang, Liu, Mei, Nakajima, & Yin, 2012). Nano-sized droplets

are weakly scattered light, which gives a transparent appearance (Mason, Wilking,

Meleson, Chang, & Graves, 2006) and allow to use for this type of food products.

Zambrano-Zaragoza et al. (2014) noticed that particle size of the emulsion droplets is

an important parameter to control the browning index of fresh-cut apples. They also

observed nanoemulsion coating containing α-tocopherol was able to reduce the

browning index significantly compared to conventional emulsion. Jo et al. (2014)

reported that carnauba-shellac wax based nanoemulsion containing lemongrass oil

coating was effective to reduce the colour changes of ‘Fuji’ apples during 5 months

storage.

2.2.3 Texture enhancers

A decrease in consumer acceptability of fresh-cut fruits is also related to losses of

texture and cell wall integrity during storage (Salvador, Varela, & Fiszman, 2007). The

texture losses of fresh-cut fruits are due to the enzymatic degradation of cell wall

(Alandes, Hernando, Quiles, Pérez-Munuera, & Lluch, 2006). The surface of fresh-cut

fruits are damaged during mechanical operation, which help to release enzymes, and to

spread them through the tissues to come into contact with their substrate, leading to

loss of texture (Alandes et al., 2006). Currently, fresh-cut fruits are treated with calcium

salts such as calcium chloride (Quiles, Hernando, Pérez‐Munuera, Llorca, Larrea, &

Ángeles Lluch, 2004; Soliva‐Fortuny, Lluch, Quiles, Grigelmo‐Miguel, & Martín‐Belloso,

2003), calcium lactate (Manganaris, Vasilakakis, Diamantidis, & Mignani, 2005),

calcium ascorbate (Fan, Niemera, Mattheis, Zhuang, & Olson, 2005; H. Wang, Feng, &

Luo, 2007), and calcium propionate (Quiles, Hernando, Pérez‐Munuera, & Lluch, 2007)

to control the loss of texture and preserve cell wall structure. Calcium ions may interact


40

with pectin polymers by pectinmethylesterase to form a cross-linked network (insoluble

calcium pectate), which strengthen the structure of the cell wall, thus delays

senescence and physiological disorders of fruits (Alandes et al., 2006; Poovaiah, 1986).

The innovative way to apply calcium salts to fruits is their incorporating into coating

systems for enhancing the beneficial effects on texture retention and minimized

softening of fresh-cut fruits. The incorporation of calcium salts into edible coating may

be helped using cross-linking of carbohydrate polymers such as alginate, gellan or

pectin (Oms-Oliu et al., 2010; Rojas‐Graü et al., 2008; Rojas-Graü, Soliva-Fortuny, &

Martίn-Belloso, 2009). Calcium interacts with cell wall component pectic acids to form a

cross-linked polymer network that increases mechanical strength making molecular

bonding between cell wall constituents (Soliva‐Fortuny, Lluch, Quiles, Grigelmo‐Miguel,

& Martín‐Belloso, 2003). In addition, water losses of fresh-cut fruits are one of the major

problems, which contribute to softening of fruits. Coating treatments also control the

water losses acting as a barrier on the fruit surface, which may decrease water vapour

pressure rate and prevent the textural decay (Olivas & Barbosa-Cánovas, 2005; Oms-

Oliu et al., 2010).

Recently, some studies demonstrated that nanoemulsion may have novel application

for modifying texture of foods and food products due to its very small size of droplets

(McClements, 2011). The nano-sized droplets of nanoemulsion may act in the

molecular level of cell wall of fresh-cut fruits, which give better functionality in

comparison with conventional coatings (Salvia-Trujillo et al., 2015b; Zambrano-

Zaragoza et al., 2013). Moreover, the possibility to modulate nanoemulsion rheological

characteristics incorporating substances known as stabilizers (xantham, pectin,

carrageenan, alginate, sorbitol, glycerol, gelatin, whey protein isolate, esters gum) is an

important feature when applied as texture enhancer for fresh cut fruits (McClements,

2011; McClements & Rao, 2011; McClements, 2011; Qian & McClements, 2011).

There are several applications of nanoemulsion on fresh-cut fruits containing texture

enhancers. As an example, xanthan gum, a carbohydrate based texture enhancer, has

been used to formulate nanoemulsion. Results showed that nanoemulsion enriched

with xanthan gum was effective to retard firmness loss of fresh-cut apples than normal

nanoemulsion (Zambrano-Zaragoza et al., 2014). Eshghi et al. (2013) also found that


41

chitosan based nanoemulsion was effective to prevent the firmness loss of strawberry.

Xanthan gum and carnauba wax based nanoemulsion was able to retain the texture of

guava up to 30 days at 10 °C and 85 % relative humidity (Zambrano-Zaragoza et al.,

2013). The study of Zambrano-Zaragoza et al. (2014) also showed that

polymethylesterase and polyphenoloxidase activity decreased in fresh-cut apples

coated with nanoemulsion enriched with α-tocopherol and nopal mucilage extracts

compared to the control sample. In the same study, it was also demonstrated that

nanoemulsion was able to maintain fresh-cut apples firmness during storage. Similar

results were obtained by Salvia-Trujillo et al. (2015b) and Jo et al. (2014) applying

nanoemulsion enriched in texture enhancers on fresh-cut apples.

2.3 Nanoemulsion formulation

Nanoemulsion is a heterogeneous system (Figure 2.3) consisting of, at least, two

immiscible liquids, one being dispersed into other in small droplets size ranging from 10

to 100 nm (Mason, Wilking, Meleson, Chang, & Graves, 2006; McClements, 2005;

Tadros, Izquierdo, Esquena, & Solans, 2004). A typical nanoemulsion contains oil

phase, aqueous phase and an emulsifier. The addition of an emulsifier is critical for the

creation of small sized droplets as it decreases the interfacial tension i.e., the surface

energy per unit area, between the oil and water phases of the emulsion (Acosta, 2009;

McClements et al., 2007). Aqueous phase is mainly obtained with water, but it may also

be prepared with other polar compounds including co-solvents (simple alcohols and

polyols), carbohydrates, proteins, minerals, acids and bases (McClements, 2011;

McClements & Rao, 2011). Oil phase can be prepared by formulating with various non-

polar component such as triacylglycerols, diacylglycerols, monoacylglycerols, free fatty

acids, flavour oils, essential oils, mineral oils, fat substitutes, waxes, weighting agents,

oil-soluble vitamins, and different lipophilic compounds (e.g. carotenoids, curcumin,

phytosterols) (McClements & Rao, 2011). In order to avoid rapid breakdown of

nanoemulsion due to different mechanisms such as flocculation, coalescence, Ostwald

ripening and gravitational separation, various food grade stabilizers, emulsifier or

surfactants (see Table 1) can be added to this system. Nanoemulsion stability is an

important prerequisite for a more efficient encapsulation of functional compounds

(Grigoriev & Miller, 2009). Therefore, the selection of an appropriate emulsifier is one of

most important aspect for the proper design of nanoemulsion. The adsorption of the


42

emulsifier or surfactants at the interface between droplets and dispersion medium,

reduce the interfacial tension facilitating droplets disruption and protecting droplets

against aggregation (Kralova & Sjöblom, 2009; McClements, 2004). Some studies

demonstrated that the formation and stability of nanoemulsion could often be improved

by using combinations of emulsifiers, rather than using a single emulsifier (McClements

and Rao, 2011).

Figure 2.3: Schematic diagram of nanoemulsion formation using oil phase and aqueous

phase

Nanoemulsion can be produced using two different approaches that can be divided in

high-energy and low-energy methods. High-energy methods apply intense disruptive

forces to break up macroscopic phases or droplets into small droplets, and are able to

intermingle immiscible liquids using mechanical devices as high-pressure valve

homogenizers, microfluidizers, and sonicators (Gutiérrez, González, Maestro, Sole,

Pey, & Nolla, 2008; McClements, 2011; Velikov & Pelan, 2008). On the other hand,

low energy methods depend on the spontaneous formation of small droplets within

mixed oil-water-emulsifier systems when the solution or environmental conditions are

altered as phase inversion and solvent mixing methods (Anton, Benoit, & Saulnier,

2008; Bouchemal, Briançon, Perrier, & Fessi, 2004; Chu, Ichikawa, Kanafusa, &

Nakajima, 2007; Yin, Chu, Kobayashi, & Nakajima, 2009). In Table 1, the most relevant

published studies using different techniques to formulate nanoemulsion incorporating

functional compounds are reported. Moreover, the disperse phase, the type of

emulsifier or surfactant used, the particle size of nanoemulsion and the functional

compounds incorporated are also listed.


43

Table 1. Summary of different functional components used as nano-carriers in nanoemulsion or microemulsion coatings and their

applications.

Technique Materials Functional Ingredient

Benefits Droplet Size (nm)

Applications

References

Ultra-Turrax Disperse phase: Beewax lipid, glycerol

Emulsifier: Whey protein isolate

Bee wax oil Antimicrobial & Antiobrowning

Micro-emulsion

Fresh-cut apple

(Perez‐Gago et al., 2003)


Emulsifier: Whey protein concentrate

Bee wax oil Antimicrobial & Antibrowning

Micro-emulsion

Fresh-cut apple

(Perez-Gago et al., 2006)


Emulsifier: pectin

Bee wax oil and pectin

Antimicrobial & texture

enhancer

Micro-emulsion

Avocado (Maftoonazad et al., 2007)

Ultra-Turrax Disperse phase: Beewax lipid, glycerol, Oleic acid

Emulsifier: Tween 80

Chitosan Antimicrobial & texture

enhancer

Micro-emulsion

strawberry (Velickova et al., 2013)

Ultra-Turrax Disperse phase: Carnauba wax, mineral oil


Lemongrass oil & carnauba

wax


enhancer

56-88 Grape berry (Kim et al., 2014)

Ultra-Turrax Disperse phase: Sunflower oil Emulsifier: Tween 20

Mandarin essential oil

Antimicrobial 176 Green bean (Donsì et al., 2015)

Ultrasound Disperse phase: Basil oil Emulsifier: Tween 80

Oregano oil Antimicrobial 148 Fresh lettuce

(Bhargava et al., 2015)

Ultra-Turrax Disperse phase: Polylene glycol with thymol

Emulsifier: Lecithin

Thymol Antimicrobial 84-558 Milk and Cantaloupe

juice

(Xue et al., 2016)

Ultra-Turrax Disperse phase: Sunflower oil Emulsifier: Span®85/

Tween® 80

α-tocopherol Antioxidant & texture

enhancer

174-240 Fresh-cut apple

(Zambrano-Zaragoza et al., 2014)

Sonication Disperse phase: Sage oil Emulsifier: Span®85/Tween® 80

Sage oil Antimicrobial 200 Food formulation

(Moghimi et al., 2016)

Ultra-Turrax Disperse phase: Cadelilla wax, Cadelilla wax Texture Micro- Guava (Tomás et al., 2005)


44

miniral oil

and Mesquite guminate

enhancer emulsion

Ultra-Turrax Disperse phase: Sunflower oil Eumsifier: Whey protein isolate

Gellan gum, Sodium alginate

Texture enhancer

Micro-emulsion

Fresh-cut apple

(Rojas-Graü et al., 2008)

Ultra-Turrax Disperse phase:Oleic acid Eumsifier: Tween 80

Chitosan Antimicrobila & Texture

enhancer

Micro-emulsion

Strawberry apple

(Vargas et al., 2006)

Ultra-Turrax Disperse phase: Candeuba wax Emulsifier: Pluronic F127

Candeuba wax &

xanthan gum

Texture enhancer

300 Guava (Zambrano-Zaragoza et al., 2013)

High-pressure homogenization

Disperse phase: Functional Lipid Emulsifier: Tween 20

β-carotene Antioxidant 132-178 Food formulation

(Tan & Nakajima, 2005b)


Disperse phase: Medium chain tryglyceride oil

Emulsifier: Tween 20/80


(Yuan et al., 2008)



Emulsifier: Soy lecithin

Anise oil Antimicrobial 276 Food formulation

(Topuz et al., 2016)


Disperse phase: Carnauba-shellac wax, mineral oil


Lemongrass oil & carnauba

wax


enhancer

195 Apple (Jo et al., 2014)

Microfluidization Disperse phase: Fish oil Emulsifier: Emulsifier: Tween 80

Lemongrass oil

Antimicrobial 5.50 Fresh-cut apple

(Salvia-Trujillo et al., 2015b)



Emulsifier: Modified starch

Peppermint Antimicrobial 200 Fresh-cut apple

(Liang et al., 2012)

Ultrasonication Disperse phase: Plant essential oil Emulsifier: Tween 80

Oregano Antimicrobial 180-250 sliced Bread (Otoni et al., 2014)




Curcumin Anti-inflammation, Antioxidant & Antimicrobial

160 Food & drug formulation

(Wang et al., 2008)


Disperse phase: Carnauba-wax solution

Lemongrass oil &

Antimicrobial 56-87 Plum (Kim et al., 2013)


45

Emulsifier: Tween 80 carnauba-wax Microfluidizer Disperse phase: Medium chain

tryglyceride oil Emulsifier: soybean lecithin

α-tocopherol Antioxidant & Antimicrobial

80-400 Drug formulation

(Hatanaka et al., 2010)

High pressure valve

homogeniser

Disperse phase: Soybean oil Emulsifier: Decaglycerol monoleate

β-carotene Antioxidant 45-183 Drug formulation

(Wang et al., 2012)

Solvent displacement

Disperse phase: carotene in hexane



(Tan & Nakajima, 2005a)

Spontaneous emulsification

Disperse phase: Medium chain tryglyceride oil, castor oil

Emulsifier: Soybean lecithin, Polysorbate 80

Carbamazepine

Medicine (anticonvulsant

drug)

150-200 Drug formulation

(Kelmann et al., 2007)


Disperse phase: Olive oil & soybean oil Emulsifier: Polysorbate 80

Thalidomide Immunomodulatory agent

200 Drug formulation

(Araújo et al., 2011)


Disperse phase: Octyldodecanol Emulsifier: Egg-lecithin

Flavonoid Antioxidant & antimicrobial

300 Food a & drug

formulation

(Fasolo et al., 2007)


Disperse phase: Capric triglyceride Emulsifier: Span®85/Tween® 20

α-tocopherol Antioxidant & Antimicrobial

171 Drug formulation

(Bouchemal et al., 2004)


Disperse phase: Medium chain tryglyceride oil, castor oil

Emulsifier: Tween 20, 40, 60, 80 & 85

Carvacrol Antimicrobial 55 Food formulation

(Chang et al., 2013)

Sonication emulsification

Disperse phase: Seama oil Emulsifier: Tween 20 & 80

Eugenol Antimicrobial 20-191 Fruit juice (Ghosh et al., 2014)


46

2.4 Nanoemulsion characterization

After formation of nanoemulsion, various properties such as droplet size, composition,

stability and crystallinity are studied. Therefore, a number of techniques are applied to

characterize nanoemulsion. For instance, Dynamic Light Scattering (DLS) is a technique

used for the determination of the size distribution profile of small particles in solutions or

suspensions. DLS measures Brownian motion of the particles, which relates to the size of

the particles by Stokes–Einstein equation:

D =𝑘𝑇

6𝜋𝜂𝑅 (1)

Where, D is the coefficient of translation diffusion, R is the radius of the particles, k is the

Boltzmann’s constant, T is the absolute temperature and η is the viscosity of the medium.

By illuminating the particles with a laser and investigating the intensity fluctuations in the

scattered light, DLS enables calculating the size of the particles (Araújo et al., 2011; Preetz,

Hauser, Hause, Kramer, & Mäder, 2010; Silva et al., 2011). In this system, protons are

scattered only by the sample before being detected. Zambrano-Zaragoza et al. (2013),

Salvia-trujillo et al. (2013), Salvia-Trujillo et al. (2015b), Jo et al. (2014), Kim et al. (2014),

Otoni et al. (2014) determined average particle size and size distribution of nanoemulsion

droplets by DLS using Zetasizer aparatus. The droplet size of the nanoemulsion observed

by them ranges from 239 to 326 nm for xanthan gum based solid-lipid nanoparticles; 23 to

56 nm and 100 to 135 nm for lemongrass-oil incorporating sodium alginate based

nanoemulsion; 74 to 195 nm and 56 to 87 nm for lemongrass-oil incorporating carnauba

based nanoemulsion; and 45 to 280 nm for pectin/papaya puree/cinnamaldehyde loaded

nanoemulsion, respectively.

Physical stability and average particle size of nanoemulsion can be addressed by Multiple light scattering. This technique follows the principle that droplets in a suspension are able

to scatter the radiation based on their particles size, shape and composition (Mengual et al.,

2000). In most of the published studies, measurements are performed with an optical

analyser called Turbiscan® (Formulation, L’Union, France). The system consists of stations

where cylindrical glass cells containing nanoemulsion can be loaded. The detection head is

composed of a pulsed near-infrared light source ( = 880 nm) and two synchronous

transmission (T) and back scattering (BS) detectors. The T detector receives the light,

which crosses the sample (at 180° from the incident beam), while the BS detector receives


47

the light scattered backwards by the sample (at 45° from the incident beam). The

measuring principle is based on the particle migration and variation on particle size,

resulting in discrepancy of BS and T signals (Lemarchand, Couvreur, Vauthier, Costantini,

& Gref, 2003; Roland, Piel, Delattre, & Evrard, 2003). More in detail, BS is linked to the

particle mean diameter and particle volume fraction through the following equations:

𝐵𝑆 = 1(λ∗)1/2⁄ (2)

Where:

λ∗(𝑑, ϕ) = 2𝑑[3ϕ(1 − 𝑔)𝑄𝑠]⁄ (3)

and * (µm) is the photon transport length, d (µm) is the particle mean diameter, ϕ (%) is

the particle volume fraction, g and Qs, are two optical parameters derived from the Lorenz–

Mie theory (Azema, 2006). As concern T values, they are derived from the Lambert-Beer

law, which relates the quenching of light to the concentration of the material through which

the light is passing, as follow:

𝑇(λ, 𝑟𝑖) = 𝑇0 𝑒−3𝑟𝑖ϕ𝑄𝑠/𝑑 (4)

Where T0 is transmission value at time zero and ri is the internal radius of the cylindrical

vials in which the sample is loaded for the measurement.

In Figure 2.4, an example of backscattering spectra acquired by multiple light scattering are

reported. We investigated the behaviour of oil in water (10 %) emulsion formulated with

octhenyl succinic anhydride starch (3 %) as emulsifier and prepared with a microfluidizer at

50 MPa.


48

Figure 2.4: Superimposition of backscattering scans with time for oil in water emulsion with

3 % octhenyl succinic anhydride starch as emulsifier.

The results showed the unstable behaviour of the emulsion and the instability phenomena

generated during the storage for 2 days at 25 °C. The spectra show profiles migration and

local variation of the concentration of particles along the height of the vial in which the

emulsion was stored. The backscattering level decreases in the bottom part (left part of the

graph), hence a clarification phenomenon occurred, while it increases at the top part (right

part of the graph) associated to a creaming phenomenon probably associated to the

particles movements of the starch and oil.

Yuan et al. (2008), Venturini et al. (2011), Kim et al. (2014), Mehta et al. (2015) invetigated

the stability of nanoemulsion by Turbiscan. Kim et al. (2014) confirmed the high stability of

the dynamic high pressure processed nanoemulsion containing 0.5 g carnauba wax in 100

g lemongrass-oil by backscattering measurement compared to 4.0 g carnauba wax in 100 g

lemongrass-oil based nanoemulsion.

Zeta Potential is a technique that measures the electro-kinetic potential difference among

the dispersion medium and the stationary layer of fluid attached to the dispersed particle. A

measurement of 30 mV (negative or positive) can be carried out as the arbitrary value that

Height (mm)

0 10 20 30 40

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scat

teri

ng (%

)

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0

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splits low-charged surfaces from highly charged surfaces (Preetz et al., 2010). Zeta

potential value may be linked with the stability of colloidal dispersions, demonstrating the

degree of repulsion between adjacent and equally charged particles in dispersion. For

particles and molecules that are small enough, a high zeta potential will indicate stability,

meaning that dispersion or solution will act against aggregation. When the potential is low,

magnetism exceeds repulsion and the dispersion will break and particles will flocculate.

Therefore, colloids with high zeta potential (positive or negative) are electrically stabilised

whereas colloids with low zeta potentials tend to flocculate or coagulate. Briefly, zeta

potentials from 0 to ± 30 mV suggest instability, whereas zeta potentials higher than ± 30

mV indicate stability (ASTM, 1985). Several researcher such as Araújo et al. (2011), Gao et

al. (2011), Preetz et al. (2010), Zambrano-Zaragoza et al. (2013), Salvia-trujillo et al.

(2013), Salvia-Trujillo et al. (2015b) investigated nanoemulsion’s stability based on zeta

potential measurements.

Other methods such as Differential Scanning Calorimetry (DSC) may be used to detect

the phase transitions, i.e., the melting of crystalline regions, and analyse the amount of

solid fat or ice crystals in emulsions (Thanasukarn, Pongsawatmanit, & McClements, 2004).

Thanasukarn et al. (2004) demonstrated that fat crystallization influences the emulsion

stability based on the emulsifier used and that thermal decomposition follows the melting of

the drug capsulated. Đorđević et al. (2013) and Strasdat & Bunjes (2013) used this

technique to characterise lipid nanoparticle dispersion into calcium alginate beads. They

observed that the small size of the lipid nanoparticles lead to a typical melting behaviour

characterised by the occurrence of multiple discrete melting peaks that enabled the

characterisation of the lipid particles within the microbeads.

Fourier Transform Infrared based technique (FTIR) can measure the quantity of

components in a mixture and determine the consistency or quality of a sample. It is a very

sensitive method, comparatively simple to work with and internally calibrated. These

benefits make measurements reproducible and accurate (Griffiths & De Haseth, 2007;

Nicolet, 2001). Araújo et al. (2011) used this technique for the process of crystallization of

encapsulated thalidomide in nanoemulsion. It was observed that regardless of the

polymorph employed (α- or β-), drug crystallization occurred in α- form. Zhang and Zhao


50

(2015) used FTIR spectrum to examine the tea polyphenol-Zn complex interaction

mechanisms with β-chitosan based nanoparticle structures and found the well-incorporated

characteristic peaks of tea polyphenol and tea polyphenol-Zn complex in β-chitosan

nanoparticles.

X-Ray Diffraction (XRD) is a non-destructive analytical techniques that provides details

information of crystallo-graphic structure, physical properties and chemical composition of

materials (Shannon, 1976). It is also usable for recognition of single phase materials and

crystal structure, for identification and structural analysis of samples, for determination of

crystallite size and shape, and for studying the thermal evolution of crystal structures

(Connolly, 2007; Johansson, Palm, & Werner, 1980). Araújo et al. (2011) used this

technique to characterise the crystals of nanoemulsion. Mulik et al. (2010) analysed the

structure of solid lipid nano-particles inclusion with curcumin using this technique and found

spherical nature of this particles and entrapment of curcumin in nanoparticacles in

amorphous form.

Small-Angle X-ray Scattering (SAXS) is a method for the study of the structure of colloidal

size particles where the elastic scattering of X-rays, measured at extremely low angles

(typically 0.1 – 10 o), by a sample are inhomogeneities. This angular range provides

information of size and shape of macromolecules, characteristic distances of partially

ordered materials, pore sizes, etc. between the wavelength of 5 and 25 nm (Kratky, 1982).

Jenning et al. (2000) analysed the effects of the oily constituent of their particles on the

subcell parameters and long spacing of solid ‘Compritol nanocyrstals’, to confirm the

polymorphism behaviour discovered by DSC method. Venturini et al. (2011) also

demonstrated that sorbitan monostearate was interacting with the oily phase of the solid–

lipid nanoparticle.

Microscopy may be utilized as a direct imaging method to analyse nanoemulsion;

however, the kind of microscopy used relies on the kind of matrix to be analysed. The

technique allows obtaining detail about the shape, size and aggregation state of

nanoemulsion. One of the imaging methods applied for the characterization of


51

nanoemulsion is Transmission Electron Microscopy (TEM). This is broadly used in the

study of materials for metallurgy/science and biological sciences; in every case, the

samples should be extremely thin and able to withstand the high vacuum present inside the

instrument (Luykx, Peters, van Ruth, & Bouwmeester, 2008; Z. L. Wang, 2000). Araújo et

al. (2011) and Bouchemal et al. (2004) investigated the structure and morphology of the

nanoemulsion by TEM; the combination of bright field imaging at increasing magnification

and diffraction modes were used to analyse the size and form of the oil in water

nanoemulsion and to find out the crystalline or amorphous character of the components.

The direct observation allowed the possibility to perform selected area electron diffraction to

check the crystallinity of the emulsion core elements. Yaun et al. (2008) and Chu et al.

(2007) investigated the microstructure and the particle-size distribution of nanoemulsion,

remarking that β-carotene particles showed spherical morphology with a mean diameter of

20 nm, confirming the findings obtained by DLS. Salvia-Trujillo et al. (2013) confirmed that

the lemongrass oil-alginate based nanoemulsion was in the nano –range (droplet sizes

between 10 nm and 250 nm of diameter) using TEM. Several researchers such as Zhang

and Zhao (2015), Hatanaka et al. (2010), Singh et al. (2011) used this technique to

examine the morphology of nanoemulsion. Scanning Electron Microscopy (SEM) is also

an imaging technique (Luykx et al., 2008; Reimer, 2000) capable of producing high-

resolution images, which have three-dimensional appearance and are useful for evaluating

the structure of surface. In general, TEM resolution is higher than SEM resolution; however,

SEM image depends on the surface processes rather than transmission. It is capable of

imaging bulky samples, has a much greater depth of field, and, so, can make images that

are a better illustration of the 3D structure of the sample. The combination of higher

magnification, larger depth of field, greater resolution, and ease of sample examination

make SEM one of the most greatly used methods in today’s research sectors. Zhou et al.

(2008) synthesised the carbonated hydroxyapatite nanospheres using oil in water

nanoemulsion for producing composite tissue engineering scaffold. After the process, they

confirmed spherical morphology of spheres using this technique and that their particles

were in the nano-meter range. The most recently developed imaging microscopy technique

is Atomic Force Microscopy (AFM) (Edwards & Baeumner, 2006; Luykx et al., 2008;

Ruozi, Tosi, Forni, Fresta, & Vandelli, 2005). The high resolution (± 0.1 nm) achieved using

AFM allows viewing directly single molecules or atoms that have dimensions of few

nanometers. AFM depends on the raster scanning of a nanometer-sized sharp probe over

a sample that has been trapped onto a carefully selected surface (glass or mica), resulting


52

in a high-resolution three-dimensional profile of the surface under study. The surface

irregularities observed in SEM are absent by AFM investigation. AFM can be applied on

any type of material’s surface and images obtained by AFM are complementary to other

established techniques. AFM may be utilized for the structural characterization of, e.g.,

polysaccharides, proteins and liposomes (Luykx et al., 2008; Moraru et al., 2003). Preetz et

al. (2010) and Zhang & Zhao (2015) demonstrated the differences among nanocapsules

and nanoemulsion by studying the shape, mechanical and morphology properties of the

capsule and emulsion shell through AFM. They also showed that the shell around an oil

droplet solidified with enhancing amounts of polyelectrolytes. Singh et al. (2011) and Salvia-

trujillo et al. (2013), confirmed the characteristics of nanoemulsion’s droplet by this

technique.

2.5 Conclusion

The development of food grade nanoemulsion able to encapsulate, and improve

functionality of some types of active ingredients is an evident interest for new generation.

Antimicrobials, antioxidants and texture enhancers from natural sources are potential

alternative of chemical additives that can be incorporated within foodstuff, representing a

promising strategy to satisfy the consumer’s claim. This review points the potential benefits

of using nanoemulsion over conventional emulsion for coating of fresh-cut fruits to improve

their quality, safety and shelf life. However, most of the studies discussed in this review

have been performed at laboratory scale. Therefore, further researches are needed at

commercial scale in order to provide more realistic information using nanoemulsion coating

on fresh-cut fruits. In addition, natural functional compounds from plant extracts such as

essential oils, organic acids and salts, reducing agent and others need to be characterised

in order to understand their interactions with nanoemulsion compositions and their

application on fruit surface. Despite these limitations, food industries are looking for such

innovative technology that can be used on a broad spectrum of foods adding value to their

products, while maintaining their quality with extended shelf life.

There are different methods available for producing and characterising of nanoemulsion,

some of them have been shown more suitable than others. For instance, DLS (Dynamic


53

light scattering) may quickly determine the diameter of nanoparticles in nanoemulsion,

Turbiscan, zeta potential can indicate the stability of nanoemulsion, TEM (Transmission

Electron Microscopy) may be used to have general image of the nanoemulsion structure

and confirm the hydrodynamic diameter given by DLS technique.


54

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3 Food and ascorbic scavengers of hydrogen peroxide

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CHAPTER III

3 Food and Ascorbic Scavengers of Hydrogen Peroxide: A Reaction Calorimetry Investigation


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Abstract

Common spectrometric methods to determine hydrogen peroxide scavenging activity

require time consuming extraction protocols and become poorly reliable because of

the sample turbidity or the presence of UV absorbing compounds. The present work

suggests the use of reaction calorimetry (RC) to determine the antioxidant capacity

of fruit juices, fruit puree, tea, coffee and alcoholic beverages, like wines. This

experimental approach, that does not imply the above drawbacks and does not

require any extraction protocol, allows the direct monitoring of the reaction between

food beverages and H2O2. The overall exothermic effect reflects the extent of the

scavenging activity of the samples versus hydrogen peroxide. The reliability of the

approach is assessed through the study of the reaction between hydrogen peroxide

and ascorbic acid at different concentrations and pH at room temperature.

Key words: reaction calorimetry; antioxidant capacity; hydrogen peroxide; food;

peroxide scavenging capacity.

This work has been published to the journal of thermal analysis and calorimetry


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3.1 Introduction

Antioxidants in food products contribute to a number of important peculiar processes,

like slowing oxidation rate (Shahidi, 2000), preserving nutritional quality (Nicoli,

Anese, Parpinel, 1999) and promoting the health-status (Frankel and Meyer, 2000).

Because of the multi-component and multi-phase nature of most food products, a

short–cut evaluation of food antioxidants is not easily achievable. In most cases the

assessment of the antioxidant content of a given food comes from experimental data

drawn with independent methods, each accounting for different aspects related to

the antioxidant functionality (Frankel and Meyer, 2000). The current practice sees

two main kinds of experimental approaches. One includes methods that measure the

capacity of transferring hydrogen atoms (HAT), such as DPPH, ORAC and TEAC

assays. The second deals with the measurement of the electron transfer (ET)

capacity, such as Folin and FRAP (Prior, Wu and Schaich, 2005). Recently, we

proposed a third method that allows the simultaneous determination of the HAT and

ET capacity through an electrochemical approach (Lemma, Schampicchio,

Bulbarello, Mason, 2014).

All these methods require preliminary time-consuming treatments of the samples and

show serious drawbacks when applied to opaque or viscous systems. The present

work aims to overcome these difficulties applying a calorimetric approach. Either

differential scan calorimeters (DSC) or reaction calorimeters (RC) indeed allows

determination of the reaction heat, regardless to the physical form of the sample and

without the need of sample pre-treatments.

Some difficulties are still to overcome. For instance, during DSC investigations with

open pans, the water contained in the sample evaporates with a large endothermic

effect that conceals any other thermal signal. Another limit of the DSC approach

comes the small size of the cells that makes difficult the addition of in situ peripherals

for dosing, sampling or stirring. Therefore, fast reactions following the mixing of

reagents are normally difficult to monitor with this technique. RC instruments too

show some drawbacks. They generally host large-volume samples (typically more

than one liter) and therefore require extensive calibration checks throughout the


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reaction time span to compensate baseline shifts. Their response depends on the

liquid level, sample viscosity and stirring speed (Nilsson and Hess, 2008).

An innovative chemical process analyzer CPA202 allows overcoming such

drawbacks. In the present investigation the instrument allowed detection of the heat

flux (HF) released during the reaction between antioxidants and hydrogen peroxide.

The choice of the hydrogen peroxide comes from the knowledge of its involvement in

the oxidative stress of plants and human cells and the initiation of many degradation

mechanisms in food products (Sroka and Cisowski, 2003).

The paper presents examples of the peroxide scavenging activity of various

beverages and food preparations (Veal, Day, Morgan, 2007). Our hypothesis is that

the higher the scavenging activity, the larger the heat of reaction.

3.2 Materials and methods

3.2.1 Materials

Ascorbic acid (AA) was of analytical grade (Sigma Aldrich). Stock solutions (5, 10,

25, 50, 100, 200, 500 and 1000 mM) were prepared by dissolving the desired

amount in distilled water (18 M). For the IC investigations 1mL of each solution was

mixed with 1 mL of H2O2 (30%, w/w) and added to 100 mL of an aqueous buffer.

Therefore the starting concentrations of AA were 100 times smaller. Britton-

Robinson (boric acid, phosphoric acid, acetic acid, Sigma Aldrich) buffer solutions

were prepared adjusting the pH NaOH 8 M (Sigma Aldrich).

Fruit juice samples (apricot, apple, and grapefruit), wine (Chianti 2010, Lagrein 2011

and 2012), baby fruit puree (apple, banana and plum), tea (green and white), and

coffee (with and without caffeine) were purchased from local market. Tea and coffee

beverages were prepared by soaking the powder sample (contained in a paper bag,

net content, 1 g) in 50 mL of boiling water for 5 min. All the other samples were used

as received. 1 mL or 1 g of food sample was diluted (dispersed) either in 100 mL of

distilled water or in 100 mL of Britton-Robinson buffer at pH 6.5. The AA solutions

were studied in buffered conditions, at various pH.


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not need calibration throughout the experiment. The highly stable baseline returns to

±0.0001 W level at the end of the reaction. The generation of an electrical power

(from 0.007 to 0.04 W, provided by an internal electrical power heater) allows,

periodically, the check of the performance of the instrument. The accuracy always is

higher than 99%. The time constant is around 20 s (according to the producer),

although a little larger value (about 35 s) affects the data of the present work.

3.2.3 Optimized procedure

Immersion of the CPA reactor into a water-bath thermostat set at 25°C allows

achievement of isothermal conditions. Prior of the analysis, the two syringes hosting

the sample and the hydrogen peroxide, respectively, attain the desired temperature

after immersion in the same thermostat. Before each measurement, the reactor cell,

filled with 0.1 L of a buffer solution (Britton-Robinson, pH = 6.5), de-aerated and

tightly closed with a lid, is immersed in the thermostat. The stirrer rate is set at 200

rpm. Once attained the thermal equilibrium (reflected by a stable baseline:

background signal ≤ 0.1 mW), 1 mL of sample is injected in the reactor cell. After

stabilizing rest (~30 min), 1 mL of H2O2 (30%w/w) is added into the reactor cell and

the resulting thermal power signal (W) monitored. The integral of the HF- vs- time

record yields the heat of the reaction (J).

3.3 Results

3.3.1 Solutions of Ascorbic Acid

The RC records collected from AA buffered (pH = 6.5) solutions are reported in

Figure 3.2. The overall thermal effect is exothermic (positive sign throughout the text

and figures).


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Figure 3.2. Isothermal RC records from buffered solutions of AA at different starting

concentrations (0.05, 0.1, 0.25., 0.5, 1.0, 2.0, 5.0, 10.0 mM, namely, after a 100 time

dilution of the original solution in the calorimetric cell). The HF values therefore refer

to a 1 mL of the original AA solution.

Figure 3.3. Integrated IC signals show a straight-line increasing trend with a slope

that corresponds to 782.1 kJ per mole of ascorbic acid.


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Figure 3.2 suggests that the overall thermal effect (areas beneath the signals) may

be proportional to the starting content of AA, no matter the amount of H2O2 available.

Integration of the signals confirms this impression (Figure 3.3).

When the starting concentration is fixed, [AA]0 = 0.5 mM, and the pH of the solution

is varied (2, 4, 5, 6.5, 7), the overall thermal effect increases with increasing pH

(Figure 3.4).

Figure 3.4. Isothermal RC records from solutions of AA (0.5 mM in the calorimetric

cell) at different pH (2, 4, 5, 7).

3.3.2 Scavenging Properties of Some Food Products.

Many food products that contain antioxidant compounds behave like AA when

treated with H2O2. Some products, like various kinds of wine, tea, coffee, fruit juices

and purée, allow an IC investigation and a direct comparison with the AA. The

relevant IC records are indeed similar to those collected for AA, namely, a sharp

single peak that ends in less than one hour (Figure 3.5).


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Figure 3.5. Isothermal calorimetric records from various food samples.

All baby foods, normally fortified with ascorbic acid, show the highest thermal

powers. The overall thermal effects calculated by integration of the IC records

increases with the order reported in the following histogram (Figure 3.6).

Figure 3.6. Histogram of the overall thermal effects evaluated by integration of the IC

records collected from various food diluting (dispersing) 1 mL or 1 g in 100 mL of

distilled water, (heat units: J/ mL and J/g, respectively).


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3.4 Discussion

Figure 3.3 reflects a purely phenomenological evidence that can be someway

misleading, as the same data, once scaled with respect to [AA]0 (Figure 3.7), show

that the overall thermal effect decreases with increasing [AA]0. This result suggests

that the overall heat exchanged per mole of AA is the result of a balance between

endo- and exothermic effects and that the underlying mechanism of the oxidation

may change with the AA/H2O2 molar ratio.

Figure 3.7. The integrated HF data (Figure 3.2) referred to [AA]0 (in the calorimetric

cell).

Looking at the literature (Deutsch, 1998; Wilson, Beezer, Mitchell, 1995), a likely

kinetic model for the whole process would include free radicals. More specifically,

J.C. Deutsch (1998) reported that AA and dehydro-ascorbic acid, DHA, undergo

oxidation along the same reaction pathway and that the early oxidation of AA occurs

via the formation of an intermediate radical species, namely, the semi-dehydro-

ascorbic radical, (DHA∙). This radical would undergo a further oxidation step to form

DHA that, after hydrolysis to di-ketogulonic acid, DKGA, would also react with H2O2


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�̇� = �̇�1 + �̇�2 = �̇�𝐴𝐴Δ𝐻1 + �̇�𝑇𝐻𝐷𝐻𝐴Δ𝐻2

= [𝑘1𝑒−𝑘1𝑡]Δ 𝐻1 + [𝑘1𝑘2

𝑘2 − 𝑘1

(𝑒−𝑘1𝑡 − 𝑒−𝑘2𝑡)] Δ𝐻2 =

= [𝑘1 Δ 𝐻1 +𝑘1𝑘2

𝑘2−𝑘1Δ𝐻2] 𝑒−𝑘1𝑡 − [

𝑘1𝑘2

𝑘2−𝑘1Δ𝐻2] 𝑒−𝑘2𝑡 = 𝑎𝑒−𝑐𝑡 − 𝑏𝑒−𝑑𝑡 (3.4)

A couple of exponentials should therefore satisfactorily fit the calorimetric trace,

provided that the earliest part of the record (about 50 s) is neglected, as it reflects

the lag time of the calorimeter (Schiraldi, 2003). Integration of such expression

between the limits (0, ∞) leads to

𝑄 = (Δ 𝐻1 + Δ 𝐻2) (3.5)

The quantities in equation (3.5) are in Joule units; when multiplied by V×[AA]0 (where

V = 0.1 L is the sample volume), they should correspond to the experimental

evidence (see Figure 3.3).

The four fitting parameters, a, b, c and d, allow evaluation of the kinetic constants

and the enthalpies:

(a-b)/c = ΔH1; b(d-c)/dc = ΔH2; c = k1 and d = k2.

Table 3.1 reports the values of the relevant enthalpies and kinetic constants and

Figure (3.8) shows the fits obtained.

Table 3.1.Kinetic constants and reaction enthalpies from the fits of the experimental

data according to equation (3.4).

[AA]0/mM [H2O2]/ [AA] k1/10-3s-1 k2/10-4s-1 ΔH1/kJ mol-1

SD ΔH2/ kJ mol-1

SD

0.05 1764 4.35 5.98 790.91 57.1 -982.58 70.9

0.1 882 4.37 5.61 539.82 34.8 -868.52 56.0

0.25 353 4.81 3.04 247.23 13.7 -949.91 52.8

0.5 176 7.99 3.36 82.90 4.11 -976.19 48.4

1 88 10.20 1.58 31.05 1.38 -914.10 40.5

2 44 2.04 2.76 -31.37 1.24 -880.37 34.8

5 18 3.03 1.49 1.29 0.04 -803.42 27.4

10 9 1.17 1.36 14.43 0.44 -707.53 21.5

The calculated value of ΔH1 and ΔH2 comes from equation (4) taking into account

that the volume of the solution is 0.1 L. AA concentrations are those in the


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calorimetric cell. Standard deviation (SD) of enthalpy drops in the neighbor side

columns.

Figure 3.8. Isothermal calorimetric records from buffered (pH = 6.5) aqueous AA

(0.05, 0.1, 0.25, 0.50 and 1.00 mM in the calorimetric cell) in the presence of excess

H2O2. Dashed lines are drawn according to equation (3.4).For larger [AA]0 the fit is

still satisfactory (R2 > 0.98), but can be improved adding a further step to the kinetic

model assumed (see below).

In spite of the satisfactory appearance of the fits, the calculated values of the kinetic

constants and the enthalpy changes with [AA]0 (see Table 3.1) and do not lead to an

unequivocal interpretation. Would the model reflect the true mechanism of the

process, the values of the kinetic constants and reaction enthalpy should not change

with [AA]0. While the values of k1 and k2 coming from the best fitting treatment may

allow a likely averaging (at least for [AA]0 < 1 mM), namely, 6.4 10-3 s-1 and 3.9 10-4

s-1, respectively, this is not the case of ΔH1 and ΔH2. The consistency of the model

seems rather poor. However, one has to take into account the oversimplification

assumed: in particular, the first step of the model actually includes several

concomitant processes related to the reactions of the free radicals, some of which,


78

like the cleavage of H2O2, imply endothermic effects that partially counterbalance the

exothermic ones.

The ΔH1 values in Table 3.1 actually tend to vanish with increasing [AA]0. This trend

suggests that endo- and exothermic effects related to the first step tend to achieve a

zero balance for [AA]0 > 1 mM, namely, for a H2O2/[AA]0 < 86.5 ([H2O2] = 86.5 mM).

The overall exothermic effect determined with IC therefore tends to coincide with the

reaction enthalpy of the second step.

This could be the reason why other authors (Deutsch, 2000), based on calorimetric

evidences collected in the presence of a poorer oxidant (either O2 or H2O2)

concentration, were inclined to describe the oxidation of AA as a pseudo first order

process, namely with a single main thermal effect.

As for the ΔH2, a reasonable average value may be -929 kJ mol-1 for [AA]0 < 5 mM.

At larger AA the IC signal shows an extra shoulder (mainly at [AA]0 = 10 mM) which

suggests the occurrence of a further thermal effect to account for.

Figure 3.9 shows the split of the overall recorded heat flux that reflects the two

consecutive steps of the assumed kinetic model. Figure 3.10 reports the

corresponding trend of the molar fractions of the species involved. The intermediate

species DKGA goes through a maximum, its formation from AA being rather fast with

respect to its oxidation to THDHA. This means that, in the presence of excess H2O2,

the observed thermal effect would mainly deal with the oxidation of DKGA.

Figure 3.9. Split of the recorded HF data (left, 0.05 mM; right, 0.5 mM in the

calorimetric cell) in two contributions related to the consecutive steps of the assumed

kinetic model. The first contribution is endothermic, while the second is exothermic.


79

The signal collected for [AA]0 ≥ 5 mM shows a shoulder that looks like a double peak

for [AA]0 = 10 mM. The fitting model used for lower AA concentration cannot

reproduce this kind of trend that suggests the occurrence of a third thermal effect.

Such an addition implies a kinetic model with three consecutive steps, the third of

which has a rate that can be predicted accurately (Schiraldi, 2003). For the present

scope, namely, just the fit of an experimental record, it is enough to say that the

corresponding trend is close to that of a broad Gaussian peak.

Figure 3.10. Trend of the molar fraction of the compounds involved in the scavenging

process according to a two-consecutive-step model (equation 3.4).

If one adds such a function to the expression of equation (3.4) and assumes a

naught neat enthalpy for the first step (see above), the obtained fits are very

satisfactory (Figure 3.11), although the adjustment accounts only for about 2% of the

overall thermal effect. The reason for such extra step may be related to oxygen-

accepting antioxidants, like threonic and oxalic acids, that arise during ascorbate

oxidation (Willson, Beezer, Mitchell, Loh, 1995; Deutsch, 1998).


80

Figure 3.11. Fit of the IC records collected from [AA]0 = 5 and 10 mM (in the

calorimetric cell) buffered solutions according to a modified kinetic model (see text).

When [AA]0 is fixed (e.g. 0.5 mM) and the pH is changed, the IC record shows a

sharp and small-area peak at pH = 2, while the signal becomes broader and larger at

higher pH (Figure 3.12). Taking into account the equilibrium of dissociation of AA

(pKa = 4.17) and that ([AA-]+[AA]) = [AA]0, one can easily obtain

[𝐴𝐴−] = [𝐴𝐴]0𝐾𝑎

𝐾𝑎+10−𝑝𝐻 (3.6)

[AA-] is therefore supposed to increase with increasing pH, which suggests that the

truly reactive species may be the ascorbate anion, the signal from low-pH solutions

being small since the ascorbate is a small fraction of [AA]0. One simply has to

replace [AA]0, with [AA-] in equation (3.4) to apply the model of two consecutive

steps described above and fit the IC records (Figure 3.12), while Figure 3.13 shows

the trend of the overall oxidation heat, Q.


81

Figure 3.12. Fits (dashed lines) of the IC traces of aqueous AA solutions ([AA]0 = 0.5

mM in the calorimetric cell) at different pH ( 2, 4, 5, 7).

Figure 3.13. Overall heat of oxidation of AA in the presence of excess H2O2, at

various pH levels.

The sigmoid trend of Q indeed parallels that of the [AA-]/[AA]0 molar ratio and implies

a low-pH and a high-pH steady levels, at about 100 and 1000 kJ per mole of AA,

respectively. At pH = 7, the anion AA- practically accounts for 100% of the ascorbic


82

species. The corresponding oxidation heat accordingly is the largest one for [AA]0 =

0.5 mM. At pH = 2, the anion [AA-] is l% of [AA]0 and the corresponding Q value

reflects the lowest scavenging effect for [AA]0 = 0.5 mM. This finding seems in line

with the evidence of low pH increase oxidative deterioration of fish oil in the

presence of ascorbate (Borsook, Davenport, Jeffreys, Warner, 1937).

The fit of the experimental IC records with a two consecutive process model allows

evaluation of the relevant parameters. k1 remains around 3.5 ±1 × 10-3 s-1 with a

mean endothermic effect of - 155 kJ mol-1, at every pH considered, while k2 and the

related enthalpy drop depend on pH. Figure 3.14 shows the overall picture that

reflects these evidences. The broader signal observed at larger pH is therefore

related to a smaller rate of the second step, that however implies a larger thermal

effect.

Figure 3.14. Trends of the molar ratios of AA and DKGA at various pH. Kinetic

constant and enthalpy drop of the second step of the two consecutive step model.

As for the food products considered in this work, no simple kinetic model seems

appropriate to describe their scavenging activity, inasmuch as each food contains

different antioxidant components (Jacobsen, Timm, Meyer, 2001; Frankel,

Waterhouse, Teissedre, 1995; Hernandez, Melgarejo, Tomas-Barberan, Artes, 1999;

Ozkan, Kirca, Cemeroglu, 2004) and, above all, metal ions that can catalyze the

oxidation (Deutsch, 2000; Marti, Perez-Vicente, Garcia-Viguera, 2002). Most of them

show a scavenging effect that follows a first order law, which is a purely empirical


83

evidence with no physical meaning. Table 3.2 reports the kinetic constants of the

scavenging process and the concentration of ascorbic acid at pH 6.5 that would

release the same heat per mL.

It is worth mentioning that the pH conditions experienced by any food through the

gastro-intestinal tract change passing from the mouth to the great intestine. Our

results from buffered AA solutions suggest that the respective scavenging potential

would be smaller in the acidic tracts (because of the slow reaction rate), and much

larger in the basic tract of the gut where most of the adsorption takes place.

Table 3.2. Apparent kinetic constant (first order) and heat released per mL or per g

of food.

Food

k1/10-3 s-1

Q/(J/mL, or J/g)

[AA] / mM equivalent

Chianti_2010 3.79 2.80 4.23

Lagrein_2011 3.59 3.52 5.10

Apricot 3.67 3.59 5.53

Lagrein_2012 3.74 4.16 6.09

Decaff Coffee 3.60 4.83 7.32

White_tea 3.74 4.95 7.38

Coffee 3.62 5.62 8.26

Apple 3.77 5.66 8.45

Green_tea 4.09 5.91 8.48

Banana_puree 3.80 6.07 8.72

Apple_puree 3.26 6.81 10.23

Grape_fruit 3.88 7.46 11.12

Plum_puree 3.53 10.06 14.24

Column 4 reports the concentration (mM) of ascorbic acid that would release the

same heat per mL at pH 6.5 (after dilution in the buffer).


84

This seems indeed the case also for the antioxidants present in some food samples.

When the same amounts of food are diluted (dispersed) in 100 mL of buffer at pH =

6.5, a significant shift toward higher scavenging activity occurs for wines and

grapefruit juice, while other remain almost unchanged or even a little reduced (Table

3.3).

3.5 Conclusions

Although many innovations have been so far proposed to assess the scavenging

properties of natural antioxidants present in many food (Grinstead, 1960; Buratti,

Scampicchio, Giovanelli, Mannino, 2008), the use of reaction calorimetry performed

with a suitable instruments offers a further possibility, based on the determination of

the heat released (exothermic effect) by systems that contain ascorbic acid or other

natural antioxidants. A tentative kinetic model, including two or three consecutive

first-order steps, allows a satisfactory fit of the experimental IC records. However,

the values of the relevant kinetic constants and reaction enthalpies actually are the

result of the combination of intermediate steps involving free radicals that imply a

balance of endo- and exothermic effects that can depend on the H2O2/antioxidant

molar ratio. The pH conditions have a substantial effect on the scavenging power of

AA solutions: this increases with increasing pH and attains its maximum for pH = 6.5,

i.e., when ascorbate anions represent almost 100% of the ascorbic species.

Assuming that the overall heat released reflects the scavenging capability of the

system considered, one may define the antioxidant potentiality of several food

products with reference to that of a buffered (pH = 6.5) AA solution that releases the

same amount of heat. The scavenging capability of the food products considered

seems enhanced (with few exceptions) when buffered at pH 6.5, namely, above the

original value. Since the pH conditions experienced by any food change along the

gastro-intestinal tract, the respective scavenging potential would be larger in the

basic tract of the gut.


85

Table 3.3. Heat released by food samples diluted in distilled water or in buffer at pH

6.5.

Food

Q /(JmL-1 or g-1)

diluted in water

Q /(JmL-1 or g-1)

at pH 6.5

Plum_puree 11.14 10.35

Grapefruit juice 8.69 10.51

Apple_puree 8.00 9.68

Apple juice 6.82 9.92

Banana_puree 6.63 6.83

Green_tea 6.61 7.92

Coffee 6.46 5.53

White_tea 5.78 6.81

Dec Coffee 5.72 5.40

Lagrein_2012 4.76 9.05

Apricot juice 4.32 5.66

Lagrein_2011 3.98 8.92

Chianti_2010 3.31 6.43


86

3.6 References Borsook, H., Davenport, H. W., Jeffreys, C. E., & Warner, R. C. (1937). The

oxidation of ascorbic acid and its reduction in vitro and in vivo. Journal of Biological Chemistry, 117(1), 237-279.

Buratti, S., Scampicchio, M., Giovanelli, G., & Mannino, S. (2008). A low-cost and low-tech electrochemical flow system for the evaluation of total phenolic content and antioxidant power of tea infusions. Talanta, 75(1), 312-316.

Deutsch, J. C. (1998). Ascorbic acid oxidation by hydrogen peroxide. Analytical biochemistry, 255(1), 1-7.

Deutsch, J. C. (2000). Dehydroascorbic acid. Journal of Chromatography a, 881(1), 299-307.

Deutsch, J. C. (1998). Oxygen-accepting antioxidants which arise during ascorbate oxidation. Analytical biochemistry, 265(2), 238-245.

Frankel, E. N., Waterhouse, A. L., & Teissedre, P. L. (1995). Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density lipoproteins. Journal of Agricultural and Food chemistry, 43(4), 890-894.

Frankel, E. N., & Meyer, A. S. (2000). The problems of using one‐dimensional methods to evaluate multifunctional food and biological antioxidants. Journal of the Science of Food and Agriculture, 80(13), 1925-1941.

Gaisford, S., Hills, A. K., Beezer, A. E., & Mitchell, J. C. (1999). Thermodynamic and kinetic analysis of isothermal microcalorimetric data: applications to consecutive reaction schemes. Thermochimica acta, 328(1), 39-45.

Grinstead, R. R. (1960). The oxidation of ascorbic acid by hydrogen peroxide. Catalysis by ethylenediaminetetraacetato-iron (III). Journal of the American Chemical Society, 82(13), 3464-3471.

Hernandez, F., Melgarejo, P., Tomas-Barberan, F. A., & Artes, F. (1999). Evolution of juice anthocyanins during ripening of new selected pomegranate (Punica granatum) clones. European Food Research and Technology, 210(1), 39-42.

Jacobsen, C., Timm, M., & Meyer, A. S. (2001). Oxidation in fish oil enriched mayonnaise: ascorbic acid and low pH increase oxidative deterioration. Journal of agricultural and food chemistry, 49(8), 3947-3956.

Lemma, S. M., Scampicchio, M., Bulbarello, A., Mason, M., & Schweikert, L. (2014). Concerted determination of the hydrogen atom and electron transfer capacity of lipid soluble reducing agents. Electroanalysis, 26(7), 1582-1587.

Martí, N., Pérez‐Vicente, A., & García‐Viguera, C. (2002). Influence of storage temperature and ascorbic acid addition on pomegranate juice. Journal of the Science of Food and Agriculture, 82(2), 217-221.

Nicoli, M. C., Anese, M., & Parpinel, M. (1999). Influence of processing on the antioxidant properties of fruit and vegetables. Trends in Food Science & Technology, 10(3), 94-100.

Nilsson, H., & Hess, U. (2008). Introduction of a calibration-free reaction calorimeter that combines the benefits of DSCS and reaction calorimeters. Journal of Thermal Analysis and Calorimetry, 93(1), 219-224.

Özkan, M., Kırca, A., & Cemeroǧlu, B. (2004). Effects of hydrogen peroxide on the stability of ascorbic acid during storage in various fruit juices. Food chemistry, 88(4), 591-597.

Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of agricultural and food chemistry, 53(10), 4290-4302.


87

Scampicchio, M., Wang, J., Blasco, A. J., Sanchez Arribas, A., Mannino, S., & Escarpa, A. (2006). Nanoparticle-based assays of antioxidant activity. Analytical chemistry, 78(6), 2060-2063.

Schiraldi, A. (2003). Phenomenological Kineticsan; an alternative approach. Journal of thermal analysis and calorimetry, 72(3), 885-900.

Shahidi, F. (2000). Antioxidants in food and food antioxidants. Food/Nahrung, 44(3), 158-163.

Sroka, Z., & Cisowski, W. (2003). Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food and Chemical Toxicology, 41(6), 753-758.

Wilson, R. J., Beezer, A. E., & Mitchell, J. C. (1995). A kinetic study of the oxidation of L-ascorbic acid (vitamin C) in solution using an isothermal microcalorimeter. Thermochimica Acta, 264, 27-40.

Willson, R. J., Beezer, A. E., Mitchell, J. C., & Loh, W. (1995). Determination of thermodynamic and kinetic parameters from isothermal heat conduction microcalorimetry: applications to long-term-reaction studies. The Journal of Physical Chemistry, 99(18), 7108-7113.

Veal, E. A., Day, A. M., & Morgan, B. A. (2007). Hydrogen peroxide sensing and signaling. Molecular cell, 26(1), 1-14.

4 Effects of ascorbic acid and light on reactions in fresh-cut apples by microcalorimetry

88

Chapter IV

4 Effects of Ascorbic Acid and Light on Reactions in Fresh-cut Apples by Microcalorimetry


89

Abstract

During the manufacturing of fresh-cut apples, a number of biochemical events,

overall exothermic, contribute to increasing the reaction rate of the fruit and the

browning of its wounded surface. This work applied isothermal microcalorimetry to

compare the overall effect of such complex events before and after treatments with

ascorbic acid solutions, pulsed lights or UV-C lights. Briefly, apple samples were cut

into cylinders and dipped in solutions containing ascorbic acid (0-2.5%) or exposed

to high energy doses of light (from 6 to 175 kJ/m2). In general, the heat-flow signal

recorded by microcalorimetry was inversely proportional to the intensity of the

applied treatment. In case of treatments with ascorbic acid, the heat-flow signal was

empirically deconvoluted in three distinctive signals, respectively, (I) an exponential

decay, (II) a gaussian central curve and (III) a final logistical function. The first and

the third functions were constant regardless of the concentration of ascorbic acid

used. Only the second Gaussian function was correlated with the concentration of

ascorbic acid and the area was used to evaluate the efficacy of the process. Overall,

this work contributes to the understanding of the heat produced by fruit after

wounding and, from a practical standpoint, can help compare the effects of different

treatments on fresh cut fruits.

Keywords: Microcalorimetry, fresh cut fruit; apple; ascorbic acid; UV-C light; pulsed

light;

This work has been published to the thermochemica acta


90

4.1 Introduction

Fresh-cut products are defined as fruits or vegetables that are processed (i.e.

washed, trimmed, peeled, cut, etc.) into high quality and ready-to-eat products (Rico,

Martin-Diana, Barat, and Barry-Ryan, 2007). Their quality is typically assessed by

external attributes, such as size, shape, color, glossiness, surface cleanliness and

absence of defects (Barrett, Beaulieu, and Shewfelt, 2010). Following cutting

operations, the rate of fruit respiration, ethylene production and enzymatic browning

increases due to tissue wounding (Kader 2002). The rate of these physiological

events affects the product shelf-life (Cortellino, Gobbi, Bianchi, and Rizzolo, 2015).

To control respiration and extend shelf life, cut fruit can be submitted to different

chemical or physical treatments (Laurila et al. 1998; Jiang et al. 2004). The former

includes traditional dipping of apple slices into a solution containing antioxidants (e.g.

ascorbic acid) and/or chelators (e.g. citric acid) and/or salts (i.e. CaCl2) More

recently, to control fruit respiration, some innovative technologies have been also

proposed, such as the application of electromagnetic waves in either continuous or

pulsed mode. For instance, the exposure of fruits to UV-C and pulsed light has been

demonstrated to inactivate polyphenoloxidase, minimizing enzymatic browning

reactions in cut apples (Gómez-López et al. 2007; Aguiló-Aguayo et al. 2013; Ignat

et al. 2014).

To evaluate the effects of such treatments on fruit respiration, the rate of change of

some quality attributes (i.e. colour, acidity, pH, soluble solids, texture, water loss,

phenolic profile, vitamin C, carotenoids or the sensorial profile) is generally

monitored. This approach makes use of destructive measurements that may require

the application of time consuming and expensive analytical techniques (i.e.

chromatographic analysis of phenols, vitamins and carotenoids) on pre-treated

samples (Gil, Aguayo, and (Kader 2002).

Among the techniques used for measuring respiration rate in foods, animal and

vegetable tissues, microcalorimetry has played a crucial role. One of the main


91

advantages of this technique is that it measures the rate of heat production (or heat-

flow) of samples contained in an ampoule, regardless of their physical state (i.e.

liquid, solid or gas), non-destructively and without any previous pre-treatment. A

number of studies have applied this technique to the measurement of the metabolic

rates in fruits under different conditions (Wadsö et al. 2004). Other applications

include the evaluation of the shelf life (Riva, Fessas, and Schiraldi, 2001) and fresh-

cut fruits metabolism ( Wadso and Galindo, 2009).

Despite the importance of microcalorimetry, there has been very little research

directly investigating the possibility to use heat flow data to discriminate the

contribution of different metabolic mechanisms occurring on fruit samples after

cutting. Accordingly, this work aimed at applying the deconvolution technique to

isothermal microcalorimetry data to separate the contribution to the overall heat flow

from respiration metabolism of the wounded tissue, polyphenol oxidaase activity and

anaerobic metabolism. This approach has been applied on fresh-cut apples (Malus

domestica cv. Golden Delicious) subjected to different stabilization treatments, such

as dipping with ascorbic acid solutions, exposure to UV-C and pulsed light. The

experimental work presented here contributes to extending the possible use of

microcalorimetry and provides an opportunity to advance our knowledge on the

effect of traditional and innovative technologies on foods.

4.2 Methods

4.2.1 Fresh cut apple samples

Golden delicious apples (Malus domestica cv. Golden Delicious) were purchased

from the local market. Apples of uniform size and color were used in the

experiments. Fruits were washed with tap water, rinsed and air-dried. All cutting

utensils were sanitized with ethanol (99.8%) prior to use. The washed-apples were

cut into 55±2 mm long cylinders of 5 mm diameter, using a sharp stainless steel

corer. The length of the cored apple cylinder was cut to 35 mm. The cylindrical apple

samples from different apples were treated with dipping treatments or light

treatments. Each experiment was replicated 3 times.


92

4.2.2 Dipping treatments

Dipping treatments were carried out at room temperature in aqueous solutions

containing increasing concentrations of ascorbic acid from 1 to 3 % (w/v) (Sigma

Aldrich, Steinheim, Germany). Additional control samples were dipped in distilled

water. After 60 s dipping, samples were removed from the solution. Excess surface

moisture was removed by drying with a cold air blower. Samples were then

introduced in the calorimetric ampoules and hermetically sealed.

4.2.3 Pulsed light treatments

Pulsed light (PL) treatments were carried out by using a pulsed light mobile

decontamination unit (Claranor, Rouaine, France) equipped with 4 xenon lamps (JA

series, Verre et Quartz, Bussy Saint Georges, France) with maximum emission in

the range 200–1000 nm (200–400 nm: 41%; 400–700 nm: 51%; 700–1000 nm: 8%).

Apple samples were placed on a 5 mm thick quartz plate at a distance of 10 mm

from the lamps positioned above, below and at the two sides of the sample, and

exposed at increasing light fluence up to 175.0 kJ/m2, by modifying capacitor voltage

(1000-3000 V). Each light pulse had a duration of 50 µs and a frequency of 0.5 Hz.

After treatments, samples were incubated for 8 hours in the icebox, then, introduced

into the calorimetric ampoules and hermetically sealed.

4.2.4 UV-C treatments

UV-C light treatments were carried out using 15 W lamps (OF, OSRAM, GmbH,

Germany) with a maximum emission of 253.7 nm. UV-C lamps were positioned into

a thermostated cell (Climacell 222, MMM Medcenter, Einrichtungen GmbH,

Graefelfing, Germany) operating at 8 °C and equipped with a system of air moisture

control settled at 95% ERH to avoid sample dehydration during the treatment. Apple

samples were exposed between two parallel UV-C lights for increasing time up to

120 min. Relevant fluence on the samples was equal to 6, 12 and 24 kJ/m2. After

treatments, samples were incubated for 8 hours in the icebox, then, introduced into

the calorimetric ampoules and hermetically sealed.


93

4.2.5 Isothermal microcalorimetry

A TAM III isothermal heat conduction calorimeter (TA instruments, New castle,

Delaware, USA) was used. The instrument is a multichannel microcalorimeter able to

simultaneously analyse 24 ampoules. The 4 ml glass ampoules were used. Apple

samples were placed inside the ampoules, crimp sealed, and positioned inside the

microcalorimeter. After an equilibration time of 45 min, the heat flow signal emitted

from the ampoule containing the sample was measured at 30 °C until the signal

dropped to approximately zero. The heat flow data for each treatment were

normalized on the basis of the sample weight.

The enthalpy change (ΔH) of the overall metabolic response can be estimated from

integration of the heat flow ( ) during the experiment time t (Wadsö et al. 2004).

reactionofmole

dtH

t

t t 0

(4.1)

Finally, with the knowledge of the enthalpy of the process, the heat flow curve

provides a direct estimate of the rate of the process (r) (O’Neill et al. 2004):

Hr

(4.2)

4.2.6 Oxygen measurement A Fibox 4-trace fiber-optic oxygen meter (PreSens GmbH, Regensburg, Germany)

equipped with inner pressure sensors (10-1200 mbar), 5mm Pst3 luminescence

oxygen sensors, resistance temperature detectors PT 100 (0-50°C) and 2 mm

PMMA fibers was used to measure the oxygen consumption in the same ampoules

as used in the calorimeter. The Pst3 oxygen sensors were used for oxygen

concentration ranges from 0-100% with LOD 0.03% and response time < 6 s. The

sensors were glued to the inner surface of the ampoule with silicone (RS

components, Mörfelden-Walldorf, Germany) at ½ height between the bottom and

neck before the experiment. Two-point calibration in oxygen-free environment and

air-saturated environment was used. Oxygen free water (100 mL) was obtained by

mixing 1 g sodium sulfite (Na2SO3) and 50 µL cobalt nitrate (Co(NO3)2) standard

solution (1000 mg/L in nitric acid 0.5 mol/L). Air saturated water was obtained by

bubbling air, while stirring the solution. To prove the accuracy of the sensors, oxygen


94

measurements were performed in ampoules filled with nitrogen and ambient air prior

to measurement.

Cylindrical apple samples were placed into the ampoule and sealed with an

aluminum cap to monitor the oxygen kinetics. The calorimetric ampoules were kept

in a water bath at controlled temperature (30.0°C ± 0.3). The polymer optical fibers of

Fibox 4-trace were fixed perpendicularly on the sensors spot from the outside of the

ampoule. The Fibox 4-trace is completely stand-alone device, which was controlled

with supervisory PC. Oxygen concentration inside the ampoule was recorded in PC

with an interval of 30 minutes during the measurement using data manager software

version 2.0.0.57 (©GmbH, Germany).

4.3 Results and Discussions

4.3.1 Calorimetric signal

Figure 4.1 shows the heat flow curves apple cylinders, after treatment with a solution

of ascorbic acid.

Figure 4.1. Calorimetric heat flow from fresh-cut apple samples after dipping in

ascorbic acid (1%) solution for 1 min.


95

The heat-flow signal of Figure 4.1 must be viewed as the result of a set of complex,

and still not well understood, reactions, involving phenol metabolites, peroxidases,

ethylene and other factors such as plant hormones, overall controlled by

environmental factors(Ke and Saltveit 1989; Peña-Cortés and Willmitzer 1995;

Tomás-Barberán et al. 1997; Saltveit 2000; Chung and Moon 2009). For the sake of

simplicity, the calorimetric signal was described by the sum of three contributions,

namely, two oxidative and one anaerobic processes. The first contribution (a – b)

fades exponentially. The early events after cutting include an increase in respiration,

sugar content and production of ethylene (Martinez and Whitaker 1995). Also, the

browning of the wounded tissue suggests the contribution of phenolic compounds

and polyphenol oxidase (Murata et al. 1995; Rocha and Morais 2002; Holderbaum et

al. 2010). . The second contribution to the overall calorimetric signal is a shoulder in

b – c. A similar behavior of fresh-cut carrots was reported by (Wadsö and Gómez

Galindo 2009) and by Galindo on potato slices (Galindo et al. 2005) and described

heat flow as proportional to the wounded surface. Finally, the last portion of the

thermograms reveals a sudden decline of the calorimetric signal that reaches a

minimum thermal power (d). This is likely associated with the remaining activity of

the fruit under near anaerobic conditions see Figure 4.2.

Figure 4.2. Rate of oxygen consumption (solid line) as determined from the thermograms of Figure 4.1 by equation (4.3 in Figure it is 3). Dashed lines: calculated concentration (%) of O2. Solid circles: measured concentration of O2. inset, the correlation of calculated and measured concentrations of O2 (r2 = 0.99).


96

4.3.2 Oxygen consumption

Hansen and Criddle proposed a simple relationship based on the Thornton’s rule

that correlated the rate of respiration with the thermal power (Criddle and Hansen

1999; Hansen et al. 2004).

mPTR

HrO

9106.32

(eq. 4.3)

where r is the rate of respiration expressed as mL of O2 per kg of fruit per hour, is

the thermal power in Watts, ΔH is the heat generated from the tissue in agreement

with the Thornton’s rule (4.55 105 J mol-1), R is the gas constant (0.082 atm L / K

mole), T is the temperature (303 K), P is the pressure (1 atm) and m is the mass of

sample (0.722 g). The factor 3.6 109 is a proportionality factor to express the rate in

mL of O2 per kg of fruit per hour. Their relationship assumes the main reaction

involved in the process is O2 reduction toH2O by organic substrates. Figure 4.2

compares the rate of oxygen consumption derived from eq. 4.3 and that obtained by

direct oxy-metric measurements. The capacity of calorimetry to predict oxygen

consumption is very good (r2 = 0.99), as shown in the inset of Figure 4.2.

The consumption of O2 (Figure 4.2) in a – b is up to 35 mL / kg h, consistent with that

reported by (Tappi et al. 2014) with fresh cut apples in similar conditions. According

to the literature, one of the most immediate responses to wounding in fruit tissues is

an increase in respiration, ethylene production and oxidation of the pre-existing

phenols (Toivonen and Brummell 2008).

Later, in b – c, the calorimetric signal formed a shoulder, when oxygen consumption

is maintained at a rate about 24 mL / kg h and the O2 concentration dropped from

15% to 8%. It is not clear which biochemical events are likely to occurs in this tract.

However, Toivonen has recently identified a secondary browning period in fresh-cut

apples (Toivonen 2004). In contrast to the primary browning that occurs within hours

of cutting, secondary browning begins to appear in fresh-cut fruit at any time after 1

to 3 weeks in storage, the exact time being governed by the temperature history of

the product.


97

In the last period, the content of O2 in the ampoule is lower than 5% (Figure 4.2) and

the rate of O2 consumption is as low as 2.5 mL / kg h, consistent with anaerobic

metabolism (Mathooko 1996; Yearsley et al. 1996; Fagundes et al. 2013).

4.3.3 Fitting of the experimental points

The modelling of fruit metabolism is of main interest for the manufacturer since it is

an expedient to improve process conditions and the quality of the fresh-cut fruit. An

empirical approach to model calorimetric signals is based on fitting the data to well-

known equations. Obviously, such an approach does not explain the actual

mechanism of the processes, but is useful from a practical standpoint. To describe

the early rapid decay of the calorimetric signal (a – b), an exponential decay function

was selected (Willson et al. 1995; Riva et al. 2001).

The last part of the calorimetric signal was attributed to anaerobic metabolism of the

fruit. After about 20 h from the insertion of the fruit into the ampoule, oxygen reached

values below 5%, and, the tissue enters a regime of anaerobic metabolism

(Jacobsson 2004). To account for the heat flow generated by this last process, a

sigmoidal curve, such as obtainable with a Gompertz function, was chosen.

After subtraction of these two functions, the remainder of the heat flow was fit by a

single Gaussian function, as depicted in Figure 4.1 (see functions I, II and III). In

particular, the Gaussian function accounted for the degradation reactions occurring

in the central part of the thermograms, from 4 to 20 h. The sum of these functions

gave a reasonable fit to the observed values (r2 = 0.995).

The observed heat is not a result of microbiological spoilage. The contribution of

microorganism growth to the calorimetric signal was neglected because the

preparation of the samples (washing, cutting, dipping, drying and storing in closed

ampoules) was performed under sterile conditions. Moreover, the pH of the fruit

(~3.5), the natural content of organic acids and the presence of ascorbic acid greatly

decrease the chance of spoilage and hamper germination of potential spores. For

these reasons, under the timeframe of the experiments, the calorimetric signal was

associated only with biochemical events in the fruit tissues.


98

4.3.4 Effect of Ascorbic acid

The effect of ascorbic acid on the calorimetric signal is shown in Figure 4.3, together

with the changes in the overall heat (see inset). From comparison of the observed

data, the main effect of ascorbic acid is to reduce the extent of the calorimetric signal

and, consequently, the overall amount of reaction.

Figure 4.3. Heat flow from apple samples dipped in aqueous solutions containing

increasing concentrations of ascorbic acid (AA: 0, 1.0, 1.5, 2.0, 2.5 and 3.0%). Solid

lines: experimental measurements. Circles: fitted values. The arrow indicates

increasing concentration of ascorbic acid. Inset: enthalpy values as a function of

ascorbic acid concentration (r2 = 0.96).

Figure 4.3 also shows (as circle points) the values obtained by fitting the observed

data by the three functions previously described an iterative non-linear regression

procedure. Interestingly, regardless of the concentration of ascorbic acid used in the

dipping treatment, the areas of the exponential decay (function I) and the logistic

function (function III) are near constant see Figure 4.4. However, the Gaussian

function showed a trend that is proportional to the concentration of the ascorbic acid

used in the dipping treatment.


99

Figure 4.4. Enthalpy values from the (I) exponential, (II) Gaussian and (III) sigmoidal

fitting curves of Figure 4.3 as a function of ascorbic acid concentration.

Figure 4.5 shows the Gaussian function derived from the fittings of Figure 4.3. The

dimensions of the Gaussian function become progressively reduced as the ascorbic

acid concentration increased.

Figure 4.5. Curves of function II obtained after deconvolution of the heat flow curves

of Figure 4.3. Letters from a to f correspond the concentration of ascorbic acid used


100

in the treatment of fresh cut apple samples, respectively, 0, 1.0, 1.5, 2.0, 2.5 and

3.0%.

There is a significant correlation between the areas of the Gaussian function and the

concentration of ascorbic acid used in the dipping treatment. Since the fitting

functions I and II were near constant, the Gaussian area is a direct estimate of the

capacity of ascorbate to prevent browning reactions (El-Shimi 1993). Such capacity

is supported by a considerable amount of literature (Zanoni et al. 1995; Schiraldi

2003), that explains how ascorbate ions decrease browning incidence by reducing o-

quinones back to phenolic compounds prior to their polymerization into colored

pigments ( oli a- ortun and art n-Belloso 2003). However, what is striking here is

that the empirical deconvolution of the heat flow signal allows to describe the

contribution of ascorbic acid without the need of any assumption on the kinetic

theory of the process.

Similar results were found by Gómez (Gómez et al. 2004) who reported the heat-flow

of respiration of carrots before and after blanching. Blanching with hot water (100°C)

inactivates the polyphenol oxidase. In that work, the heat flow signal of carrot slices

was very similar to that observed in Figure 4.1 on fresh cut apples. In particular, the

calorimetric signal reached a plateau between 8-16 h, followed by a last anaerobic

period, similar to the case discussed here. However, when the samples were

blanched for even 5 s, the intensity of the plateau region greatly decreased, with an

effect equivalent to that observed in the present work with ascorbic acid. Again, the

control of browning reactions resulted in the suppression of the calorimetric signal.

4.3.5 Application of light treatments

Microcalorimetry was tentatively applied on apple samples treated by UV-C light and

pulsed light. Both are physical treatments that are plied on fresh cut fruits with the

purpose inactivating enzymatic activity on the surface of the fruit tissues. Figure 4.6

shows the resulting calorimetric signal of apple samples treated with pulsed light or

UV-C. Both treatments have a lower signal than the control. Consistent with the


101

literature, this research found that exposures to UV-C light and pulsed light reduce

fruit respiration (Manzocco et al. 2011, 2013).

When samples were irradiated with 175 kJ/m2 of pulsed light, the resulting heat flow

lower than that observed for samples irradiated with 87.5 kJ/m2 and much lower than

the control experiment (Figure 4.6). Exposure to higher irradiance is thus associated

with a substantial decrease in the reaction rate, likely through the inactivation of

reactions involved in the browning of the tissues (Ignat et al. 2014).

Figure 4.6: Heat flow profiles of apple (samples) after treatment with UV light and

pulsed light (PL). The inset shows the total amount of heat production during

calorimetric analysis.

However, when the apple samples were irradiated with increasing doses of UV-C

light, the resulting apple reaction lasted longer. In particular, the treatment with the

lowest fluence (6.0 kJ/m2) led to samples producing the highest heat flow but shorter

reaction time. Treatments with a higher fluence resulted in a reduction of the heat

flow, but associated with a longer exothermic process.


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The Gaussian function (II) used previously is no longer applicable. The process

observed here has the effect of extending the exothermic event, although reducing

the total heat released. Such behavior is different from that observed with the dipping

treatment with ascorbic acid (Ignat et al. 2014). Accordingly, for these specific cases,

it is impossible to apply the three fitting functions used for the case of ascorbic acid,

since, in this case, the physical nature of the treatment is completely different.

4.4 Conclusion

This work described the use of microcalorimetry as a mean of quantifying the

processes on cut fruits. When the cut fruits were treated with ascorbic acid, pulsed

light or UV-C treatments, a sharp decrease in the heat flow occurred. This change on

the heat flow signal was proportional to the concentration of ascorbic acid or pulsed

light dose used. The combination of microcalorimetry data with those obtained with

other techniques (ethylene production, pH, color, texture, taste. etc.) can be of great

interest for the producer because this will contribute to gain necessary knowledge for

process development, optimization and computer simulation in the food industries

predicting and ensuring the quality and shelf life of this type of foods.

4.5 Acknowledgments

We acknowledge the industry Fructus Vilpiano, Italy for their technical support. We

thank the Province of Bolzano for financial support (Landesregierung mittels

Beschluss Nr. 1472, 07.10.2013).


103

4.6 References

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Rico, D., Martin-Diana, A. B., Barat, J. M., & Barry-Ryan, C. (2007). Extending and measuring the quality of fresh-cut fruit and vegetables: a review. Trends in Food Science & Technology, 18(7), 373-386.

Chung, H. S., & Moon, K. D. (2009). Browning characteristics of fresh-cut ‘Tsugaru’apples as affected b pre-slicing storage atmospheres. Food Chemistry, 114(4), 1433-1437.

Criddle, R. S., & Hansen, L. D. (1999). Calorimetric methods for analysis of plant metabolism. Handbook of thermal analysis and calorimetry, 4, 711-763.

El-Shimi, N. M. (1993). Control of enzymatic browning in apple slices by using ascorbic acid under different conditions. Plant Foods for Human Nutrition, 43(1), 71-76.

Fagundes, C., Carciofi, B. A. M., & Monteiro, A. R. (2013). Estimate of respiration rate and physicochemical changes of fresh-cut apples stored under different temperatures. Food Science and Technology (Campinas), 33(1), 60-67.

Galindo, F. G., Rocculi, P., Wadsö, L., & Sjöholm, I. (2005). The potential of isothermal calorimetry in monitoring and predicting quality changes during processing and storage of minimally processed fruits and vegetables. Trends in food science & technology, 16(8), 325-331.

Gómez, F., Toledo, R. T., Wadsö, L., Gekas, V., & Sjöholm, I. (2004). Isothermal calorimetry approach to evaluate tissue damage in carrot slices upon thermal processing. Journal of Food Engineering, 65(2), 165-173.

Gomez-Lopez, V. M., Ragaert, P., Debevere, J., & Devlieghere, F. (2007). Pulsed light for food decontamination: a review. Trends in food science & technology, 18(9), 464-473.

Hansen, L. D., Macfarlane, C., McKinnon, N., Smith, B. N., & Criddle, R. S. (2004). Use of calorespirometric ratios, heat per CO 2 and heat per O 2, to quantify metabolic paths and energetics of growing cells. Thermochimica Acta, 422(1), 55-61.

Holderbaum, D. F., Kon, T., Kudo, T., & Guerra, M. P. (2010). Enzymatic browning, polyphenol oxidase activity, and polyphenols in four apple cultivars: dynamics during fruit development. HortScience, 45(8), 1150-1154.

Ignat, A., Manzocco, L., Maifreni, M., Bartolomeoli, I., & Nicoli, M. C. (2014). Surface decontamination of fresh-cut apple by pulsed light: effects on structure, colour and sensory properties. Postharvest Biology and Technology, 91, 122-127.

Jacobsson, A. (2004). Quality aspects of modified atmosphere packaged broccoli. Annelie Jacobsson, Lokföraregatan 15C, 222 37 Lund, Sweden, or SIK, Box 5401, 40229 Göteborg, Sweden,.

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Kader, A. A. (2002). Quality parameters of fresh-cut fruit and vegetable products. Fresh-cut fruits and vegetables, 11-20.

Ke, D., & Saltveit, M. E. (1989). Wound‐induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce. Physiologia Plantarum, 76(3), 412-418.

Laurila, E., Kervinen, R., & Ahvenainen, R. (1998). The inhibition of enzymatic browning in minimally processed vegetables and fruits. Postharvest news and information, 9(4), 53-66.

Manzocco, L., Panozzo, A., & Nicoli, M. C. (2013). Inactivation of polyphenoloxidase by pulsed light. Journal of food science, 78(8), E1183-E1187.

Manzocco, L., Da Pieve, S., Bertolini, A., Bartolomeoli, I., Maifreni, M., Vianello, A., & Nicoli, M. C. (2011). Surface decontamination of fresh-cut apple by UV-C light exposure: Effects on structure, colour and sensory properties. Postharvest Biology and Technology, 61(2), 165-171.

Martinez, M. V., & Whitaker, J. R. (1995). The biochemistry and control of enzymatic browning. Trends in Food Science & Technology, 6(6), 195-200.

Mathooko, F. M. (1996). Regulation of respiratory metabolism in fruits and vegetables by carbon dioxide. Postharvest Biology and Technology, 9(3), 247-264.

Murata, M., Tsurutani, M., Tomita, M., Homma, S., & Kaneko, K. (1995). Relationship between apple ripening and browning: changes in polyphenol content and polyphenol oxidase. Journal of Agricultural and Food Chemistry, 43(5), 1115-1121.

O’Neill, . A. A., Beezer, A. E., itchell, J. C., Orchard, J., & Connor, J. A. (2004). Determination of Michaelis–Menten parameters obtained from isothermal flow calorimetric data. Thermochimica acta, 417(2), 187-192.

Peña-Cortés, H., & Willmitzer, L. (1995). The role of hormones in gene activation in response to wounding. In Plant hormones (pp. 395-414). Springer Netherlands.

Riva, M., Fessas, D., & Schiraldi, A. (2001). Isothermal calorimetry approach to evaluate shelf life of foods. Thermochimica Acta, 370(1), 73-81.

Rocha, A., & Morais, A. M. (2002). Polyphenoloxidase activity and total phenolic content as related to browning of minimall processed ‘Jonagored’apple. Journal of the Science of Food and Agriculture, 82(1), 120-126.

Saltveit, M. E. (2000). Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biology and Technology, 21(1), 61-69.

Schiraldi, A. (2003). Phenomenological Kineticsan; an alternative approach. Journal of thermal analysis and calorimetry, 72(3), 885-900.

Soliva-Fortun , . C., art n-Belloso, O. (2003). New advances in extending the shelf-life of fresh-cut fruits: a review. Trends in Food Science & Technology, 14(9), 341-353.

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(2014). Atmospheric gas plasma treatment of fresh-cut apples. Innovative Food Science & Emerging Technologies, 21, 114-122.

Toivonen, P. M., & Brummell, D. A. (2008). Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biology and Technology, 48(1), 1-14.

Toivonen, P. T. A. (2004). Postharvest storage procedures and oxidative stress. HortScience, 39(5), 938.

Tomás-Barberán, F. A., Loaiza-Velarde, J., Bonfanti, A., & Saltveit, M. E. (1997). Early wound-and ethylene-induced changes in phenylpropanoid metabolism in harvested lettuce. Journal of the American Society for Horticultural Science, 122(3), 399-404.

Wadsö, L., Gomez, F., Sjöholm, I., & Rocculi, P. (2004). Effect of tissue wounding on the results from calorimetric measurements of vegetable respiration. Thermochimica acta, 422(1), 89-93.

Wadsö, L., & Galindo, F. G. (2009). Isothermal calorimetry for biological applications in food science and technology. Food Control, 20(10), 956-961.

Willson, R. J., Beezer, A. E., Mitchell, J. C., & Loh, W. (1995). Determination of thermodynamic and kinetic parameters from isothermal heat conduction microcalorimetry: applications to long-term-reaction studies. The Journal of Physical Chemistry, 99(18), 7108-7113.

Yearsley, C. W., Banks, N. H., Ganesh, S., & Cleland, D. J. (1996). Determination of lower oxygen limits for apple fruit. Postharvest biology and technology, 8(2), 95-109.

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5 Free-radical scavenging capacity using Fenton reaction by reaction calorimetry

106

Chapter V

5 Free-radical Scavenging Capacity using Fenton reaction by Reaction Calorimetry


107

Abstract

A reaction calorimetric investigation was performed to determine antioxidant free-

radical scavenging capacity. A major cause of food oxidation by free-radical

generated via Fenton reaction. Here, reaction calorimetry assay (measurement of

heat flow and heat evolved during the reaction between food antioxidants and

oxidant (i.e. H2O2)) has been modified by adding Fe2+ (0.25 mM), which makes the

oxidation reaction ten times faster. Such an assay can stimulate the oxidation

reaction in foods and food ingredients in presence of Fe2+. It thus may be more

relevant than other assay of antioxidant capacity, as it measures the heat flow by

direct monitoring the reaction between foods and hydrogen peroxide without time-

consuming sample pre-treatment protocol. This assay allows to measure the free-

radical scavenging capacity of antioxidant compounds, fruit juices, fruit purees, tea

and coffee. The acceptability of the assay was assessed through the study of the

reaction between ascorbic acid and hydrogen peroxide in presence of Fe2+ at

different concentrations and pH at 250C.

Key words: reaction calorimetry; antioxidant capacity; hydrogen peroxide (H2O2);

food; peroxide scavenging capacity.

This work is under submission to a scientific journal


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5.1 Introduction

Free radicals are produced from oxidation in foods, chemical and/or living systems

(McCord, 2000). Several literatures are reported the role of free radicals, involved in

both positive and negative function in biological system such as act in cell signalling,

defence against infectious agents and cause cellular damages (Poli, Leonarduzzi,

Biasi, & Chiarpotto, 2004; Valko, Rhodes, Moncol, Izakovic, & Mazur, 2006; Van

Campenhout et al., 2006). Among different free radicals, hydroxyl radical is

extremely reactive and short-lived species. The system containing metal in the

reduced form with H2O2 (Fenton type system) may lead to the production of a strong

oxidant (i.e. hydroxyl radical, •OH).

𝐹𝑒2+ + 𝐻2𝑂2 → 𝐹𝑒3+ + 𝐻𝑂− + 𝐻𝑂∙ (5.1)

The Fenton reaction is selected as radical source to measure the antioxidant

capacity in a manner similar to the in vivo generation of hydroxyl radical. This

approach is an extensively researched issue due to its application in biochemistry

and medicine (Halliwell & Gutteridge, 1990; Lubec, 1996), foods (Donnelly &

Robinson, 1995; Yu, Xu, Lei, Li, & Li, 2008) and environment chemistry (Bolobajev,

Trapido, & Goi, 2015; Sun & Pignatello, 1992), and several reviews (Sánchez-

Moreno, 2002; Vanderhaegen, Neven, Verachtert, & Derdelinckx, 2006; Wardman &

Candeias, 1996) are available on this subject. In this study, the free-radical

scavenging capacity may be used to assess the antioxidant capacity in foods and

food ingredients.

Antioxidants, which scavenge free radical (Wood, Gibson, & Garg, 2006), retard

oxidation reaction (Shahidi, 2000), preserve nutritional quality (Nicoli, Anese, &

Parpinel, 1999), and promote health status (Frankel & Meyer, 2000). Several

methods have been developed to measure antioxidant capacity of foods and food

ingredients. Depending on the antioxidant functionality, three main analytical

methods are developed for measuring the antioxidant capacity in foods or biological

systems. First, the measurement of the hydrogen atom transfer (HAT) capacity of

samples, such as DPPH (1,1-diphenyl-2picrylhydrazyl), ORAC (Oxygen radical

absorbance capacity) and TEAC (Trolox equivalent antioxidant capacity) assays

(Miller, Rice-Evans, Davies, Gopinathan, & Milner, 1993; Ou et al., 2002). Second,

the measurement of the electron transfer (ET) capacity include Folin and FRAP


109

(Ferric reducing antioxidant power) assays (Prior, Wu, & Schaich, 2005). A third type

of method based on electrochemical kinetic that allows the simultaneous

determination of the HAT and ET capacity (Lemma, Scampicchio, Bulbarello, Mason,

& Schweikert, 2014). However, all these methods are required time-consuming

extractions, pre-treatments protocol, as well as they depend on the nature of the

samples such as turbidity and viscosity. In addition, experimental conditions may

vary from one to another (i.e pH, solvents, wavelength of the measurement), yielding

different results, which are main drawbacks.

Recently, we proposed reaction calorimetry method using innovative chemical

process analyser (CPA202) instrument based on heat released from the system for

measuring antioxidant capacity of foods that overcome such drawbacks (Kamrul,

Schiraldi, Cosio, & Scampicchio, 2015). This instrument was originally developed by

ChemiSense® (Nilsson & Hess, 2008). We applied the concept of measuring heat

flow (W) and heat (J) evolved during the reaction between food antioxidants and

oxidant (i.e. hydrogen peroxide). We have modified this assay as iron-catalysed

antioxidant activity to measure the heat flow in the oxidation reaction by adding

ferrous ions. The presence of metal (Fe2+) in the system may greatly influence the

antioxidant activity foods, involving promotion of free-radical production due to its

chelation properties (Puppo, 1992). With this modified assay, we have measured

free-radical scavenging capacity based on heat released from the system. The

resulting heat is used an index of antioxidant capacity of foods. Foods with the

highest scavenging activity is produced the highest heat of reaction when mixed with

oxidant. This work presents radical scavenging capacity of some antioxidant

compounds and commercial food products. Our hypothesis is to evaluate that

highest the heat of reaction, higher the free-radical scavenging capacity.

5.2 Materials and methods

5.2.1 Materials

Ascorbic acid (AA) was of analytical grade (Sigma Aldrich). Stock solutions of

ascorbic acid (5, 10, 25, 50, 100, 200, 500 and 1000 mM)) were prepared by

dissolving the desired amount in distilled water (18 MΩ). For the RC investigation

1ml of each solution was mixed with 1ml of H2O2 (30% w/w ≈ 9.8 M) and added to


110

100ml of aqueous buffer. Therefore, the stating concentrations of sample were 100

times smaller. The Britton-Robinson buffer in the pH ranges of 2 – 7 was prepared

from an equal mixture of 0.04 M boric acid, 0.04 M phosphoric acid and 0.04 M

acetic acid. The pH value was adjusted using a 0.2 M NaOH solution. Reagents

were purchased from Sigma Aldrich.

Fruit juice samples (apple, orange, and apricot), baby fruit puree (plum and apple),

tea (green and white) and coffee were purchased from local market. Tea were

prepared by soaking the powder sample (contained in a paper bag, net content, 1 g)

in 50 mL of boiling water for 5 min. Gallic acid, caffeic acid, vanillic acid, catechin,

quercetin, oleuropein, and trolox were of analytical grade from Sigma Aldrich. The 25

mM stock solutions of these antioxidants were prepared by dissolving the desired

amount in distilled water (18 MΩ) and ethanol (Sigma Aldrich, 99.8%). 1ml or 1 g of

all these samples were diluted (dispersed) in 100 ml Brittion-Robinson buffer at pH

6.5.

5.2.2 Reaction calorimetry apparatus

The reactor calorimeter CPA 202 (ChemiSens AB, Lund, Sweden) developed by

ChemiSense (Nilsson & Hess, 2008), supresses uncontrolled temperature

differences between the content of the reactor and its surrounding jacket. This

means that all the heat from the reactor to the thermostat bath flows through the heat

transducer, which is located between the bottom of the reactor and a Peltier element

used as a heat pump CPA 202 performs rather well with either solid or liquid

samples, without requiring any sample pre-treatment (Nilsson & Hess, 2008).

The reaction cell (250 mL) is made by a double-walled pyrex glass, a base of

Hastelloy and a lid of tightly-proof. The reactor was equipped with a stirrer, a torque

transducer and a true-heat flow sensor. The heat-flow signal is the on-line output

monitored with the help of ChemiCall v2 ProFind® software. The instrument is

calibration-free throughout the experiment. The highly stable baseline returns to

±0.0001 W at the end of the reaction. The generation of an electrical heat (from

0.007 to 0.04 W, provided by an internal electrical power heater) was used, from


111

time to time, to verify the performance of the instrument. The accuracy was always

higher than 99%. The time constant is around 20 s.

5.2.3 Optimized procedure

Isothermal condition was achieved by immersing the CPA reactor into a

thermostated bath set at 25°C. Prior of the analysis, the samples and the hydrogen

peroxide reagent were contained in a syringe, whose temperature was equilibrated

by immersing it in the same thermostated bath. Before each measurement, the

reactor cell was filled with 0.1 L of buffer solution containing 0.25mM FeSO4 (Britton-

Robinson, pH 6.5), de-aerated and tightly closed with lid, and immersed into the

thermostated bath. The stirrer rate of the reactor was set at 200 rpm. Once attained

the thermal equilibrium (indicated by a stable baseline: background signal ≤ 0.1

mW), 1 mL of sample was injected in the reactor cell with a syringe. After

stabilisation rest (~30 min), 1 mL of H2O2 (30%w/w) was added into the reactor cell

and the resulting thermal power signal (W) monitored. The integral of the HeatFlow-

vs-time yield the heat of the reaction (J).

5.3 Results and discussions

5.3.1 Reaction calorimetry of the Fenton reaction

Mixing of H2O2 (98 mM) with increasing concentrations of ascorbic acid (0.05 – 10.0

mM) in buffered solutions (pH 6.5) containing a fixed amount of Fe2+ (0.25 mM) led

to generate the heat flow profile shown in Figure 5.1. The resulting heat flow profile

was ten times faster that the heat flow observed in buffer solution without Fe2+ (data

not shown). The concentration of Fe2+ showed the catalytic effect on the system, this

takes into account that metal ions are known to accelerate the oxidation of AA

sustained by H2O2, likely because they take a role in the formation of free radicals.


112

Figure 5.1: Isothermal RC records of different concentration of AA (r2 = 0.998)

The heat flow (HF) signal increases with increasing the concentration of ascorbic

acid (AA). The inset of Figure 5.1 shows the corresponding integral value (Q/J) of

heat flow-vs-time curve. The overall heat (integral value) is proportional (r2 = 0.998)

to the stating concentration of AA with or without Fe2+ (inset). The overall thermal

effect is exothermic (positive sign throughout the text and figures). However, when,

the heat flow data are normalized with the mole of AA, the overall heat effect

decreases with increasing concentration of AA (Figure 5.2). This result suggests that

the decomposition of AA is associated with larger endothermic reaction in smaller

concentration of AA, and the overall heat exchanged per mole of AA is the result of a

balance of endo- and exothermic effects that underlying the mechanism of the

oxidation with AA/H2O2 molar ratio (Kamrul et al., 2015).


113

Looking at the literature (Bolobajev, Trapido, & Goi, 2015; Burkitt & Gilbert, 1990),

AA undergo oxidation to yield dehydroascorbic acid (DHA) through the formation of

intermediate radical species in presence of Fe2+/H2O2. Probably, ascorbate

monoanion of ascorbic acid in water undergoes a two-step oxidation to yield

dehydroascorbic acid through the formation of semidehydroascorate and ascorbyl

radical, which are responsible for autocatalytic transformation of Fe2+. A rapid

reducing ability of AA related to ferric iron may allow the formation of hydroxyl radical

according to the Fenton mechanism (Haber & Weiss, 1932). The suggested reaction

scheme from these authors may be correspond to the following scheme (a):

Figure 5.2: The integrated heat flow data of Figure 5.1


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Indeed, DHA can undergo further oxidation to form other molecule, like dikegulonic

acid, oxalic acid, tetradydroxydiketohexanoic acid, etc (Deutsch, 1998a). These

steps are much slower than the early oxidation of AA and may be overwhelmed by

the formation of free radicals. Since the calorimetric signal is very neat and looks like

the results of one single process, and the sum of a number of endo- and exothermic

effects of calorimetric outputs are difficult to single out. Assuming that the major

thermal effects come from the first step of AA oxidation, a reasonable kinetic model

for the whole process may correspond to the following simple scheme (b):

Where, k is known as apparent kinetic constant of the oxidation product of AA. In

presence of Fe2+/H2O2, a pseudo-first order kinetic may possibly describe this step.

The assumed kinetic model implies the following equations:

𝑑[𝐴𝐴]

𝑑𝑡= −𝑘[𝐴𝐴] with [𝐴𝐴] = exp(−𝑘. 𝑡) . [𝐴𝐴]0 (5.2)

Let’s define the heat flow signal according to following equation:

𝑃 = 𝑟𝑎𝑡𝑒. 𝑒𝑛𝑡ℎ𝑎𝑙𝑝𝑦 (5.3)

Where, P (dq/dt) is the heat flow signal (J/s), rate (d[AA]/dt) is conversion of AA

(mol/s) and enthalpy (ΔH) is the amount of heat exchanged per mole of AA

(kJ/mol)). Therefore, the relevant heat flow (Willson, Beezer, Mitchell, & Loht, 1995)

equation is:

𝑑𝑞

𝑑𝑡=

𝑑[𝐴𝐴]

𝑑𝑡 . ∆𝐻 = −𝑘. [𝐴𝐴]0. exp(−𝑘. 𝑡) . ∆𝐻 (5.4)

AA DHA k

Fe3+ Fe2+

H2O2 HO- + HO•

OHOH

HO

OH

O

O

O O

O

HO

OH

O


115

Now, the exponential decay of heat flow should allow satisfactory fit the calorimetric

signal, provided the earlier part of the record (50 s) is neglected, as reflect the lag

time of the calorimeter. The equation (5.4) has two variable (k and ΔH), which can be

obtained from the fitting of calorimetric trace. Table 5.1 reports the values of the

kinetic constants and reaction enthalpies and Figure 5.3 represents the fit obtained.

Figure 5.3: Fitted heat flow records collected from AA (0.05,0.10,0.25,0.50,1,00 and

2.00 mM in the calorimetric cell) according to model (r2 = 0.98) equation (5.4). For

larger concentration of AA, the fits are still satisfactory (r2 = 0.98).

Despite the satisfactory appearance of the fits, the estimated values of the kinetic

constants and enthalpy changes with AA (Table 5.1) do not lead to straightforward

interpretation. The values of the kinetic constants and enthalpy changes should not

change with concentration of AA. While the values of k coming from the best fitting

treatment may allow a likely be an average of 2.61 10-3 s-1, this is not the case of ΔH.

However, as for ΔH, a reasonable average value may be 3672 ± 213 kJ/mol. One

other possibility can try assuming a two consecutive process model. Adding such a

function to the expression of equation (5.4) may also allow to evaluate the relevant


116

model parameters. The valuves of ΔH2 shown in Table 5.1 tend to vanish with

increasing AA. This trend suggests that endothrimic effects related to the second

step tend to achive a counterbalance the exothermic ones for AA< 2.0 mM. On the

other hand, the the values of ΔH1 return approximately same like single step kinetic

model. Therefore, the overall thermal effect determined with RC tends to coincide

with the reaction enthalpy of fisrt steps. For this reason, other authors (Deutsch,

2000; Wilson, Beezer, Mitchell, 1995) suggest to describe the oxodation of AA as a

pseudo first order process with a single main thermal effect based on calorimetric

evidence collected in the presence of poorer oxidant concentration (either O2 or

H2O2). Figure 5.4 shows the corresponding trend of the molar fractions of the

species involved in the reaction.

Table 5.1: Kinetic constants and reaction enthalpies from the fits (r2 = 0.98) of the

experimental data according to equation (5.4).

AA H2O2/AA Single step Two steps

k/

10-3 s-1

ΔH/

kJ/mol

SD k1/

10-3 s-1

k2/

10-4 s-1

ΔH1/

kJ/mol

ΔH2/

kJ/mol

0.05 1960 4.407 5788 585 4.10 2.00 4907 -336

0.10 980 3.731 3202 356 3.65 2.20 2959 -592

0.25 392 2.843 2049 189 2.92 3.60 1919 -310

0.50 196 2.359 1695 135 2.52 3.80 1732 -57

1.00 98 2.039 1422 129 2.25 2.70 1515 -22

2.00 49 1.862 13092 125 2.21 2.50 1492 -14

5.00 19.6 1.635 1123 95 2.49 2.90 1214 -465

10.00 9.8 1.997 1005 91 2.90 3.50 1045 -792

The calculated value of k and ΔH comes from equation (5.4) taking into account that

the volume of the solution is 0.1L. AA concentrations are those in the calorimetric

cell. Standard deviation (SD) of enthalpy drops in the neighbour side columns.


117

Figure 5.4: Trend of the molar fraction of the compounds (AA = 0.50 mM) involved in

the free-radical scavenging process according to model equation (5.4)

5.3.2 pH effect on the rate of the Fenton Reaction

The total oxidation of AA in the Fenton system may depend on many factors, such

as pH, concentration of Fe2+ and H2O2. The effect of pH on the oxidation of AA is

shown in Figure 5.5 in which the heat flow signal collected from buffered solution

containing fixed amount of AA (0.50 mM), Fe2+ (0.25 mM) and H2O2 (98 mM) with

varying the pH (2 to 7). At lower pH = 2, Isothermal RC records show a sharp and

small peak, while the signal becomes broader and larger with increasing pH.

0

0.2

0.4

0.6

0.8

1

1.2

0

0.02

0.04

0.06

0.08

0.1

0.12

0 500 1000 1500 2000 2500

Mol

ar fr

actio

n

HF/

W

Time/ s

HF Fit AA DHA


118

Figure 5.5: Isothermal RC records from buffered solutions of AA at different pH

Once data are normalised with respect to per mole of AA, the overall heat (Q/ kJ.mol-

1) shows sigmoidal trend (Figure 5.6) as a function of pH. The overall oxidation heat

increases with increasing pH, and attains its maximum when pH = 7. This result

suggests that the scavenging activity is associated with different ionised form of AA

at a given pH. The ascorbic acid has two acidic protons with pKa 4.04 and 11.34

(Williams & Yandell, 1982), and the ascorbate anion (AscH-) may be truly reactive

species of the ascorbic acid. Where, AscH- may supposed to increase with

increasing pH, the signal from low pH being small since the fraction of AscH- may be

small. This is taken into account that the equilibrium of dissociation of AA (pKa =

4.04) and that ([AscH-] + [AA]) = [AA]0, can easily calculate

[𝐴𝑠𝑐𝐻−] = [𝐴𝐴]0 𝐾𝑎

𝐾𝑎+10−𝑝𝐻 (5.5)

The molar ration of AscH-/AA0 is parallel (Figure 5.6) with the sigmoidal trend of Q

that indicates a steady level of heat released at low and high pH. At pH 7.0, where,

the AscH- practically accounts for 100% of the ascorbic species, and the

corresponding Q values reflect the highest scavenging activity. This finding seems in

line with the low pH increase in oxidative deterioration of fish oil in presence of

ascorbate (Jacobsen, Timm, & Meyer, 2001). The resulting ascorbyl radical anion

0

0.02

0.04

0.06

0.08

0.1

0.12

0 500 1000 1500 2000 2500

HF/

W

Time/s

pH 2.0pH 4.0pH 5.0pH 6.0pH 7.0Fits


119

(𝐴𝑠𝑐 ∙−) (𝐹𝑒3+ + 𝐴𝑠𝑐𝐻− → 𝐹𝑒2+ + 𝐴𝑠𝑐 ∙− + 𝐻+ ) may be expected to disproportionate

and may form DHA by following the reduction of Fe3+ by ascorbate (Bielski, Allen, &

Schwarz, 1981; Macartney & McAuley, 1981).

Figure 5.6: The overall heat of AA at different pH levels.

5.3.3 Iron (Fe2+) effect on the rate of the Fenton Reaction

Since AA is found to be an effective iron-chelating agent to form hydroxyl radicals

(Mcnaught & Wilkinson, 2003), we studied the effects of Fe2+ on heat released in the

proposed Fenton-based approach. Figure 5.7 shows the heat flow recorded from

buffered solution (pH 6.5) containing fixed amount of AA (0.50 mM) and H2O2 (98

mM) with increasing concentration of Fe2+ (0.01 - 2.00 mM). The resulting heat flow

signal increases with increasing concentration of Fe2+. Inset of Figure 5.7 shows the

corresponding integral value (Q/J) of heat flow-vs-time curve, which shows a straight

line with r2 = 0.93. When the data are scaled with respect to per mole of Fe2+, the

overall thermal effect decreased as the Fe2+ concentration increased (inset). These

results reflect same interpretation that the overall heat exchanged per mole of Fe2+ is

a balance of endo- and exothermic reaction like the effects of AA in calorimetric


120

approach. However, the overall heat released per mole of Fe2+ is larger in

comparison of per mole of H2O2 (see the effect of H2O2 below). This result suggests

that AA/Fe2+ complex is able to generate hydroxyl radical, and larger heat is

associated with the reaction between hydroxyl radical and Fe2+ compared the

reaction between hydroxyl radical and H2O2. Similar conclusion also reported on

Fenton based oxidation system (Neyens & Baeyens, 2003; Yu, Xu, Lei, Li, & Li,

2008).

Figure 5.7: Isothermal RC signal from buffer solution containing fixed amount of AA

(0.50 mM) and H2O2 (98 mM) at different concentration of Fe2+. Dashed lines are

drawn according to equation (5.4).

5.3.4 Hydrogen peroxide (H2O2) effect on the rate of the Fenton Reaction

The effects of H2O2 was also studied on Fenton calorimetric system, where the heat

flow signals were collected from buffer solution contained fixed amount of AA

(0.50mM) and Fe2+ (0.25mM) with increasing concentration of H2O2 . Figure 5.8


121

shows the heat flow signal increases with increasing the concentration but signal

drop down faster with higher concentration of H2O2 indicates shorter time of reaction.

The integral value (Q/J) of heat flow-vs-time curve shows a straight line with r2 = 0.91

(inset). Once data are scaled with respect to per mole of H2O2, the overall thermal

effect decreased with the concentration increase (inset). However, the overall heat

exchanged per mole of H2O2 is smaller, and this values lie within the limit of standard

error when we compared the overall heat exchanged per mole of AA and Fe2+.

Therefore, we may consider the effect of H2O2 is negligible on Fenton system based

on calorimetric study whatever the amount is used (Schiraldi, 2003).

5.3.5 Free-radical scavenging activity of antioxidant compounds

We applied the proposed method to study the free-radical scavenging behaviour of

different antioxidant agents. We studied both hydrophilic and lipophilic antioxidant

Figure 5.8: Isothermal RC signal from buffer solution containing fixed amount of AA (0.50 mM) and Fe2+ (0.25 mM) with different concentration of H2O2


122

compounds. Figure 5.9 shows the recorded heat flow signal from various

antioxidants. Each antioxidants exhibited different heat flow profile because of their

various radical scavenging activity level. The amount of heat released (Q/kJ.mol-1)

reflects the corresponding free-radical scavenging index of various antioxidants

(Table 5.2). The free-radical scavenging capacity follows the trend: quercetin>

ascorbic acid > gallic acid > oleuropein > trolox > vanillic acid > catechin > caffeic

acid. This trend correlates with the reports of free-radical scavenging capacity of

antioxidants obtained by spectrophotometric analysis with Rhodamine B with the

reference of AA (Yu et al., 2008) and by DPPH method (TEC50) with the reference of

AA (Villano, Fernández-Pachón, Moyá, Troncoso, & García-Parrilla, 2007).

Figure 5.9: Isothermal RC record from buffered solution containing fixed amount of

Fe2+ (0.25 mM) and H2O2 (98 mM) for different antioxidants.

We also studied the effect of solvent (Ethanol) on oxidation of lipophilic compounds

in Fenton calorimetry system. We noticed that increasing the amount of solvent,

increasing the heat flow signal (data not shown). This result suggests that solvent

may increase the solubility of antioxidants that may enhance the free radical

scavenging activity, resulting increased in heat flow. Table 5.2 shows the free radical

scavenging capacity of different antioxidant agents with their empirical kinetic

constants.

0

0.01

0.02

0.03

0.04

0.05

0.06

0 500 1000 1500 2000

HF/

W

Time/s

QuercetinGallic acidCatechinVanllic acidTroloxOleuropeinCaffeic acid


123

5.3.6 Free-radical scavenging capacity of food products

Next, the assay was applied on commercial beverage product fruit juice, baby fruit

puree, tea and coffee to investigate the free radical scavenging activity. The free

radical scavenging activities are expressed as the heat released from the system.

The heat flow profiles are shown in Figure 5.10. The overall heat obtained by

integrating of heat flow signal (Table 5.2), which reflects the radical scavenging

capacity. Among four commercial foods, the radical scavenging activity of plum

puree was highest followed by orange juice, coffee, apple juice green tea, apple

puree, apricot juice and white tea (Table 5.2). Plum contains higher amount of total

phenolics and flavonoids than apple (Kim, Jeong, & Lee, 2003) shows highest

scavenging activity.

Figure 5.10: Isothermal RC record from buffered solution containing fixed amount of

Fe2+ (0.25 mM) and H2O2 (98 mM) for different food products

In comparison of the two tea, green tea contain higher amount of polyphenol,

flavonoids and catechins than white tea (Unachukwu, Ahmed, Kavalier, Lyles, &

Kennelly, 2010). They also noticed that certain white teas have comparable

quantities of total catechins to some green teas but lower antioxidant capacity. Our

0

0.01

0.02

0.03

0.04

0.05

0.06

0 500 1000 1500

HF/

W

Time/s

Orange juiceApricot juiceApple juicePlum pureeApple pureeCoffeeGreen TeaWhite Tea


124

result shows the highest radical scavenging activity in green tea compared to white

tea, and this may be correlated with their content above-mentioned compounds. Our

results may also agree with that obtained by Yamaguchi et al. (1998) and Yen &

Chen (1995) who observed the radical scavenging effect of tea extracts on DPPH by

HPLC and spectrophotometric measurement. However, in case of coffee, there are

some losses of polyphenol compounds during roasting process, which could be the

reason for having lower antioxidant capacity of coffee than green tea (Richelle,

Tavazzi, & Offord, 2001).

Fruit juices are widely consumed as a daily beverage because of rich source of

vitamin C and total phenols which are known as powerful antioxidants (Gardner,

White, McPhail, & Duthie, 2000). In our results, orange juice and apple juice

produced highest heat of reaction than apricot juice, and this may correlates with

higher amount of vitamin C and total phenols in orange and apple juice (Klimczak,

Małecka, Szlachta, & Gliszczyńska-Świgło, 2007; Loots, van der Westhuizen, &

Jerling, 2006; Mahdavi, Nikniaz, Rafraf, & Jouyban, 2010). Other studies have also

shown that increase levels of total phenolic content bring increased antioxidant

ability (Proteggente, Saija, De Pasquale, & Rice-Evans, 2003).

No simple kinetic model seems appropriate to describe the radical scavenging

activity of foods because each food contains different antioxidant compound

(Gardner et al., 2000; Özkan, Kırca, & Cemeroǧlu, 2004; Unachukwu et al., 2010).

However, metal ions can catalyse the oxidation of all foods (Grinstead, 1960; Willson

et al., 1995). Most of the foods show scavenging effects that follows a first order

kinetic, which is purely empirical evidence with no physical meaning. Table 5.2

shows the kinetic constants of the free radical scavenging process.


125

Table 5.2: The free-radical scavenging capacity of antioxidant compounds and food

samples

Antioxidant Fitting results (r2 = 0.997) Scavenging capacity

[AA]/ µM equivalent

Pmax (W) k (10-3 s-1)

A (J) Qtotal

Antioxidant compounds

Quercetin 0.045 1.36 51.5 1997 ± 100 312

Ascorbic acid 0.076 2.86 40.0 1599 ± 90 250

Gallic acid 0.036 2.13 31.7 1271 ± 82 199

Oleuropein 0.052 3.68 17.2 687 ± 70 107

Trolox 0.050 3.43 16.3 652 ± 65 102

Vanillic acid 0.037 3.61 13.2 527 ± 58 82

Catechin 0.038 3.68 12.9 517 ± 54 81

Caffeic acid 0.031 3.63 10.2 408 ± 42 64

Product samples

Plum puree 0.051 3.24 18.2 17.3 ± 2.0 108

Apple puree 0.046 3.07 16.8 15.5 ± 1.7 107

Orange juice 0.049 3.07 18.3 17.1 ± 2.0 103

Apple juice 0.055 3.60 17.7 16.5 ± 1.8 98

Apricot juice 0.043 3.34 14.6 13.8 ± 1.6 97

Green tea 0.038 2.63 16.2 15.5 ± 1.5 96

White tea 0.029 2.64 12.2 11.7 ± 1.5 86

Coffee 0.032 2.20 15.9 15.4 ± 1.7 73

The free-radical scavenging capacity of antioxidant compounds expressed as kJ/mol

and for food samples J/ml or J/g.

5.4 Conclusion

The results reported an example of the potential use of a reaction calorimetric

method using an innovative instrument CPA 202 for the investigation of the free

radical scavenging capacity of foods samples. The method has been tested with

standard antioxidant compounds, and directly applied to commercial foods without


126

sample preparation steps such as pre-treatment and extraction. The results of this

assay can correlate with other assay such as spectrophotometry, ORAC, DPPH,

HPLC. The main advantage of the assay can apply on any kind of biological

samples, regardless to their physical form such as solid, liquid or viscous without

need of preliminary sample preparation steps. The method showed great potential to

test a number of materials, such as additives, food ingredients and pharmaceuticals.


127

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6 Conclusions and future prospects

131

Chapter VI

6 Conclusions and Future Prospects


132

6.1 General conclusion

In this thesis, first, the research was focused on the development of a simple method

based on reaction calorimetry using innovative instrument named ‘chemical process analyser (CPA 202). The CPA 202 is constructed with the heat flow

sensor. The principle of this method is based on the measurement of the heat flow

transferred from the cell, where the reaction takes place at isothermal condition. The

method was performed for monitoring the reaction between food containing

antioxidants and an oxidant reagent (i.e. hydrogen peroxide) to investigate the

antioxidant capacity of foods. The instrument measures the heat flow signal (W)

released during the reaction. Such heat flow as well as its integral yields the heat (J)

of the reaction. The overall heat was used as index to express the antioxidant

capacity of the food samples.

The method was initially performed with the mixture of ascorbic acid (AA) as model

compound and hydrogen peroxide (H2O2) as oxidant to monitor the reaction. This

study was carried out with different concentrations of ascorbic acid and pH. Then,

the method was applied to real samples, such as fruit juice, fruit puree, tea, coffee

and wine. The results were allowed to evaluate the antioxidant capacity of real

samples without extraction or any pre-treatment steps. The results of these analysis

revealed that the overall thermal effect was proportional to the concentration of

ascorbic acid (r2 = 0.998). The heat flow profile was affected by pH. The pH

conditions have a substantial effect on the scavenging power of AA solutions: this

increases with increasing pH and attains its maximum for pH = 6.5, i.e., when

ascorbate anions represent almost 100% of the ascorbic species. A tentative kinetic

model, including two or three consecutive first-order steps, allows a satisfactory fit of

the reaction calorimetric experimental records. However, the values of the relevant

kinetic constants and reaction enthalpies actually are the result of the combination of

intermediate steps involving free radicals. These imply a balance of endo- and

exothermic effects that can depend on the H2O2/antioxidant molar ratio.


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Lastly, the use of reaction calorimetry performed with innovative instrument offers a

further possibility to assess the scavenging properties of antioxidants present in

foods among other novel technique. Assuming that the overall heat released reflects

the scavenging capability of the system considered, one may define the antioxidant

potentiality of several food products.

Second, the research works aimed at evaluating the efficiency of traditional and

innovative preservation treatments on fresh-cut fruits using developed calorimetric

approach. Processing operations trigger a number of physiological responses that

affect the fresh-cut fruit’s quality through changes in respiration, colour, texture,

aroma, etc. The research was focused for minimizing damages caused by

processing operations and for reduction of the rate of quality losses in fresh-cut

fruits. Fresh-cut apples (Malus domestica cv. Golden Delicious) were subjected to

different stabilization treatments, such as dipping with ascorbic acid solution,

exposure to UV-C and pulsed light. The rate of reaction of treated fresh-cut apples

was investigated with microcalorimetry. When the cut fruits were treated with

ascorbic acid, pulsed light or UV-C treatments, a sharp decrease in the heat flow

occurred, confirming the reduction of fruit reaction and control. This changes of the

heat flow signal was proportional to the concentration of ascorbic acid or pulsed light

dose used, but was not linearly proportional to the fluence of the UV-C treatment.

The dipping of fresh-cut apples in solution with increasing concentration of ascorbic

acid reduces progressively the polyphenol activity. Exposure to light on the apple

slices is also associated with the inactivation of enzymes that involved in the

browning reaction of the tissues. However, when the apple samples were irradiated

with increasing fluence, the resulting apple reaction lasted longer. Moreover, the

heat flow data from microcalorimetric measurements were also used to discriminate

the contribution of different metabolisms (aerobic metabolism, polyphenol oxidase

activity and anaerobic metabolism) occurring on fruits samples after processing. The

findings of this study suggest that innovative surface treatments based on the

irradiance of light were able to preserve and enhance the stability of fresh-cut

apples. In addition, it’s also confirmed the suitability of microcalorimetry to

investigate the reaction in fresh-cut fruits.


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Furthermore, the developed reaction calorimetry method was extended with Fenton

type reaction, where, the oxidation reaction in samples induced by ferrous iron and

hydrogen peroxide. Fenton type reaction is very important for faster oxidation of

foods and food ingredients with the formation of free-radicals. Here, reaction

calorimetry antioxidant assay was modified as iron-catalysed antioxidant activity in

the oxidation reaction adding ferrous ions. With this modified assay, I have

investigated free-radical scavenging capacity of foods based on heat released from

the system. The resulting heat is used an index of antioxidant capacity of foods.

Foods with the highest scavenging capacity is produced the highest heat of reaction.

The reliability of this method was performed with the mixture of ascorbic acid (AA)

used as model compound, hydrogen peroxide (H2O2) as oxidant and Ferrous iron

(Fe2+) as catalyst like the previous application of this method. These studies were

carried out with different concentrations of ascorbic acid, ferrous iron, hydrogen

peroxide, and the effects of ascorbic acid on pH (2-7). Then, first, the method was

applied to antioxidant compounds such as quercetin, gallic acid, vanillic acid,

catechin, oleuropein, trolox, and caffeic acid. Next, this method was applied to real

samples, such as fruit juice, fruit puree, tea, and coffee. The antioxidant capacity of

antioxidant compounds and food samples was derived according to their reaction

with H2O2 in presence of Fe2+. The results were promising, which made ten times

faster of the oxidation of antioxidant compounds (i.e. ascorbic acid) and food

samples. The overall thermal effect was proportional to the concentration of ascorbic

acid, ferrous iron and hydrogen peroxide. The heat flow profile was affected by pH.

Moreover, a tentative kinetic model based on single step first-order law applied to get

the insights mechanism of the reaction. The model was allowed to fit the

experimental heat-flow traces satisfactorily and provided relevant kinetic parameters

and enthalpies of the reaction.

In conclusion, calorimetry is a great technique to evaluate the antioxidant capacity of

foods without requiring any sample pre-treatment protocol. The data obtained by this

instrument also provide satisfactory fitting for reaction kinetics analysis. In addition,

calorimetry is also very useful tool to determine the stability of fresh-cut apples.


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6.2 Future prospects

There is new trend to use coupled and/or integrated systems for performing evolved

gas analysis in relation to heat flow signal; for example, thermogravimetry coupled

with mass spectrometry is commonly used today. Therefore, the combination of data

from calorimetric process analyser with those obtained with other techniques (for

oxidation, respiration, ethylene production, pH, colour, texture, taste, etc.) can be of

great interest for the food producer, because, this will contribute to gain necessary

knowledge for process development, optimization and computer simulation in the

food industries predicting and ensuring the quality and shelf life of foods.

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Acknowledgement

In the name of Allah, the Most Gracious and the Most Merciful

Alhamdulillah, all praises to Allah for the strengths and His blessing in completing this

thesis. I would like to express my sincere gratitude to Professor Matteo Scampicchio for

providing the thesis topic and opportunity to do research on his laboratory. Beside the

valuable advice, suggestion and inspiration, he provided me a great research experiences.

I am grateful to Professor Alberto Schiraldi, University of Milan and Prof.ssa Tanja Mimmo,

Free University of Bolzano, Italy for their willingness and support to be my co-supervisor.

Also, I wish to express my warm and sincere thanks to Dr. Md. Sazzat Hossain Sarker,

Associate Professor in the Department of Food Engineering and Technology at Hajee

Mohammad Danesh Science and Technology University, Bangladesh for his valuable

advice, friendly help during the work in his group as research period abroad.

I would like to forward my gratitude to Prof.ssa Lara Manzocco and Prof.ssa Maria Cristina

Nicoli for collaboration with the University of Udine, Italy. Furthermore, I am grateful to my

colleague Marco Mason and all members of our research group for their kind support

during my entire experiments in the laboratory.

Of course, this PhD dissertation could not be completed without the kind assistance of

several individuals and financial support of the Free University of Bolzano, Italy that are

gratefully acknowledged here.

Finally, I am grateful to my lovely and sweetie wife Sharmin Khan and my family in

Bangladesh for their prayers, constant support and endless love which kept me always

striving and sane to finish this tough journey. Again, all praises to Allah for blessing me a

baby boy M Farhan Hasan during this time.

Hasan S. M. Kamrul

December, 2016

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4 Effects of ascorbic acid and light on reactions in fresh-cut apples