Accepted Manuscript

Title: Effects of Ascorbic Acid and Light on Reactions in

Fresh-Cut Apples by Microcalorimetry

Authors: S.M. Kamrul Hasan, Lara Manzocco, Ksenia

Morozova, Maria Cristina Nicoli, Matteo Scampicchio

PII: S0040-6031(17)30018-7
DOI: http://dx.doi.org/doi:10.1016/j.tca.2017.01.008
Reference: TCA 77668

To appear in: Thermochimica Acta

Received date: 5-8-2016

Revised date: 13-1-2017
Accepted date: 18-1-2017

Please cite this article as: S.M.Kamrul Hasan, Lara Manzocco, Ksenia Morozova,
Maria Cristina Nicoli, Matteo Scampicchio, Effects of Ascorbic Acid and Light
on Reactions in Fresh-Cut Apples by Microcalorimetry, Thermochimica Acta
http://dx.doi.org/10.1016/j.tca.2017.01.008

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Effects of Ascorbic Acid and Light on Reactions in Fresh-Cut Apples

by Microcalorimetry

S. M. Kamrul Hasan1, Lara Manzocco2, Ksenia Morozova1, Maria Cristina Nicoli2, Matteo

Scampicchio1*
1
Free University of Bolzano, Faculty of Science and Technology, Piazza Università, 1, 39100

Bolzano, Italy
2
Dipartimento di Scienze degli Alimentari, University of Udine, via Sondrio 2/a, 33100

Udine, Italy

* Corresponding author: matteo.scampicchio@unibz.it ; Tel.: (+39) 04710 17 210

Graphical abstract

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Highlights

 Microcalorimetry measures the effects of ascorbic acid and light treatments on fresh-
cut apples
 Ascorbic acid or light dose decrease the heat flow generated by fresh-cut fruit
 The heat was inversely proportional to the intensity of the applied treatments

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; Ascorbic acid; UV-C light; Pulsed light;

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

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 [1]. Their quality is

typically assessed by external attributes, such as size, shape, color, glossiness, surface

cleanliness and absence of defects [2]. Following cutting operations, a number of biochemical

events, overall exothermic, contribute to increasing the reaction rate of the fruit and the

browning of its wounded surface [3]. The rate of these physiological events affects the

product shelf-life [4]. Especially the browning of fruits is a major problem in the fresh-cut

fruit industry and is believed to be one of the main causes of quality loss.

To control the occurrence of these reactions, fresh-cut fruits can be submitted to different

chemical or physical treatments [5]. The former includes traditional dipping of apple slices

into a solution containing antioxidants (e.g. ascorbic acid) and/or chelants (e.g. citric acid)

and/or salts (i.e. CaCl2). More recently, to control fruit reactions, 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 polyphenol oxidase, minimizing enzymatic

browning reactions in cut apples [6 - 7].

To evaluate the effects of such treatments on fruit reaction, the rate of change of some quality

attributes (i.e. color, 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 time consuming and expensive analytical

techniques (i.e. chromatographic analysis of phenols, vitamins and carotenoids) on pre-

treated samples [8].

Among the techniques used for measuring reaction rate in foods, animal and vegetable

tissues, microcalorimetry has played a crucial role. One of the main advantages of this

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

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 metabolic rates in fruits under different conditions [9]. Other applications

include the evaluation of the shelf life [10] and fresh-cut fruits metabolism [11].

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 the

reactions occurring on fruit samples after cutting. Accordingly, this work aimed to apply

isothermal microcalorimetry to evaluate the effect of ascorbic acid on the resulting heat of

reaction of fresh-cut apples (Malus domestica cv. Golden Delicious). Also, microcalorimetry

was applied to evaluate the effect of UV-C and pulsed-light treatments. The experimental

work presented here contributes to extending the possible uses of microcalorimetry and

provides an opportunity to advance our knowledge on the effect of traditional and innovative

technologies on foods.

2 Methods

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.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

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.

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.

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

5
Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

and 24 kJ/m2. After treatments, samples were incubated for 8 h in the icebox, then,

introduced into the calorimetric ampoules and hermetically sealed.

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 analyze 24

ampoules simultaneously. 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 (φ) during the experiment time t [9].

H 
t 0
t  dt
(1)
mole  of  reaction

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) [12]:


r (2)
H

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

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

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 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, controlled by PC and data manager software (version

2.0.0.57, ©GmbH, Germany). Oxygen concentration inside the ampoule was recorded every

30 minutes.

3 Results and Discussions

3.1 Calorimetric signal

Figure 1 shows the heat flow curves from apple cylinders, after treatment with a solution of

ascorbic acid.

< Figure 1 about here >

The heat-flow signal of Figure 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[13–17]. 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 [18]. Also, the browning of the wounded tissue suggests the

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

contribution of phenolic compounds and polyphenol oxidase [19–21] . 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 [11] and by Galindo on potato slices [22]. Both

reported that the heat flow was 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 2).

< Figure 2 about here >

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 [23 - 24].

 3.6  109  R  T
r O2   (eq. 3)
H Pm

where r is the rate of respiration expressed as mL of O2 per kg of fruit per hour,  is the

thermal power in Watts, H 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 to

H2O by organic substrates. Figure 2 compares the rate of oxygen consumption derived from

eq. 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 2.

The consumption of O2 (Figure 2) in a – b is up to 35 mL / kg h, consistent with that reported

by [25] with fresh cut apples in similar conditions. According to the literature, one of the

8
Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

most immediate responses to wounding in fruit tissues is an increase in respiration, ethylene

production and oxidation of the pre-existing phenols [26].

Later, in b – c, the calorimetric signal formed a shoulder, when oxygen consumption is

maintained at a rate of about 24 mL / kg h and the O2 concentration dropped from 15% to

8%. It is not clear what biochemical events occur in this region. However, Toivonen [27] has

recently identified a secondary browning period in fresh-cut apples. 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.

In the last period, the content of O2 in the ampoule is lower than 5% (Figure 2) and the rate

of O2 consumption is as low as 2.5 mL / kg h, consistent with anaerobic metabolism [3], [28 -

29].

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 [10; 12].

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 [30]. To account for the

heat flow generated by this last process, a sigmoidal curve, such as a Gompertz function, was

chosen.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

After subtraction of these two functions, the remainder of the heat flow was fit by a single

Gaussian function, as depicted in Figure 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.

3.4 Effect of Ascorbic acid

The effect of ascorbic acid on the calorimetric signal is shown in Figure 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 3 about here >

Figure 3 also shows (as circle points) the values obtained by fitting the observed data with

the three functions previously described by 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). However, the Gaussian function showed a trend that is proportional

to the concentration of the ascorbic acid used in the dipping treatment.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 5 shows the Gaussian function derived from the fittings of Figure 3. The dimensions

of the Gaussian function become progressively reduced as the ascorbic acid concentration

increased.

< Figure 5 about here >

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 [31]. Such capacity is supported by a considerable amount of

literature [32-33], that explains how ascorbate ions decrease browning incidence by reducing

o-quinones back to phenolic compounds prior to their polymerization into colored pigments

[34]. 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.

< Figure 4 about here >

Similar results were found by Gómez [35] who reported the heat-flow of wounding stress 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 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.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

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 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 literature, this research

found that exposures to UV-C light and pulsed light reduce fruit reaction [36-37].

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 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 [7].

< Figure 6 about here >

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.

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 [7]. 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.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

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.

Acknowledgments

We acknowledge Peter Theiner (Fructus Meran, Italy) and the Province of Bolzano for

financial support (Landesregierung mittels Beschluss Nr. 1472, 07.10.2013).

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 1. Calorimetric heat flow from fresh-cut apple samples after dipping in

ascorbic acid (1%) solution for 1 min.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 2. Rate of oxygen consumption (solid line) as determined from the

thermograms of Figure 1 by equation (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).

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 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).

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 4. Enthalpy values from the (I) exponential, (II) Gaussian and (III) sigmoidal

fitting curves of Figure 3 as a function of ascorbic acid concentration.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 5. Curves of function II obtained after deconvolution of the heat flow curves of

Figure 3. Letters from a to f correspond the concentration of ascorbic acid used in

the treatment of fresh cut apple samples, respectively, 0, 1.0, 1.5, 2.0, 2.5 and 3.0%.

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Hasan et al. 2016 Fresh-cut apples by Microcalorimetry

Figure 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.

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