333

External Quality Aspects in Relation to Internal Product Physiology Maarten L.A.T.M. Hertog K.U. Leuven, Lab. Postharvest Technology W. de Croylaan 42, B-3001 Leuven Belgium E-mail: maarten.hertog@agr.kuleuven.ac.be Keywords: apple, avocado, Belgian endive, gas exchange, mechanistic model, metabolic

rate Abstract

This paper deals with the link between effects of storage conditions on metabolic rate on one hand and the effects on external quality aspects on the other hand. Mechanistic models are used as a first step in interpreting experimental data to determine the likelihood of the possible underlying mechanisms and to direct future research to elucidate these mechanisms at a physiological and biochemical level. Even though details of the underlying processes are still to be unravelled, the simplified approach of directly linking metabolic rate to the rate of quality breakdown is proven to be successful in describing the effects of modified atmosphere on external quality attributes through their known effects on metabolic rate. GENERAL INTRODUCTION

Quality of horticultural product is largely based on subjective consumer evaluation of a complex of quality attributes (like taste, texture, colour, appearance), which are based on specific product properties (like sugar content, volatile production, cell wall structure. These product properties are generally changing during time, as part of the normal metabolism of the product. Storage is based on the principle that manipulating or controlling storage conditions (temperature, humidity, gas conditions) affects the metabolism of the packaged product, such that the ability to retain quality of the product can be optimised. To understand the mode of action for a specific product, a good understanding of how relevant product properties depend on storage conditions is required. This paper will deal with the link between the effects of storage conditions on metabolic rate on one hand and the effects of storage conditions on external quality aspects on the other hand.

Temperature is the main factor affecting all biochemical processes through its effects on activation enthalpy and entropy of the underlying reactions. ATP demanding processes are also indirectly affected by temperature through the effect of temperature on respiration and fermentation, the main ATP producing processes. The levels of O2 and CO2 inhibiting respiration also affect the amount of ATP available. Those quality changes that are either directly influenced by O2 or CO2 or driven by the ATP supplied by respiration or fermentation will all be affected by modified atmosphere (MA) conditions. This is the base of success of MA (whether that is MA packaging, controlled atmosphere (CA) storage or ultra low oxygen (ULO) storage). Some quality degrading processes are affected more than others due to the way they depend on atmospheric conditions. Although much work has been done on showing the general effects of MA conditions, quantification of the effects of gas conditions on changes in quality attributes is still limited.

Such a relationship between rate of gas exchange and rate of quality loss has been seen in broccoli (Polderdijk et al., 1995), where the rate of discoloration of buds depended on atmospheric composition. Also, deterioration of asparagus spears appeared to be strongly related to the products own metabolic rate (Brash et al., 1995). Tijskens (1996) suggested using metabolic rate as a rate index for quality changes and Hertog et al. (1999) applied this approach to explain the effects of MA on spoilage of strawberries.

Proc. Int. Conf. Quality in Chains Eds. Tijskens & Vollebregt Acta Hort. 604, ISHS 2003

334

This paper will discuss recent work on the effects of MA storage conditions on the rate of gas exchange on one hand and the change in quality attributes of apple, avocado and chicory on the other hand. APPLE FIRMNESS Introduction

Early studies on the effect of MA found that apples softened slower in MA than in regular air (Magness and Diehl, 1924; Kidd and West, 1936; Smock and Blanpied, 1963). Temperature as such is also known to affect the rate of softening in apples (Landfald, 1966). The combined effect of atmospheric composition and temperature on the gas exchange rate has been extensively characterised for ‘Golden Delicious’ apples (Hertog et al., 1998). However, there have been few studies quantifying the effects of MA on both gas exchange and fruit softening rates and quantitatively relating the two. Knee (1980) investigated the effects of a limited number of O2 levels on the rate of gas exchange and ripening processes of ‘Cox’s Orange Pippin’ apples, revealing a clear parallel between responses to the applied MA conditions. Hertog et al. (2001) extensively described the effects of O2 and CO2 on both gas exchange and firmness loss of Braeburn apple stored for 55 days at 0 °C (for experimental details see Hertog et al. 2001). Gas exchange

Both decreasing levels of O2 and increasing levels of CO2 inhibited O2 consumption rates (rO2). The oxidative CO2 production was inhibited accordingly with fermentative CO2 production (rCO2) developing at the low O2 levels (Fig. 1). The experimental data was described using Michaelis-Menten type gas exchange models with the uncompetitive type of CO2 inhibition to describe oxidative respiration (Eq. 1).

(f)O

O

f)(COOCO

CO

COOO

OOO

2

2

2

22

2

2

22

22

2

1

1

Kmcp

rrRQr

Kmup

pKm

prr

max

max

++⋅=

+⋅+

⋅=

(1)

Firmness

Softening showed a clear response to the various MA conditions applied (Fig. 2). Firmness changes were assumed to be linear for the relatively short storage period (55 days) at low temperature (0 °C). Firmness loss decreased with decreasing levels of O2. At high O2 levels a small inhibiting effect of CO2 became visible. More unexpectedly, a clear increase in softening was observed at extremely low O2 levels. Similar, but less pronounced effects of low O2 levels have been observed in Cox’s Orange Pippin (Stow, 1989) and Fuji apples (Fan et al., 1997). The observed effect of low O2 on softening corresponds with what was seen for the gas exchange, with oxidative respiration declining with declining O2 levels and increasing CO2 levels, and fermentation increasing with declining O2 levels (Fig. 1). This suggests a strong link between gas exchange and firmness loss. Integrated approach

To explore the suggested functional relationship between gas exchange and fruit

335

softening the complete set of data on gas exchange and fruit softening was analysed explicitly defining this relationship. Gas exchange was again described according Eq. (1) while the rate of firmness breakdown was described following:

(f)O

O

max(f)CO

C

2

2

22

22

1//

KmcprC

rCdtdFdtdF CO

O

rOr

+

⋅+⋅+=

(2)

where

2OrC and

2COrC were factors directly relating the softening process to the rate of respiration and fermentation respectively. By allowing these factors to vary the model could account for a differential dependency of the softening process on fermentation and respiration. This model approach was able to describe 88% of the observed variation assuming that the effects of O2 and CO2 on the rate of firmness loss can be explained through their effects on respiration and fermentation. From the values of

2OrC and

2COrC it could be concluded that the softening process related more closely to oxidative respiration than to anaerobic fermentation. This can be explained from the less efficient character of fermentation in terms of the amount of energy produced per mole of CO2 released through gas exchange. In spite of the high CO2 production during fermentation only a small amount of energy is being fixed as ATP to drive the fruit’s metabolism. Overall, these findings support the empirical approach taken by Tijskens et al. (1997) to model the softening of ‘Elstar’ apples under modified atmospheres using relative respiration as a rate index for softening. AVOCADO FIRMNESS Introduction

Meir et al. (1995, 1997) have done some work on the effect of MA on quality changes of avocado fruit, but they did not link the two in a quantitative way. Although the relationship between gas exchange rates and rates of quality decay will be much more complex, and for climacteric fruit should take into account the involvement of ethylene, a first approach can be taken by linking the rates of gas exchange directly to the rate of colour change.

Hertog et al. (2003) extensively described the effects of O2 and CO2 on both gas exchange and firmness of ‘Hass’ avocado stored for 32 days at 7 °C (experimental details to be published in Hertog et al. 2003). Gas exchange

The averaged gas exchange data (Fig. 3) showed typical Michaelis-Menten type behaviour. The rate of O2 consumption increased with increasing levels of O2 and decreased with increasing levels of CO2. The fact that the inhibiting effect of CO2 did not decrease with increasing O2 levels indicated an uncompetitive type of CO2 inhibition (Hertog et al., 1998). The avocado fruit did not show a significant increase in their CO2 production, as would be expected assuming alcoholic fermentation at low O2 levels.

The CO2 production at high CO2 levels at aerobic conditions was suppressed much more than O2 consumption resulting in a reduced respiratory quotient (RQ). This change in RQ depending on CO2 was explained by mitochondrial respiration shifting to the alternative respiratory pathway increasing the O2 consumption as a result of an up-regulation of alternative oxidase proteins. The data were analysed using Michaelis-Menten type gas exchange models using the uncompetitive type of CO2 inhibition (Hertog et al., 1998). Under the assumption the change in RQ was due to activation of the

336

alternative respiratory pathway, the gas exchange model was extended to include this phenomenon (Hertog et al., 2003). Gas exchange of avocado was described according the formulation of Eq. 3 in combination with the formulation of rO2 from Eq. 1.

22

222

222

222

2

OCO

OOO

COOO

COO,

OO

rRQrrrr

ppKmppr

r

AOXtot

AOX

maxAOXAOX

⋅=+=

⋅+⋅⋅

=

(3)

Firmness

Firmness of avocado fruit (F) was measured using the HandyHit device that gives a reading between 0 (firm) and 100 (soft; F+∞). The sensitivity of the device is such that it will only start to respond at the stage the fruit starts to turn black. On average, control fruit stored in air (Fig. 4A) did not soften sufficiently to reach a firmness of 100. A simple logistic model was used to describe the experimental firmness data as function of time:

0

01)(

FFFe

FtFtkF

−⋅+=

∞+⋅−

∞+ (4)

Analysing the control data with this model (Fig. 4A) resulted in values for the rate

constant kF valid for air conditions at 7 °C of RAFk =0.1633 day-1 and a value for F0 of

0.36. Integrated approach

A comparable approach as applied to the apple data was followed by making the rate of softening kF at the different MA conditions dependent on the relative metabolic rate based on the normal oxidative respiration as this is the part of the metabolism responsible for ATP production:

RAO

MA ORA

2

2

rr

kk FF ⋅= (5)

It needs to be emphasised that in this model approach to describe softening at MA

conditions, the model was not fitted to the data but the model was applied using the estimated parameter values on the gas exchange data in combination with the estimated parameter values on the softening of the air stored control fruit, assuming the rate of softening of the MA stored fruit depended on the relative metabolic rate according to Eq. (5). The model fitted the data quit well (Fig. 4B).

This model used to describe softening is a crude simplification of the whole softening process ignoring all details on the involvement of ethylene and a whole range of enzymes. However, the message that can be learned from this simplified approach is that MA was inhibiting the rate of softening to the same extent as it was inhibiting the metabolic rate as expressed by oxidative respiration.

337

CHICORY STEM GROWTH Introduction

Consumers are generally looking for tight chicory heads. Postharvest growth of the central stem loosens the heads enhancing evaporation from the now exposed leaves. The market has decided that stem length should not exceed 75% of the crop length.

The gas exchange of chicory has been extensively characterised (Hertog et al., 1998) and recently some extensive work has been done on the effect of MA on the change in quality of chicory heads (Vanstreels et al., 2002). Vanstreels et al. focused on red discoloration but they also collected an extensive set of destructive data on stem growth (for experimental details see Vanstreels et al., 2002). Integrated model approach

A simple model was applied assuming postharvest stem growth is the result of growth (cell division followed by cell elongation) where cell material is reallocated from the leaves to the stem. This mechanism can be simplified and represented by the following scheme:

stemk

leaves MM g→ (6) The overall mass of the heads is assumed to be constant, neglecting water and

respiration losses. Assuming stem growth is an energy demanding process, the rate constant is linked to the metabolic rate using the relative respiration rate of chicory. Approaching the stem as a cylinder, stem length can be calculated according:

( )

RAO

MA O

20

2

2

rr

RR

rπρMM-eM

lstem

stem, headtRR-k

headstem

g

=

⋅⋅−⋅

=⋅⋅

(7)

The developed model (Fig. 5) was used to analyse data from 3 seasons on storage

of chicory heads at a range of temperatures and gas conditions. The model was able to describe most of the observed behaviour of chicory stem growth. Besides the temperature effect there was a clear effect of O2 on stem growth, almost completely inhibiting stem growth at 2 kPa O2. Stem growth was not affected by CO2 levels up to 19 kPa. This was in agreement with the lack of any effect of CO2 on the rates of gas exchange (Fig. 6).

Given the destructive nature of the measurement, large variation was present in the dataset. To improve the model and its predictive value, non-destructive data is needed to allow monitoring of single chicory heads during time. APPLE AROMA Introduction

Aroma release from stored apples is known to be subject to the preceding storage conditions (Fellman et al. 2003). Modified atmospheres can be detrimental to post storage volatile production when compared to regular air storage. Also time to regenerate aroma profiles after removal from modified atmospheres can be affected. However, when comparing post storage production of the different volatile compounds, the timelapse for the different compounds can vary largely (Fellman et al., 2003; Saevels et al., 2003 – see Fig. 7).

338

Integrated model approach A simple model was proposed consisting of three consecutive reactions describing

how a substrate, via one intermediate compound is transformed into an aroma compound that subsequently evaporates from the fruit.

→→→ 321 kkk compoundAromateIntermediaSubstrate (8)

The flow of volatiles leaving the fruit is what was measured using headspace

analysis of the fruit during two weeks of shelflife. The model was used to describe the overall fruit history of storage and shelflife to explain differences in shelflife behaviour of fruit stored at 1, 3 or 21 kPa O2. All three rate constants were assumed to depend on temperature, while as a first approach, only the first rate constant was assumed to depend on the gas conditions. In contrast to the previous examples no a priori assumption was made on how this rate constant would relate to the gas conditions. Instead, separate values were estimated for the three O2 levels applied (Fig. 8).

The results clearly show how a relative simple generic model can be used to describe the wide range of observed time lapses for the different aroma compounds. There is a clear effect of the O2 level during storage on the biosynthesis of the different aroma compounds. However, the effect varies with the compound. While in most cases the effect of O2 on k1 is linear, in some cases a Michaelis-Menten like effect can be observed (compound 15, 18, 19 and 22), while in other cases the minimum value of k1 is observed during CA storage at 3 kPa O2 (compounds 6 and 17). These different ways of how volatile production depends on O2 levels should be related to their different biochemical pathways, their dependence on ATP availability and their dependence on O2 as a possible reactant directly or indirectly involved in their biosynthesis. GENERAL CONCLUSION

To improve the understanding of the behaviour of certain external quality aspects a good understanding of the underlying product physiology is needed. The examples outlined clearly illustrate the link between the effects of storage conditions on metabolic rate on one hand and the effects of storage conditions on external quality aspects on the other hand. Subsequent evidence needs to be gathered to identify the exact relationships, whether gas conditions affect quality-degrading processes directly through their involve-ment as a reactant, or indirectly through their involvement in ATP production.

Mechanistic models are only the first step in interpreting experimental data to determine the likelihood of the possible underlying mechanisms and to direct future research to elucidate these mechanisms at a physiological and biochemical level. Even though exact details are still to be unravelled, the simplified approach of directly linking metabolic rate to the rate of quality breakdown has already proven successful in describing the effects of modified atmosphere on external quality attributes through their known effects on metabolic rate. Literature Cited Brash, D.W., Charles, C.M., Wright, S. and Bycroft ,B.L. 1995. 'Shelf-life of stored

asparagus is strongly related to postharvest respiratory activity', Postharvest Biol. Technol. 5,77-81.

Fan, X., Mattheis, J.P., Patterson, M., Fellman, J.K. 1997. Optimum harvest date and controlled atmosphere storage potential of 'Fuji' apples. In: Proc. 7th Int. Contr. Atm. Res. Conf., UC Davis, CA, Vol. 2: Apples and Pears, pp. 42-49.

Fellman, J.K., Rudell, D.R., Mattinson, D.S., Mattheis, J.P. 2003, Relationship of harvest maturity to flavor regeneration after CA storage of ‘Delicious’ apples. Postharvest Biol. Technol. 27,39-51.

Hertog, M. L. A. T. M. Nicholson, S. E. Banks, N.H. 2001. The effect of modified atmospheres on the rate of firmness change in 'Braeburn' apples. Postharvest Biol. Technol. 23,175-184.

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Hertog, M.L.A.T.M., Nicholson, S.E., Whitmore, K. 2003. The effect of modified atmos-pheres on the rate of quality change in 'Hass' avocado. Postharvest Biol. Technol. (in Press).

Hertog, M.L.A.T.M., Boerrigter, H.A.M., Boogaard, G.J.P.M. van den, Tijskens, L.M.M., Schaik, A.C.R. van 1999. Predicting keeping quality of strawberries (cv. 'Elsanta') packed under modified atmospheres: an integrated model approach. Postharvest Biol. Technol. 15,1-12.

Hertog, M.L.A.T.M., Peppelenbos, H.W., Evelo, R.G., Tijskens, L.M.M 1998. A dynamic and generic model of gas exchange of respiring produce: the effects of oxygen, carbon dioxide and temperature. Postharvest Biol. Technol. 14,335-349.

Kidd, F., West, C., 1936. Gas storage of fruit IV. Cox's Orange Pippin apples. J. Pomol. Hortic. Sci. 14,276-294.

Knee, M. 1980. Physiological responses of apple fruits to oxygen concentrations. Ann. appl. Biol. 96,243-253.

Landfald, R. 1966. Temperature effects on apples during storage. Bull. Int. Instit. Refrig. Ann. 1966-1, 453-460.

Magness, J.R., Diehl, H.C. 1924. Physiological studies on apples in storage. J. Agric. Res. 27,1-38.

Polderdijk, J.J., Boerrigter, H.A.M. and Tijskens, L.M.M. 1995. 'Possibilities of the model on keeping quality of vegetable produce in controlled atmosphere and modified atmosphere applications', Proceedings of the 19th International congress of refrigeration, volume II, 318-323.

Saevels, S., Lammertyn, J., Berna, A.Z., Veraverbeke, E.A., Di Natale, C. Nicolaï, B.M. 2003. Aroma assessment of apple during shelf-life exposure by means of electronic Noses. Postharvest Biol. Technol. (Subm.)

Smock, R.M., Blanpied, G.D. 1963. Some effects of temperature and rate of oxygen reduction on the quality of controlled atmosphere stored McIntosh apples. Proc. Am. Soc. Hortic. Sci. 83,135-138.

Stow, J.R. 1989. The response of apples cv. Cox's Orange Pippin to different concentrations of oxygen in the storage atmosphere. Ann. App. Biol. 114,149-156.

Tijskens, L.M.M. 1996. A model on the respiration of vegetable produce during postharvest treatments, in: G.R. Fenwick, C. Hedley, R.L. Richards, and S. Khokhar (eds.), Agri-food quality, an interdisciplinary approach, The Royal Society of Chemistry, Cambridge, UK 1996.

Tijskens, L.M.M., Van Schaik, A.C.R., Hertog, M.L.A.T.M. and De Jager, A. 1997. Modelling the firmness of Elstar apples during storage and transport, Acta Horticulturae, 485,363-371.

Meir, S., Naiman, D., Akerman, M., Hyman, J.Y., Zauberman, G. and Fuchs, Y. 1997. Prolonged storage of ‘Hass’ avocado fruit using modified atmosphere packaging. Postharvest Biol. Technol. 12,51-60.

Meir, S., Akerman, Fuchs, Y. and M., Zauberman, G. 1995. Further studies on the controlled atmosphere storage of avocados, Postharvest Biol. Technol. 5,323-330.

Van streels, E., Lammertyn, J., Verlinden, B.E., Gillis, N., Schenk, A., Nicolaï, B.M. 2002. Red discoloration of chicory under controlled atmosphere conditions. Post-harvest Biol. Technol. 26,313-322.

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Notation Symbol Description Unit Gas Exchange

2Or oxidative O2 consumption rate mol⋅kg-1⋅s-1

2COr CO2 production rate mol⋅kg-1⋅s-1 )(CO2 fr fermentative CO2 production rate mol⋅kg-1⋅s-1

2OKm

Michaelis constants for respiration kPa

2COKmu Michaelis constants for uncompetitive inhibition of respiration by CO2 kPa )(Kmc fO2 Michaelis constants for the inhibition of fermentation by O2 kPa

RQ respiratory quotient for oxidative respiration -

2Op ,2COp O2 , CO2 partial pressure kPa

Apple firmness DF/dt rate of softening N.s-1 DF/dtc correction factor to compensate for the negative offset in the softening data N.s-1 C factor relating rate of softening to rates of gas exchange - Avocado firmness F Firmness HandyHit unitsF+∞ asymptotic firmness values (=100) at plus infinite time HandyHit unitskF rate constant for firmness breakdown day-1

Chicory stem length L stem length m M Mass kg RR relative respiration rate - kg rate constant for stem growth day-1 ρ density of the stem kg.m-3 R stem radius m

Subscripts Superscripts Head referring to intact chicory head Max maximum rate unconstrained by O2 or CO2 Leaves referring to leaves of chicory head AOX related to alternative oxidase Stem referring to stem of chicory head RA valid for regular air conditions 0 initial value at time=0 MA valid for modified atmosphere conditions

341

Figurese

A.

01

2

0 5 10 15 20

10

20

30

40r O

2 (nm

ol.s

-1.k

g-1)

O2 (kPa)

CO 2

(kPa

)B.

01

2

0 5 10 15 20

10

20

30

40

r CO

2 (nm

ol.s

-1.k

g-1)

O2 (kPa)

CO 2

(kPa

)

Fig. 1. O2 consumption (A.) and CO2 production (B.) of ‘Braeburn’ apples (nmol.s-1.kg-

1) at 0°C as a function of O2 and CO2. Data points plotted represent the measured data while the plane represents the fitted model from Eq. (1).

01

2

0 5 10 15 20

-2-10123456semean

∆ Fi

rmne

ss (N

)

O2 (kPa)

CO 2

(kPa

)

Fig. 2. Total firmness loss of ‘Braeburn’

apples (N) at 0°C as a function of O2 and CO2 over 55 days of storage. Data points plotted show the average change in firmness for the 20 fruit packed in each atmosphere. The error bar represents the average standard error of these means. The plane represents the model from Eq. (2).

342

A.

05

1015

0 5 10 15 20

100

200

300r O

2 (nm

ol.s

-1.k

g-1)

O2 (kPa)

CO 2

(kPa

)

B.

05

1015

0 5 10 15 20

100

200

300

r CO

2 (nm

ol.s

-1.k

g-1)

O2 (kPa)

CO 2

(kPa

)

Fig. 3. O2 consumption (A.) and CO2 production (B.) of ‘Hass’ avocado fruit (nmol.

s-1.kg-1) stored at 7°C as a function of O2 and CO2. Data points plotted represent the averaged measured data while the plane represents the model from Eq. (3).

A.

0 10 20 30 400

20

40

60

80

100

Firm

ness

(Han

dyH

it un

its)

time (days)

B.

05

1015 0

510

15 20

10

20

30

40

50

Firm

ness

(Han

dyH

it un

its)

O2 (kPa)

CO2 (kPa)

Fig. 4. Firmness (HandyHit units) of the stem end of ‘Hass’ avocado fruit stored at 7°C

with (A.) representing the observed time course for the control fruit and (B.) the final firmness of the MA stored fruit as a function of the MA conditions applied. The plotted symbols represent the average of the measured data with the error bars representing the standard deviation due to fruit-to-fruit variation. The line from (A.) is fitted using the model from Eq. (4) while the plane (B.) represent the final firmness prediction based on the modelled gas exchange rates (Eqs. 3, 4, 5).

343

stem

leng

th (m

m)

O2 (kPa)

time (days) 05

1015

200

510

15

20

60

70

80

90

100

110

120

Fig. 5. Modelled stem length of chicory

(mm) as function of time, O2 and temperature according to Eq. 7. The three planes represent stem length at different temperatures (from bottom to top respectively 5, 12 and 20°C).

Fig. 6. The effect of O2 and CO2 on gas

exchange rate of chicory at 21°C; O2 consumption (rO2, ) and CO2 production (rCO2,−−). Redrawn after Hertog et al., 1998.

344

ULO CA RA

0 5 10 15 0 5 10 15

0 5 10 15

0 5 10 15

0 5 10 15

0 5 10 15

Vol

atile

pro

duct

ion

rate

time (days)

Fig. 7. The release of 22 different volatiles during two weeks shelflife at 20°C, after 8

months storage at either ULO, CA or RA at 0°C. (data from Saevels, 2003).

0 5 10 15 200.0

0.2

0.4

0.6

0.8

1.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

k 1

O2 level (kPa)

Fig. 8. Value of k1 as function of storage O2 level applied, expressed relative to the value at 21 kPa O2. Each line represents the values for a different aroma compound.