Postharvest Biology and Technology 101 (2015) 58–65

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Optical properties, ethylene production and softening in mango fruit

Paola Eccher Zerbini a, *, Maristella Vanoli b,c , Anna Rizzolo c , Maurizio Grassi c,
Rodrigo Meirelles de Azevedo Pimentel d , Lorenzo Spinelli e , Alessandro Torricelli b
a
Horticulture and Product Physiology (Horticultural Supply Chains), Wageningen University, Droevendaalsesteeg 1, 6708 PD Wageningen, The Netherlands
b
Dipartimento di Fisica, Politecnico di Milano, Piazza L. Da Vinci, 32 – 20133 Milano, Italy
c
Consiglio per la Ricerca e Sperimentazione in Agricoltura – Unità di ricerca per i processi dell'industria agroalimentare (CRA-IAA), via Venezian 26 – 20133
Milano, Italy
d
Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG), Av. José Cândido da Silveira, 1647 – Cidade Nova, Belo Horizonte, Minas Gerais, Brazil
e
Istituto di Fotonica e Nanotecnologie, CNR, Piazza L. Da Vinci, 32 – 20133 Milano, Italy

A R T I C L E I N F O A B S T R A C T

Article history: Firmness decay, chlorophyll breakdown and carotenoid accumulation, controlled by ethylene, are major
Received 21 July 2014 ripening events in mango fruit. Pigment content and tissue structure affect the optical properties of the
Received in revised form 4 November 2014 mesocarp, which can be measured nondestructively in the intact fruit by time-resolved reflectance
Accepted 6 November 2014
spectroscopy (TRS). This work is aimed at improving the maturity assessment in mango (Mangifera indica
L. cv Haden) from Brazil, using TRS absorption in both the carotenoid and chlorophyll regions in order to
Keywords: develop a model for fruit ripening. Scattering and absorption in the 540–900 nm spectral range by TRS,
Mango
ethylene production and respiration rate, and firmness, were measured in one day on each individual
Maturity
Ethylene
fruit of a sample covering the range of maturity. The fruit displayed a variability which was attributed to
Absorption the different biological age. Absorption spectra showed two peaks at 540 and 670 nm, corresponding
Scattering respectively to the tail of carotenoid absorption and to chlorophyll-a absorption. Carotenoids increased
Time-resolved reflectance spectroscopy substantially only in fruit where chlorophyll had almost disappeared. The absorptions at 540 and 670 nm,
which described the maturity state of each fruit relative to the range of each wavelength, were combined
in one index of biological age (biological shift factor) for each fruit and used in logistic models of ethylene
increase and firmness decay respectively. The model explained about 80% of the variability in ethylene
production rate. A similar result was obtained for firmness when scattering was added in the model. The
combination of absorption at 540 and 670 nm measured by TRS in the intact fruit can be used to classify
mango fruit according to maturity and to predict the ripening of individual fruit.
ã 2014 Elsevier B.V. All rights reserved.

1. Introduction (Vásquez-Caicedo et al., 2006). This process is accompanied by

The ripening process of climacteric fruit such as mango
(Mangifera indica L.) is regulated by genetic and biochemical events
that result in changes in color, texture, aroma, nutritional content
and flavor of the fruit (Giovannoni, 2004). Ethylene plays a major
role in controlling these events. During ripening, ethylene produc-
tion becomes autocatalytic, being stimulated by ethylene itself. Loss
of firmness (softening), change of exocarp and mesocarp color, and
development of volatiles are among the most obvious symptoms of
ripening. During fruit ripening, chloroplasts differentiate into
chromoplasts by disintegration of the thylakoid membranes and
by the development of new pigment-bearing structures, as
observed in pepper (Camara and Brangeon, 1981) and mango
* Corresponding author.
E-mail address: paola.zerbini@wur.nl (P. Eccher Zerbini).
biochemical changes such as degradation of chlorophyll and
accumulation of carotenoids, which cause the characteristic bright
yellow–orange mesocarp coloration in ripening mangoes (Vásquez-
Caicedo et al., 2005). Ethylene accelerates chlorophyll breakdown
and stimulates biosynthesis of carotenoids and their precursors
(Rodrigo and Zacarias, 2007; Montalvo et al., 2009). Ethylene and
carotenoid synthesis and chlorophyll degradation pathways are
integrated in that they share some common regulating factors
(Lee et al., 2012; Luo et al., 2013). The most abundant carotenoids in
mango are all-trans-b-carotene, all-trans-violaxanthin and
9-cis-violaxanthin. Ripe ‘Haden’ fruit has been characterized by a
high content of all-trans-b-carotene and all-trans-violaxanthin as
compared to other cultivars (Ornelas-Paz et al., 2007). The
concentrations of these carotenoids increased in an exponential
manner during fruit ripening and were highly correlated with the
color coordinate a* (positive) and H
(negative) values of the
mesocarp (Ornelas-Paz et al., 2008).

http://dx.doi.org/10.1016/j.postharvbio.2014.11.008
0925-5214/ ã 2014 Elsevier B.V. All rights reserved.
 P. Eccher Zerbini et al. / Postharvest Biology and Technology 101 (2015) 58–65
Mango, as other climacteric fruit, is generally harvested at the
preclimacteric, mature-green stage, and its ripening process is
completed in the postharvest phase. Fruit harvested in a ripe
condition has better quality for direct consumption, but a shorter
shelf-life. For long supply chains, the maturity stage at harvest
must prevent ripening during transport, while ensuring acceptable
potential for subsequent ripening. Fruit harvested too early may be
unable to ripen, as the ripening ability of a fruit is acquired on the
tree (Joas et al., 2012).
The determination of reliable maturity indexes is therefore of
paramount importance for the mango fruit industry. Commonly, in
practice the shape and appearance of the fruit is used, which can be
subjective. According to Kienzle et al. (2011), titratable acidity,
mesocarp yellowness and dry matter are the most useful indices to
specify harvest maturity. Exocarp color changes with maturity, but it
is not always well correlated to the other maturity indices, which are
related to the mesocarp properties. The best tools to assess changes
in the mesocarp during ripening are the penetrometer, followed by
flesh a* value and total soluble solids content (Padda et al., 2011).
Unfortunately all these measurements are destructive, so they can
be applied only to a sample of a fruit batch. To overcome the
problem, and make possible the individual measurement of each
fruit in a batch, nondestructive methods are needed, recently
reviewed by Nicolaï et al. (2014). Among optical methods,
continuous wave NIR or Vis–NIR spectroscopy has been widely
used to estimate established maturity parameters of mango,
following them during fruit ripening: e.g., dry matter, starch
(Saranwong et al., 2004), total soluble solids, color (Subedi et al.,
2007), firmness (Subedi and Walsh, 2009) and a combined maturity
index (Jha et al., 2014) were predicted from NIR spectra using partial
least squares regression. The latter approach predicts maturity
parameters indirectly, and the robustness of the prediction model
depends on the extent and variation of the population used for
calibration. The direct nondestructive measurement of relevant
parameters in the mesocarp by means of time-resolved reflectance
spectroscopy (TRS) could be an improvement to the standard Vis–
NIR approach. TRS and space-resolved reflectance spectroscopy
(SRS), differently from continuous wave methods, can separate the
effects of light absorption (due to chemical compounds such as
pigments or water) and light scattering (due to microscopic changes
in refractive index caused by membranes, air, vacuoles, starch
granules, organelles, etc.). With TRS, the absorption (ma) and
reduced scattering (ms0 ) coefficients are quantified by measuring
photon time-of-flight distribution with picosecond temporal
resolution. In nectarines, the absorption at 670 nm (ma670), near
the chlorophyll-a peak, has been shown to be an effective maturity
index, which, at harvest, allowed the prediction of softening rate
Fig. 1. Scheme of the TRS instrumental setup. TCSPC: time-correlated single-photon counting board; SYNC: synchronization signal; CFD: constant fraction discriminator.
59
during ripening (Eccher Zerbini et al., 2006). An interesting feature
of TRS is that it measures optical properties at a depth of 1–2 cm in
the sample with no or limited influence from the skin (Cubeddu
et al., 2001; Torricelli et al., 2008), while continuous wave
spectrophotometers have a useful penetration depth of a few
mm, depending on wavelength (Lammertyn et al., 2000). When
measurements of peeled and intact mangoes were compared, TRS
absorption spectra were fairly identical in the two cases, and
correlated with spectrophotometric measurements only in peeled
fruit, indicating that TRS measured internal properties, while
spectrophotometer measured rather superficial features (Spinelli
et al., 2012, 2013; Vanoli et al., 2011a, 2013). Scattering spectra can
be interpreted with Mie theory: under the hypothesis that the
scattering centers are homogeneous spheres behaving individually,
Mie theory predicts the wavelength dependence of the scattering
and the relation between scattering and sphere size and density.
Scattering has been related to textural properties of fruit. In apple
fruit ms0 at 750 and 780 nm were positively correlated to the
intercellular space volume, and negatively to firmness (Vanoli et al.,
2007), and were related to pectin composition showing a high and
positive correlation to galacturonic acid content in water soluble
pectin fraction, and a negative correlation to residue insoluble
pectin and protopectin index (Vanoli et al., 2009). A significant
positive correlation was found between firmness and ms0 880 in
ripening ‘Tommy Atkins’ mangoes (Vanoli et al., 2013). TRS
measurements in the chlorophyll absorption region (near
670 nm) have been used to obtain a direct indication of harvest
maturity and to model fruit ripening on an individual fruit basis,
taking into account the variability of fruit. In fact fruit maturity at the
tree level is heterogeneous owing to variations in flowering time as
well as to variability in environmental conditions of the fruit-
bearing branches (Léchaudel and Joas, 2007). This variance may be
seen as a disadvantage for fruit industry which looks for uniform
batches of produce; however, it can also be managed in order to treat
each fruit in the most suitable way, e.g., directing the less mature
fruit to long distance transport and the more mature ones to direct
consumption in the near or gourmet markets. In the last decade,
biological variation has been studied by many authors (Hertog,
2002; Tijskens et al., 2003; Hertog et al., 2004; Schouten et al., 2004;
De Ketelaere et al., 2006). The concept of biological shift factor
allows reducing many different aspects of variation in postharvest
behavior to that of a different biological age of individuals which
share a common behavior at constant conditions (Tijskens et al.,
2005). In nectarines ma670, decreasing with maturity, was
considered an index of the fruit biological age (Tijskens et al.,
2007) and, converted into the biological shift factor, was
successfully used to predict fruit softening rate during shelf-life,
60 P. Eccher Zerbini et al. / Postharvest Biology and Technology 101 (2015) 58–65
and, hence, to select fruit for different market destinations (Eccher
Zerbini et al., 2009). Previous work on ‘Tommy Atkins’ mango fruit
showed that ma630 (related to chlorophyll-b content) could be used
to predict softening rate, but the model explained only 70% of the
variation in firmness decay rate (Pereira et al., 2010).
The aim of this research was to improve the maturity
assessment in mango by considering simultaneously TRS
absorption in both the carotenoid and chlorophyll regions, and
to develop a model for fruit ripening in relation to the maturity
determined from the two absorption coefficients, using the
biological shift factor theory.
2. Material and methods
2.1. Time-resolved reflectance spectroscopy
The schematic of the TRS setup developed at Politecnico di Milano
and used for measurements is shown in Fig. 1 (Spinelli et al., 2012).
The light source was a supercontinuum fiber laser (SC450-6W,
Fianium, UK) providing white-light picosecond pulses, adjustable in
power by a variable neutral-density attenuator. A filter wheel loaded
with 14 band-pass interference filters was used for spectral selection
in the range 540–940 nm. Light was delivered to the sample by
means of a multimode graded-index fiber. Diffuse remitted light was
collected by 1 mm fiber, placed at 1.5 cm distance from the
illumination point. A second filter wheel identical to the first one
was used for cutting off the fluorescence signal originated when
illuminating the fruit in the visible spectral region. The light then was
detected with a photomultiplier (HPM-100-50, Becker&Hickl,
Germany) and the photon distribution of time-of-flight was
measured by a time-correlated single-photon counting board
(SPC-130, Becker&Hickl, Germany). A model for photon diffusion
in turbid media was used to analyze TRS data to assess the bulk
optical properties of samples (Martelli et al., 2009) to obtain the
estimates of ma and ms0 at each wavelength. An approximation of
Mie theory: ms0 = A (l/l0)B, where l is wavelength, A the scattering
coefficient at the reference wavelength l0 = 600 nm, and B is a
parameter related to the equivalent size of the scattering centers
(Mourant et al., 1997; Nilsson et al., 1998) was used to relate ms0 to
the structural properties of the medium (density and size of
scattering centers).
2.2. Fruit
Mango fruit (cv Haden) harvested in a commercial orchard in
Minas Gerais, Brazil, was immediately transported by plane to
Milan, Italy. At arrival, 60 fruit without defects were selected and
individually measured by means of the TRS set-up for the ma650, as
the signal-to-noise ratio observed at 670 nm (i.e., on the
chlorophyll-a peak) was too low to guarantee reliable TRS
measurements. Each fruit was measured on two opposite sides
and the results were averaged per fruit, and mangoes were then
sorted by decreasing ma650, i.e., increasing maturity and stored at
20
C.
After two days at 20
C, a subsample of 20 fruit, covering the
whole range of ma650, was selected and measured for ethylene
production rate and respiration. The optical properties in the 540–
900 nm spectral range were measured by means of the TRS set-up
on two opposite sides in the equatorial region of each intact fruit
and, at the same positions, flesh firmness was assessed after all
nondestructive measurements. One fruit was discarded because it
was decayed.
In this paper the results relative to this subsample are reported,
while the global results have been presented by Vanoli et al. (2012)
and Spinelli et al. (2013).
2.3. Ethylene and respiration measurement
Ethylene production rate (EP) and respiration were measured
by putting fruit in 1.7 L gas tight glass jars (one fruit per jar) for 2 h
at 20
C; then, for the determination of the ethylene content, the
headspace gas was sampled using a syringe, injected into a gas
chromatograph (DANI Instruments 86.10, Monza, Italy) fitted with
a VU65 switching valve equipped with a 1 mL loop and analyzed
using a deactivated aluminum oxide F1 (80–100 mesh) column
(1/8 in.  200 cm, Alltech Italia, Sedriano, MI, Italy) at a column
temperature of 100
C and FID detection. Quantitative data were
obtained by relating the ethylene peak area to that of a 10 mL L1
standard and were expressed as pmol kg1 s1. GC data were
corrected for fruit mass, empty volume of the jar and time of
production. The results of four fruits were missing due to problems
in the analysis.
For the analysis of respiratory gases (CO2, O2), the jar was
directly connected via a 30 cm  1/16 in. O.D. tubing bearing a
70 mm  0.63 mm O.D. side hole/dome needle to the MicroGC MTI
(model P-200, Hewlett-Packard) fitted with two columns in
parallel: a MS5A column (4 m  0.32 mm ID, 30 mm) at 45
C and
an OV-1 column (4 m  0.15 mm ID, 1.2 mm) at 40
C, each equipped
with a thermal conductivity detector and using helium at
8 mL min1 as carrier gas. GC data were corrected for fruit mass,
void volume, temperature and pressure of the jar and the time of
production to express CO2 production and O2 uptake rates as
nmol kg1 s1 in standard conditions. Respiratory quotient (RQ)
was computed as the ratio between CO2 production and O2 uptake
rates.
2.4. Firmness
Flesh firmness was measured using a penetrometer (Instron
UTM model 4301, crosshead speed 200 mm min1, 8 mm diameter
plunger) after skin removal by a slicer, in position corresponding to
the TRS readings.
2.5. Ethylene production model
It was assumed that EP during mango ripening is autocatalytic,
following a sigmoid curve increasing with biological age of fruit
from zero to a maximum production rate (EPmax):
EP max
EP ¼
 (1)
1 þ exp DtEP 
where DtEP  is the biological shift factor (BSF) for ethylene, which
accounts for the different age of individual fruit in regard to
ethylene production rate (Tijskens et al., 2005). The variability of
maturity in the batch of mangoes, which were measured at one
time, represents a set of different biological ages, so each individual
fruit represents one biological age and will have its biological shift
factor. By this model it was also assumed that all fruit in the batch,
grown in the same orchard and conditions, had the same behavior
as regards EP in the course of ripening. Each fruit, with its BSF,
represents a different step in the same process. DtEP  is a stochastic
variable that contains all the information concerning maturity for
each individual fruit in the whole batch, expressed in standardized
dimensionless time (Tijskens et al., 2007). The BSF is the shift of
individual fruit maturity in relation to the intermediate maturity
(BSF = 0) corresponding to EP equal to half of the maximum. The
BSF for ethylene is an index of the fruit age in terms of its ethylene
biosynthesis, which is known to increase during fruit ripening. The
age of fruit can also be described in terms of the stage of
chlorophyll breakdown and/or of carotenoid accumulation. Both
processes characterize fruit ripening and can be assessed by
absorption at 670 and 450 nm respectively. In our experiment we
 P. Eccher Zerbini et al. / Postharvest Biology and Technology 101 (2015) 58–65 61

could not perform measurements at 450 nm, however even at
540 nm the effect of carotenoids was appreciable (see Section 3.1),
and Azzollini (2012) showed that ma540 was positively correlated
to total carotenoids in mango with correlation coefficient ranging
from 0.84 (cv Tommy Atkins) to 0.91 (cv Haden). Tijskens et al.
(2006) showed that ma670 in nectarines followed a logistic decay
during ripening, both on and off the tree. The concentration of
carotenoids was found to increase exponentially during mango
ripening (Ornelas-Paz et al., 2008), however, it is reasonable to
assume that the increase may not be infinite and growth rate
decays towards a maximum. In fact preliminary analysis showed
that ma670 followed a logistic decay also in mango, similar to that
of nectarines, and ma540 followed a logistic but increasing trend
(data not shown). Both for chlorophyll degradation and for
carotenoid accumulation each fruit is characterized by its
individual BSF. Since both these biochemical processes are related
to ethylene biosynthesis, it can be assumed that the BSF for
ethylene is linearly related to those of chlorophyll and of
carotenoids. So it was assumed that the BSF for ethylene (DtEP 
in Eq. (1)) could be expressed as a function of the measured ma540
(m540
a ) and ma670 (ma ) relatively to their range:
670
! !
m540  m540 m670  m670
DtEP  ¼ a540 log a;540max 540a; 0 þ a670 log a;670max 670a; 0 þ b
ma; 0  ma; min ma; 0  ma; min
(2)
where a540, a670, and b are parameters to be estimated. The index
0 indicates the absorption measured in each fruit by TRS on the
same day as EP measurement. The indices max and min indicate
the maximum and minimum values ever possible (at plus and
minus infinite time). They were fixed at the maximum and
minimum values found in this (m540
a;max and ma;min ) or other (ma;min
670 540
of ethylene: it accounts for the different age of individual fruit in
regard to firmness decay. Firmness decay during ripening parallels
chlorophyll degradation and carotenoid accumulation, as all these
processes are dependent on ethylene, so it can be assumed that the
BSF for firmness (DtF  in Eq. (4)) is linearly related to the BSFs of
chlorophyll and of carotenoids and can be expressed as a function
of the measured absorptions at 540 and 670 nm (Eq. (4)). In the
Eq. (4) also two terms related to scattering (the Mie’s A and B
estimated from scattering spectra) were added, assuming that
firmness decay is paralleled by a change in scattering:
! !
m540  m540 m670  m670
DtF  ¼ aF;540 log a;540max 540a; 0 þaF;670 log a;670max 670a; 0
ma; 0  ma; min ma; 0  ma; min
þ bF þ kA A þ kB B (4)
where ma symbols and values are the same indicated for Eq. (2),
while aF,540, aF,670, bF, kA and kB are parameters to be estimated.
2.7. Statistical analysis
EP and firmness data were analyzed by non-linear regression
(PROC NLIN, SAS/STAT, SAS Institute Inc., Cary, NC, 2002) based on
model (1) combined with Eq. (2) for EP, and on model (3) combined
with Eq. (4) for firmness. In this way, EP and firmness were
represented as functions of fruit maturity at time of measurement,
as assessed by selected optical properties.
3. Results
3.1. Optical properties

and ma;max ) experiments with mango fruit, where we could find

670 Absorption spectra in the range 540–900 nm showed two main
fruit with extreme values (see Table 1). peaks (Fig. 2). The variation was very high in the 540–580 nm
range, near the carotenoid absorption peak, while it was still
2.6. Firmness decay model remarkable, but less high in the 650–690 nm range, in the region of
chlorophyll-a absorption. There was also a slight contribution of
A similar approach was also applied to firmness. A model for water absorption in the 800–900 nm region. In Fig. 2, a high
firmness decay was developed by Tijskens et al. (2007). That model absorption at 670 nm corresponded to a low absorption at 540 nm.
is used here to relate firmness to biological shift factor for firmness With increasing absorption at 540 nm, that at 670 nm decreased.
as assessed by ma540 and ma670. Firmness, in mango and other Only when the absorption at 540 nm was very high, the tail of this
fruits, decays to a minimum value without reaching zero: peak affected the absorption at 670 nm, which increased slightly.
At wavelengths higher than 730 nm there were no differences
F max  F min between fruit. The relation between ma540 and ma670 is made
F ¼ F min þ
 (3)
1 þ exp DtF  clear in Fig. 3, left. Absorption of carotenoids (ma540) remained
where F is firmness and Fmax and Fmin its maximum and minimum around 0.2 cm1 as long as chlorophyll absorption (ma670) was
values ever possible (at minus and plus infinite time). The present. When chlorophyll disappeared, ma670 did not become
biological shift factor DtF* has the same meaning as in the case zero, but remained around 0.03 cm1, which can be ascribed to the
background absorption due to the many absorbing compounds in
Table 1 the tissue, other than chlorophyll. Carotenoids (ma540) increased
Parameters of the non-linear regression model for ethylene production rate in only where ma670 was below 0.04 cm1.
function of ma540 and ma670 (Eq. (1) and (2)). Maximum and minimum values of ma Scattering (Fig. 2) decreased with increasing wavelength, as
were fixed.
predicted by Mie theory. The range of variation was quite high
Parameter Estimate Approx. std error Approx. 95% among fruit, as regards both the average level (related to parameter
or fixed value confidence limits A) and the slope (related to parameter B) (Fig. 3, right).
m540
a;max 0.84
3.2. Respiration
m 540
a;min 0.05

m670
a;max 0.65
Oxygen uptake rate ranged between 360 and 570 nmol kg1 s1.
The range of CO2 production rate was slightly higher (400–
m670
a;min 0.025 670 nmol kg1 s1). Respiration data in relation to ma540 show that
CO2 production was similar to O2 uptake when ma540 was low, but
EPmax 0.48 0.05 0.38 0.58
with ma540 > 0.8 cm1 the CO2 production rate was higher than
a540 0.73 0.16 1.08 0.38
a670 0.37 0.11 0.14 0.61
oxygen uptake rate (Fig. 4, left). This was reflected in the
respiratory quotient, which increased above 1 when ma540 was
R2adj 0.80 high (Fig. 4, right).
62 P. Eccher Zerbini et al. / Postharvest Biology and Technology 101 (2015) 58–65

Fig. 2. Absorption (left) and scattering (right) spectra measured on 20 mango fruit cv Haden covering the whole range of maturity.

Fig. 3. Relation between ma540 and ma670 (left) and Mie’s A and B (right) in mangoes cv Haden.

Fig. 4. Oxygen uptake and CO2 production rate (left) in mango fruit cv Haden and their respiratory quotient (right) in function of ma540.

3.3. Ethylene production rate
EP ranged from 0.1 to 0.5 pmol kg1 s1. EP increased with
increasing ma540, and with decreasing ma670 (Fig. 5). The results
of modelling EP in function of maturity (expressed as biological
shift factor derived from ma540 and ma670) are reported in Table 1
and Fig. 6. The b parameter was not significant so it was dropped
from the model. The approximate standard error was low for all the
parameters. The estimated EPmax was similar to the measured
maximum EP (0.496 pmol kg1 s1). The coefficients a540 and a670
had a low negative correlation, and had obviously opposite sign, as
ma540 increased and ma670 decreased with increasing EP. This
model explained 80% of the variation of EP in the batch of fruit.
The same model was run considering only one wavelength at a
time: when only ma540 or ma670 was considered, R2adj became
0.61 and 0.49 respectively, indicating that both wavelengths
should be considered together to obtain a better index of fruit age
in relation to ethylene biosynthesis.
3.4. Firmness
Firmness indicated that most fruit was in an advanced maturity
stage (Fig. 7). Even if firmness varied from 5 to 70 N, the majority of
the mangoes had firmness lower than 25 N, which is characteristic
of ready to eat or ripe fruit. Firmness decreased with decreasing
ma670, and was already low when ma540 increased above 0.2 cm1.

Fig. 5. Ethylene production rate (EP) in function of ma540 (left) and ma670 (right).
 P. Eccher Zerbini et al. / Postharvest Biology and Technology 101 (2015) 58–65 63

Fig. 8. Measured data (diamonds) and predicted firmness decay (line) in function of
Fig. 6. Measured data (diamonds) and predicted (line) ethylene production rate the biological shift factor (DtF  ) as assessed by ma540, ma670 and Mie’s B,
(EP) in function of biological shift factor (DtEP  ) as assessed by ma540 and ma670 according to model (Eq. (3) and (4)) and parameters in Table 2.
according to model (Eq. (1) and (2)) and parameters in Table 1.

Fig. 7. Firmness in function of ma540 (left) and ma670 (right).

The results of modelling firmness in function of maturity as 4. Discussion

assessed by absorption at 540 and 670 nm and by scattering are
reported in Table 2 and Fig. 8. The bF and kA parameters were not 4.1. Optical properties
significant and were omitted. To avoid over parameterization,
parameters Fmax and Fmin were fixed at the maximum and The most interesting features were absorptions related to the
minimum firmness measured, and aF,540 was fixed at 1.4, based main pigments in the fruit, i.e., chlorophyll and carotenoids, which
on some preliminary calculations. The approximate standard error decrease and increase respectively with fruit ripening, as already
of kB was relatively high, but the presence of the scattering found in other mango cultivars (Spinelli et al., 2012). The
parameter B in the model raised the adjusted R2 to 0.80, while differences of absorption in fruit could be attributed mainly to a
without it the R2adj was lower (0.75). different content of chlorophyll (670 nm) and of carotenoids
If either wavelength was omitted from the model, R2adj (540 nm). We found that ma540 was higher than 0.3 cm1 only in
was obviously lower; when only ma670 was used, kB was not fruit with ma670 lower than 0.04 cm1. The different content was
significant, and hence also B could be omitted as its effect in this assumed to be due to a different biological age of fruit, which had
restricted model was near zero (R2adj = 0.49), while using only undergone a more or less advanced stage of ripening at the time of
ma540 the model could not fit, unless also B was considered examination. With this assumption, it seems that carotenoids
(R2adj = 0.58). increased substantially only when chlorophyll had almost
disappeared. Pigments in mango mesocarp were measured by
Kienzle et al. (2011, 2012),) who found that, during postharvest
storage, chlorophyll a and b decreased from 3.3 and 2.2 mg hg1
DW, respectively, to not detectable, while all-trans-b-carotene
increased from 0.4 to 4.9 mg hg1 DW; interestingly, the carotene
increased only when chlorophyll was very low or not detectable, in
Table 2
Parameters of the non-linear regression model for firmness in function of accordance with our results. The mechanism of this synchroniza-
ma540 and ma670 and of Mie’s B (Eq. (3) and (4)). Fmax,Fmin and aF,540 were fixed. tion between chlorophyll degradation and carotenoid accumula-
Maximum and minimum values of ma were fixed and are the same reported in tion has been particularly studied in tomato. STAY-GREEN (SGR)
Table 1. proteins, which play important roles in the regulation of
Parameter Estimate Approx. std error Approx. 95% chlorophyll degradation, can also regulate and inhibit lycopene
or fixed value confidence limits and b-carotene accumulation through direct interaction with
Fmax 65 – phytoene syntase, a key carotenoid synthetic enzyme (Luo et al.,
Fmin 5 – 2013). It seems that high levels of SGR induce chlorophyll
aF,540 1.4 – breakdown, while carotenoid accumulation is inhibited, until
aF,670 0.53 0.14 0.23 0.83
the SGR decreases so allowing carotenoid synthesis and plastid
kB 5.41 1.29 2.69 8.12
conversion. Synchronization and balance between chlorophyll
R2adj 0.80
breakdown and lycopene accumulation have been studied at a
quantitative level in tomato using a kinetic model by Schouten
64 P. Eccher Zerbini et al. / Postharvest Biology and Technology 101 (2015) 58–65
et al. (2014), who found that they depend on temperature and
cultivar. At microscopic level, Vásquez-Caicedo et al. (2006) found
a very dynamic interconversion of the plastid structures in the
mango mesocarp tissue (cv Tommy Atkins), where no sequential
pattern could be clearly established between chloroplasts and
chromoplasts. In contrast, in our study, optical absorption
measurements, which respond to pigment concentration,
appeared to show a clear sequence in that the increase of
ma540 only occurred after the complete decrease of ma670.
As regards scattering, the differences in ms0 reflect the changes
occurred in the mesocarp structure due to the mango softening. A
decrease of scattering spectra of ‘Tommy Atkins’ mangoes, as well
as of the parameter related to density of the scatterers was found
during shelf-life (Vanoli et al., 2013). Softening is due to the
enzymatic cell wall breakdown which may decrease the density of
the scattering particles in the mesocarp so leading to less
scattering events in the tissue, as found also in tomatoes, apples
and plums (Qin and Lu, 2008; Vanoli et al., 2011b; Seifert et al.,
2014). This suggests that fruit with lower ms0 had a more advanced
cell wall breakdown. In fact in ripening mango the appearance of
the flesh changes becoming translucent (i.e., less scattering) and
juice is easily expressed from the fruit, indicating cell rupture due
to weakening of cell wall. A relation of scattering with pectic
composition was demonstrated in apples (Vanoli et al., 2009). The
same could occur in mango, however also starch, which is present
in immature mango, and starch hydrolysis during ripening might
influence scattering, as well as the amount of intercellular spaces.
The combined effect of such different processes and properties on
scattering makes it difficult to relate scattering to firmness in a
simple manner.
4.2. Respiration and ethylene production rate.
The values of oxygen uptake, CO2 production and EP rates were
similar to those reported in literature (Lalel et al., 2003; Zheng et al.,
2007; Zaharah and Singh, 2011). The respiratory quotient in normal
aerobic conditions is around 1 (0.8–1.2, depending on the substrate
used for energy production). A higher value suggests a change from
aerobic to anaerobic respiration, which occurs when oxygen in the
tissue is insufficiently available so that the energy requirements
cannot be fulfilled. This may occur in climacteric fruit when
ethylene triggers many simultaneous ripening processes which
require energy, and at the same time the modifications and
breakdown of cell walls and membranes can reduce the perme-
ability to gases. It can be assumed that fruit in this condition was
already in the overripe, senescent phase.
4.3. Modelling
The model for EP in function of ma540 and ma670 explained 80%
of the variation of EP in the batch of fruit. The model for firmness in
function of ma540, ma670 and Mie’s B gave a similar result, despite
softening already having occurred at a certain extent in the batch of
fruit. This confirmed the assumption that there was a common
behavior as regards chlorophyll degradation, carotenoid accumu-
lation, ethylene biosynthesis and firmness decay among mango
fruit grown in the same orchard: the individual differences of
maturity were different steps in the same process, and were taken
into account by the biological shift factors DtEP  and DtF  for
ethylene and firmness respectively. Both Dt*s indicate that most
fruit, having a positive biological shift factor, were beyond the
intermediate maturity corresponding to the inflection point of the
curve. To express Dt* in time dimension, this variable should be
divided by the range and by the rate constant at the desired
temperature. However the latter information is missing, having
performed the experiment in one time. Further research is under
way to study EP and firmness decay rate over time.
The models show that both wavelengths should be considered
together to obtain a better index of fruit age in relation to ethylene
biosynthesis and firmness. In fact the sequence of carotenoid
accumulation following the chlorophyll breakdown makes the two
processes little overlapping and almost mutually exclusive (Fig. 3,
left), so that, depending on the fruit age, either one is prevalent.
The synchronization between variation in ma540 (carotenoids) and
ma670 (chlorophyll) and EP or firmness could be explained by the
manifold effects of SGR proteins (see Section 4.1), which affect also
ethylene signal transduction by altering the expression of ethylene
receptor genes and of ethylene induced genes, such as
polygalacturonase and pectinesterase (Luo et al., 2013), which have
important effects on fruit texture and firmness during ripening.
The model confirms that the relation between the absorption of
carotenoids and chlorophyll and ethylene biosynthesis or firmness
follows a logistic curve. The combination of ma540 and
ma670 could be used as an index to classify fruit according to
maturity. In the firmness model also the Mie parameter related to
the size of scattering centers was significant, confirming that
scattering is related to textural properties.
When further confirmed by the analysis of more samples over
time, the model could be used to predict the ripening of individual
fruit, and to classify fruit according to the expected time to reach
eating quality.
5. Conclusions
The measurement of absorption coefficients by TRS allowed
detecting the ripening state of each fruit, by assessing the extent of
chlorophyll decay and carotenoid accumulation through
ma670 and ma540 respectively, in a nondestructive way. The
optical properties, respiration, EP and firmness all showed that the
fruit displayed a variability of ripening stages, with some fruit
definitely ripe or overripe. Carotenoids increased substantially
only in fruit where chlorophyll had almost disappeared. The
absorptions at the two wavelengths 540 and 670 nm, combined in
a logistic model, defined an index of biological age (biological shift
factor) of each fruit which explained about 80% of the variability in
the ethylene production rate. A similar result was obtained for
firmness when scattering was also added in the model. The
combination of optical absorption and scattering at selected
wavelength measured by TRS in the intact mango provides an
assessment of the biological age of individual fruit (maturity index)
that can be used to manage the biological variation found in a batch
of fruit due to their different age at harvest, to select fruit according
to maturity and to predict the ripening of individual fruit.
Acknowledgements
This work was partially supported by Regione Lombardia (Italy)
and Region Minas Gerais (Brazil) (Progetto di Cooperazione
Scientifica e tecnologica “Approccio multidisciplinare per
l’innovazione della filiera di frutti tropicali – TROPICO” ID 17077,
Rif.n
AGRO-16).
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