Scientia Horticulturae 260 (2020) 108879

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Scientia Horticulturae
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Collaborative analysis on difference of apple fruits flavour using electronic T

nose and electronic tongue
⁎ ⁎
Danshi Zhu , Xiaojun Ren, Liwei Wei, Xuehui Cao, Yonghong Ge, He Liu, Jianrong Li
College of Food Science and Technology, Food Safety Key Lab of Liaoning Province, National & Local Joint Engineering Research Center of Storage, Processing and Safety
Control Technology for Fresh Agricultural and Aquatic Products, Bohai University, Jinzhou, Liaoning, 121013, China

A R T I C LE I N FO A B S T R A C T

Keywords: The flavour difference and other quality indicators among five popular apple varieties (‘Ralls’, ‘Jonagold’, ‘Orin’,
Apple variety ‘Indo’, and ‘Hanfu’) were evaluated mainly using electronic nose and electronic tongue coupled with gas
Flavour chromatography-mass spectrometry (SPME/GC–MS). By linear discriminant analysis (LDA) of E-nose output
Electronic nose data, the aroma of ‘Orin’ was found to be quite different from other varieties. There were obvious differences on
Electronic tongue
sourness, saltiness, and umami among the five apple varieties in E-tongue analysis. For five apple varieties, 45
SPME/GC–MS
volatile compounds were identified, and their respective concentrations quantified using SPME/GC–MS, and
which showed that, the most abundant volatile compounds in apples were esters. By principal component
analysis (PCA), hexyl butanoate, (E)-2-hexenal and α-farnesene were the important volatile compounds in ap-
ples. The hierarchical cluster analysis (HCA) according electronic nose and electronic tongue analysis and other
quality indicators showed that the flavour of ‘Hanfu’ was closely related to ‘Jonagold’, while ‘Indo’ and ‘Ralls’
flavour were closely related to ‘Orin’. The collaborative analysis method of combining electronic nose and
electronic tongue had potential significance on flavor difference evaluation.

1. Introduction cultivation practices, fruit maturity stage on harvest, storage and

Apple belongs to the Rosaceae family, and it is one of the most
widely grown and most frequently consumed fruits (Yi et al., 2016). At
present, Fuji ranks first in terms of apple acreage in China (Heng et al.,
2008). Newer varieties such as ‘Hanfu’ (Dongguang × Fuji) have shown
good performance in some apple-production areas. Some other in-
troduced varieties also showed high market share, including ‘Ralls’ and
‘Jonagold’ (originally from the USA), as well as ‘Orin’ and ‘Indo’ (ori-
ginally from Japan).
As a general rule, the first purchase of fruit is mainly dependent on
aspects of appearance, such as, colour and texture; while, continuous
purchase of fruits are determined by internal quality traits, such as,
odour and taste. Some research focused on the correlation between
sensory attributes and other parameters, such as texture or taste (Costa
et al., 2011). However, few studies investigated odour and flavour at-
tributes of sensory quality (Karlsen et al., 1999). The flavour is a result
of two sensations: taste and odour (Abrodo et al., 2010). Taste is the
feeling that the tongue gets when it comes into contact with something.
Odour is sensed through people's olfactory organs. The identities and
concentrations of volatile organic compounds-responsible for the fla-
vour depend on the variety, region where grown and climate,
transport conditions, and processing.
Volatile compounds are directly related to perceived odour and
flavour attributes. Flavour or eating quality depends upon sweetness,
acidity, astringency, and aroma. Most published studies describe fla-
vour by a single generic attribute such as “sensory flavour” or “apple
flavour” (Altisent et al., 2011). A complex mixture of organic com-
pounds, such as esters, alcohols, aldehydes, among others, comprise the
volatile profile of apple (Both et al., 2017). The determination of vo-
latiles from apple requires a suitably selective, sensitive analytical
method. Solid-phase microextraction (SPME) has become popular for
studying the aroma profile of fruits. Also, it isolates volatiles and semi-
volatiles and can be suitable for defining marker compounds for the
discriminating between samples (Lasekan et al., 2013). The most
commonly employed technique coupled with SPME is gas chromato-
graphy-mass spectrometry (GC–MS). In addition, the electronic nose
and tongue also make significant contributions to the determination of
flavour, which have been inspired by the way in which mammals re-
cognise samples via olfaction and taste, respectively. They offer a fast,
comprehensive, and easy-to-handle alternative to assess food quality
(Escuder-Gilabert and Peris, 2010). At present, there are few studies on
the fusion of electronic nose and tongue sensors to analyze sample

⁎
Corresponding authors at: School of Food Science and Technology, Bohai University, 121013, Jinzhou, Liaoning Province, China.
E-mail addresses: zhudanshi1978@163.com, zdsruby@126.com (D. Zhu), lijr6491@163.com (J. Li).

https://doi.org/10.1016/j.scienta.2019.108879
Received 3 August 2019; Received in revised form 19 September 2019; Accepted 20 September 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
D. Zhu, et al. Scientia Horticulturae 260 (2020) 108879

Fig. 1. Colour difference and texture characteristics of five apple varieties. Colour difference of L* value (I), a* value (II), b* value (III), Hue angle (h°) and Chroma
(C*) (IV), peel hardness and flesh hardness (V), and profile pictures (VI) of five apple varieties. In figure (IV), different lowercase letters (a–d) meant significant
differences (p < 0.05) in hue angle (h°), and different uppercase letters (a–d) meant significant differences (p < 0.05) in chroma (C*). Data of (I–V) were the mean
values of six different fruits and vertical bars indicate ± SD (p < 0.05).

a a IIIa Fig. 2. The difference on sugar, acid and starch

12 I TSS 140 II a 0.45
a
A contents of five apple varieties: total soluble
Starch Content (mg/mL)

b TAC
10 0.4 120 b solids (TSS) and titratable acid content (TAC)
c
0.40 c c
(I), sugar acid ratio (SAR) (II), starch content
TAC (%)
TSS (%)

8 0.3 100
B d (III). In fig. (I), different lowercase letters (a–d)
SAR

6 80 b 0.35
C 0.2 meant significant differences (p < 0.05) on
D 60 c TSS, and different uppercase letters (A–E)
4
E 0.1 40 d 0.30 meant significant differences (p < 0.05) on
2
20 e TAC. Data are the mean of six different fruits
0 0.0 0.25 and vertical bars indicate ± SD (p < 0.05).
Ralls Jonagold Orin Indo Hanfu Ralls Jonagold Orin Indo Hanfu Ralls Jonagold Orin Indo Hanfu

quality changes (Zhi et al., 2017), classify samples (Dong et al., 2017), difference of flavour were evaluated by electronic nose and electronic
and model construction (Qiu et al., 2015). The two technologies of tongue. The correlations of flavour and quality indices (colour differ-
electronic nose and electronic tongue work together, would be more ence, texture and sugar acid ratio) were also analysed in the five apple
comprehensive and more accurate to distinguish fruit flavour. varieties. This work aimed at providing a reference for establishing
In this study, the contents of volatile compounds in apples of five more comprehensive rapid identification method by flavour char-
main cultivated varieties were determined using SPME/GC–MS, and the acteristics of apple varieties.

2
D. Zhu, et al.
Fig. 3. Flavour analysis of five apple varieties: linear discriminant analysis (LDA) of volatile flavour by E-nose (A), radar image of taste determination by E-tongue
(B), and analysis on the relationship between the main taste indices (C–E) of E-tongue.
2. Materials and methods
2.1. Materials and experimental conditions
Mature apple fruits (Malus pumila) of five cultivars, ‘Ralls’,
‘Jonagold’, ‘Orin’, ‘Indo’, and ‘Hanfu’ were hand-harvested from a
commercial orchard in Jinzhou, Liaoning Province, China, in the ma-
ture season of October 2017. After harvest, apples were transported to
the laboratory of Bohai University on the same day. Apple maturity was
determined by starch content. The apples with the same weight and
approximately equal radius of curvature of the same variety were
placed in a constant temperature and humidity box (Shanghai, China)
and stored at (20 ± 1) °C, and a relative humidity of 80–85%. After the
temperature was stabilized, each apple cultivar was randomly sampled
at the same time to measure the indices (colour difference, texture, total
soluble solids (TSS), titratable acidity (TAC), starch contents and fla-
vour analysis). All indexes were measured within a week, and the de-
tections of electronic nose and electronic tongue analysis were executed
at the same day.
2.2. Colour difference
The colour difference of apple was determined using CR-400
chroma meter (Konica Minolta Sensing, Ink., Tokyo, Japan). Colour was
expressed as L*, a*, and b*, indicating luminosity, chromaticity on a
green (−) to red (+) axis, and chromaticity on a blue (−) to yellow
(+) axis, respectively. Chroma and hue angle were calculated from a*
and b* values, and the formulars were C* = (a*2+b*2)1/2 and h° =
(arctan b*/a*), respectively (Holzwarth et al., 2012). Angular values
representing colour are defined as follows: red-purple: 0°, yellow: 90°,
bluish-green: 180°, and blue: 270°. 5 points evenly distributed on the
surface of sample were selected for colour difference analysis, and
measurement repeated 3 times for each point.
2.3. Texture
The texture of apple was measured using TA-XT-PLUS Texture
Analyser (Stable Micro Systems, Scarsdale, NY). Sample processing and
test parameters are the same as those described by Zhu et al. (2018).
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Scientia Horticulturae 260 (2020) 108879
Apple samples with skin were cut into 1.5 × 1.5 × 2 cm portions for
testing. The puncture test was performed with a 2 mm diameter flat-
head stainless steel cylindrical probe (P/2), and the texture parameters
were set as follows: pre-test velocity 1.00 mm/s, test speed 1.00 mm/s,
post-test uplink velocity 1.00 mm/s, and measurement distance 10 mm.
Two maximum forces were registered in this test, corresponding to the
maximum force required to penetrate the skin and the flesh, which
represent peel hardness and flesh hardness, respectively. The experi-
mental samples were measured six times.
2.4. Total soluble solids (TSS), titratable acidity (TAC) and starch contents
The total soluble solid (TSS) content of apple was determined by
PAL-3 hand-held sugar meter (ATAGO, Japan). The titratable acidity
content (TAC) of apples was based on the principle of acid and alkali
neutralisation. The titratable acid content in apples is calculated based
on the consumption of sodium hydroxide. The conversion coefficient of
malic acid is 0.067. The sugar-acid ratio (SAR) was calculated by total
soluble solids content divided by titratable acid content. And the de-
termination of starch contents was refer to the study (Kasemsuwan
et al., 1995).
2.5. Electronic nose analysis
The smell characteristics of apples were analyzed using a portable
PEN 3 E-nose (Insent Company, Japan). The system consists of a sam-
pling apparatus, a detector unit containing an array of ten different
metal oxide sensors, and pattern recognition software (Win-Muster) for
data recording and analysis. The sensor array contains 10 metal oxide
gas sensors, which can detect olfactory cross-sensitive information
(Men et al., 2017). The response characteristics of each sensor were
shown as follows: W1C (aromatic compounds); W5S (nitrogen oxide);
W3C (ammonia and aromatic compounds); W6S (hydrogen); W5C
(olefin and aromatic compounds); W1S (hydrocarbons); W1W (hy-
drogen sulphide); W2S (alcohols and partially aromatic compounds);
W2W (aromatic compounds and organic sulphides); W3S (alkanes
(methane, etc.).
Apples were pulped and then filtered through a mesh cloth. After
that, 15 mL apple juice was placed in a 50 mL centrifuge tube. The tube
D. Zhu, et al. Scientia Horticulturae 260 (2020) 108879

Table 1
Volatile components of five apple varieties using SPME/GC–MS.
Volatile compound RIa Content (μg·kg−1)

Ralls Jonagold Orin Indo Hanfu

Esters
Ethyl butyrate 800 NDb ND 159.98 ± 10.12c ND ND
Butyl acetate 819 ND ND ND 85.54 ± 9.43 ND
2-methylbutyl acetate 856 ND 7.75 ± 1.62 ND 32.34 ± 8.01 ND
Propyl butyrate 868 ND ND ND ND 18.97 ± 4.25
Ethyl valerate 870 ND ND 4.13 ± 0.92 ND ND
Butyl propionate 879 ND 0.80 ± 0.12 ND ND ND
Ethyl 3-hydroxybutyrate 933 ND ND 2.29 ± 0.23 ND ND
Butyl butyrate 958 ND 10.96 ± 2.01 2.94 ± 0.43 ND ND
Hexanol acetate 973 11.62 ± 3.01 438.66 ± 32.43 ND 712.78 ± 42.16 14.44 ± 2.86
Butyl 2-methylbutyrate 996 3.60 ± 0.61 4.45 ± 1.02 ND ND 13.92 ± 2.93
Ethyl hexanoate 1003 ND 4.54 ± 0.98 322.24 ± 21.61 23.10 ± 3.76 ND
Octyl formate 1023 ND ND ND ND 8.35 ± 2.13
Heptyl acetate 1054 ND 1.07 ± 0.23 136.31 ± 14.02 2.38 ± 0.24 ND
Amyl-2-methylbutyrate 1073 0.34 ± 0.07 ND ND ND 2.09 ± 0.11
Hexyl butyrate 1118 74.41 ± 9.12 36.61 ± 5.67 11.28 ± 2.72 13.11 ± 2.79 79.01 ± 9.21
Hexyl isobutyrate 1119 ND ND ND ND 4.18 ± 1.06
Ethyl octanoate 1121 ND ND 6.24 ± 1.50 ND ND
Octyl acetate 1130 ND ND ND 1.49 ± 0.42 ND
Hexyl methylbutyrate 1148 19.30 ± 3.68 ND 4.77 ± 0.95 8.64 ± 2.35 52.90 ± 6.87
Ethyl trans-2-octenoate 1157 ND ND 2.02 ± 0.19 ND ND
Amyl hexanoate 1187 ND ND ND ND 2.78 ± 0.53
Ethyl trans-4-decenoate 1269 ND ND 1.93 ± 0.37 ND ND
Hexyl hexanoate 1275 15.29 ± 3.37 ND 3.58 ± 0.78 4.47 ± 1.13 17.23 ± 3.28
Nerolidyl acetate 1329 ND ND ND ND 0.70 ± 0.21
Ethyl-(E,Z)-2,4-decadienoate 1331 ND ND 3.76 ± 1.02 ND ND

Aldehydes
Hexanal 799 136.04 ± 10.31 30.29 ± 7.06 ND 37.85 ± 5.38 528.86 ± 72.03
(E)-2-hexenal 856 183.95 ± 14.93 73.14 ± 10.28 95.95 ± 10.21 249.61 ± 17.48 282.62 ± 25.67
Nonanal 1047 ND 0.98 ± 0.21 2.11 ± 0.42 3.13 ± 0.29 4.52 ± 1.05
Decanal 1125 ND ND ND 0.89 ± 0.23 ND

Ketones
Geranyl acetone 1324 ND 2.67 ± 0.62 ND ND 1.57 ± 0.38

Alcohols
1-Butanol 675 ND 1.16 ± 0.12 ND ND 13.05 ± 2.45
2-Methylbutanol 739 ND ND ND ND 33.24 ± 6.97
1-Hexanol 844 45.26 ± 10.23 9.71 ± 2.18 19.54 ± 3.68 ND 233.72 ± 18.59
1-Heptanol 938 ND ND ND ND 2.78 ± 0.46
2-Ethylhexanol 987 4.28 ± 1.11 ND ND ND 11.49 ± 2.78
1-Octanol 1022 3.40 ± 0.63 1.60 ± 0.37 4.95 ± 1.23 1.94 ± 0.22 ND

Alkenes
Dipentene 982 1.97 ± 0.32 ND 2.38 ± 0.48 ND 4.18 ± 1.10
α-Farnesene 1361 10.67 ± 2.72 0.80 ± 0.13 14.95 ± 3.27 35.02 ± 5.21 21.58 ± 3.42

Alkanes
Tetradecane 1475 ND ND 0.64 ± 0.06 ND ND
Octadecane 1544 ND ND ND 0.60 ± 0.03 ND

Aromatics
Toluene 773 9.17 ± 1.72 2.41 ± 0.52 0.92 ± 0.10 5.51 ± 1.21 20.71 ± 3.67
p-Xylene 858 4.69 ± 1.02 ND 0.92 ± 0.08 ND ND
m-Xylene 860 ND ND 1.83 ± 0.49 ND 12.36 ± 1.85
Naphthalene 1106 142.27 ± 11.02 ND ND ND ND
Estragole 1126 148.56 ± 10.56 ND 19.08 ± 3.81 ND 2.96 ± 0.32

a
RI (Retention indices): Standards of n-alkanes (C8-C20) were used for RI conversion.
b
ND: Not detected.
c
Values represent as mean ± standard deviation (p < 0.05).

was then closed and the headspace therein equilibrated for 30 min.
During the measurement phase, the headspace gas was pumped
through the sensor array at 300 mL/min, which induced a change in
sensor conductance. The sensor response was defined as the ratio of
conductance G/G0 (G0 and G are the conductances of the sensor before,
and after, exposure to the gas samples, respectively). The measurement
phase lasted for 120 s, a period of time sufficient for the sensors to reach
a stable responsive value. When the measurement was completed, the
acquired data were stored for later use. Then, a stand-by phase was
activated (100 s), during which, clear air desorbed remaining volatiles
from the sensors and circuitry, and then the signals return to their
baseline values before the next test. To reduce experiment error, the
environmental temperature was kept at 25 °C.
2.6. Electronic tongue analysis
Taste analysis was performed using SA402B e-tongue (Insent
Company, Japan). The taste sensor consists of five test sensors and two
reference sensors. The five test sensors were expressed as CA0, CT0,
AAE, C00, and AE1, which represent sour, salty, fresh, bitter, and

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D. Zhu, et al.
Compounds
Es13 Es11
Es1,5,7,17,20,22,25,Alka1
Alke1
0.5
Alc6
Ar3
Alc1 Alc3
Es4,12,14,16,21,24,Alc2,4
Es23 Es10
Es19
Ar5 Ald3 Alc5
0.0 Ar2
Es8 Es15 Ar1
Ar4 K
Es6 Alke2
Ald2
-0.5
Es2,18,Alka2
Ald4
Es9 Es3
-1.0
-1.0 -0.5 0.0 0.5 1.0
Fig. 4. Principal component analysis (PCA) of volatile compounds for five apple
varieties.
astringent tastes, respectively. The instrument has a total of 36 sample
tanks, which can be used to measure 10 samples, and the remaining
sample tanks contained positive and negative electrode liquid and re-
ference liquid to reduce measurement error. It is necessary to activate
the sensor and reference sensor 24 h in advance of any determination.
When the taste substances are absorbed by artificial bimolecular
membranes, the potential difference between the working electrode and
the reference electrode is measured.
When testing, 70 mL of apple pulp was divided evenly into two
parts, and were injected into two 50 mL sample cups respectively for
detection. Then three cleaning operations were performed, each lasting
90 s, 120 s, and 120 s, respectively. After cleaning, the sensor balance
lasted 30 s with samples before measurement and then 30 s for mea-
surement. Then we cleaned the sensor twice, and returned the reference
solution to measure the aftertaste value, each measurement being
completed once. After each measurement, the sensor cleaning runs
automatically (Men et al., 2017). The potentiometric difference be-
tween each individually coated sensor and the Ag/AgCl reference
electrode in the equilibrium state was measured and recorded at room
temperature. Four replicated measurements were run for each sample.
The first measurement cycles were discarded due to instability, and
only the later three stable sensor responses were used as original data.
2.7. SPME and GC–MS analysis
The 50/30 μm divinylbenzene/carboxen/polydi-methylsiloxane
(DVB/CAR/PDMS) fibre (Supelco, Bellefonte, USA) was used for vola-
tile extraction by Solid-phase microextraction (SPME). The extract
method was slightly modified from the procedure of Zhu et al. (2013).
5 g apple samples spiked with 20 μL (0.473 mg/mL) cyclohexanone
(internal standard solution) were placed into 20 mL amber glass vials
and equilibrated at 50 °C under continuous stirring for 30 min. The
SPME fibre was first cleaned at 250 °C for 10 min and then the head-
space extracted for 30 min in each sample vial. Standards of n-alkanes
(C8-C20) (Sigma-Aldrich Co.) were used for RI conversion.
The interface temperature of chromatography-mass spectrometry
(Agilent Technologies 7890A/5975C) was 280 °C, the ion source tem-
perature was set as 230 °C, and the quadrapole temperature was 150 °C.
The ionisation mode was EI, the electron energy was 70 eV, and mass
scanning range was 30–550 amu. After extraction, the SPME fibre was
inserted into the GC injection port set at 250 °C for 5 min in splitless
mode. The flow rate of the helium on the HP-5MS capillary column
(30 m×0.25 mm×0.25 μm) was 1.2 mL min−1. The temperature re-
gime was as follows: the initial temperature of the column was 40 °C for
5 min, raised to 120 °C at a ramp rate of 4 °C min−1, then increased to
230 °C at 10 °C min−1, then held for 6 min at the final temperature.
Identification of the volatile compounds detected by GC–MS ana-
lysis was based on computer matching with the reference mass spectra
Scientia Horticulturae 260 (2020) 108879
of the MS library of NIST 11, Wiley 7.0 and retention times (RTs). Semi-
Esters quantitative determinations were carried out using cyclohexanone as an
Aldehydes
internal standard. The content of volatile compounds in the samples
Ketones
Alcohols
was calculated by comparing the peak area of each compound with that
Alkenes of the internal standard. All samples were prepared in triplicate, and the
Alkanes values were reported as mean ± standard error.
Aromatics
2.8. Statistical analysis
One-way ANOVA test, correlation analysis (Pearson correlation)
and hierarchical cluster analysis of samples were performed by SPSS
Statistics at test significant differences (p < 0.05) to evaluate significant
difference between groups, correlation between indexes, and flavour
hierarchical cluster. Linear discriminant analysis (LDA) was analysed
using Win-Muster software from the PEN3 electronic nose. Principal
component analysis (PCA) data were analysed using Origin 8.5 soft-
ware.
3. Results and discussion
3.1. Colour difference and texture characteristics of apples
The sensory characteristics were quite different among the five
apple cultivated varieties. The results of colour difference were shown
in Fig. 1(I–IV), and the profile pictures were shown in Fig. 1(VI). L*
could express the level of luminosity on apple epidermis, as shown in
Fig. 1(I). The apple peel colour of ‘Orin’ was much brighter than that of
other varieties, and there showed a significant difference (p < 0.05).
Compared with other varieties, the brightness of ‘Ralls’ and ‘Jonagold’
were lower. The fruit ground colour of ‘Ralls’ is red-green, ‘Jonagold’
and ‘Hanfu’ were red, or with red stripes. ‘Orin’ was a yellow-green
colour variety, while ‘Indo’ was green-yellow. The index of a* was used
to evaluate the peel chromaticity on a green to red axis. ‘Hanfu’, Ralls
and ‘Jonagold’ were significantly redder (p < 0.05), while ‘Orin’ and
‘Indo’ were greener (p < 0.05) (Fig. 1(II)). The b* value was used to be a
measurement of yellowness. The b* value of green-yellow variety ‘Indo’
was higher (Fig. 1(III)).
Hue angle is an important parameter to evaluate fruit colour
(Nguyen et al., 2013). Fig. 1(IV) showed that there was significant
difference on hue angle for the two green varieties, ‘Orin’ and ‘Indo’
(p < 0.05), and the values were 109.44 and 117.92 respectively.
Chroma (C*) described chromaticity, and it was an indicator of colour
intensity and visual properties as associated with colour. With the sa-
turation being higher, the colour intensity was brighter. It was shown
that chroma of ‘Indo’ samples was higher than that of others
(Fig. 1(IV)).
Texture was an evaluation index used to assess fruit sensory quality
(Nguyen et al., 2013). The hardness of apple flesh was between 200 g
and 400 g (Fig. 1(V)). The higher flesh hardnesses were found on ‘Ralls’,
‘Orin’, ‘Indo’ and ‘Hanfu’, and there was no significant difference
therein (p > 0.05). As far as pericarp was concerned, the peel hard-
ness of ‘Ralls’, ‘Orin’, and ‘Indo’ were higher than others. On the whole,
the flesh hardness and peel hardness of ‘Jonagold’ were lower.
3.2. Differences in TSS, TAC and starch contents
Starch is the most abundant carbohydrate of storage organs. A large
amount of starch will accumulate in the apple fruit during the growth
and development. The fruit retain starch until commercial maturity,
and its postharvest degradation is essential for consumer acceptance
(Hu et al., 2016). The fruit flavour can be affected by amounts of sev-
eral constituents, including sugars, organic and amino acids, and vo-
latile aromatic compounds. In particular, the taste of the fruit flesh is
highly dependent on the balance between soluble sugars and non-vo-
latile organic acids. Sugars of apples present different sweetness levels
and organic acids give a different perception of acidity. Consequently,
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D. Zhu, et al.
Fig. 5. Correlation analysis (A) and hierarchical cluster analysis (B) based on flavour and quality characteristics of five apple varieties.
the composition of these sugars and organic acids, as well as sugar/acid
balance, probably influences the taste of the apple. The soluble solids
content, 98.8% of which are sugars, is a standard index of fruit maturity
(Odeyemi et al., 2018). The total soluble solids (TSS), titratable acid
(TAC), and sugar acid ratio (SAR) of the five apple varieties were shown
in Fig. 2. The total soluble solids (TSS) in ‘Jonagold’ and ‘Orin’ were
higher than in others. TSS in ‘Hanfu’ and ‘Ralls’ were lower (Fig. 2(I)),
6
Scientia Horticulturae 260 (2020) 108879
and the two species were predominant cultivated in northern China.
The index of TSS is an important internal quality attribute in de-
termining planting condition, fruit maturity, and harvesting time. The
difference on TSS of the five apple varieties might due to the different
geographical location and the different nutrients absorbed by the fruit
from the soil (Melke and Fetene, 2014). Under high-temperature con-
ditions, trees tend to stop producing sugars as a result of cessation of
D. Zhu, et al.
photosynthetic activity (Melke and Fetene, 2014).
As far as titratable acid (TA) was concerned, there showed sig-
nificant difference among the five varieties (p < 0.05) (Fig. 2(I)). TAC
in ‘Ralls’ was higher, and that in ‘Orin’ was lower. Therefore, the sugar
acid ratio (SAR) of ‘Orin’ was the highest (reached 131.78), while, SAR
of ‘Ralls’ was the lowest (Fig. 2(II)). Which showed that SAR was mainly
dependent on TAC for apples. The composition of sugars, organic acids
and volatile compounds in fruit play a key role in flavour development,
and can affect the chemical and sensory characteristics of fruit. TSS and
TAC are the most important factors influencing the taste of apple. The
higher TAC and lower SAR in ‘Ralls’ gave an intense sour taste. In ad-
dition, the peel hardness and flesh hardness of ‘Ralls’ were higher, and
the fruits were smaller (Fig. 1), therefore, ‘Ralls’ seemed more suitable
for processing into apple juice. In contrast, ‘Orin’ had a higher TSS and
SAR, and this sweeter variety might be more suitable for fresh eating.
Starch content of ‘Ralls’ (Fig. 2(III)) was comparable to ‘Orin’, and there
was no significant difference (p > 0.05) between them. Starch content
of ‘Indo’ was similar to that of ‘Hanfu’, and the value was about 0.39
mg/mL.
3.3. Electronic nose analysis
Appearance and texture influence the subjective evaluation of fla-
vour, but the underlying chemistry can be reduced to molecules that
interact with taste and olfactory receptors. Olfactory sensations are
evoked by a complex array of volatile substances. The E-nose, an ana-
lytical instrument that mimics the human olfactory system, is a non-
destructive method whose detection process is simple and fast (Sun
et al., 2016). Linear Discriminant Analysis (LDA) is a common classi-
fication procedure: the method maximises the variance between cate-
gories and minimises the variance within categories. LDA emphasizes
the spatial distribution of and distances between of apple aroma com-
ponents. Fig. 3(A) showed the LDA analysis of aroma components of
five apple varieties using the e-nose. According to the discreteness of
the response values, LDA could be used to distinguish aroma among the
five apple varieties. The contribution rates of LD1 and LD2 were
88.47% and 9.00%, respectively, and the total contribution rate was
97.47%. The greater the degree of dispersion between data acquisition
points, the better differentiation of the groups. There was no obvious
overlap of volatile flavour data collection points among different apple
varieties, which indicated that LDA could better distinguish the scent. It
could be seen that the position of ‘Orin’ was close to the origin of the
horizontal coordinate axis, which was significantly different from other
varieties on a volatile flavour scale.
3.4. Electronic tongue analysis
More than 300 compounds have been identified in the volatile
fraction of apple fruit, only a few dozen of them have been recognised
as significantly contributing to the perceived fruit aroma. Studies on
fruit have found that some volatiles are correlated with acceptability, as
well as sweet taste, and sour taste. Fig. 3(B) shows the radar image of
taste determination using electronic tongue. There were no obvious
differences in bitterness, astringency, richness, aftertaste-astringency
(aftertaste-A), and aftertaste-bitterness (aftertaste-B) among these five
apple varieties.
However, for sourness, saltiness, and umami, there were significant
differences among the five varieties of apples. Glutamic acid is re-
sponsible for the characteristic umami taste of the present-day culti-
vated apple fruit (Bemer et al., 2012). The relationships between
sourness, saltiness, and umami among the five apple varieties were
shown in Fig. 3(C–E): the umami taste of five kinds of apple (in des-
cending order) was: ‘Indo’, ‘Orin’, ‘Hanfu’, ‘Jonagold’, and ‘Ralls’
(Fig. 3(C)). The sourness positively correlated to umami in all cultivars
(R2 =0.839, p < 0.01). The critical point of sourness is -13 in e-
tongue analysis. The sourness of ‘Orin’ was -11.92, which was close to
7
Scientia Horticulturae 260 (2020) 108879
the critical point. So, the lowest sourness was found in ‘Orin’, and the
highest sourness was in ‘Ralls’, which was in accordance with the TAC
results (Fig. 2). Fig. 3(D) showed that the correlation of sourness and
saltiness taste was not remarkable among these apples. The critical
point for saltiness is -6, which meant the saltiness taste of ‘Ralls’ was
higher, and that of ‘Indo’ was lower. According to Fig. 3(D–E), the
umami and sourness of ‘Jonagold’ and ‘Hanfu’ were quite different,
although their saltiness was similar. In general, the taste of ‘Orin’ and
‘Hanfu’ were relatively moderate.
3.5. Analysis of volatile profiles of apples
Flavour is a complex trait resulting from combinations of sugars,
acids, and many volatile chemicals (Du and Rouseff, 2014). To identify
the difference of volatile components in different apple samples, the
aroma components of apple were analyzed and identified using SPME
coupled with GC–MS. There were about 45 kinds of volatile compounds
detected in apples with matched-degree greater than 80% (Table 1).
Esters have distinctive fruit-like odours and are important in apple
fruits. Among these five varieties of apples, the most volatile com-
pounds were 25 types of esters. These results were consistent with the
research of Satish Kumar et al (Kumar et al., 2015). ‘Orin’ had abundant
volatile flavour substances, especially for esters. ‘Hanfu’ possessed more
species of alcohols.
From Table 1, the esters, aldehydes, alcohols, and aromatics were
the main compounds in apple, followed by ketones, alkanes, and al-
kenes. In total, 25 kinds of esters were identified by SPME/GC–MS in
these apples. Among them, the contents of hexanol acetate in ‘Jonagold’
(438.66 μg/kg) and ‘Indo’ (712.78 μg/kg) were higher than that of the
other varieties. The ethyl hexanoate content in ‘Orin’ was the highest
(322.24 μg/kg) among the varieties. Ethyl hexanoate is a volatile that
provides the fruity aromas detected in many fruits such as pineapple,
pear, mango, and strawberry. There were 7 kinds of esters (ethyl bu-
tyrate, ethyl valerate, ethyl 3-hydroxybutyrate, ethyl octanoate, ethyl
trans-2-octenoate, ethyl trans-4-decenoate and ethylene-(E,Z)-2,4-dec-
adienoate) only in ‘Orin’.
A total of 4 kinds of aldehydes were identified. Among them, the
content of (E)-2-hexenal in ‘Hanfu’ was higher, and those in ‘Jonagold’
and ‘Orin’ were little. (E)-2-hexenal confers a green leafy sensorial at-
tribute to apple flavour (Amaro et al., 2012). The hexanal content in
‘Hanfu’ was much higher than that in other varieties. Hexanal is an
important contributor to the characteristic fish-like, sweet odours, and
which also confers a green aroma to apples. Nonanal provides a strong
smell of grease and a sweet orange flavour (Gabler et al., 2010), which
was not detected in ‘Ralls’. Decanal was just detected in ‘Indo’, and the
content therein was also lower than that in other varieties.
Ketones can be generated from the emzymatic degradation of
polyunsaturated fatty acids (PUFAs), amino acid degradation, Maillard
reaction, or microbial oxidation. Ketones contribute to the floral and
fruity sweet flavour (Odeyemi et al., 2018). Among all the volatile
components, the variety of ketone was much lower than that of other
components, and only geranyl acetone was detected.
A total of 6 kinds of alcohols were identified. Alcohols have low
odour activity and are present in low concentrations as glycosidically
bound forms in fruit (Nikfardjam and Maier, 2011). The content of 1-
hexanol in ‘Hanfu’ (233.72 μg/kg) was higher than that of other vari-
eties, and the content of hexanal in this cultivar was also the highest
(528.86 μg/kg). Presumably, ‘Hanfu’ relies more on a pathway that
converts 1-hexanal to 1-hexanol via the enzyme alcohol dehydrogenase
(ADH) (Schaffer et al., 2007). Other alcohols in all cultivars were lower.
Hydrocarbons including alkenes and alkanes were also ubiquitous in
apples. 2 kinds of alkenes and 2 kinds of alkanes were identified in the
five kinds of apples, respectively. Terpenes are naturally occurring or-
ganic compounds, produced by a variety of plants, often with a strong
aroma (Schmutzer et al., 2014). Among alkenes, α-farnesene was the
most abundant terpene detected in the five kinds of apples. Related
D. Zhu, et al.
research showed that, apples contained a large concentration of α-far-
nesene, a sesquiterpene responsible for the characteristic green apple
odour (Jaros et al., 2009). Esters are the most abundant volatile com-
pounds emitted by apple, and together with α-farnesene, have been
proposed for cultivar classification (Doron et al., 2005; Young et al.,
2004). 5 kinds of aromatic compounds were also found in the apples,
including toluene, p-xylene, naphthalene, estragole and m-xylene.
These aromatic contents differed across the various apple varieties, and
they imparted a particular aroma to each variety.
There were common volatile compounds in these apples, such as
hexyl butyrate, (E)-2-hexenal, α-famesene, and toluene. Several acetate
esters have been identified as main contributors to the overall apple
aroma (Plotto et al., 1999; Young et al., 2015), while hexanal and (E)-2-
hexenal have been described as green apple-like odours (Fuhrmann and
Grosch, 2015). (E)-2-hexenal is commonly known as acanthaldehyde,
which is named after its rich, fresh fruit, green leaf aroma. (E)-2-hex-
enal has been associated with green apple-like odour in a previous re-
port on apple aroma. Flavour is a somewhat subjective phenotype that
integrates multiple sensory systems, ultimately manifesting itself at
brain level (Kumar et al., 2015). Other research (Karlsen et al., 1999)
showed that the main volatile components contributing to the sensory
flavour profiles of some common apple cultivars were: propyl acetate,
butyl acetate, 2-methylbutyl acetate, hexyl acetate, ethyl acetate, ethyl
butanoate, 1-butanol, ethanol and α-farnesene.
3.6. Principal component analysis
Principal component analysis (PCA) is used to provide a partial vi-
sualisation of data in a reduced-dimension plot. It is a mathematical
procedure used to resolve sets of data into orthogonal components, in
which linear combinations (principal components) approximate the
original data to any desired degree of accuracy (Delgado et al., 2010).
Fig. 4 showed the loadings plots in PCA for the difference apple volatile
compounds (Table 1) by the first two principal components (PC1 and
PC2), which contribution rate was 47.48% and 25.19%, respectively.
By principal component analysis, PC1 mainly represented esters and
alcohols, while PC2 mainly represented esters, and the two kinds of
substances had an important effect on the volatile flavour of the apples.
There were 10 kinds of esters and 5 kinds of alcohols in the first
quadrant, and they might more have a great influence on apple flavour.
By GC–MS (Table 1), the four flavour substances of Es15 (hexyl buty-
rate), Ald2 ((E)-2-hexenal), Alke2 (α-farnesene), and Ar1 (toluene)
were found in the five apple varieties. From Fig. 4, Es15 (hexyl buty-
rate), Ald2 ((E)-2-hexenal), Alke2 (α-farnesene) and Ar1 (toluene) were
located in the positive region of PC1. Related research also showed that
hexyl butyrate (Scalzo et al., 2003), (E)-2-hexenal (Rizzolo et al., 2010),
α-farnesene (Lara et al., 2006) were important volatile compounds in
apples. This results were consistent with theirs.
3.7. Correlation analysis and hierarchical cluster analysis
Flavour is one of the sensory qualities of apple fruits, and appear-
ance and texture also influence the subjective evaluation. The correla-
tion analysis of sensory indicators of apple varieties was conducted in
Fig. 5(A). The brightness value of L* was significantly correlated to
other indices except saltiness. It was negatively correlated with a*,
which expressed the redness and greenness of apples. a* value was
significantly negatively correlated with b*, flesh hardness, SAR, and
umami (p < 0.01). b* was significantly negatively correlated with all
response values (W1C, W1S, W2S, and W2W) of the e-nose (p < 0.05),
but positively correlated with SAR and umami (p < 0.01). That meant,
when the b* value increased, the colour of the apple became yellower,
the smell faded, and the apple turned sweeter, and exhibited an in-
creased umami taste. The response values of the four sensors in e-nose
analysis were significantly positively with each other (p < 0.01), and
only W2W was negatively correlated with umami. There was no
8
Scientia Horticulturae 260 (2020) 108879
significant correlation between SAR and the fresh hardness or saltiness
(p > 0.05).
Based on the correlation analysis, the hierarchical cluster analysis
(HCA) of five kinds of apple fruits was conducted. HCA revealed re-
lationships between apple varieties by the distance between quality and
flavour indicators of the samples. The HCA grouped samples in clusters,
based on their similarities calculated from distances between samples,
using all variables simultaneously (Khakimov et al., 2016). This ap-
proach clustered sensory evaluations from the five varieties and pro-
vided an overview (dendrogram) of their similarities (Fig. 5(B)). In
terms of their sensory and flavour profiles, ‘Hanfu’ was most similar to
‘Jonagold’, while ‘Indo’ and ‘Ralls’ were more close to ‘Orin’.
4. Conclusions
‘Hanfu’, ‘Ralls’ and ‘Jonagold’ were red apple varieties, while ‘Orin’
and ‘Indo’ were green varieties. The hue angle and chroma of ‘Orin’
were lower, but its sugar acid ratio was higher. The flesh hardness and
peel hardness of ‘Jonagold’ were lower. An electronic nose could dis-
tinguish the smell difference among apple varieties, and the aroma of
‘Orin’ was much different from other varieties as evinced by LDA of E-
nose. Electronic tongue analysis showed that, sourness, saltiness, and
umami, there were obvious differences. By SPME/GC–MS analysis,
there were common volatile flavour compounds in these apples, such
as, hexyl butyrate, (E)-2-hexenal, α-famesene, and toluene. The most
abundant volatile compounds in apples were esters. Volatile com-
pounds were abundant in ‘Orin’. The results of PCA showed that,
nonanal, dipentene and m-xylene were the most important volatile
compounds in apples. HCA, based on electronic nose, electronic tongue
and other quality indicators, showed that ‘Hanfu’ was closely related to
‘Jonagold’, while ‘Indo’ and ‘Ralls’ were closely related to ‘Orin’.
Acknowledgments
This work was financed by the National Key Technologies Research
and Development Programme (2017YFD0400106) of China and
National Natural Science Foundation of China (31701618). We thank
Dr. Danshi Zhu and Dr. Jianrong Li (Bohai University, CN) for their
critical review of the manuscript.
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