Food Chemistry 141 (2013) 34023408

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The effect of cryogenic grinding and hammer milling on the avour

quality of ground pepper (Piper nigrum L.)
Hong Liu a,1, Fankui Zeng b,1, Qinghuang Wang a,, Shiyi Ou c, Lehe Tan a, Fenglin Gu a
a

Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning 571533, China
Research and Development Center for Eco-Material and Eco-Chemistry, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China
c
Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China
b

a r t i c l e

i n f o

Article history:
Received 22 February 2013
Received in revised form 31 May 2013
Accepted 12 June 2013
Available online 21 June 2013
Keywords:
Cryogenic grinding
Hammer milling
Sensory property
Flavour quality
Storage

a b s t r a c t
In this study, we compared the effects of cryogenic grinding and hammer milling on the avour attributes
of black, white, and green pepper. The avour attributes were analysed using headspace solid-phase
micro-extraction (HS-SPME) and gas chromatography-mass spectrometry (GC/MS), sensory evaluation
and electronic nose (e-nose) analysis. Cryogenic grinding resulted in minimal damage to the colour, avour, and sensory attributes of the spices. Cryogenic grinding was also better than hammer milling at preserving the main potent aroma constituents, but the concentrations of the main aroma constituents were
dramatically reduced after storing the samples at 4 C for 6 months. Pattern matching performed by the
e-nose further supported our sensory and instrumental ndings. Overall, cryogenic grinding was superior
to hammer milling for preserving the sensory properties and avour attributes of pepper without significantly affecting its quality. However, we found that the avour quality of ground pepper was reduced
during storage.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Considered to be The King of Spices, pepper shares a prominent place on most dinner tables next to salt. Black, white, and
green peppers are all derived from the fruits of Piper nigrum L., a
plant species cultivated throughout the tropical world. Black pepper is harvested before it reaches full maturity, while green pepper
is produced from unripe green fruits. Because it is derived from unripe fruit, green pepper has a fresh, herbal avour. In contrast,
white pepper is made from fully ripe pepper fruits, following the
removal of their outer skin. White pepper is typically described
as having a milder avour than black pepper. Attributes such as avour, taste, and pungency vary among cultivars and varieties
(Friedman, Levin, & Lee, 2008; Mamatha, Prakash, Nagarajan, &
Bhat, 2008). However, the methods used to process (Murthy, Rrni,
& Rao, 1999; Plessi, Bertelli, & Miglietta, 2002; Waje, Kim, Kim,
Todoriki, & Kwon, 2008; Zhong, Akai, Osaki, & Iwabuchi, 2003)
Abbreviations: BPCM, black pepper ground by cryogenic grinding; BPHM, black
pepper ground by hammer milling; WPCM, white pepper ground by cryogenic
grinding; WPHM, white pepper ground by hammer milling; GPCM, green pepper
ground by cryogenic grinding; GPHM, green pepper ground by hammer milling.
Corresponding author. Tel.: +86 898 66962942; fax: +86 898 66962948.
E-mail addresses: hongliu1982@yahoo.com (H. Liu), zengfk@licp.cas.cn (F. Zeng),
wangqh1967@yahoo.com (Q. Wang).
1
These authors contributed to the work equally and should be regarded as co-rst
authors.
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.06.052

and preserve the spice (Nisha, Singhal, & Pandit, 2009; Rao et al.,
2011; Steinhaus & Schieberle, 2005; Suresh, Manjunatha, & Srinivasan, 2007) have been identied as key factors that affect the
characteristic pungent taste and avour of pepper.
The objective of grinding spices is to obtain a powder with the
smallest particle sizes that also has excellent quality attributes,
such as aroma and avour. Spices can be ground in a hammer
mill (Singh & Goswami, 1999), an attrition mill (Goswami &
Singh, 2003), or a freezer mill (also called cryogenic grinding)
(Murthy & Bhattacharya, 2008). In modern spice milling, cryogenic grinding is generally used because it performs better than
other methods, given that liquid nitrogen, at 195.6 C, provides
the refrigeration needed to pre-cool spices and also keeps them at
a low temperature. In addition, the vaporisation of liquid nitrogen
to the gaseous phase creates an inert, dry environment that helps
further protect the spice quality. Pre-cooling raw spices and
maintaining the appropriate low temperature within the mill reduces the loss of volatile oils, which are responsible for the
strength of a spices avour (Murthy & Bhattacharya, 2008; Singh
& Goswami, 1999, 2000). It has previously been reported that
cryogenically ground spices retained their colour better than
spices ground in a traditional mill at ambient conditions (Pesek
& Wilson, 1986). In addition, cryogenic grinding of black pepper
in a laboratory-scale grinding system resulted in better product
characteristics than were found in pepper that was not cryogenically ground (Murthy, Krishnamurthy, Ramesh, & Srinivasa Rao,

H. Liu et al. / Food Chemistry 141 (2013) 34023408

1996). However, the effects of cryogenic grinding on the quality

of green and white pepper have not yet been reported. In the
present study, we analysed the avour quality of black, white,
and green pepper crushed using different techniques. The spices
underwent cryogenic freezing and were then hammered into
250-lm sized particles. The particles were analysed using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC/MS), sensory evaluation, and an
electronic nose (e-nose). The effect of storage on the avour quality of each spice was also assessed.

2. Materials and methods

2.1. Materials
Fresh berries of the pepper variety Reyin2 were collected in
May 2012 at the Spice and Beverage Research Institute at the Chinese Academy of Tropical Agricultural Sciences in Hainan, China.
The pepper variety Reyin2 is a new pepper variety that has been
identied by the scientists from Spice and Beverage Research
Institute.
Black pepper was produced from fully-mature green fruits,
which were dried in a cross-ow dryer at 50 C, resulting in fruits
with a brownish-black colour and a shrivelled surface. White pepper was produced from ripe orange-to-red fruits, which were dried
in a cross-ow dryer at 50 C after the outer skin was removed.
Green peppers was produced from unripe green fruits by blanching
them in boiling water for 10 min in order to inactivate the browning enzymes and then drying them in a cross-ow dryer at 50 C
(Gopinathan & Manilal, 2004; Mangalakumari, Sreedharan, &
Mathew, 1983; Variyar, Pendharkar, Banerjee, & Bandyopadhyay,
1988). Broken and immature seeds, as well as foreign matter, were
removed from the samples prior to all treatments (Murthy & Bhattacharya, 1998).

2.2. Standards
Chemical standards of a-pinene, b-pinene, b-myrcene, d-3-carene, limonene and linalool were purchased from Dr. Ehrenstorfer
GmbH (Augsburg, Germany). Standards of b-caryophyllene were
purchased from SigmaAldrich (Steinheim, Germany). The purity
of all standards was >90%. These chemicals were chosen as standards for this study because they were the most abundant compounds in pepper aromas (Jagella & Grosch, 1999a, 1999b,
1999c; Jirovetz, Buchbauer, Ngassoum, & Geissler, 2002) .

2.3. Cryogenic grinding

Samples of black, white, and green peppercorns were ground
separately using a cryogenic grinder (SPEX 6870; SPEX Metuchen,
NJ). First, peppercorns were pre-frozen in a liquid nitrogen bath
( 210 to 196 C) for 10 min and then pulverised with a magnetically driven impactor at 10 s 1 in the liquid nitrogen bath. Peppercorns were subjected to ve pulverisation cycles, each of which
consisted of a 2-min grinding period followed by a refreezing period. The refreezing step was done to ensure that pepper samples
were maintained at proper cryogenic temperatures at all times because grinding caused the temperature of the spice to increase. The
ground samples were passed through 250-lm sieves, resulting in
black, white, and green pepper powders, which were stored separately in black packaging bags in a 4 C refrigerator for 6 months
until analysis.

3403

2.4. Hammer milling

Samples of black, white, and green peppercorns were ground at
ambient temperature using a hammer mill (IKM-310 system, China) and following standard protocols. The temperatures of samples
before hammer milling were 29.530 C (12 C higher than room
temperature), and the temperatures of samples after hammer milling were 3740 C. The opening sizes of the hammer-mill screens
used were 250 lm. In addition, peppercorns were passed through
the hammer mill two or three times to produce particles that were
comparable in size to those generated using cryogenic grinding
(see Section 2.3). The hammer mill was cleaned between passes
to avoid cross-contaminating different samples. The resulting
black, white, and green pepper powders were stored separately
in black packaging bags in a 4 C refrigerator for 6 months until
analysis.
2.5. Determination of Hunters colour values
The Hunters colour L (lightness), a (redness), and b (yellowness) values were determined using a colorimeter (X-Rite SP62; XRite, Grand Rapids, MI). Each ground pepper sample was distributed on a piece of paper and measurements were taken at three
random locations. The L, a and b values of the standard plate
were 95.02, 1.04, and 0.05, respectively.
2.6. Analysis of avour components by solid-phase microextraction
(SPME)
Headspace volatiles were collected using a 50/30 lm divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) StableFlex SPME bre assembly A carefully ground quantity (1.0 g) of
each sample was placed in a 10.0-mL glass vial, and the vial was
closed using a septum. The sample was then heated to 60 C, and
the SPME bre was exposed to the headspace for 30 min. The SPME
bre was desorbed at 250 C for 5 min in the injection port of an
Agilent 7890A gas chromatograph coupled to an Agilent 5975C
quadrupole mass spectrometer (Agilent Technologies, Santa Clara,
CA) in electron ionisation mode (EI) at 70 eV. The ion source and
quadrupole temperatures were 230 C and 150 C, respectively,
and the mass spectrometer scanned from m/z 500 to m/z 30. Helium (99.99% purity) was the carrier gas at 1.0 mL/min. Volatile
compounds were separated on a non-polar HP-5 ms fused capillary
column (length, 30 m; inner diameter, 0.25 mm; lm thickness,
0.25 lm) and on a polar DB-Wax fused capillary column (length,
30 m; inner diameter, 0.25 mm; lm thickness, 0.25 lm). The
injection port was operated in splitless mode and the oven temperatures were programmed with the following methods. For HP5 ms: 50 C (0 min), 5085 C (15 C/min), 85140 C (8 C/min),
140200 C (4 C/min), 200280 C (15 C/min). For DB-Wax:
40 C (0 min), 40200 C (4 C/min), 200 C (5 min). Each pepper
powder sample was analysed in triplicate. The individual components were identied on the basis of retention indices (I) with reference to a homologous series of n-alkanes (C8C24) on both
columns, by matching the mass spectra of our samples to spectra
contained in the National Institute of Standards and Technology
(NIST) spectral library. In addition, we compared the fragmentation
patterns in the mass spectra of our samples with those reported in
the literature (Cardeal, Gomes da Silva, & Marriott, 2006). Component relative percentages were calculated based on GC/MS peak
areas with area normalisation method.
For the quantitative determination of the main pepper aroma
constituents, standard solutions (in chromatographic purity n-hexane) of a-pinene, b-pinene, b-myrcene, d-3-carene, limonene, linalool and b-caryophyllene were diluted to yield suitable
concentrations of 0.1, 1.0, 10.0, 100.0 and 1000.0 ppm. The

3404

H. Liu et al. / Food Chemistry 141 (2013) 34023408

recovery trials using HS-SPME were carried out at different concentrations for standards. A 1.0-mL aliquot of each standard was
transferred to a 1.5-mL vial, capped and analysed by GC/MS (Mazida, Salleh, & Osman, 2005) with HP-5 ms column; the method was
as same as that reported above. The volume injected of standard
solutions was 1.0 lL; each samples concentration was determined
in triplicate. The calibration curves for sample concentrations were
plotted using OriginPro 8.0 software (OriginLab Corp., Northampton, MA), which calculated the concentration of each pepper aromas constituents based on its peak area.

2.9. Statistical analysis

Data were expressed as mean S.D. Statistical analysis was performed using a one-way analysis of variance (ANOVA). A p-value of
0.05 or less was considered to be statistically signicant. Statistical
calculations were performed using SPSS software (Version 13.0;
SPSS Inc., Chicago, IL) for the Windows operating system.
3. Results and discussion
3.1. Hunters colour values

2.7. Sensory evaluation

Samples were also subjected to sensory assessment for colour,
odour, and pungency of taste by 15 trained judges (both male
and female) between 25 and 35 years old. Approximately 2.0 g of
each sample were placed in a disposable petri dish and given to
each panellist. The single-blind study was performed by labelling
each sample with a unique three-digit number. The panellists were
instructed to record their ratings using a 10-point hedonic scale.
The mean quality ratings were designated as follows: 2 was considered not usable, 34 was poor, 5 was average, 6 was fair,
78 was good, and 910 was very good (Narasimhan, Chand,
Rajalakshmi, & Indiramma, 1990). Colour was determined under
ordinary light conditions. Odour and pungency of taste were evaluated using a pepper powder fusion, which was prepared by dissolving 1.0 g of powder in 100 mL of 60 C water (Waje et al.,
2008).

2.8. Measurements using an e-nose

An e-nose (a-Gemini, Alpha M.O.S. SA, Toulouse, France) assessment was performed using metal oxide semiconductor (MOS)
chambers equipped with six sensors (LY2/AA, LY2/gCT, T30/1, PA/
2, P30/2, T70/2) in the auto-sampler setting (CTC 6500; CTC Analytics AG, Zwingen, Switzerland). An e-nose is able to detect a
change in the amount of a chemical compound because the mass
change causes the sensors electrical resistances to change accordingly, which is then measured.
Each sample (0.2 g) was placed in a 10-mL sample vial. Using a
crimping tool, each sample vial was sealed with a tted cap and
septum. Each vial was sealed tightly to prevent leaks, which would
articially decrease the signal strength. Then, the sample vials
were placed in the instrumental tray for analysis. The samples
were measured in standard headspace vials in triplicate. Standard
headspace auto-sampling was used in this study. First, a sample
was incubated at 35 C for 2.0 min. Then, 800 lL of the sample
was injected and measured for 3.5 min. After each measurement,
the reference gas was streamed through the sensor array for
1.0 min to clean it. The data collected from e-nose measurements
were analysed using principle components analysis (PCA) to differentiate the odours of different ground pepper samples.

Noticeable colour differences were observed in black, white,

and green pepper samples (Table 1). When considering the two different grinding methods, we found that hammer milling resulted
in a signicantly greater colour change than cryogenic grinding.
The pepper products obtained after hammer milling exhibited reduced L and b values, indicating that pepper samples darkened
during milling. Furthermore, the degree of redness in white and
green pepper became more intense (higher a values), while a values of black pepper decreased after hammer milling. Compared
with cryogenic grinding, hammer milling caused more discoloration in black, white, and green pepper samples. The colours of
cryogenically ground pepper samples were less affected because
cooling them with liquid nitrogen decreased their temperature
and reduced their exposure to oxygen. Our colour results are consistent with the ndings of a previous study (Pesek & Wilson,
1986), which noted that cryogenically ground spices were lighter
in colour than ambient milled spices, and untrained panellists
were able to detect those colour differences. In addition, higher
temperatures have been reported to promote colour degradation
in spices (Almela, Nieto-Sandoval, & Lpez, 2002; Waje et al.,
2008). Storing samples under refrigeration (4 C) for 6 months resulted in a slight darkening of black and green pepper samples,
indicated by their increased a and b values; these ndings are
also consistent with previous studies (Waje et al., 2008).
3.2. Flavour components
The percentages of avour compounds in cryogenically ground
and hammer-milled black, white, and green pepper are presented
in Table 2. Forty-one compounds were identied, which represent
more than 93.2% of the total avour compounds found in the pepper samples. The primary monoterpene compounds of all three
pepper aromas were a-pinene, b-pinene, d-3-carene, limonene, terpinolene, and 4-carene, while their sesquiterpene compounds were
d-elemene, a-copaene, b-caryophyllene, and a-caryophyllene.
These compounds accounted for 81.3094.97% of the total aroma
compounds in both cryogenically ground and hammer-milled samples. Although b-myrcene and linalool were relatively minor constituents, they are the most potent odorants of black pepper
(Jagella & Grosch, 1999a; Jagella & Grosch, 1999b). We found
marked differences in the compositions of volatile aroma

Table 1
Hunters colour valuesa from cryogenic grinding and hammer milling of black, white, and green pepper stored at 4 C for 0 months and 6 months.
Hunters parameterb

Months

BPCM

BPHM

WPCM

WPHM

GPCM

GPHM

0
6
0
6
0
6

56.33 1.31bx
59.33 0.26by
2.31 0.21cx
2.39 0.04ex
17.36 0.47bx
19.83 0.30cy

53.58 0.70ax
54.15 0.14ax
2.09 0.09cx
2.17 0.00dx
11.55 0.34ax
16.53 0.08ay

75.17 0.66fx
76.32 0.28ex
0.96 0.14bx
0.56 0.07cy
20.60 0.13cx
20.12 0.06cx

73.14 0.91ey
75.73 0.39ex
1.12 0.12bx
0.11 0.06by
20.40 0.59cx
17.65 0.12by

68.30 0.26dx
68.97 0.57dx
-0.75 0.12ax
-0.62 0.05ax
27.06 0.25dx
28.54 0.22ey

65.71 0.80cx
65.24 0.22cx
-0.56 0.10ax
-0.66 0.07ax
20.69 0.35cx
23.39 0.27dy

a
Values represent means S.D. (n = 3). Means followed by the same letters within a row (af) and within a column per parameter (x and y) are not signicantly different
(p < 0.05).
b
Hunters parameter: L, degree of lightness (white + 100?0 black); a, degree of redness (red + 100? 80 green); b, degree of yellowness (yellow + 70? 80 blue).

925
942
960
975
981
990
1007
1015
1024
1028
1038
1048
1063
1071
1087
1092
1102
1192
1202
1233
1342
1352
1378
1385
1396
1416
1435
1445
1469
1487
1495
1498
1501
1509
1514
1521
1533
1564
1580
1587
1609

1019
1033

0.11 0.01
7.73 0.25
0.29 0.02
ND
18.26 0.24
0.30 0.07
0.39 0.04
22.08 1.24
ND
ND
18.78 0.06
0.27 0.01
0.43 0.07
ND
1.48 0.21
2.78 0.25
0.43 0.11
ND
0.10 0.00
ND
3.88 0.48
0.29 0.04
ND
3.03 0.23
0.90 0.07
0.26 0.02
13.69 0.42
1.32 0.01
0.12 0.01
0.15 0.01
0.17 0.01
0.10 0.00
ND
0.24 0.01
ND
0.21 0.01
0.53 0.09
0.27 0.01
0.30 0.01
0.11 0.01
0.12 0.01
99.12
73.43
25.69
2.86

6 Months
0.16 0.12
6.63 1.15
0.22 0.12
0.35 0.00
11.68 1.55
0.59 0.42
0.37 0.08
17.75 2.70
0.36 0.00
0.86 0.44
12.74 1.47
0.18 0.06
0.37 0.17
ND
1.53 0.37
1.52 0.63
0.27 0.04
ND
0.10 0.01
ND
7.60 0.87
0.48 0.03
0.12 0.01
4.62 0.47
1.61 0.26
0.34 0.02
18.99 2.44
2.58 0.30
0.09 0.01
0.18 0.02
0.16 0.02
0.10 0.01
0.15 0.02
0.48 0.08
ND
0.57 0.04
1.42 0.14
0.79 0.07
0.78 0.08
0.29 0.01
1.08 0.03
98.11
55.68
42.43
1.31

0 Months

BPHM

ND
14.19 0.69
0.56 0.01
ND
21.83 1.04
0.44 0.05
0.41 0.03
21.96 2.10
ND
ND
17.29 0.56
ND
0.30 0.05
ND
3.09 0.70
0.86 0.47
0.34 0.06
ND
ND
ND
0.92 0.04
0.22 0.01
ND
2.59 0.11
0.52 0.04
0.25 0.05
10.56 0.42
1.02 0.06
ND
0.14 0.01
0.18 0.02
0.14 0.04
ND
0.22 0.01
ND
0.36 0.04
0.44 0.03
0.22 0.01
0.26 0.02
0.12 0.02
0.03 0.01
99.46
81.27
18.19
4.47

6 Months
0.25 0.16
7.63 0.70
0.31 0.19
0.26 0.04
13.77 0.88
0.12 0.05
0.14 0.05
17.34 4.72
5.09 0.00
1.44 0.72
15.03 0.62
ND
0.44 0.04
0.12 0.02
1.55 0.35
1.26 0.47
0.36 0.04
0.10 0.01
0.12 0.01
0.13 0.01
5.52 0.35
0.26 0.01
0.06 0.01
2.69 0.18
1.08 0.09
0.14 0.01
18.95 2.50
1.33 0.17
ND
0.13 0.01
0.12 0.01
0.10 0.01
0.18 0.02
0.17 0.03
0.09 0.01
0.17 0.02
0.96 0.08
0.23 0.02
0.22 0.02
0.15 0.01
1.65 0.13
99.66
65.46
34.2
1.91

0 Months

WPCM

0.06 0.01
4.96 0.17
0.83 0.01
0.16 0.03
15.18 0.20
0.30 0.01
0.34 0.00
22.17 0.29
ND
0.12 0.00
22.78 0.39
ND
0.35 0.01
0.12 0.01
1.70 0.03
2.86 0.05
0.53 0.03
0.13 0.01
0.15 0.01
0.16 0.01
3.17 0.06
0.20 0.01
ND
2.27 0.02
0.74 0.02
0.14 0.01
16.03 0.18
1.50 0.08
ND
0.15 0.01
0.20 0.06
0.10 0.01
ND
0.29 0.01
ND
0.11 0.01
0.39 0.04
0.17 0.01
0.17 0.02
ND
0.24 0.02
98.77
72.9
25.87
2.82

6 Months
0.16 0.06
6.69 1.09
0.25 0.12
0.40 0.22
12.07 1.65
0.11 0.06
0.20 0.12
16.73 2.71
ND
2.57 1.53
13.44 1.92
ND
0.28 0.08
0.12 0.03
1.99 0.52
0.71 0.27
0.40 0.07
0.12 0.03
0.13 0.04
0.15 0.03
6.52 0.65
0.28 0.01
0.07 0.01
2.91 0.27
1.23 0.17
0.16 0.01
17.52 1.41
2.72 0.33
ND
0.18 0.03
0.13 0.02
0.11 0.01
0.22 0.01
0.24 0.05
0.11 0.01
0.26 0.04
1.21 0.16
0.29 0.04
0.34 0.05
0.19 0.02
1.96 0.17
93.17
56.52
36.65
1.54

0 Months

WPHM

Values represent means S.D. (n = 3). ND = not detected.

a
The relative percentage of the avour compounds was calculated in peak areas using a normalisation method without using correction factors.
b
I means retention indices according to the literature (Cardeal, Gomes da Silva, & Marriott, 2006).

0.14 0.06
6.80 1.09
0.19 0.06
0.38 0.00
11.74 1.50
0.62 0.30
0.26 0.06
18.10 2.80
0.38 0.00
0.95 0.40
12.91 1.57
0.21 0.07
0.50 0.03
ND
1.18 0.77
1.97 0.93
0.27 0.03
ND
0.10 0.04
ND
7.36 0.75
0.56 0.02
0.13 0.01
4.95 0.48
1.63 0.23
0.26 0.03
19.04 2.53
2.51 0.24
0.11 0.01
0.17 0.02
0.17 0.01
0.11 0.01
0.15 0.02
0.30 0.07
ND
0.64 0.08
1.47 0.11
0.74 0.06
0.74 0.07
0.29 0.01
1.09 0.05
99.12
56.70
42.42
1.34

0 Months

930
939
956
975
980
995
1003
1031
1025
1025
1037
1050
1060

BPCM

DB-Wax

Lit.b

HP-5MS

Camphene
Sabinene
1156
b-Pinene
1140
b-Myrcene
1197
a-Phellandrene
1224
d-3-carene
1180
a-Terpinene
1236
p-Cymene
1290
Limonene
1229
E-b-Ocimene
1280
c-Terpinene
1275
q-Cresol
Terpinolene
1088
1307
4-Carene
1097
Linalool
1105
1551
Terpinen-4-ol
1197
1597
a-Terpineol
1213
1682
Eucarvone
1233
d-Elemene
1344
1515
a-Cubebene
1351
1517
Cyclosativene
1371
a-Copaene
1387
1522
b-Cubebene
1394
1524
a-Gurjunene
1421
b-Caryophyllene
1431
1604
a-Caryophyllene
1440
1699
a-Humulene
1469
1650
c-Muurolene
1487
1729
a-Curcumene
1495
Germacrene D
1496
1690
d-Selinene
1501
1756
b-Selinene
1507
1698
b-Guaiene
1514
b-Bisabolene
1520
1740
d-Cadinene
1533
1772
a-Calacorene
1560
Germacrene B
1578
1805
b-Calacorene
1585
Caryophyllene oxide 1605
1986
Total
Monoterpenes
Sesquiterpenes
Ratio of monoterpenes and sesquiterpenes

a-Thujene
a-Pinene

Compounds

0.42 0.05
8.29 0.12
ND
1.91 0.12
21.24 1.92
0.41 0.02
0.51 0.01
22.67 0.42
ND
ND
19.27 0.11
ND
0.50 0.02
0.12 0.01
1.65 0.08
2.91 0.11
0.43 0.03
0.11 0.01
ND
ND
0.95 0.03
0.10 0.00
ND
1.42 0.08
0.33 0.03
ND
11.69 0.31
1.17 0.05
ND
0.32 0.10
0.12 0.05
0.15 0.05
ND
0.11 0.03
ND
0.23 0.03
0.33 0.02
0.22 0.02
0.13 0.02
0.40 0.27
0.05 0.01
98.16
80.44
17.72
4.54

6 Months

Table 2
The percentagesa (%) of avour components from cryogenic grinding and hammer milling of black, white, and green pepper stored at 4 C for 0 months and 6 months.

0.10 0.04
6.74 0.50
0.19 0.02
0.45 0.22
12.12 1.23
0.25 0.02
0.29 0.04
18.03 5.40
0.28 0.05
0.67 0.45
11.89 2.00
ND
0.40 0.15
ND
1.30 0.44
1.78 0.60
0.36 0.06
ND
0.15 0.05
ND
7.15 0.46
0.69 0.05
0.15 0.01
5.83 0.35
1.95 0.28
0.46 0.02
19.14 2.04
1.51 0.24
0.13 0.01
0.24 0.03
0.27 0.01
0.13 0.01
0.27 0.03
0.44 0.08
0.10 0.01
0.54 0.08
2.48 0.37
0.99 0.12
1.04 0.14
0.38 0.03
0.58 0.05
99.47
55.00
44.47
1.24

0 Months

GPCM

ND
8.07 0.23
0.27 0.00
ND
16.31 0.29
0.22 0.02
0.35 0.01
23.89 0.13
ND
ND
16.71 0.08
ND
0.40 0.02
ND
3.52 0.19
0.28 0.01
0.39 0.05
ND
ND
ND
1.61 0.04
0.33 0.01
0.12 0.01
3.77 0.06
0.97 0.04
0.46 0.08
15.65 0.41
1.65 0.05
0.18 0.01
0.12 0.00
0.41 0.01
0.15 0.01
0.13 0.01
0.13 0.01
0.38 0.01
0.27 0.01
0.88 0.13
0.41 0.00
0.45 0.01
0.23 0.08
0.37 0.06
99.08
70.41
28.67
2.46

6 Months
0.17 0.11
4.92 0.61
0.26 0.00
1.06 0.00
11.49 1.29
0.22 0.00
0.18 0.09
16.04 2.62
0.34 0.00
0.99 0.00
11.75 1.65
ND
0.50 0.16
ND
2.07 0.91
1.40 1.15
0.21 0.13
ND
0.07 0.03
ND
7.83 1.08
0.60 0.04
0.12 0.01
4.99 0.28
1.48 0.26
0.39 0.02
18.08 2.25
2.99 0.29
0.11 0.00
0.19 0.02
0.23 0.02
0.11 0.00
0.31 0.05
0.34 0.07
0.12 0.01
0.45 0.03
1.93 0.17
0.79 0.07
0.83 0.09
0.31 0.01
0.67 0.04
94.54
51.67
42.87
1.21

0 Months

GPHM

0.15 0.00
17.63 1.20
ND
ND
24.83 0.12
0.30 0.08
0.49 0.06
21.24 0.91
ND
ND
15.48 0.37
ND
0.55 0.08
ND
1.31 0.26
2.43 0.42
0.29 0.03
ND
ND
ND
1.83 0.39
0.20 0.04
ND
1.57 0.28
0.58 0.04
0.14 0.02
7.91 0.34
0.74 0.05
0.12 0.05
ND
0.18 0.02
0.03 0.00
0.04 0.01
0.04 0.00
0.16 0.01
0.13 0.04
0.25 0.05
0.08 0.00
0.12 0.00
0.10 0.03
0.12 0.04
99.04
84.7
14.34
5.91

6 Months

H. Liu et al. / Food Chemistry 141 (2013) 34023408

3405

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H. Liu et al. / Food Chemistry 141 (2013) 34023408

Table 3
The calibration curves of standard compounds.
Compounds

a-Pinene
b-Pinene
b-Myrcene
d-3-Carene
Linalool
Limonene
b-Caryophyllene

Line equation
8

Y = 3.58  10 + 1.08  10 X
Y = 1.69  109 + 9.61  106X
Y = 2.07  108 + 6.84  105X
Y = 2.70  108 + 1.36  106X
Y = 4.84  107 + 3.42  105X
Y = 3.28  108 + 1.12  106X
Y = 2.68  109 + 1.15  107X

0.9355
0.9078
0.9332
0.9583
0.9847
0.9287
0.9294

0.0195
0.0331
0.0205
0.0102
0.0023
0.0226
0.0223

Range of concentrations (ppm): 0.1, 1.0, 10.0, 100.0 and 1000.0 (in triplicate).

compounds when black, white, and green pepper were ground

using either cryogenic grinding or hammer milling. Compared with
hammer-milled samples, the percentages of a-pinene, b-pinene, d3-carene, b-caryophyllene and limonene in black pepper were increased by 2.56%, 0.51%, 1.97%, 0.26% and 1.33%, respectively, when
pepper was cryogenic ground. Those same compounds in white
pepper were increased by 14.05%, 14.08%, 3.65%, 11.83% and
8.16%, respectively, when cryogenic grinding was performed. In
green pepper, those same aroma compounds were increased by
36.99%, 5.48%, 12.41%, 1.19%, and 5.86%, respectively, when cryogenic grinding was used. In addition, some very potent and key
odorants, such as limonene, a-pinene, and b-pinene (Jagella &
Grosch, 1999a, 1999b, 1999c), were present in higher percentages
in cryogenically ground samples than in hammer-milled samples.
We did not detect 3-methyl-1-butanol or 2-methyl-1-propanol in
any of our samples even though both of these compounds were
previously found in black pepper after performing an aroma extract dilution analysis (Jagella & Grosch, 1999a, 1999b, 1999c). Furthermore, we did not nd b-damascenone, eugenol, skatole,
guaiacol, or piperonal in any of our samples even though they were
found after performing an aroma extract dilution analysis on a distillate of Indonesian white pepper (Nitz, 1996). These differences
may have arisen because of different varieties and methods of
analysis employed here and in their study. It was previously reported that the faecal off-avour of white pepper is enhanced by
the presence of q-cresol (Jagella & Grosch, 1999c), which we identied within white pepper samples ground by both cryogenic
grinding and hammer milling in our study; storage did not significantly affect this compound.
The avour compositions and their amounts were signicantly
different in cryogenically ground and hammer-milled samples. The
percentages of monoterpenes in unstored black, white, and green
pepper processed by cryogenic grinding were 56.7%, 65.5% and

55.0%, respectively, which were higher than the corresponding percentages found in hammer-milled samples. The reduced amounts
of monoterpenes in hammer-milled samples may be attributed
to the higher temperature that the peppercorns experience during
hammer milling compared with cryogenic grinding (Murthy &
Bhattacharya, 2008). However, the percentage of sesquiterpenes
in samples stored for 0 months was fairly constantwe found almost no difference in sesquiterpene content when black, white,
and green pepper samples were either ambiently or cryogenically
ground, possibly because sesquiterpene compounds are not very
sensitive to high temperatures (Murthy & Bhattacharya, 2008).
The ratios of monoterpenes to sesquiterpenes varied from 1.24 to
1.91 in cryogenically ground samples stored for 0 months, while
in hammer-milled samples the ratio was lower, between 1.21
and 1.54. This discrepancy shows that cryogenic grinding technique is superior to hammer milling when considering the content
of monoterpenes in the aroma compounds within ground pepper.
After storing under refrigeration (4 C) for 6 months, the percentages of monoterpenes in black, white, and green pepper all markedly increased, which may be because some sesquiterpenes were
lost or degraded as a result of browning or oxidative decomposition of the spices during storage.
For the purpose of quantitative comparison, we monitored only
the seven main pepper aroma compoundsa-pinene, b-pinene, d3-carene, limonene, b-myrcene, linalool, and b-caryophyllene. The
aroma prole used serial dilutions of the seven standard compounds as external standards, and the results can be seen in Table 3. The mean recoveries using HS-SPME for a-pinene, bpinene, b-myrcene, d-3-carene, limonene, linalool and b-caryophyllene were 93.9%, 91.2%, 97.1%, 96.2%, 96.6%, 96.9%, and
94.7%, respectively. Table 4 is based on the calibration curves,
and shows the concentration changes of the main aroma compounds after cryogenically ground and hammer-milled samples
had been stored at 4 C for 6 months. The results show that the
quantities of a-pinene, b-pinene, d-3-carene, limonene, b-myrcene,
linalool, and b-caryophyllene were signicantly higher in cryogenically ground samples than in hammer-milled samples, which
matches well with the ndings of previous studies (Balasubramanian, Gupta, & Singh, 2012; Jacob, Kasthurirengan, & Behra, 2000;
Pesek, Wilson, & Hammond, 1985). These data demonstrate that
the cryogenic grinding technique employed in our study was superior to ambient grinding in terms of the amounts of main aroma
compounds recovered in the ground powder. However, there were
extreme variations of concentration for some samples; this may be
due to the heterogeneity of the pepper samples, different grinding
methods and storage. In addition, we found that the potent

Table 4
The concentrationa (ppm) of the main avour components from cryogenic grinding and hammer milling of black, white and green pepper stored at 4 C for 0 months and
6 months.

a-Pinene
b-Pinene
b-Myrcene
d-3-Carene
Limonene
Linalool
b-Caryophyllene

Months

BPCM

BPHM

WPCM

WPHM

GPCM

GPHM

0
6
0
6
0
6
0
6
0
6
0
6
0
6

25.39 13.67bx
7.67 0.79cy
33.69 12.51cx
24.06 8.71cx
20.99 3.04bx
5.43 0.46ey
71.72 18.43ax
26.24 2.07cy
42.48 12.00abx
17.30 2.12cy
2.93 1.63ax
2.27 0.83bcx
52.28 16.75bx
5.39 0.19ay

15.31 4.12abx
5.18 0.47aby
12.16 1.31ax
7.09 0.85ax
10.27 2.60ax
3.23 0.73cy
60.43 26.80ax
20.42 1.83by
38.57 8.91abx
12.53 1.15by
2.57 0.95ax
1.39 0.32abx
40.38 0.31abx
8.91 1.34bcy

24.69 3.94bx
6.24 0.21bcy
35.10 5.81cx
16.55 1.72bcy
10.66 0.00ax
4.22 0.24dy
83.12 15.04ax
29.58 1.00dy
51.95 3.33bx
19.57 0.45cy
2.59 0.13ax
1.78 0.51abcx
42.93 0.24abx
8.30 0.13by

13.74 3.99abx
3.11 0.17ay
23.73 7.24abcx
9.90 0.33aby
7.41 0.00ax
2.85 0.13bcy
64.86 19.04ax
24.31 0.58cy
40.81 5.28abx
19.47 0.16cy
2.48 0.85ax
1.13 0.21ay
37.59 2.11ax
9.78 0.33cy

19.46 0.82abx
15.18 2.86dy
28.38 2.95bcx
24.30 6.47cx
10.07 1.05ax
2.28 0.49aby
78.83 39.42ax
40.49 2.36ey
35.63 5.96ax
22.99 1.77dy
3.37 0.99ax
2.48 0.52cx
42.63 0.83abx
8.37 1.01by

10.23 2.12ax
3.10 0.20ay
17.76 5.30abx
6.58 0.57ay
8.95 2.60ax
1.71 0.27ay
59.91 16.28ax
16.24 1.07ay
33.47 8.33ax
8.80 0.66ay
2.83 2.20ax
2.48 0.13cx
38.53 0.41ax
5.85 0.27ay

a
Values represent means S.D. (n = 3). Means followed by the same letters within the row (ae) and within the column per parameter (x and y) are not signicantly
different (p < 0.05).

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H. Liu et al. / Food Chemistry 141 (2013) 34023408

Table 5
Sensory propertiesa of cryogenically ground and hammer-milled black, white, and green pepper stored at 4 C for 0 months and 6 months.
Sensory parameter

Months

BPCM

BPHM

WPCM

WPHM

GPCM

GPHM

Colour

0
6
0
6
0
6

7.55 0.02bx
7.02 0.06ay
7.25 0.03cx
7.05 0.04cy
8.95 0.10fx
6.91 0.07by

7.12 0.03ax
6.99 0.13ax
7.20 0.05cx
6.98 0.09bcy
8.02 0.04ex
6.87 0.13aby

8.62 0.07dx
8.46 0.14cx
7.02 0.01bx
6.92 0.08bx
7.42 0.01bx
6.85 0.12aby

8.21 0.10cx
8.12 0.06bx
6.45 0.08ax
6.35 0.08ax
7.01 0.05ax
6.74 0.07ay

9.65 0.05fx
8.70 0.16dy
8.36 0.02ex
8.22 0.06ey
7.89 0.07dx
6.85 0.16aby

9.02 0.05ex
8.54 0.07cy
8.15 0.05dx
8.02 0.06dy
7.53 0.02cx
6.85 0.03aby

Odour
Pungent taste

Sensory evaluation was conducted by 15 panellists using a 10-point hedonic scale, the overall quality grading indicate that the mean score of up to 2 was considered not
usable, 34 was poor, 5 was average, 6 was fair, 78 was good, 910 was very good.
a
Values represent means S.D. (n = 15). Means followed by the same letters within the row (ae) and within the column per parameter (x and y) are not signicantly
different (p < 0.05).

Fig. 1. Electronic nose pattern matching of cryogenically ground and hammer milled pepper samples stored at 4 C for 0 months and 6 months (PC1, principal component 1;
PC2, principal component 2).

odorants of pepper produced through cryogenic grinding yielded

better-preserved products compared with samples processed by
conventional grinding techniques. This likely occurred as a result
of the heat-induced chemical alterations that spices undergo,
including browning and oxidative decomposition, during conventional ambient grinding techniques, which cause signicant losses
in the potent odorants of pepper. After storing samples under
refrigeration (4 C) for 6 months, the concentrations of the main
avour constituents were dramatically reduced, which demonstrated that, regardless of grinding technique, the aroma compositions of pepper suffer some degree of loss during storage.
A survey of the literature indicated that the typical d-3-carene
found in pepper has spicy notes and rened-limonene-like notes.
Limonene has citrus-like, mild lemon-notes and orange-notes,
while b-caryophyllene has spicy notes, and a-pinene has pine-needle-like and sharp notes. The compound b-pinene has spicy and
dry-woody notes, while b-myrcene has sweet and balsamic notes,
and linalool has fresh and oral notes (Jirovetz et al., 2002). Here, it
was observed that the high content of a-pinene, b-pinene, d-3-carene, limonene, and b-caryophyllene tended to contribute to the
sensory perceptions in cryogenically ground samples, which were
reported to have spicy, limonene-like, woody, and citrus-like notes.
3.3. Sensory analysis
The mean values and standard deviations of the sensory attributes of black, white, and green pepper ground by cryogenicand hammer-based methods are shown in Table 5. The sensory
scores for colour, odour and pungent taste were all lower for hammer-milled samples than for cryogenically ground samples. The
differences in odour may have been due to the disappearance of
some volatile aroma compounds from pepper during hammer

milling (Lilie, Hein, Wilhelm, & Mueller, 2007), which is consistent

with our results. After storing pepper samples at 4 C for 6 months,
all of the corresponding sensory scores for black, white, and green
pepper powder treated by both grinding methods were lower,
which demonstrated that storing pepper samples resulted in an
overall reduction in avour quality.
3.4. Electronic nose (e-nose) analysis
An e-nose data analysis was carried out on all samples, which
measured the value of maximum change in the sensors resistance.
Principal component analysis (PCA) was carried out using the
built-in software supplied by the e-nose manufacturers. The results were shown as a two-dimensional PCA plot. The e-nose pattern matching was able to discriminate between the twelve
pepper samples by comparing PC1 and PC2. The PCA patterns of
the e-nose data for the various powders were shown in Fig. 1.
PC1 accounted for 81.5% of the variance, while PC2 accounted for
18.4%. As expected, the PCA analysis was able to discriminate between black, white, and green ground pepper powders, and it
was also able to distinguish between the grinding techniques applied to each sample. While the PCA analysis was not able to discriminate between corresponding samples stored at 4 C for
0 months and 6 months, it was possible that the aromas of corresponding samples did not undergo signicant changes even though
the intensities of the aromas were lower after storage. White pepper samples (both cryogenically ground and hammer milled) had
distinct sensory avour perceptions and GC/MS patterns, and were
therefore grouped into a completely separate quadrant than the
other samples. We also found unique PCA signatures for ground
black pepper and green pepper samples using the e-nose analysis.
From these tests, we concluded that the e-nose, because it is so

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H. Liu et al. / Food Chemistry 141 (2013) 34023408

easy to use, has the potential to become a widespread method for

determining the pungency of black and green pepper powder with
no sample preparation. Therefore, we propose that e-nose analyses
can be used as a rapid method to test the avour quality of spices.
4. Conclusion
The percentages and concentrations of the main aroma constituents in black, white, and green pepper samples were signicantly
higher in cryogenically ground samples than in hammer-milled
samples. The cryogenic grinding technique employed here was
superior to hammer milling because it afforded good preservation
of monoterpenes, retained better odorant potency, colour and sensory attributes in the ground pepper samples. These results likely
arose from the signicant losses of pepper characteristics, particularly odorant, that occurred during hammer milling. Hammer milling has been known to cause heat-induced chemical alterations in
spices, such as browning and oxidative decomposition. Although
the percentages of monoterpenes in the aroma compounds were
dramatically increased, the concentrations of the main aroma constituents and the sensory scores of all samples were decreased
after storage at 4 C for 6 months. By all measures, we found that
the avour quality of cryogenically-ground samples was signicantly better than that of hammer-milled samples, but the corresponding avour qualities of black, white, and green pepper were
all decreased after storage. These alterations to the pepper components, including browning and oxidative decomposition, decreased
the quality of the resulting product. The e-nose pattern matching
further supported our sensory and instrumental data, and highlighted the superiority of cryogenic grinding over traditional grinding methods.
Cryogenic grinding clearly improves the sensory properties and
avour attributes of ground pepper. The use of this method for the
preparation of pepper will likely continue to be an important approach for ensuring the highest quality pepper products.
Acknowledgements
The authors gratefully acknowledge Mr. Huang and Mr. Shao at
National Institute of Metrology P.R.China for providing the cryogenic grinding and hammer milling for the study. This study was
funded by The National Key Technology R&D Program of China
(2012BAD36B03).
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