Assessment of the N-nitrosopiperidine formation risk from piperine and
piperidine contained in spices used as meat product additives
Eveline De Mey1*, Hannelore De Maere1,2, Lore Dewulf1, Hubert Paelinck1, Mieczysław Sajewicz3, Ilse Fraeye1,
Teresa Kowalska3
1
Research Group for Technology and Quality of Animal Products, KaHo Sint-Lieven, , Gebr. Desmetstraat 1,
9000 Ghent, Belgium, member of Leuven Food Science and Nutrition Research Centre (LFoRCe), Department
M2S, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
2
ISA, Food Quality Laboratory, Boulevard Vauban 48, F-59046 Lille Cedex, France
3
Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40 006 Katowice, Poland
Corresponding Author: Eveline De Mey
E-mail address: eveline.demey@kuleuven.be
Tel: +32(0)9 265 86 10
Fax: +32(0)9 265 87 24
1
Abstract
The N-nitrosopiperidine formation in blends of spices and nitrite curing salt was investigated
in relation with the piperine and piperidine contents in spices. Firstly, two analytical methods
were developed. Piperine was extracted with dichloromethane by means of accelerated
solvent extraction, and determined by HPLC-DAD (λ=343 nm). A selective hydroextraction
of piperidine using ASE and its quantification by HPLC-ELSD was applied. Both methods
were sufficiently sensitive and accurate (limit of detection, limit of quantification and
recovery: 0.28 µg, 0.84 µg, and 98.9±2.6% for piperine, and 5.76 µg, 17.45 µg, and
95.9±2.9% for piperidine, respectively). Secondly, both compounds were quantified in
commercial samples (black and white pepper, paprika, chili pepper, allspice and nutmeg). The
maximum amount of piperine (21.12 mg g-1) was found in pepper, while the other spices
contained only traces. Piperidine was detected mainly in the pepper samples, whereby the
highest concentration was found in the white pepper extract (11.42 mg g-1). Thirdly, during
the storage of spices blended with nitrite curing salt, the N-nitrosopiperidine content was
determined, using a gas chromatograph coupled with a thermal energy analyzer. Against our
expectations, no N-nitrosopiperidine formation was observed in the curing mixture which
contained white pepper extract. This result remains in contrast with the white pepper mixture,
in which the N-nitrosopiperidine content significantly increased from not detected to
9.80±0.41 ng g-1 after the two months storage period. In conclusion, high amounts of piperine
or piperidine in spices do not systematically result in the formation of N-nitrosopiperidine,
when blended with nitrite curing salt.
2
Key words: piperyol-piperidine, piperidine, N-nitrosopiperidine, accelerated solvent
extraction, spices, nitrite curing mixtures.
3
1. Introduction
Piperidine is a cyclic secondary amine, which can be considered as a parent molecular
structure for many plant alkaloids, and piperine (1-piperyol-piperidine) is the main pungent
compound of pepper (Piper nigrum, Piperaceae) (Figure 1). White and black pepper,
produced from the dried fruit without the pericarp and the dried whole, unripe berry,
respectively [1], are used for many culinary purposes. The pungency, and thus the quality, of
pepper (P. nigrum) are related to the amount of piperine [2]. However, this quality can be
influenced by the hydrolysis of piperine, resulting in the cleavage of the piperidine ring [3].
In food (e.g, in spice premixes and the meat products), simultaneous presence of nitrite
(as a curing agent) and pepper can give rise to the formation of the carcinogenic Nnitrosopiperidine (NPIP). This formation can occur either by the oxidative cleavage of the
amide bond of piperine and a subsequent nitrosation of piperidine [4], or by a direct
nitrosation of the already available piperidine [5,6]. In order to evaluate a risk of Nnitrosamine formation, it would be interesting to determine the piperine and piperidine
contents in spices.
Normally, piperine and piperidine are extracted from ground pepper with use of
organic solvents (e.g., methanol, ethanol, hexane, and dichloromethane). However, large
quantities of these volatile solvents are consumed during the long extraction [7,8]. An
optimized Soxhlet apparatus technique was proposed [9], but several alternative techniques
have also been developed. For instance, high solubility of piperine in glacial acetic acid can
accelerate the extraction [10], while the microwave-assisted extraction (MAE) enhances
4
solvent penetration by breaking the cell structure of pepper [11]. Furthermore, ionic liquids
can be used in ultrasonic assisted extraction (UAE) [12]. Although accelerated solvent
extraction (ASE) is known to be a powerful technique to reduce the time and solvent
consumption, reports concerning the extraction of piperidine alkaloids from spices are scarce.
In fact, Rajopadhye et al. [13] described an ASE procedure for the extraction of piperine from
the roots of Piper longum, while no reports were found about an application of this technique
to isolation of piperidine from P. nigrum.
The most frequently used analytical techniques are thin-layer chromatography (TLC)
and high performance liquid chromatography (HPLC). TLC is carried out on silica plates,
occasionally treated with silver nitrate, and usually with n-hexane or ethyl acetate as eluents
[14,15]. Although TLC is a relatively cheap method, it is less sensitive, as compared with
HPLC. For the separation of the piperine isomers by HPLC, a C18 column is recommended
[16]. Piperine analogues can directly be detected by the UV absorption spectroscopy.
However, piperidine is often derivatized with ninhydrin [17] or dansyl-chloride [18,19], due
to the lack of a proper chromophoric group.
The aim of this study was threefold. Firstly, the methods for the determination of
piperine and piperidine in spices were developed, in order to assess the quality of the
commercial pepper samples. Secondly, the piperine and piperidine concentrations in a
selection of culinary spices were measured. And finally, the N-nitrosopiperidine formation
was evaluated during the storage of certain spices in combination with the nitrite curing salt.
5
In that way, possible risk of the N-nitrosopiperidine formation was assessed, caused by
piperine and piperidine contained at different concentration levels in spices.
2. Experimental
2.1 Spices and nitrite curing mixtures
Different quality grades of the white and black pepper samples (P. nigrum) were selected.
They also differed in particle diameter (e.g., the whole peppercorns, the cracked pepper with
the 12 mm particle diameter, and the pepper powder with the ca. 0.3 mm average particle
diameter were analyzed). The commercial powder was either produced by the fine grinding,
or by evaporating ethanol from the spice extract. Besides of the pepper samples, some other
spices were also purchased, namely paprika (Capsicum annuum) and chili pepper (Capsicum
frutescens), both belonging to the plant family of the nightshades (Solanaceae), and also
allspice (Pimenta dioica, Myrtaceae) and nutmeg (Myristica fragans, Myristicaceae). The
spice samples were purchased either from the local supermarket (the black pepper powder and
peppercorns came from Delhaize, Brussels, Belgium), or directly from the two suppliers.
Nutmeg powder, white pepper powder and white pepper extract came from Raps (Kulmbach,
Germany). The paprika, chili and allspice powder, and also the cracked white pepper and the
Muntok white peppercorns originated from Rejo (Nazareth-Eke, Belgium).
In order to evaluate the risk of the N-nitrosopiperidine formation, meat curing mixtures were
prepared. These premixes consisted of 3 g of one of the above mentioned spices in 100 g
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nitrite curing salt (NaCl + 0.6% NaNO2, Rejo). These mixtures were vacuum packed and
stored in the darkness at 25°C. For tracing the N-nitrosopiperidine formation, the mixtures
were sampled (n=3) after one day, one week, and two months storage period.
2.2 Chemicals and standards
Hydrochloric acid (37% HCl), potassium hydroxide pellets (KOH), and antifoam silicone
were all obtained from VWR International (West Chester, PA, USA). Methanol and
dichloromethane (DCM) were purchased from Merck (Darmstadt, Germany). All chemicals
were of analytical grade. Water used in the experiment was de-ionized and double distilled in
our laboratory by means of the Elix Advantage model Millipore system (manufactured in
Molsheim, France). In order to prepare 3M KOH, a suitable amount of potassium hydroxide
(KOH) was dissolved in de-ionized water. The 6M HCl solution was prepared by diluting a
suitable volume of hydrochloric acid (HCl) with de-ionized water.
The
piperine
(≥97%),
piperidine
(≥99%),
N-nitrosopiperidine
(NPIP),
and
N-
nitrosodipropylamine (NDPA) standards were purchased from Sigma-Aldrich (St Louis, MO,
U.S.A.). For the purpose of the HPLC analyses, piperine was dissolved in DCM at the
concentration of 0.10 mg mL-1, and piperidine was dissolved in water at the concentration of
1.00 mg mL-1. NPIP was analyzed with a gas chromatograph coupled with a thermal energy
analyzer (GC-TEA). For this purpose, the NPIP standard was dissolved and diluted in DCM
to obtain a concentration of 0.25 µg mL-1. Quantification was carried out using NDPA as an
internal standard (IS) at a concentration of 1 µg mL-1 DCM.
7
2.3 Accelerated solvent extraction
Prior to the HPLC analysis of piperine and piperidine, the ASE 200 model extractor
(manufactured by Dionex, Sunnyvale, CA, U.S.A.) was used to carry out the accelerated
solvent extraction on the spice samples. A 0.5-g portion of the dry spice was carefully
weighed and placed in the stainless steel cell and underwent three consecutive extraction runs.
A pressure of 100 atm was applied. The remaining working parameters were, as follows: the
solvent volume, 24 mL; static time, 15 min; number of extraction cycles within one extraction
run, 2. Three 24-mL portions of extract obtained from a single spice sample were merged in a
100-mL calibrated volumetric flask and filled up to 100 mL with DCM or water, respectively.
These solutions were used for the chromatographic quantification of piperine and piperidine
in the investigated spices.
In order to optimize the extraction temperature for piperine with DCM as an extraction
medium, the white pepper powder from Raps (containing a high level of piperine) was used as
a model spice at six different working temperatures (50, 60, 65, 70, 75 and 80°C). For the
isolation of piperidine, the temperature of hydroextraction was optimized and this time, the
white pepper extract from Raps (containing a high level of piperidine) was used as a model
spice at six different working temperatures (40, 45, 50, 55, 60, and 65°C).
2.4 Recovery study
8
In order to evaluate the efficiency of the ASE procedure, a recovery study was performed.
Therefore, the ground black pepper fruit was used as a model sample for the preparation of
the blank plant matrix sample. In order to exhaustively extract piperine and piperidine from
this sample, the aforementioned ASE procedure with DCM and H2O, respectively, as a
solvent was repeated six consecutive times.
One series of the blank matrix samples was spiked with piperine (and extracted with DCM),
and the other series of the blank matrix samples was spiked with piperidine (and extracted
with H2O). The obtained concentrations per one gram of the blank plant matrix were specified
in Table 1. From each dried and spiked matrix, four 1-gram portions were weighed out and
each portion underwent a single ASE extraction run. Then the four extracts obtained in
parallel from the four equally spiked matrix samples were condensed in the flow of nitrogen
at 40°C to the volume of 1 mL each, with use of the TurboVap LV model evaporator
(manufactured by Zymark, Hopkinton, MA, U.S.A.). These condensed solutions were then
utilized for the chromatographic recovery studies and standard deviation was calculated from
the recovery repetitions.
2.5 Determination of piperine and piperidine by HPLC
The HPLC analyses were carried out using a Varian model 920 liquid chromatograph (Varian,
Harbor City, CA, USA) equipped with a Varian 900-LC model autosampler, a gradient pump,
a Varian model 330 DAD detector, a Varian 380-LC model ELSD detector, and the Galaxie
software for data acquisition and processing. The analyses were carried out in the isocratic
9
mode, using a Pursuit 5 C18 (5 µm particle size) column (250 mm × 4.6 mm i.d.; Varian). As
mobile phase, we employed methanol at a flow rate of 0.5 mL min-1 in the isocratic mode.
The retention times (tR) of piperine and piperidine were equal to 5.9 and 10.9 min,
respectively.
The presence of an aromatic chromophore in piperine offers a possibility of using a diode
array detector (DAD). For the calibration curve purpose, the peak heights at the wavelength of
343 nm (i.e., at the absorption maximum of piperine) were employed. The calibration curve
for piperine was obtained for the aliquots ranging from 0.20 to 1.50 µg piperine (in the
intervals of 0.10 µg). The chromatographic peak heights were plotted against the microgram
amounts of piperine injected on to the column. For each individual aliquot, six repetitions
were performed (n=6). The limit of detection (LOD) and the limit of quantification (LOQ)
were calculated using the formulas LOD = 3.3 × SD/a, and LOQ = 10 × SD/a, where SD is
standard deviation of the peak height (n=6) taken as a measure of noise, and a is the slope of
the corresponding calibration curve (y = ax + b).
The HPLC results valid for piperidine were derived from the ELSD detector. The calibration
curve for piperidine was obtained for the aliquots ranging from 2 to 7 μg piperidine (in the
intervals of 1 μg piperidine). This time, the chromatographic peak heights were plotted
against the microgram amounts of piperidine. For each individual aliquot, six sampling
repetitions were performed (n=6). The calibration curve and the LOD and LOQ values were
determined, as described earlier.
10
2.6 Determination of NPIP by GC-TEA
In order to determine the contents of N-nitrosopiperidine, the method according to DrabikMarkiewicz et al. [20] was used, with some modifications. The spice samples (in the 5-g
portions) were spiked with 500 µL NDPA, and mixed with 200 mL 3 M KOH and 4 mL
antifoam. The volatile N-nitrosamines were extracted from the spices by means of vacuum
distillation from the alkaline solution. Subsequently, the distillates were extracted with DCM
and washed with 6 M HCl. Finally, the extract was concentrated to 100 µL in a KudernaDanish apparatus (Sigma Aldrich). For the detection and quantification, a GC-TEA (Thermo
Electron Cooperation, Rodano, Italy) was used. The 5-µL aliquots of the concentrated extracts
were injected on a packed column (10% Carbowax 20M + 2% KOH on Chromosorb WAW,
80/100 mesh, 1.8m, 2mm i.d. Varian, Middelburg, The Netherlands) and a chromatographic
separation was carried out with use of argon as a carrier gas (25 mL min-1). The injection port
was set at 175°C and the oven temperature was increased from 110°C to 180°C at the 5°C
min-1 rate. The temperature of the interface and the pyrolizer of TEA were set at 250°C and
500°C, respectively. The retention times (tR) of NDPA and NPIP were equal to 6.3 and 10.4
min, respectively. The calibration curves for NPIP were obtained from 7 aliquots ranging
from 0.625 to 12.5 ng with NDPA as an internal standard. The relative chromatographic peak
areas (area NPIP/area NDPA) were plotted against the relative amounts of NPIP (µg NPIP/µg
NDPA). For each individual aliquot, six repetitions were performed (n=6). The limit of
detection (LOD) and the limit of quantification (LOQ) were 0.68 and 2.04 ng, respectively.
11
2.7 Statistical analyses
The results were expressed as the means ± standard deviation. The linear least-square
regression was used to calculate the intercepts (a), slopes (b) and coefficients of
determinations (r2) of the respective calibration curves (Microsoft Office Excel 2007,
Microsoft Corporation, Redmond, WA). Significant differences among the extraction
temperatures were assessed with a one-way analysis of variance (ANOVA, PASW Statistics
19.0.0, SPSS Inc.). Piperine and piperidine contents of the spices and the accumulation of the
NPIP content in the blends were also subjected to one-way ANOVAs. Tukey’s honestly
significant difference criterion (p<0.05) was used to compare the means.
3. Results and discussion
3.1 Evaluation of the HPLC procedure
The chemical structure of the two investigated alkaloids proved a decisive factor for the
choice of the most suitable detection and thus, the quantification mode also. The presence of
an aromatic chromophore in piperine allowed the use of a diode array detector (DAD). As a
result, good sensitivity was obtained and since the wavelength of 343 nm was proven to be
very selective, no other interfering peaks could be seen in the chromatograms (Figure 2).
The lack of a chromophore in the chemical structure of piperidine excludes a possibility of
using DAD, and enforces an employment of the universal ELSD detector, which is sensitive
to the analyte’s molecular weight. In that way, an additional derivatization step, as applied by
12
several authors [17-19] could be avoided. Although the LOD and LOQ values for piperidine
are by one magnitude order higher than those valid for piperine, it can be concluded that all
the results concerning method characteristics are satisfactory from the analytical point of view
(see Table 2).
Most frequently, piperine and piperidine are extracted from the plant material by the Soxhlet
extraction. In this study, ASE was applied as an alternative extraction technique, in order to
reduce the analysis time and the solvent consumption. As it can be seen from Figure 3, the
extraction yield of piperine (measured after a single extraction run) increased, when elevating
the working temperature of the ASE extraction. At the temperatures higher than 70°C, no
more significant improvement of the extraction yield could be observed, so it was decided to
employ the temperature of 70°C for the DCM extraction of piperine. Due to an excellent
solubility of piperidine and practical insolubility of piperine (and the other alkaloids) in water,
a selective hydroextraction method for the isolation of piperidine was elaborated. Complete
extraction of piperidine was observed over the whole range of the employed temperatures
(results not shown). For practical reasons, the temperature of 50°C was chosen for the
hydroextraction of piperidine (as the lowest working temperature of the ASE 200 apparatus is
set on 40°C). Thus, the hydroextraction of piperidine could be done at a temperature
considerably lower than that employed for the extraction of piperidine with DCM.
In order to evaluate the accuracy and precision of both extraction procedures, the recovery
studies were performed as well. For that purpose, a blank plant matrix was prepared from the
black pepper powder by thoroughly extracting piperine with DCM and piperidine with water,
13
using the ASE technique. After the six consecutive runs, neither of the two compounds could
be chromatographically detected in the final extract. Hence, after drying the extracted plant
material, it could be used as a blank plant matrix for the recovery study. The obtained results
showed an excellent performance of the ASE extraction system and practically full recovery
of the two compounds of interest from the spiked plant matrix (Table 1). Moreover, the
recovery precision was confirmed by a good repeatability of the recovery results (RSD of ca.
23%). This successful outcome allowed using the calibration curves derived for the piperine
and piperidine standards from the HPLC quantification experiments (without a tedious
spiking of the blank plant matrix, in order to prepare the plant-material-based calibration
curves).
3.2 Piperine and piperidine content in spices
In Table 3, the results of the piperine and piperidine analyses for the commercially available
spices are given. In the white and black pepper samples (P. nigrum), a maximum of 21.12 mg
g-1 piperine was measured, although in the literature, the piperine contents usually show
within the range of 3080 mg g-1 [21,22]. Nevertheless, the lower amounts have been reported
as well, especially due to the ageing of the pepper samples [2]. In the white pepper extract, a
significantly lower amount of piperine was found, although it was much higher than the
amounts extracted from the whole peppercorns. In the course of the ASE procedure, the
compounds present inside the cells of the non-ground botanical matrix do not come in a
sufficient contact with the extraction solvent, which results in the lower extraction yields. In
14
fact, the whole peppercorns certainly contain higher levels of piperine than those measured in
this study. Nevertheless, these results are interesting since they suggest a possibility of a
reduced release of piperine from the non-ground spices in the curing mixtures and / or in the
cured meat products. In the other investigated spices, only the trace amounts (± 2 mg g-1) of
piperine were detected, and no significant difference among these samples could be observed.
In the pepper samples (P. nigrum), the piperidine content was in most cases a fraction of the
piperine content, except for the black peppercorns, in which the piperidine level exceeded the
low piperine content. An industrial extract of white pepper contained the highest level of
piperidine among all tested spices, while its piperine content was rather low. Although no
information is available about an industrial production of the spice extract, it is clear that the
composition of the white pepper extract is affected by the industrial extraction process. The
piperine content apparently decreased, while the piperidine content was remarkably higher in
the white pepper extract than in the pulverized white pepper samples. Considering the
reversed ratio of the two alkaloids, it can be expected that piperine degrades during the
production of the extract, e.g., under the influence of heat [23].
In the other spices, only traces (paprika and chili), or very low amounts (allspice and nutmeg)
of piperidine were detected. As checked in the literature, no written evidence could be found
on any meaningful amounts of piperidine or piperidine alkaloids contained in the spices from
the families Solanaceae, Myrticaceae and Myristicaceae, so that our results remain in
conformity with the literature data.
15
3.3 Evaluation of the NPIP formation in the nitrite curing mixtures
Four spice samples were then tested for the risk of the NPIP formation. As it can be seen from
Table 4, in none of these spices NPIP could initially be detected, which suggested their safe
use in the food products. After one day from the preparation of the nitrite curing salt blended
with these spices, no detectable amount of NPIP was found in any of them. After one week,
however, the curing mixture containing the white pepper powder showed a NPIP
contamination, which significantly increased after the two months storage period. In an
agreement with the results of the earlier studies [24,25], the presence of the secondary amines
(such as piperidine), can cause a risk of the N-nitrosamines formation. For this reason, the
FDA strongly discourages combination of nitrites and nitrates with spices [26]. Although
NPIP was easily formed in the curing mixture containing the white pepper powder, all the
other curing mixtures remained free from NPIP. As anticipated, no NPIP was formed in the
mixtures prepared with nutmeg and paprika, due to very low concentrations of piperine and
piperidine in these spices. However, based on an elevated piperidine level, the NPIP
formation was expected in the mixture produced with use of the white pepper extract.
Probably in that case, the lack of the NPIP formation can be linked with an altered
composition of white pepper in the course of the industrial extraction process [27]. It is
known from the literature that many spices contain antioxidants, such as polyphenols and the
nitrite scavenging compounds [28,29], and the activity of these compounds is higher in the
extracts than in the unprocessed spices [30]. In that case, antioxidants and the nitrite
scavenging compounds can probably inhibit the N-nitrosamines formation [30]. Finally, it has
to be emphasized that no information is available on the production process of the white
16
pepper extract and on the polyphenolic composition contained therein, and a further research
is needed to reveal the inhibitory effects of this extract.
4. Conclusions
Rapid and accurate accelerated solvent extraction procedures were developed to facilitate
quantification of piperine and piperidine in spices by means of HPLC. For the determination
of piperine, high selectivity was obtained by using the DAD detector at the specific
wavelength of 343 nm, after the ASE extraction with DCM (70°C) as an extraction solvent.
For the determination of piperidine and due to the lack of a chromophoric group, the universal
ELSD detector was used. In that case, the selectivity was obtained by an application of a
hydroextraction at 50°C. For both alkaloids, good recovery was obtained by means of ASE.
The piperine contents in the pepper samples were considerably higher than the trace levels in
the spices belonging to the botanical plant families of Solanaceae, Myrtaceae, and
Myristicaceae. The piperidine content in the extract of the white pepper powder was
considerably higher than that found in the pulverized samples. As predicted by the low
piperine and piperidine levels, nitrite curing mixtures prepared with nutmeg and paprika did
not show any NPIP contamination. Against our expectations, the use of the white pepper
extract, containing a high piperine concentration, did not induce the NPIP formation. In
contrast, the use of the white pepper powder in the curing mixtures caused an increase of the
N-nitrosopiperidine content from not detected to 9.80±0.41 ng g-1 after the two month storage
period. In conclusion, high amounts of piperine and piperidine in the spices do not necessarily
17
result in the NPIP formation, which depends on the total composition and the industrial
processing of the spices and extracts.
Acknowledgements
This work was performed in the framework of the MeCagrO2 project “Safe products,
sustainable processes and employment increased attractiveness for companies from the 2 Seas
agro food Area”. Note: “The document reflects the authors’ views. The interreg IVA 2 Seas
Programme Authorities are not liable for any use that may be made of the information
contained therein.”
18
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21
Table 1 The recovery (T%) and the repeatability (r) expressed as RSD (%) of piperine and
piperidine from 1 gram dry blank plant matrix derived from the ground black pepper fruit and
spiked with four different concentrations of the test compounds (n=4).
Compound
Piperine
Piperidine
Spiked amount
Recovery
T (%)
RSD (%)
(mg/g)
(mg/g)
0.100
0.098
98.4
2.80
0.200
0.198
98.8
2.76
0.500
0.496
99.2
2.89
1.000
0.989
99.2
2.11
0.100
0.092
92.3
3.54
0.200
0.189
94.5
3.11
0.500
0.491
98.2
2.74
1.000
0.985
98.5
2.79
22
Table 2 Calibration curves obtained for piperine and piperidine by HPLC, and the respective
LOD and LOQ values (n=6)
Compound
Calibration curve
r2
SD
y= ax + b
LOD
LOQ
(g)
(g)
Piperine
y =554.296x–08.602
0.9565
46.7470
0.28
0.84
Piperidine
y=40.145x64.001
0.9835
70.0700
5.76
17.45
23
Table 3 Contents (mg g-1) piperine and piperidine in the spice samples (mean±standard
deviation; n=6)
Piperine
Piperidine
powder (Raps)
20.65±0.84c,d
3.70±0.11c
cracked peppercorns (Rejo)
19.18±0.66c
0.06±0.00a
powder, Muntok (Rejo)
20.80±0.52d
5.83±0.17e
peppercorns, Muntok (Rejo)
2.51±0.10a
0.08±0.00a
extract (Raps)
12.12±0.31b
11.42±0.29g
powder (Delhaize)
21.12±0.75d
6.49±0.21f
peppercorns (Delhaize)
3.32±0.12a
4.22±0.18a
1.96±0.06a
0.02±0.00a
1.97±0.08a
0.04±0.00a
2.11±0.08a
0.66±0.03b
2.17±0.06a
0.69±0.03b
Spice sample
White pepper (P. nigrum)
Black pepper (P. nigrum)
Paprika (C. annuum)
powder (Rejo)
Chili pepper (C. frutescens)
powder (Rejo)
Allspice (P. dioica)
powder (Rejo)
Nutmeg (M. fragrans)
powder (Raps)
Different letters in the same column indicate significant differences at p<0.05 (Tukey's post
hoc test)
24
Table 4 Contents (ng g-1) NPIP in the spice samples and during the storage of the nitrite
curing mixtures (mean±standard deviation; n=3)
Spice
Spice sample
Curing mixture
1 day
1 week
2 months
White pepper (P. nigrum)
powder (Raps)
nd
nd
3.28±0.19a
9.80±0.41b
extract (Raps)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Paprika (C. annuum)
powder (Rejo)
Nutmeg (M. fragrans)
powder (Raps)
Different letters in the same row indicate significant differences at p<0.05 (Tukey's post hoc
test)
25
Captions to figures
Fig. 1 Chemical structure of piperine (A) and piperidine (B)
Fig. 2 HPLC-DAD chromatogram of piperine extracted from the white pepper powder
sample, detected at 343 nm
Fig. 3 Effect of the extraction temperature on ASE extraction of piperine with DCM from the
white pepper powder sample (n=3). Data points with different letters are significantly
different at p<0.05 (Tukey's post hoc test)
26