Comparative study of the use of sarcosine, proline, and glycine as
acrylamide inhibitors in ripe olive processing
Antonio Higinio Sánchez, Víctor Manuel Beato, Antonio López-López and Alfredo
Montaño*
Antonio Higinio Sánchez (ahiginio@cica.es), Food Biotechnology Department,
Instituto de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Seville, Spain
Víctor Manuel Beato (vmbeagal@ig.csic.es), Food Biotechnology Department, Instituto
de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Seville, Spain
Antonio López-López (all@cica.es), Food Biotechnology Department, Instituto de la
Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Seville, Spain
* Corresponding autor. Alfredo Montaño (amontano@cica.es), Food Biotechnology
Department, Instituto de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012
Seville, Spain. Tel: +34 95 4691054, fax: +34 95 4691262
Acknowledgments
This work was supported in part by the European Union (FEDER funds) and the
Spanish government through Project AGL 2010-19178. Financial support from the
Junta de Andalucía (group AGR-208) is also acknowledged.
1
Abstract
2
The main purpose of this study was to evaluate the inhibitory effect on acrylamide (AA)
3
formation and the impact on sensory characteristics in ripe olives of three selected
4
amino acids (sarcosine, proline, and glycine), which previously showed high AA
5
inhibition rates in an olive model system. Each amino acid was separately added to
6
packing solutions to give 100 or 200 mM at equilibrium, prior to a sterilization
7
treatment at 121 ºC. The results showed that sarcosine at 100 mM may be a good
8
candidate for reducing the AA content in ripe olives with a limited effect on sensory
9
characteristics. Studies with a model solution of AA and sarcosine heated at 121 ºC for
10
30 min suggested that the main mechanism for the inhibitory effect of sarcosine on AA
11
formation was the Michael reaction.
12
13
Keywords: Acrylamide; amino acids; glycine; proline; ripe olives; sarcosine
2
14
Introduction
15
Acrylamide (AA) aroused worldwide concern after 2002 when it was found that it could
16
be formed in foods during cooking (Tareke et al. 2002). This compound has been
17
classified by the International Agency for Research on Cancer (IARC) as a probable
18
human carcinogen (IARC 1994). Particular attention has been paid to potato products
19
and bakery products (Amrein et al. 2004; Taubert et al. 2004; Claus et al. 2008) because
20
of their high AA concentrations and their high rate of consumption as a staple food. The
21
formation of AA was believed to be generally associated with the application of harsh
22
heat treatments (e.g. roasting, baking, deep-frying) with temperatures exceeding 150 °C.
23
Astonishingly, although these heat conditions do not apply to ripe olive processing, this
24
product presents high levels of AA (200-2000 ng g-1) according to earlier exploratory
25
surveys (United States Food and Drug Administration 2006) and further studies (Casado
26
and Montaño 2008).
27
Ripe olives (also called “Californian-style table olives”) are one of the most
28
prominent classes of table olive commercialized in the world. In this type of processing
29
the olives, mostly in the green and cherry stages of ripening, are usually stored in brine
30
with 4-6% NaCl for 2-8 months, depending on production demand. The brine may be
31
acidified to pH 4 with acetic acid and kept under aerobic conditions to prevent spoilage.
32
Alternatively, the usage of acidified water (2.4% acetic acid) under anaerobic conditions
33
has been proposed (de Castro et al. 2007). Once the stored fruits are sorted and graded,
34
they are treated with a series of dilute NaOH solutions (lye) to remove their natural
35
bitterness, caused by oleuropein. Between lye treatments, the fruits are aerated. During
36
this operation, the fruits are progressively darkened by means of polyphenol oxidation.
37
After the lye treatments and oxidation, the olives are washed several times with water to
38
remove most of the residual lye, reaching a final pH of around 7, and placed in 3-5%
3
39
brine with ferrous gluconate or ferrous lactate to maintain the black colour. Finally, the
40
olives are canned in brine and heat sterilized, generally at 121-126 ºC (Sánchez et al.
41
2006).
42
The use of additives for the potential inhibition of AA formation is a strategy
43
that has been investigated in different foods, including ripe olives (Casado et al. 2010).
44
Sodium bisulfite was demonstrated to be the most effective compound to eliminate AA
45
in ripe olives, but this additive is not currently permitted in the EU (European
46
Commission 2011). Proline (Pro), sarcosine (N-methyl glycine; Sar), and glycine (Gly)
47
have also been demonstrated to be effective inhibitors of AA formation in a ripe olive
48
model system, although at concentrations 10 times higher compared to sulfite (López-
49
López et al. 2014). However, the effectiveness of these amino acids in real food and
50
their impact on the sensory characteristics of olives has not been provided. Although in
51
a previous publication (Casado and Montaño 2008) we demonstrated that AA in ripe
52
olives remained stable during storage for at least up to 6 months, the AA content could
53
change with storage time in the presence of AA-reducing additives. The mentioned
54
amino acids could form amino acid-AA adducts through the Michael addition type
55
reactions thereby reducing the AA content. This has been demonstrated for Pro and Gly
56
through the identification of the corresponding Michael adducts in aqueous model
57
systems (Koutsidis et al. 2009; Liu et al. 2011), but direct evidence for AA-Sar adduct
58
formation has not been provided.
59
Therefore, the aims of the present work were (1) to assess the impact of the
60
amino acids Sar, Pro and Gly on AA contents and on the sensory characteristics of ripe
61
olives, (2) to assess the stability of AA in the presence of added amino acids during
62
storage , and (3) to study the reaction between AA and Sar in an aqueous model system
4
63
heated at 121 ºC in order to confirm the type of reaction involved based on the
64
identification of the main reaction product by LC/MS.
65
66
Materials and methods
67
Chemicals
68
Individual amino acids (Gly, L-Pro, Sar) were supplied by Sigma-Aldrich (St. Louis,
69
MO, USA). Deionised water (Milli-Q; Millipore Corp.) was used throughout. All
70
reagents and chemicals used for the AA analysis were as described in Casado and
71
Montaño (2008). All other chemical and solvents were of analytical grade from various
72
suppliers.
73
74
Preparation of ripe olives
75
Hojiblanca cultivar olives (≈ 25 kg), which had been preserved in acidified water (2.4%
76
acetic acid) for more than six months, were subjected to darkening following the
77
habitual procedure for the elaboration of ripe olives. Briefly, olives were treated in a
78
cylindrical stainless steel container with a lye solution of 3% NaOH, which
79
progressively penetrated the flesh until the alkali reached the pit (treatment time ≈ 6 h).
80
Next, the lye was removed and the olives were washed with water until the pH reached
81
8.0. During the lye treatment and washing, air was injected through the bottom of the
82
container. Then, a 0.1% ferrous gluconate solution was added to fix the black colour.
83
After the darkening step, the olives were packed in “A314” glass bottles (145 g of pitted
84
olives plus 170 mL of brine capacity) and covered with brine containing 3% NaCl,
85
0.015% ferrous gluconate, and the corresponding amino acid (Pro, Sar, or Gly). Amino
86
acids were added to give equilibrium concentrations of 100 and 200 mM, which were
87
demonstrated to be effective concentrations to inhibit AA formation in a ripe olive
5
88
model system (López-López et al. 2014). For the corresponding calculations, the
89
moisture content of the olives was assumed to be 75% (w/w). A control product was
90
prepared using the same brine without any additive. If necessary, before the olives were
91
covered, the pH of the packing brine was adjusted to 6.5-7.0 by the addition of NaOH or
92
HCl. Fifteen bottles from each treatment were sterilized in a static retort (Steriflow,
93
SAS, Paris, France)similar to that used at olive industry. In case of ripe olives a
94
minimum value for accumulated lethality of 15F0 must be reached according to Trade
95
Standard Applying to Table Olives (IOOC, 2004) and this was the intended lethality in
96
the present work. The real conditions of retort were as follows: (1) before starting the
97
retort cycle, the bottles were pre-heated at 50 ºC for 10 min; (2) in the heating phase or
98
come-up time of the retort cycle (22 min), the water process was heated by steam in the
99
primary circuit of heat exchanger; (3) in the holding phase (15 min), temperature (121
100
ºC) and pressure (2.8 bar) were stabilized; and (4) in the cooling phase (18 min), cold
101
water was injected in the heat exchanger in order to cool down the process water. The
102
cycle period was 55 min. The experimental Fo value through measurement of
103
temperature from the coldest point of the bottle during sterilization treatment was
104
calculated as 15.5. The sterilised bottles were stored at room temperature for 8 months.
105
The initial sampling was performed after 20 days (and not immediately after heating
106
treatment) to ensure that the equilibrium inside the bottles had been reached. Samples
107
were also taken at the end of the storage period to assess the effect of storage time on
108
AA level and quality parameters.
109
110
AA determination
111
The AA content was determined following a gas chromatography-mass spectrometry
112
(GC-MS) method (with previous derivatisation of AA), as described previously (Casado
6
113
and Montaño 2008). After the addition of 13C3-AA as internal standard and further
114
clean-up of the sample, bromination of the AA double bond was performed. The
115
reaction product (2,3-dibromopropionamide) was extracted with ethyl acetate and dried
116
over sodium sulphate, and the solvent was evaporated to dryness under a stream of
117
nitrogen. The derivative was then converted to 2-bromopropenamide with triethylamine,
118
and analysed by GC-MS. Acrylamide in samples was quantified using the ion at m/z
119
151 for 2-bromopropenamide and the ion at m/z 154 for 2-bromo(13C3)propenamide.
120
The detection and quantification limits were calculated to be 6 and 20 μg/L,
121
respectively, from the values obtained after performance of the analysis using standard
122
solutions (Casado and Montaño 2008).. Taking into account the sample preparation
123
used, the above values would correspond to 60 and 200 ng g-1 of olives, respectively.
124
The analytical errorunder repeatability conditions ( n = 6) for 580 ng g-1 of AA in pulp
125
of ripe olives was 2.9% (Casado and Montaño 2008).
126
127
Amino acids determinations
128
Added amino acids were analysed by HPLC after derivatisation with 9-
129
fluorenylmethylchloroformate (FMOC-Cl). Olives, after pitting, were homogenised and
130
10 g of slurry were added to 500 mL of distilled water at 70 ºC in a beaker and the
131
mixture was stirred vigorously for 10 min using a magnetic bar. The content was then
132
transferred into a 1-L flask and made up to volume with water. After mixing, an aliquot
133
(1 mL) was centrifuged at 11600g for 5 min. The supernatant, after proper dilution with
134
water, was derivatised and then analysed following the method of Montaño et al.
135
(2000) with slight modifications. A Kinetex C18 (2.6 μm, 150 x 4.6 mm i.d.;
136
Phenomenex, Torrance, CA) column, held at 45 ºC, and gradient elution with two
137
eluents (A and B) was used. Eluent A: to 3 mL acetic acid and 7 mL 1M
7
138
triethylammonium acetate were added to 900 mL of water plus 100 mL of acetonitrile,
139
and the pH was adjusted to 6.8 with 20 M NaOH. Eluent B: acetonitrile-water (90:10).
140
The gradient was: 0 to 4 min from 12% to 18% B (liner gradient), 4 to 7 min with 18%
141
B, and 7 to 14 min from 18% to 27% B (linear gradient).The flow rate was constant at
142
1.1 mL/min. To purge the column, after the elution of FMOC-amino acid, eluent B
143
percentage was increased to 80% within 5 and maintained for 5 min. The HPLC system
144
consisted of a Waters 2695 separations module (Waters Assoc., Milford, MA, USA)
145
connected to a Waters 996 photodiode array detector (wavelength 265 nm).
146
147
Quality parameters
148
The firmness of the olives was measured using a Kramer shear compression cell 10
149
blades coupled to a Texture Analyser TA.XT.Plus, (Stable Microsystems, Godalming,
150
UK) with a 50 kg load cell. A force/displacement curve was logged using the system
151
software (Texture Expert). The test speed was set at 200 mm min−1 with an acquisition
152
rate of 250 data points per second. The firmness of the olives in each bottle was
153
expressed as the mean of 10 replicate measurements, each of which was performed on 4
154
olives. The shear compression force was expressed as N/g.
155
Colorimetric measurements on the olives were made using a Colour-View
156
Model 9000 spectrophotometer (BYK-Gardner, Inc., Silver Spring, MD). Interference
157
by stray light was minimised by covering the samples with a box with a matt black
158
interior. Colour was expressed as reflectance at 700 nm (García et al. 1999). Lower
159
reflectance values indicate darker colours. The data of each measurement are the
160
average of 20 olives.
161
Sensory evaluations were performed in order to determine whether samples with
162
selected additives were significantly different from a control sample (ripe olives without
8
163
additive). The panelists (3 women and 13 men between 32 and 64 years old from our
164
Department) had been previously trained so that they could recognise the normal
165
flavour of the product. For this, the control sample was used as the reference product.
166
Evaluations were done in individual sensory booths under controlled environmental
167
conditions (20-22 ºC, incandescent lighting). The panelists were presented
168
simultaneously with two samples and were asked whether they perceived the samples to
169
be the same or different according to the so-called difference paired comparison test
170
(Lawless and Heymann 2010). Samples, coded with 3-digit numbers, were randomly
171
provided to the judges in colourless, transparent cups covered with a watch glass. Tap
172
water was offered between samples for cleansing the palate. In addition to record the
173
answers, there was space on the score sheet to write down comments. In order to
174
increase the power of the difference test, the number of judgments was increased by
175
replicate (duplicate) evaluations. Each evaluation was carried out on two separate days.
176
The Smith´s test was used for detecting differences between replicates.
177
178
Preparation of AA/Sar model system
179
An aqueous model solution (1 mL) containing equimolar concentrations of AA and Sar
180
(12.5 mM) was added to a glass pressure tube (length 10.2 cm, outside diameter 0.8 cm;
181
Sigma-Aldrich). The tube was sealed and heated at 121 ºC for 30 min in a thermostated
182
silicon oil bath. After heating, the tube was immediately immersed in an ice bath to stop
183
further reaction. The model solution before and after heating was subjected to LC/MS,
184
according to the method described by Koutsidis et al. (2009) with modifications. The
185
LC/MS system consisted of a Waters 2695 separation module connected to a Waters
186
ZMD mass detector operated in positive electrospray ionisation mode (ESI+) and
187
controlled by MassLynx software (Micromass, Wythenshawe, U.K.). Aliquots of the
9
188
samples (10 μL) were injected into a Discovery HF F5 column (150 x 4.6 mm, 3 μm)
189
(Sigma-Aldrich/Supelco) maintained at 35 ºC. The mobile phase consisted of 20:80
190
methanol:water with 0.1% formic acid, at a flow rate of 0.3 mL/min. The mass
191
spectrometer was operated at the following settings: capillary voltage, 3 kV; cone
192
voltage, 20 V; source temperature, 100 ºC; desolvation temperature, 350 ºC; desolvation
193
gas flow, 400 L/h nitrogen; and cone gas flow, 90 L/h.
194
195
Statistical analysis
196
Statistical data (mean, SD, 95% confidence interval) were calculated using an Excel
197
spreadsheet (Microsoft Excel 2010). Sensory data from the difference paired
198
comparison tests were analysed by the binomial test using statistical tables that show the
199
minimum number of correct judgments to establish significance at probability levels of
200
5 and 1% (Lawless and Heymann 2010).
201
202
203
Results and discussion
204
Effect of amino acids on AA formation
205
The selected amino acids were added to give concentrations of 100 or 200 mM in olive
206
juice or brine, after equilibrium was reached inside the bottles. Actual concentrations of
207
these amino acids (in free form) measured in olive pulp were considerably lower than
208
the above-mentioned values (Table 1). This appears to indicate that part of the amino
209
acids reacted with olive components, which would be initially present in olives or
210
would be formed during the heating treatment of sterilisation (e.g. AA). For example,
211
Maillard reaction can easily occur during heat treatment favored by the alkaline pH of
212
product (the pH of olive juice prior heating was 8.4). This reaction involves the
10
213
interaction of amino acids with sugar-derived α-dicarbonyl compounds, which implies
214
an amino acid loss (Kwak and Lim 2004). In addition, Strecker degradation of amino
215
acids during heating would be highly likely. This reaction involves the reaction of
216
amino acids with o-quinones derived from polyphenol oxidation (Rizzi 2006). The
217
darkening process in ripe olives has been mainly related to chemical oxidation reactions
218
involving the oxidation of natural o-diphenols in olives to quinones, followed by the
219
transformation of quinones into different dark compounds (Marsilio et al. 2001). The
220
concentrations of each amino acid in olive pulp did not significantly change during
221
storage (Table 1)
222
The impact of the above compounds on AA formation in ripe olives was
223
evaluated after both 20 days and 8 months of storage at room temperature (Table 2).
224
After 20 days of storage, the control product contained 598 ng g-1 of AA in the pulp. Pro
225
and Sar showed a similar behavior, both showing ~60% and ~80% reductions at
226
concentrations of 100 and 200 mM, respectively. For Gly, the reduction rates were 34%
227
and 52% for 100 and 200 mM, respectively. These findings agree with our previous
228
results in ripe olive model system (López-López et al. 2014), although the reduction
229
rates in that case were slightly higher. The above-mentioned behavior of Pro and Sar
230
suggests that the nucleophilic reactivities of Pro and Sar were closely similar and
231
significantly higher than reactivity of Gly. Brotzel and Mayr (2007) reported
232
nucleophilicity parameters (N) for protein amino acids, with Pro showing a significantly
233
higher value than that of other protein amino acids except cysteine.
234
After 8 months of storage, additional AA reductions with respect to those
235
observed after 20 days were found in all cases, with the exception of the product
236
containing 100 mM Gly which maintained the same level of AA. Consequently, the
237
AA content was lower than the detection limit (i.e. 60 ng g-1 of olives) in the products
11
238
containing 200 mM Sar and 200 mM Pro, respectively. The AA level was also low (69
239
ng g-1 pulp) in the product containing 100 mM Sar. It must also be noted that AA in the
240
control decreased by approximately 25% compared to its content after 20 days of
241
storage (from 598±61 to 459±27 ng g-1 pulp, mean±95% CI, n=6). This result is in
242
disagreement with that previously reported by Casado and Montaño (2008) , who did
243
not found any significant change of AA during storage of ripe olives, but the time
244
period studied by these authors was shorter than ours (6 vs. 8 months). Decreases of AA
245
in the control can be attributed to the reaction of AA with special food components
246
and/or reaction products during storage (Hoenicke and Gatermann 2005).
247
248
Effect of amino acids on quality parameters
249
The influence of the amino acids studied on the surface colour of ripe olives is shown in
250
Table 3. The color values (%R700) were always <10, which can be rated as “good color”
251
in the ripe olive scale established by Fernández and Garrido (1971). After 20 days
252
storage, only Sar at 100 mM and Gly at 200 mM appeared to slightly affect the R700
253
parameter, but in an opposite way. Thus, the olives containing 100 mM Sar showed a
254
positive effect on colour with a significant decrease in R700 compared to control (i.e.
255
their colour was darker than that of the control) which resulted in a R700 <6.5
256
corresponding to a “very good color” in the above-mentioned color scale. On the other
257
hand, Gly at 200 mM provoked a significant increase in R700 compared to control (i.e. a
258
loss of black colour in comparison with the control, although the effect was not great
259
enough to produce a noticeable visual difference. The different behaviour of Sar and
260
Gly with respect to colour was confirmed in determinations performed after 8 months of
261
storage. An explanation for this result cannot be readily given due to the complex
262
mechanisms involved in the browning of ripe olives (Maillard reactions, oxidative
12
263
polymerization of phenols). The type of amino acid could affect the type of melanoidin
264
formed during the Maillard reaction, which would result in varying degrees of browning
265
(Kwak and Lim 2004). Experiments using model solutions instead of using the real
266
food might provide some clarification on this subject.
267
The impact of the selected amino acids on olive firmness is shown in Table 4.
268
As expected, in general the effect of amino acids on firmness was negligible. Only the
269
sample containing Sar at 200 mM showed a significant increase in firmness compared
270
to the control after 20 days of storage. This effect was confirmed after 8 months of
271
storage, not only for 200 mM but for 100 mM Sar as well. We have not found any
272
plausible explanation for this observation. However, it must be pointed out that the
273
firmness increase with respect to control (< 2 N/g) was not large enough to produce a
274
noticeable improvement in olive texture.
275
With regards to product flavour, after 20 days of storage, paired comparison
276
tests showed significant differences in all products compared to the control (Table 5),
277
with the exception of Sar at 100 mM. It must be pointed out that the results of the
278
difference paired comparison test only indicate whether the panelists could significantly
279
discriminate between the samples (Lawless and Heymann 2010). Since the number of
280
panelists was small for this type of test, replicate (duplicate) evaluations were carried
281
out in order to increase the power of the test. The Smith´s test showed that the two
282
replications were not significantly different (data not shown), so that data were pooled
283
and analyzed as if each judgment was made by a different panelist. From the panelists´
284
comments recorded on the score sheets, it was possible to know that all amino acid-
285
containing samples had an attribute (sweetness) in common. It is known that all the
286
three studied amino acids are sweet amino acids and this taste was clearly noted by
287
panelists at the 200 mM level. Even for Sar at 100 mM level, a slightly sweet taste was
13
288
perceived by some of the panelists. However, the sample that is higher in sweetness, at
289
the same amino acid concentration, cannot be reliably identified from the difference
290
paired comparison test. A two-alternative forced choice (2-AFC) test would be more
291
appropriate for this purpose (Lawless and Heymann 2010), but unfortunately we did not
292
have enough sample bottles to perform it..
293
All in all, the above-mentioned results suggest that Sar at 100 mM may be a good
294
candidate for reducing the AA content in ripe olives with a limited or slightly positive
295
impact on quality parameters. Sar is more recommendable than Pro because its effect
296
with regard to quality parameters is more favorable. In addition, a feature of the
297
structure of Sar is that it does not present a chiral carbon atom adjacent to the carboxyl
298
group. Therefore, the possibility of amino acid racemisation (i.e. the process of
299
conversion of L-amino acid into its mirror image, named D-amino acid) during
300
sterilisation treatment of ripe olives does not apply to Sar. On the contrary, racemisation
301
could occur in case of Pro, which may impair digestibility and nutrition quality
302
(Friedman 2010). Although the toxicity of the isomer D-Pro has not been evaluated in
303
humans, some toxic effects have been reported in experiments with rats (Kampel et al.
304
1990).
305
306
Sar and AA reaction
307
Previous investigations (Koutsidis et al. 2009; Liu et al. 2011) have demonstrated that
308
some amino acids (eg Gly) can react with AA at temperatures higher than 150 ºC
309
through the Michael reaction to form the adducts shown in Fig. 1. In the case of
310
secondary amine containing amino acids such as Sar or Pro only the adduct with one
311
molecule of AA would be formed. The present study appears to confirm this mechanism
312
in the Sar/AA model system heated at l21 ºC, as one main reaction product was detected
14
313
in the heated sample with a molecular weight corresponding to that of the AA-Sar
314
adduct (MW 160). As only a quite simple model system was analysed and no complex
315
foodstuffs, the use of tandem mass spectrometry was not required. In the total ion
316
chromatogram (TIC) of the heated mixture, this reaction product coeluted with Sar (7.2
317
min) and the MS spectrum of the peak showed the possible presence of the AA-Sar
318
adduct through its molecular ion at m/z 160.9 corresponding to [M+H]+ along with the
319
sodium adduct ion [M+Na]+ at m/z 182.9 (Fig. 2). No reaction product was detected in
320
the non-heated sample.
321
Although the exact mechanism of AA formation is still unknown, several studies
322
suggest that asparagine and reducing sugars, in contrast to other heated foods (e.g. fried
323
potatoes, bread, roasted coffee and almonds), are irrelevant for AA formation in ripe
324
olives (Amrein et al. 2007; Casado and Montaño 2008). Recent investigations support
325
the role of peptides/proteins as precursors of acrylamide formation in this product
326
(Casado et al. 2013). Thus, the inhibitory effect of Sar on AA formation, rather than
327
mediated through reacting with precursors or reaction intermediates, would be mainly
328
based on its capacity to eliminate AA through adduct formation in the Michael reaction.
329
Conclusions
330
Of the studied amino acids, Sar at 100 mM had the highest impact on the AA level in
331
ripe olives along with the lowest effect on olive flavour. This amino acid appeared to
332
slightly improve the colour and firmness of olives. The AA-reducing effect of any of the
333
studied amino acids was demonstrated to be extended even to the product storage phase.
334
In conclusion, we believe that Sar could be a good candidate as an additive to reduce
335
AA contents in ripe olives. This amino acid could significantly decrease toxic
336
acrylamide content with concomitant accumulation of the Michael adduct. However, the
15
337
toxicological properties of this adduct are not known. This subject would warrant
338
further investigation. Sar has not been previously tested in any heated food to reduce
339
AA content. Therefore, the present work is the first one in which Sar is applied in foods
340
to that end.
341
342
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Figure captions
422
423
Figure 1. Michael addition reactions of acrylamide with glycine, proline, and sarcosine.
424
425
Figure 2. Total ion chromatograms obtained by LC/MS (positive ion mode) of the
426
model solution of acrylamide (AA) with sarcosine (Sar) before and after heating at 121
427
ºC for 30 min. The inserts were the MS spectra of the peak at 7.2 min.
20
Table 1. Actual concentrations of added amino acids in olive pulp after 20 days and 8
months of storage
Actual concentration (free form)a
Sample
20 days
8 months
100 mM Pro
64.9 ± 2.3
57.8 ± 7.0
200 mM Pro
133.6 ± 13.8
120.5 ± 20.0
100 mM Sar
48.7 ± 4.7
61.1 ± 8.7
200 mM Sar
101.2 ± 16.0
128.7 ± 42.7
100 mM Gly
63.2 ± 7.0
63.8 ± 4.8
200 mM Gly
126.1 ± 28.5
121.1 ± 7.2
a
Data values (in mM) are mean ± 95% confidence interval (n=3).
21
Table 2. Impact of the selected amino acids on the acrylamide content of ripe olives
after 20 days and 8 months of storagea
Storage time
8 months
459 ± 27†
Sample
Control
20 days
598 ± 61
100 mM Pro
200 mM Pro
100 mM Sar
227 ± 21*
115 ± 18*
216 ± 27*
(62)
(81)
(58)
133 ± 29*†
ND*†
69 ± 7*†
(71)
(94)
(85)
200 mM Sar
100 mM Gly
200 mM Gly
104 ± 27*
337 ± 25*
248 ± 30*
(80)
(35)
(52)
ND*†
305 ± 31*
183 ±14*†
(97)
(34)
(60)
a
Acrylamide contents in ng g-1 of pulp. Data values are mean ± 95%
confidence interval (n=6). Reduction rates (%) compared to the
corresponding control in parentheses.
*Significant difference with respect to the corresponding control.
†
Significant difference with respect to the acrylamide content at 20 days
storage.
ND = not detected (< 60 ng g-1 of pulp)
22
Table 3. Impact of the selected amino acids on the surface color of ripe olives (R700)
after 20 days and 8 months of storagea
Storage time
Sample
20 days
8 months
Control
7.06 ± 0.35
6.99 ± 0.31
100 mM Pro
7.35 ± 1.36
6.91 ± 0.30
200 mM Pro
6.80 ± 0.32
7.19 ± 0.64
100 mM Sar
5.87 ± 0.20*
6.24 ± 0.07*†
200 mM Sar
6.85 ± 0.10
6.55 ± 0.18†
100 mM Gly
6.83 ± 0.23
7.59 ± 0.04*†
200 mM Gly
7.91 ± 0.49*
8.45 ± 0.36*
a
Values are mean ± 95% confidence interval (n=3).
*Significant difference with respect to the corresponding control.
†
Significant difference with respect to R700 at 20 days storage.
23
Table 4. Impact of the selected amino acids on the firmness (N/g) after 20 days and 8
months of storagea
Sample
Storage time
20 days
8 months
Control
9.81 ± 0.49
9.60 ± 0.76
100 mM Pro
9.39 ± 0.44
9.19 ± 0.64
200 mM Pro
9.19 ± 0.62
9.59 ± 0.68
100 mM Sar
10.54 ± 0.36
11.18 ± 0.69*
200 mM Sar
11.13 ± 0.49*
11.59 ± 0.68*
100 mM Gly
10.61 ± 0.62
10.28 ± 0.86
200 mM Gly
11.04 ± 0.79
10.51 ± 0.97
a
Values are mean ± 95% confidence interval (n=3).
*Significant difference with respect to the corresponding control.
24
Table 5. Difference paired comparison tests for additive-containing samples compared
to control (without additive)
Samples compareda
No. of correct
judgmentsb
Significancec
Flavour descriptors used
by some panellists for the
additive-containing sample
100 mM Pro vs
control
24
**
Sweet, unpleasant, strange
200 mM Pro vs
control
24
**
Sweet, atypical,
unpleasant, strange
100 mM Sar vs
control
20
ns
Slightly sweet
200 mM Sar vs
control
30
**
Sweet, atypical
100 mM Gly vs
control
23
*
Sweet
Sweet, atypical,
200 mM Gly vs
24
**
unpleasant, butter odor
control
a
Pro = proline, Sar = sarcosine, Gly = glycine. b Number of judgments = 32 (2x16).
Smith´s test showed that the two replications were not significantly different, so that
data were pooled. c **, significantly different at p=0.01; *, significantly different at
p=0.05; ns, not significantly different.
25