Astaxanthin profiles and corresponding colour properties in Australian farmed black
tiger prawn (Penaeus monodon) during frozen storage
Kent J. Fanning*, Carl Paulo, Sharon Pun, Caterina Torrisi, Kerrie Abberton, Paul
Exley and Sue Poole
Crop and Food Science, Agri-Science Queensland, Department of Agriculture,
Fisheries and Forestry, 39 Kessels Rd, Coopers Plains, Queensland, Australia, 4108
*corresponding author. Telephone: +61 7 3276 6011, Fax: +61 7 3216 6591, email:
kent.fanning@daff.qld.gov.au
1
1
Abstract
2
Commercially harvested black tiger prawns (Penaeus monodon) were received from
3
farms in the major production regions of Queensland, Australia and the astaxanthin (as
4
cis, trans, mono-ester and di-ester forms) content was measured by liquid
5
chromatographic methods, together with the colour properties using a chroma meter, in
6
both the pleopods (legs) and abdominal segments. Changes in astaxanthin content and
7
colour properties were further determined during frozen storage (-20˚C) of the prawns.
8
Total astaxanthin content was seen to decrease in all samples over time, with the rate of
9
degradation being significantly greater (p<0.05) in the pleopods than the abdomen. In
10
both the pleopods and abdomen, rate of degradation of the esterified forms was
11
significantly greater (p<0.05) than that of the non-esterified forms. Hue angle
12
(increase), a* value (decrease) and L value (increase) were all seen to significantly
13
change (p<0.05) during storage, with changes being more prevalent in the pleopods.
14
15
Keywords – prawns, colour, astaxanthin, frozen storage.
16
17
2
18
INTRODUCTION
19
Prawn farming has grown to be the fourth largest aquaculture sector in Australia, by
20
quantity and value, behind salmonids, tuna and oysters (1). Total production volume of
21
farmed prawns is currently 5,381 tonnes yielding a revenue return to the industry of
22
A$77.5 million, with Penaeus monodon (black tiger prawn) being the most cultivated
23
species (1). Australian prawn aquaculture ventures are focused in Queensland, a tropical
24
region, with open pond growout systems. Production is typically presented as cooked
25
product, both chilled and frozen, into the domestic market.
26
27
Cooked black tiger prawns are characteristically red-orange in colour and the depth of
28
colour is considered a primary quality attribute driving consumer acceptance and price
29
attained (2, 3). Where prawns do not meet the consumer colour expectation for this
30
species, substantial revenue loss occurs from consequent rejection at retail markets
31
(Aqua Marine Marketing Pty Ltd, Kippa-Ring, Queensland, 2010 and Australian Prawn
32
Farmers Association, 2011, personal communication). The red-orange pigmentation in
33
black tiger prawns is associated with the carotenoid astaxanthin (3,3’-dihydroxy-β,β-
34
carotene-4,4’-dione), present in the exoskeleton and epidermal layers of the prawn (4,
35
5). Astaxanthin is accumulated through ingestion and dietary sources which include
36
both environmental algal carotenoids present in the pondwater and the applied prawn
37
feed (6, 7).
38
39
The effects of prawn maturity and feeding regimes on astaxanthin content and prawn
40
colour have been previously studied in farmed prawns, including Penaeus monodon (4,
41
6, 8) and Litopenaeus vannamei (3, 9, 10). Recent studies have demonstrated the effect
42
of environmental background colour on the mobilisation of astaxanthin isomers and
43
transition between esterified and non-esterified forms of the carotenoid (5, 11).
44
However there has been little published on colour and astaxanthin changes during post3
45
harvest storage following cooking, with only two studies found on frozen Pandalus
46
borealis (12) and dried Penaeus indicus (13).
47
48
Within Australian prawn supply chains, cooked black tiger prawns are sometimes
49
subject to long periods of frozen storage to ensure continuous availability of product.
50
During such storage, it has been observed that the prawns lose the characteristic red-
51
orange colouration and fade to a lighter, more yellow hue (Aqua Marine Marketing Pty
52
Ltd, Kippa-Ring, Queensland 2010 and Australian Prawn Farmers Association, 2011,
53
personal communication).
54
55
The present study determines colour and astaxanthin levels during long-term frozen
56
storage of black tiger prawns and highlights colour changes, along with astaxanthin
57
degradation patterns. Furthermore, it is the first study to report astaxanthin content and
58
colour of the pleopods (legs) specifically.
59
60
4
61
MATERIALS AND METHODS
62
Chemicals.
63
Hexane (Ajax, Taren Point, New South Wales, Australia) and ethanol (Ajax, Taren
64
Point, New South Wales, Australia) were analytical grade, and methyl tert butyl ether
65
(Burdick & Jackson, Muskegon, MI), methanol (Mallinckrodt, Phillipsburg, NJ) and
66
dichloromethane (Merck, Darmstadt, Germany) were HPLC grade. Trans-astaxanthin
67
and
68
Switzerland).
astaxanthin-dipalmitate
were
purchased
from
Carotenature
(Lupsingen,
69
70
Prawns for Trials.
71
Black tiger prawns (Penaeus monodon) were sourced from five geographically
72
separated commercial prawn farms in Queensland, Australia: two farms were located on
73
the southern coast, two on the central coast and one on the northern coast of
74
Queensland. Prawns were harvested, cooked and chilled under commercial operational
75
practice on farm and were then delivered on ice to the laboratory facility by refrigerated
76
transport at 1-2 days post-harvest. On arrival prawns were blast frozen to a core
77
temperature of below -20 °C. Prawns, 5kg quantities, were stored in plastic-lined
78
cardboard boxes at -20±1 °C. All 0 d (baseline) sample analyses were assessed on
79
chilled prawns shortly after arrival.
80
81
Frozen Storage Trials.
82
Table 1 provides an overview of sampling and store times within trials. In the first trial,
83
prawns were sourced from the northern farm in late June 2010. Six individual randomly
84
selected prawns were analysed for astaxanthin content and colour properties at 0, 12, 14,
85
22, 28, 56 and 119 d storage. In subsequent trials, prawns were sourced from the five
86
commercial farms during 2011.
87
5
88
Baseline data for colour properties and astaxanthin content was generated from day 0
89
prawn samples. On each sampling day, twenty randomly selected individual prawns
90
were each measured for colour properties and then prawns were pooled to create a
91
composite sample for astaxanthin content analysis. For the subsequent sampling of
92
frozen stored prawns, ten individual prawns were assessed for colour properties and
93
then a pooled sample of the ten prawns was composited for astaxanthin content at 7, 28,
94
56, 124, 187 and 371 d storage.
95
96
Sample Preparation.
97
In the preliminary storage trials, abdominal segments 2, 4 and 6 were taken (full cross
98
section of intact shell and flesh with pleopods removed) of 6 individual prawns at each
99
time point and processed individually for analysis. For baseline samples and the seven
100
additional storage trials, abdominal segments 2, 3 and 4 (as a block, including full cross
101
section of intact shell and flesh, with pleopods removed) were taken. Pleopods
102
associated with abdominal segments 1 through to 6 were removed and pooled as a
103
separate assessment sample for comparison of astaxanthin content in these structures
104
relative to the abdominal sections. This was undertaken as it was anticipated that the
105
colour and astaxanthin profiles, and degradation rates of both colour and astaxanthin,
106
would be different between the pleopods and abdomen. For the 0 d samples, abdominal
107
segments of 20 prawns were pooled together and the pleopods were similarly pooled.
108
For the 7, 28, 56, 124, 187 and 371 d time points, of all seven storage trials, a reduced
109
number of 10 prawns were used with abdominal segments and pleopods pooled
110
separately as consistent results between composite samples of 20 and 10 randomly
111
selected prawns were observed.
112
113
Pooled samples were diced, fed through a mincer and then cryogenically milled, to a
114
fine powder, using a Retsch MM301 mill (Haan, Germany). The use of a composite
6
115
sample for astaxanthin analysis was undertaken so as to allow direct correlation between
116
colour measurement values attained and astaxanthin content for the same prawns. The
117
coefficient of variation for the extraction and analysis method was determined as <5%
118
by undertaking 6 separate extractions on 6 aliquots of the same prawn pooled material,
119
for both abdomen and pleopods.
120
121
Extraction for Astaxanthin Analysis.
122
The extraction method was modified from a combination of previously described
123
methods (5, 14). Ten mL acetone, containing 0.1% butylated hydroxyl-toluene (BHT),
124
was added to 1 g of the milled prawn sample and vortexed for 20 s. Samples were stored
125
on ice throughout the extraction process. Five mL of 10% NaCl (in water) and 10 mL of
126
hexane were added followed by vortexing and centrifugation (5000 x g for 3 min, at 4
127
˚C) to separate layers, with the top layer being transferred to another clean tube (hexane
128
fraction). These steps were then repeated until the top layer was colourless. Combined
129
hexane fractions were dried in a centrifugal evaporator at 25 ˚C and then reconstituted
130
in 2-10 mL of methanol/dichloromethane (50:50, v/v), containing 0.1% BHT. Samples
131
were filtered (0.22 μm syringe filter, Grace, Sydney, Australia) and placed into HPLC
132
vials, before storing under nitrogen at -80 ˚C prior to HPLC analysis. Standard solutions
133
were prepared similarly in methanol/dichloromethane (50/50, v/v), containing 0.1%
134
BHT.
135
136
HPLC Analysis.
137
The HPLC system consisted of a SIL-10AD VP auto injector (Shimadzu, Kyoto, Japan),
138
SCL-10A VP system controller (Shimadzu), LC-10AT VP liquid chromatograph
139
(Shimadzu) and a SPD-M10 A VP diode array detector (Shimadzu). Forty μL of each
140
extract was injected onto a YMC C30 Carotenoid Column, 3 μm, 4.6 x 250 mm
141
(Waters, Milford, MA, USA), with a mobile phase consisting of 92% methanol:8% 10
7
142
mM ammonium acetate (phase A), and 100% methyl tert butyl ether (phase B). The
143
following 54 min gradient was used (15): 0 min, 80% phase A; 48 min, 20% phase A;
144
49 min, 80 % phase A; 54 min, 80% phase A.
145
146
Identification and Quantitation of Astaxanthin Forms.
147
Identification followed the LC-(APCI)MS method of Breithaupt (15), using the negative
148
ion mode. The column, mobile phase and flow were as described above in HPLC
149
Analysis.
150
151
Trans-astaxanthin was identified by comparison with the retention time and absorption
152
spectra of the standard and mass spectra (negatively charged molecular ion m/z = 596.5
153
(15)). Cis-astaxanthin peaks were identified by relative retention time, absorption
154
spectra and mass spectra (negatively charged molecular ion m/z = 596.5 (15)). The two
155
separate blocks of the various astaxanthin mono-esters and di-esters were identified by
156
relative retention time (including comparison with astaxanthin dipalmitate standard),
157
absorption spectra and mass spectra (monoesters having backbone mass of m/z 578.4
158
[negatively charged molecular ion minus the fatty acid] (15)).
159
160
A trans-astaxanthin solution of approximately 2 μg/mL was made up in hexane and
161
absorbance measured at 472 nm. The concentration of solution was determined by the
162
following formula, using A1% of 1911 (provided by Carotenature):
163
Concentration (μg/mL) = Absorbance x 10000/ A1%
164
165
The peak purity of each peak was determined. The actual concentrations of the standard
166
solutions were then calculated by multiplying the concentration determined
167
spectrophotometrically by the % peak area of the standard peak as determined by
168
HPLC. Standard curves were linear over the range 0.03 to 10 μg/mL with r2 values of <
8
169
0.999. The trans-astaxanthin standard curve was used to determine the concentration of
170
the other astaxanthin forms. Total astaxanthin content was determined as the sum of cis-
171
astaxanthin, trans-astaxanthin, mono-ester and di-ester content. Non-esterified
172
astaxanthin was determined as the sum of cis-astaxanthin and trans-astaxanthin,
173
whereas esterified astaxanthin was determined as the sum of mono-ester and di-ester
174
content.
175
176
To examine the rate of degradation of astaxanthin in solution, a commercial astaxanthin
177
supplement was purchased (Source Naturals softgel, stated to contain 2 mg astaxanthin
178
from 20 mg algae extract) and diluted in 50:50 (v/v) methanol:dichloromethane to a
179
concentration of approximately 3 µg/ml. Aliquots were placed into HPLC vials and
180
stored in the dark at 22 ˚C for 1, 2, 3, 4 and 5 weeks.
181
182
Colour Measurement.
183
Colour was measured using a Minolta Chroma meter CR-400 (Konica Minolta Sensing,
184
Inc., Osaka, Japan) fitted with a CR-A33b light projection tube. The instrument was
185
calibrated using a CR-A43 white calibration plate (illuminant C: Y = 88.5, x = 0.3157, y
186
= 0.3224) at the beginning of each sampling time point. For this study the CIELAB
187
colour space (L*a*b*) was used. Each prawn had a total of 6 colour measurements
188
taken of the abdomen and an additional 6 of the pleopods. Abdominal segments 2, 4 and
189
6 or segments 2, 3 and 4 were measured for colour as described above in Sample
190
Preparation. A single averaged measurement from the three segments of both left and
191
right sides was recorded. To measure colour of pleopods, prawn was curled to gather
192
pleopods to a centralised point. Triplicate measurements were averaged from both left
193
and right pleopods and recorded. Results reported were averaged from all prawns during
194
each sampling time. Values for chroma and hue angle were calculated by the following
195
formula:
9
196
Chroma = (a*2 + b*2)½
197
Hue angle = tan-1 (b* / a*)
198
199
Data and Statistical Analysis.
200
Results are presented as mean ± standard deviation (sd) unless otherwise specified,
201
except for Figure 1B where mean ± standard error of the mean (sem) was used for
202
clarity of the figure. Differences in astaxanthin concentration and colour properties
203
between storage times were evaluated using one-way analysis of variance (ANOVA)
204
and the Tukey HSD procedure using JMP software (Version 7). Both linear and non-
205
linear regression analysis was undertaken using Sigmaplot (Version 10).
206
207
Astaxanthin loss during storage has been shown to follow first-order reaction kinetics,
208
in dried prawn, as defined by ln (C/Co) = -kt; where C is astaxanthin content at time
209
point of interest, Co is initial astaxanthin content, k is the rate constant and t is time
210
(13). ln (C/Co) was plotted against time to calculate k, using Sigmaplot.
211
212
10
213
RESULTS
214
The total astaxanthin of abdominal segments decreased over time in the 119 d frozen
215
prawn storage trial (Fig 1A). Changes in total astaxanthin were significant (p<0.007)
216
from 22 d onwards when compared to initial levels (0 d), with mean losses of total
217
astaxanthin of 60% after 28 d, 87% after 56 d and 86% after 119 d. All four forms of
218
astaxanthin decreased with time (Fig 1B) with a significantly greater (p<0.006)
219
degradation rate for mono-ester astaxanthin (k = 3.61 ± 1.30 s-1, x102, over 56d) and di-
220
ester astaxanthin (k = 3.27 ± 1.36 s-1, x102, over 56d) than trans-astaxanthin (k = 1.19 ±
221
0.58 s-1, x102, over 56d) and cis-astaxanthin (k = 0.66 ± 0.67 s-1, x102, over 56d). This
222
resulted in a shift from a predominance of esterified forms (74% of total astaxanthin at 0
223
d) to non-esterified forms (54% of total astaxanthin at 119 d) with time. Both the L*
224
value and hue angle were significantly higher at 119 d when compared with initial
225
values (p<0.05).
226
227
The total astaxanthin content present in the prawns from the various locations ranged
228
from 5.5-11.5 ug/g, in the abdomen, and from 47.0-159.7 ug/g, in the pleopods (Table
229
2). In the abdominal segments the mean content of trans-astaxanthin and mono-ester
230
astaxanthin were similar but significantly higher than di-ester astaxanthin (p<0.03) and
231
cis-astaxanthin (p<0.0001) (Table 3). In the pleopods, mean mono-ester astaxanthin, di-
232
ester astaxanthin and trans-astaxanthin content, of the pleopods, were not significantly
233
different from each other but were all significantly greater than the cis-astaxanthin
234
content (p<0.007).
235
236
There were significant changes (p<0.05) in some of the colour properties of the
237
abdominal segments and pleopods during frozen storage with changes generally being
238
greater in the pleopods (Figure 2). In the abdominal segments significant increases in L*
239
value (becoming lighter) were seen by 124 d in 1 trial, by 187 d in 2 trials and by 371 d
11
240
in 4 trials. Significant decreases (p<0.05) in a* value were evident by 56 d in 2 trials, by
241
124 d in 4 trials, by 187 d in 2 trials and by 371 d in 2 trials. Significant increases
242
(p<0.05) in hue angle (shift from red/orange to orange/yellow) were evident by 56 d in
243
2 trials, by 124 d in 3 trials, by 187 d in 1 trial and by 371 d in 2 trials. Chroma and b*
244
value were generally constant.
245
246
In the pleopods significant increases (p<0.05) in L* value were evident by 56 d in 1
247
trial, by 124 d in 1 trial, by 187 d in 4 trials and by 370 d in 2 trials. Significant
248
decreases (p<0.05) in a* value were observed by 56 d in 4 trials, by 124 d in 6 trials and
249
in all 7 trials by 187 and 371 d. The b* value was generally constant only decreasing
250
significantly (p<0.05) in 1 trial after 371 d. The chroma decreased (p<0.05)
251
significantly in 2 trials after 28 d, in 3 trials by 56 d, in 5 trials by 124 d and 187 d and
252
in all 7 trials by 371 d. The hue angle significantly increased (p<0.05) by 56 d in 2
253
trials, by 124 d in 6 trials and in all 7 trials by 187 and 371 d.
254
255
Total astaxanthin content decreased with time, in both abdominal segments and
256
pleopods during -20˚C storage in the 371 d trials (Figure 3). The rate of astaxanthin
257
degradation was greater in the pleopods than the abdomen samples (Table 4). The
258
astaxanthin degradation followed first order kinetics up to 187d but for 371d departed
259
from first-order relationship. There was a directly proportional relationship between the
260
rate of astaxanthin loss over 187 d and initial astaxanthin content in the abdomen
261
(r2=0.86) and pleopods (r2=0.97).
262
263
Total astaxanthin versus L* value, a* value and hue angle (no correlation was seen with
264
b* value or chroma), for the combined data from all 7 of the 371d trials, fitted using an
265
exponential decay function showed poor to moderate correlations for the pleopods
266
(r2=0.34 for L*, r2=0.37 for a*, r2=0.46 for hue) and abdominal segments (r2=0.35 for
12
267
L*, r2=0.18 for a*, r2=0.41 for hue). For the pleopods, correlations from the individual
268
trials were better than the combined data. For example, for total astaxanthin versus hue
269
angle the mean r2 for the 7 trials was 0.67±0.15, with five of the trials having r2>0.71.
270
Furthermore, for total astaxanthin versus chroma the mean r2 for the 7 trials was
271
0.60±0.20. Analysis of baseline samples (0 d) of the storage trials yielded good
272
correlations between total astaxanthin and hue angle (r2=0.69, fitted using exponential
273
function) and total astaxanthin and L* value (r2=0.70, fitted using exponential function),
274
for the abdominal segments (no correlation was seen for chroma or a* value). However
275
for the pleopods there was no correlation between the initial astaxanthin content and hue
276
angle or a* value but there was between initial astaxanthin content and L* value
277
(r2=0.70, fitted using exponential function). No correlation was seen between total
278
astaxanthin and chroma.
279
280
The content of each form of astaxanthin, in both abdominal segments and pleopods,
281
decreased with storage at -20˚C. There were significant differences (p<0.05) between
282
the degradation rate of each form of astaxanthin (Table 4). In both the abdominal
283
segments and pleopods, degradation rate of mono-ester and di-ester forms were
284
significantly greater (p<0.05) than that of cis-astaxanthin and trans-astaxanthin. As with
285
total astaxanthin, the degradation, of each form, followed first-order kinetics up to 187d.
286
287
The differences in rate of degradation, of the various forms, resulted in a change in
288
astaxanthin profile (as shown in Figure 4), in both abdominal segments and pleopods.
289
The predominance of esterified forms changed to a predominance of non-esterified
290
forms. The % of cis-astaxanthin and trans-astaxanthin were significantly higher
291
(p<0.05) after storage for 371 days at -20˚C, whereas the % of mono-ester astaxanthin
292
and di-ester astaxanthin were both significantly lower (p<0.05), in both the abdominal
293
segments and pleopods. The rate of degradation of total astaxanthin, and each
13
294
astaxanthin form, in the abdominal segments and pleopods, were seen to be directly
295
proportional (using linear regression) to the initial content of that form (r2=0.86, total
296
astaxanthin in abdominal segments; r2=0.97, total astaxanthin in pleopods).
297
298
A directly proportional relationship between initial % of esterified astaxanthin and
299
initial total astaxanthin content (r2=0.74) was seen for the pleopods. A similar but
300
weaker relationship was seen for the abdominal segments (r2=0.43). There was no loss
301
of any of the forms of astaxanthin in the commercial algae supplement that was made
302
up in solution and left at 22˚C for 5 weeks.
303
304
14
305
DISCUSSION
306
Despite the commercial significance of colour loss of prawns during frozen storage,
307
there is little published information describing astaxanthin content and colour properties
308
of cooked prawns during storage. Bak and colleagues (12) investigated astaxanthin
309
content and visual appearance of wild caught and cooked cold-water prawn species
310
(Pandalus borealis) during storage at -18 ˚C. The total astaxanthin content was
311
measured in a composite of meat and surrounding shell (head, tail, legs and roe
312
removed) and had initial levels of 66 mg/kg. These were considerably higher than those
313
reported in the abdominal segments for the present work, which may have been due to
314
the higher presence of cartenoid algal species in nutrient-rich cold harvest waters off the
315
coast of Norway for Pandalus borealis compared with tropical pondwaters for black
316
tiger prawns. Despite the higher initial level of astaxanthin in the cold-water prawns, the
317
astaxanthin loss over time was greater in black tiger prawns than observed in P. borealis
318
suggesting differences in form of astaxanthin present in each of the prawn species.
319
320
The astaxanthin loss in dried prawn (Penaeus indicus) stored at 4˚C was 10% after 4
321
weeks and reached 45% after 16 weeks (13), which was higher than that seen in the
322
abdominal segments in the present study, but similar to that in the pleopods. The
323
degradation rate in dried prawn stored at 4˚C (0.60 d-1, x102) was similar to the rate in
324
black tiger abdominal segments (0.54 d-1, x102) but lower than the rate in pleopods
325
(1.15 d-1, x102).
326
327
Astaxanthin levels have been described in wild caught black tiger prawns from India
328
(16) and farmed black tiger prawns from Thailand (4, 6, 8), Indonesia (8) and
329
Philippines (8). Menasveta and colleagues gave no details of how prawns were prepared
330
for analysis but, depending on feeding regime, levels ranged from 11.1-44.8 mg/kg in
331
shell and from 4.0-13.5 mg/kg, in flesh (4). In both the shell and flesh the relative
15
332
percentage of esterified astaxanthin increased with increasing total astaxanthin levels,
333
which is similar to what was observed in the present study. Okada and colleagues
334
described the part they extracted as the “exoskeleton of tails (the edible part)”, with
335
astaxanthin content ranging from 2.3-33.1 mg/100 g (8). Non-esterified astaxanthin
336
content was the main component (61-65.6%) in the samples with lower astaxanthin
337
content but was only 30% in the samples with the highest total astaxanthin content.
338
Okada and colleagues suggested this was indicative of an upper limit of non-esterified
339
astaxanthin accumulation.
340
341
Available data for Australian-grown black tiger prawns is from recent work of Tume
342
and colleagues and Wade and colleagues who reported the influence of environmental
343
growout and background colour on the harvest colour of the prawns (5, 11). Both
344
studies describe total astaxanthin and astaxanthin profiles in the cephalothorax,
345
abdominal epidermal layer, abdominal exoskeleton and digestive gland, as well the tail
346
(combined abdominal epidermal layer and abdominal exoskeleton). The colour of
347
storage tanks was seen to significantly change the astaxanthin profile in both epidermal
348
layer and exoskeleton with prawns suggesting a mobility between non-esterified to
349
esterified forms of astaxanthin in a live animal.
350
351
It is difficult to directly compare the astaxanthin content between studies due to
352
differences in parts of prawn that were used for extraction, however, the trend identified
353
in the present study, where high initial astaxanthin levels showed a predominance of
354
esterified forms, is similar to that reported for other studies on black tiger prawns (4, 8).
355
A similar trend was also reported for Litopenaeus vannamei, with feeding regimes that
356
increased total astaxanthin content also increased the % of esterified forms in tail
357
muscle samples (9). On analysis of their data, fitted using an exponential function, good
16
358
correlations were shown between esterified (%) and total astaxanthin content in both tail
359
muscle (r2=0.73) and whole abdomen (r2=0.76).
360
361
There was a wide range of astaxanthin content (47-160 µg/g) in initial analysis of
362
pleopods observed in the present study, but there was generally little difference in
363
astaxanthin content after 124-371 d storage. The higher rate of degradation, in more
364
initially concentrated samples, was associated with a higher % of esterified astaxanthin,
365
which degraded more rapidly than non-esterified astaxanthin. However the faster
366
degradation of the esterified forms of astaxanthin as compared with the non-esterified
367
forms, observed in every storage trial undertaken, in both pleopods and abdominal
368
segments, was not expected. It has been previously reported that it is the non-esterified
369
forms of astaxanthin that are more susceptible to oxidation than esterified astaxanthin
370
forms (13, 15). Wade and colleagues have recently shown that esterified astaxanthin is
371
contained in an ‘insoluble pellet fraction of the hypodermis’ but most of the non-
372
esterified astaxanthin is in ‘the soluble protein fraction’ (11). The difference in
373
degradation rates may be due to differences in the location and binding of the different
374
astaxanthin forms. The idea of an upper limit of free astaxanthin accumulation during
375
dietary supplementation (8), above which astaxanthin is accumulated in esterified form,
376
may suggest that the more recently accumulated esterified astaxanthin would then be
377
lost first during storage. However the understanding of astaxanthin degradation in
378
frozen prawns is currently very limited and further investigation is warranted.
379
380
In the early trial (119d storage, 2010), abdominal segments of the prawns had higher
381
initial astaxanthin levels and greater degradation rates compared to the later trials
382
(sourced 2011 season). It is suggested this may be due to the 2010-harvest prawns being
383
sourced late in the harvest season at which time they would have gained sustained
384
dietary astaxanthin from applied feed and may also have suffered temperature stress. It
17
385
is recognised that stress induced by any cause (temperature, starvation, predation,
386
disease) results in attempts by the animal to ‘camouflage’ against their background and
387
within a pond environment, and they will typically ‘darken’ to hide against the pond
388
bottom (17-20). Feeding regimes, used to boost astaxanthin content in prawns for
389
improved colour and appearance, increase the % of esterified astaxanthin (4, 8). As
390
these forms degrade faster than non-esterified forms and higher initial total astaxanthin
391
content was associated with faster degradation of total astaxanthin, when stored at -20
392
°C, preservation of the astaxanthin during storage is required to gain the benefit from
393
the feeding regime.
394
395
In the 371d storage trials, the slower decrease of astaxanthin levels in abdominal
396
segments relative to the pleopods could be directly related to the relatively lower
397
proportion of esterified forms within the abdominal segments. The rapid loss of
398
astaxanthin in the pleopods compared to abdominal segments was also correlated with
399
greater change in colour parameters. This accords with visual observations that the
400
orange colouration in pleopods of stored tiger prawns fades to yellow hues more rapidly
401
than the body of the prawn. Thus it is proposed that assessment of pleopod colour is a
402
key monitoring tool for black tiger prawn quality during storage.
403
404
Astaxanthin content correlated well with certain chroma meter values obtained for black
405
tiger prawns, during individual storage trials, and this is in accord with studies for dried
406
Litopenaeus vannamei (13). However it is noted that the relationship between
407
astaxanthin content and colour appearance of prawns is complex and dependent on
408
several factors including mobility between the form and binding of astaxanthin (5).
409
Therefore a strong direct correlation between astaxanthin content in prawn structures
410
and overall colour may not always occur. In the current study, the use of hue angle
411
and/or a* value (red-yellow parameter) appear to be good predictors of astaxanthin
18
412
content in the pleopods, particularly when prawns are grown, harvested and stored
413
under the same conditions.
414
415
This study has determined astaxanthin degradation patterns within both prawn
416
abdominal segments and pleopods, with the latter structures losing astaxanthin and
417
fading in colour very rapidly during frozen storage. As the loss of bright red-orange
418
colour has direct impact on purchase choice at retail level and therefore revenue return
419
for the prawn farming industry, this work has illustrated the importance of using
420
preservation methods for the retention of astaxanthin in black tiger prawns.
421
422
Acknowledgement – Warwick Turner for his help in setting up the LC-MS system.
423
19
424
Literature cited
425
1.
426
Agricultural and Resource Economics, B. o. R. S., Australian Goverment, Ed. Canberra,
427
2010.
428
2.
429
Y., Potential use of the astaxanthin-producing microalga, Monoraphidium sp. GK12, as
430
a functional aquafeed for prawns. Journal of Applied Phycology 2010, 22, (3), 363-369.
431
3.
432
C. C.; Moreira, C. C., Preference ranking of colour in raw and cooked shrimps.
433
International Journal of Food Science & Technology 2011, 46, (12), 2558-2561.
434
4.
435
of black tiger prawn (Penaeus monodon fabricius) coloration by astaxanthin.
436
Aquacultural Engineering 1993, 12, (4), 203-213.
437
5.
438
colour on the distribution of astaxanthin in black tiger prawn (Penaeus monodon):
439
Effective method for improvement of cooked colour. Aquaculture 2009, 296, (1-2),
440
129-135.
441
6.
442
E., Effects of β-carotene source, Dunaliella salina, and astaxanthin on pigmentation,
443
growth, survival and health of Penaeus monodon. Aquac. Res. 2001, 32, (supp.1), 182-
444
190.
445
7.
446
Bate, by various pigment sources and levels and feeding regimes. Aquaculture 1992,
447
102, (4), 333-346.
448
8.
449
exoskeleton of commercial black tiger prawns. . Fisheries Science 1994, 60, (2), 213-
450
215.
ABARE, Australian fisheries statistics, 2009. In Australian Bureau of
Fujii, K.; Nakashima, H.; Hashidzume, Y.; Uchiyama, T.; Mishiro, K.; Kadota,
Parisenti, J.; Beirão, L. H.; Tramonte, V. L. C. G.; Ourique, F.; da Silveira Brito,
Menasveta, P.; Worawattanamateekul, W.; Latscha, T.; Clark, J. S., Correction
Tume, R. K.; Sikes, A. L.; Tabrett, S.; Smith, D. M., Effect of background
Boonyaratpalin, M.; Thongrod, S.; Supamattaya, K.; Britton, G.; Schlipalius, L.
Chien, Y.-H.; Jeng, S.-C., Pigmentation of kuruma prawn, Penaeus japonicus
Okada, S.; Nureborhan, S. A.; Yamaguchi, K., Carotenoid composition in the
20
451
9.
Ju, Z. Y.; Deng, D. F.; Dominy, W. G.; Forster, I. P., Pigmentation of Pacific
452
white shrimp, Litopenaeus vannamei, by dietary astaxanthin extracted from
453
Haematococcus pluvialis. Journal of the World Aquaculture Society 2011, 42, (5), 633-
454
644.
455
10.
456
Vieira, F.; Bedin, L. H.; Rodrigues, E., Pigmentation and carotenoid content of shrimp
457
fed with Haematococcus pluvialis and soy lecithin. Aquaculture Nutrition 2011, 17, (2),
458
e530-e535.
459
11.
460
Glencross, B. D., Mechanisms of colour adaptation in the prawn Penaeus monodon. The
461
Journal of Experimental Biology 2012, 215, (2), 343-350.
462
12.
463
atmosphere packaging on oxidative changes in frozen stored cold water shrimp
464
(Pandalus borealis). Food Chemistry 1999, 64, (2), 169-175.
465
13.
466
Kinetics of astaxanthin degradation and color changes of dried shrimp during storage.
467
Journal of Food Engineering 2008, 87, (4), 591-600.
468
14.
469
sweetcorn for enhanced zeaxanthin concentration. Journal of the Science of Food and
470
Agriculture 2010, 90, (1), 91-96.
471
15.
472
shrimp (Pandalus borealis) and in a microalga (Haematococcus pluvialis) by liquid
473
chromatography-mass spectrometry using negative ion atmospheric pressure chemical
474
ionization. Journal of Agricultural and Food Chemistry 2004, 52, (12), 3870-75.
475
16.
476
body components of Indian shrimps. Journal of the Science of Food and Agriculture
477
2005, 85, 167-172.
Parisenti, J.; Beirao, L. H.; Maraschin, M.; Mourino, J. L.; Do Nascimento
Wade, N. M.; Anderson, M.; Sellars, M. J.; Tume, R. K.; Preston, N. P.;
Bak, L. S.; Andersen, A. B.; Andersen, E. M.; bertelsen, G., Effect of modified
Niamnuy, C.; Devahastin, S.; Soponronnarit, S.; Vijaya Raghavan, G. S.,
Fanning, K. J.; Martin, I.; Wong, L.; Keating, V.; Pun, S.; O'Hare, T., Screening
Breithaupt, D. E., Identification and quantification of astaxanthin esters in
Sachrindra, N. M.; Bhaskar, N.; Mahendrakar, N. S., Carotenoids in different
21
478
17.
Herring, P. J., Depth distribution of the carotenoid pigments and lipids of some
479
oceanic animals. 2. Decapod crustaceans. Journal Marine Biological Association (UK)
480
1973, 53, 539-562.
481
18.
482
Canthaxanthin) Rainbow Trout (Oncorhynchus mykiss) Fillets Stored under Vacuum
483
Packaging during Chilled Storage. Journal of Agricultural and Food Chemistry 1998,
484
46, (10), 4358–4362.
485
19.
486
Extracted from Fermented Shrimp Byproducts. Journal of Agricultural and Food
487
Chemistry 2009, 57, (14), 6095-6100.
488
20.
489
deposition in the flesh of Atlantic salmon , Salmo salar L., in relation to dietary
490
astaxanthin concentration and feeding period. Aquaculture Nutrition 1995, 1, 77-84.
Gobantes, I.; Choubert, G.; Gomez, R., Quality of Pigmented (Astaxanthin and
Armenta, R. E.; Guerrero-Legarreta, I., Stability Studies on Astaxanthin
Torrissen, O. J.; Christiansen, R.; Struksnaes, G.; Eastermann, R., Astaxanthin
491
492
493
The authors gratefully acknowledge the active support of the Australian Prawn Farmers
494
Association and Aqua-Marine Marketing Pty Ltd for the open provision of industry
495
information. The generous financial support of the Australian Government through the
496
Australian Seafood Co-operative Research Centre and the Fisheries Research and
497
Development Corporation is fully acknowledged.
498
499
500
501
22
502
Tables
Preliminary trial
(n=1)
(n=7)
Region of prawn
sampled
Abdomen – segments
2,4,6
Pleopods – segments 1- 6
Baseline samples (0d)
n=6 individuals
chromameter and
astaxanthin analyses
n=20 individuals
chromameter
n=6 individuals
n=10 individuals
chromameter
Samples during
storage
Storage time (d)
503
Frozen storage trials
chromameter and
astaxanthin analyses
12 14 22 28 56 119
Abdomen – segments 2,3,4
n=20 composite astaxanthin
n=10 composite astaxanthin
7 28 56 124 187 371
Table 1. Summary of sampling and frozen storage times within trials.
504
23
505
Location in
Queensland,
Australia
Northern 1a
Northern 1b
Northern 1c
Central 1a
Central 1b
Central 2a
Southern 1a
Southern 1b
Southern 2a
Southern 2b
Harvest
Date
Astaxanthin
total (µg/g)
L* value
Abdo Pleo Abdo
men
pods men
17/03/11
11.5 125.0 49.9
± 2.0
24/03/11
10.4 80.0 50.4
± 3.0
9/06/11
7.5 59.4 55.2
± 1.3
11/03/11
7.1 75.2 52.8
± 3.1
23/03/11
5.5 47.0 53.7
± 3.3
18/03/11
9.3 88.2 49.1
± 3.0
6/04/11
10.1 124.2 48.7
± 1.9
19/04/11
9.4 159.7 48.2
± 2.5
9/03/11
6.1 77.2 50.6
± 2.6
18/03/11
5.6 57.5 53.7
± 2.6
Pleop
ods
46.3
± 3.7
44.4
± 3.0
49.9
± 3.2
47.1
± 4.1
48.5
± 4.4
45.2
± 3.8
43.1
± 2.6
38.5
± 4.1
45.3
± 4.1
47.2
± 3.8
a* value
b* value
Chroma
Hue angle
Abdo
men
32.5
± 2.4
34.0
± 3.2
28.6
± 2.0
28.8
± 3.0
28.9
± 3.5
30.1
± 3.3
28.4
± 2.3
28.1
± 2.1
32.0
± 2.4
34.1
± 2.8
Abdo
men
33.5
± 2.6
37.1
± 2.6
36.6
± 1.6
33.9
± 2.4
35.5
± 1.9
35.3
± 3.5
31.8
± 1.8
32.5
± 2.6
36.1
± 2.4
38.6
± 3.2
Abdo
men
46.8
± 2.8
50.3
± 3.1
46.5
± 2.0
44.6
± 3.4
45.8
± 3.2
46.5
± 3.7
42.7
± 2.3
43.0
± 2.8
48.3
± 2.5
51.5
± 3.6
Abdo
men
45.9
± 2.6
47.6
± 3.0
52.1
± 1.8
49.7
± 2.2
51.0
± 3.0
49.5
± 3.8
48.3
± 2.4
49.2
± 2.3
48.5
± 2.8
48.5
± 2.6
Pleop
ods
24.9
± 2.1
25.3
± 3.0
23.4
± 2.4
20.7
± 3.5
20.0
± 4.1
23.6
± 3.1
19.5
± 2.9
18.9
± 2.6
22.1
± 3.1
24.1
± 4.0
Pleop
ods
26.9
± 2.6
26.4
± 2.0
31.5
± 3.0
26.2
± 2.5
27.0
± 2.5
28.5
± 2.1
27.6
± 2.8
23.3
± 2.9
29.1
± 2.3
28.0
± 3.0
Pleop
ods
36.8
± 2.5
36.6
± 2.5
39.3
± 3.0
33.4
± 3.7
33.8
± 3.5
37.1
± 2.3
33.9
± 2.6
30.1
± 3.4
36.7
± 2.5
37.1
± 3.8
Pleop
ods
47.2
± 3.4
46.3
± 4.2
53.3
± 3.6
51.9
± 3.7
53.8
± 5.6
50.4
± 4.5
54.8
± 5.2
51.0
± 3.6
52.9
± 4.6
49.4
± 4.9
506
Table 2: Total astaxanthin content (composite of 20) and colour properties of abdominal segments and pleopods (mean±sd, n=20) of commercial black tiger
507
prawns from the major production areas of Queensland, Australia.
24
Astaxanthin form
Abdominal segments. µg/g (%)
Pleopods. µg/g (%)
Cis-astaxanthin
0.6±0.2c (7 ± 0.8)
4.6±1.8B (5 ± 1)
Trans-astaxanthin
3.4±1.0a (39 ± 8)
17.9±4.3A (22 ± 6)
Mono-esters
3.2±1.2a (35 ± 7)
47.5±26.3A (46 ± 7)
di-esters
1.7±0.6b (19 ± 4)
25.7±12.3A (26 ± 5)
508
Table 3: Mean astaxanthin content (µg/g) and astaxanthin form as a percentage mean of
509
total astaxanthin (in parentheses), present in black tiger prawns sourced from prawn
510
farms in the major production areas of Queensland (mean ± sd, n=10). Values in the
511
same column that are followed by different letters are significantly different (p<0.05)
512
Astaxanthin form
Prawn part
k (d-1, x102)
r2
Total astaxanthin
Abdominal segment
0.54 ± 0.08a
0.83
Total astaxanthin
pleopod
1.15 ± 0.40b
0.97
Cis-astaxanthin
Abdominal segment
0.39 ± 0.08B
0.89
Trans-astaxanthin
Abdominal segment
0.32 ± 0.06B
0.79
Mono-esters
Abdominal segment
0.66 ± 0.12A
0.96
Di-esters
Abdominal segment
0.57 ± 0.11A
0.91
Cis-astaxanthin
pleopod
0.92 ± 0.59b
0.99
Trans-astaxanthin
pleopod
0.58 ± 0.36b
0.99
Mono-esters
pleopod
1.31 ± 0.63a
0.98
Di-esters
pleopod
1.70 ± 0.65a
0.96
513
Table 4: Rate constants (k; mean±sd, n=7) of degradation of total astaxanthin, and each
514
astaxanthin form, in abdominal segment and pleopod. Different letters, within each
515
group, indicate statistical significance (p<0.05).
516
517
Figures
518
25
519
Graphic for table of contents
520
26