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J Sci Food Agric 1997, 75, 433È441
Formation of White Spots in the Shell of Raw
Shrimps During Frozen Storage. Seasonal
Variation and Effects of Some Production Factors
Anni Mikkelsen,1* Birgitte RÔnn2 and Leif H Skibsted1
1 Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30,
DK-1958 Frederiksberg C, Denmark
2 Department of Mathematics and Physics, Royal Veterinary and Agricultural University,
Thorvaldsendsvej 40, DK-1871 Frederiksberg C, Denmark
(Received 5 August 1996 ; revised version received 11 April 1997 ; accepted 18 April 1997)
Abstract : During frozen storage of raw pink shrimps, Pandalus borealis, calcium
carbonate tends to precipitate in the exoskeleton, giving the shrimps a spotted
appearance. Seasonal variations in the formation of white spots and the inÑuence
of shrimp size, production time, chemical treatment, and storage temperature on
white spot formation was determined. A signiÐcant e†ect (P \ 0É003) of day of
catch was observed and shrimps caught in the summer period showed a greater
tendency of white spot formation than shrimps caught during the rest of the year.
Treatment in a sulphite, a phosphate or a phthalate solution prior to freezing
retards calcium carbonate crystallisation (P \ 0É05) while treatment in a borax
solution promotes crystallisation (P \ 0É05). Prolonged time from catch to
chemical treatment and increased storage time increase the risk of white spot
formation. Storage temperature has a signiÐcant e†ect on white spot formation
for which process the rate at di†erent temperatures was described by the Arrhenius equation with a high energy of activation of 90 kJ mol~1. The size (age) of
the shrimps did not inÑuence white spot formation (P \ 0É69). From these Ðndings it is concluded that calcium carbonate precipitation resulting in white spots
in shrimp shell can be retarded or possibly prevented by short production time,
appropriate chemical treatment and very low storage temperature.
J Sci Food Agric 75, 433È441 (1997)
No. of Figures : 5. No. of Tables : 3. No. of References : 18
Key words : pink shrimp, Pandalus borealis, white spots, calcium carbonate crystallisation, frozen storage, shrimp size, chemical treatment, production time,
storage temperature
INTRODUCTION
quality problem is formation of white spots on the
shrimp shell a†ecting the appearance but not the taste
or other eating qualities of the shrimp. Among the
shrimp industry the problem is well known. However,
to the best of our knowledge, no scientiÐc papers
dealing with this topic has been published.
Pandalus borealis belongs to the class Crustacea of
the order Decapoda of the family Pandalidae (Shumway
et al 1985) and, as for other decapods, the shell consists
of chitin, protein and calcium salts (Stevenson 1985). In
order to allow growth, the exoskeleton is periodically
shed and replaced (Gnatzy and Romer 1984 ; Stevenson
Raw shrimps are a very popular and rather expensive
food product in Asia. Among other species, the pink
shrimp, Pandalus borealis (KrÔyer 1838), is processed
for this product. The shrimps are frozen shortly after
catch and kept frozen until they reach the consumer.
Raw shrimps with shell on are also frequently used for
decoration of meals and the appearance of the shrimps
is a very important quality parameter. A common
* To whom correspondence should be addressed.
433
( 1997 SCI.
J Sci Food Agric 0022-5142/97/$17.50.
Printed in Great Britain
A Mikkelsen, B RÏnn, L H Skibsted
434
1985) and the physical properties of the shell therefore
varies during the growth cycle. This might be of importance in relation to white spot formation. When the
shrimps are caught and still alive no white spots are
observed but after some time of frozen storage white
spots can develop in the shell. During continued frozen
storage the spots increase in size and consequently the
quality of the product decreases. We have recently
found that the white spots are crystals of calcite and
vaterite, two forms of calcium carbonate, in a matrix of
chitin (Mikkelsen et al 1997). Whereas the chemical
composition of the white spots is now known, the conditions triggering the precipitation of calcium carbonate
have not been identiÐed yet. From non-systematic
observations, formation of white spots in the shell of
frozen raw shrimps seems to follow a seasonal variation
and to be a natural event that, to some extent, might be
controlled during the production process and storage
conditions. The usual production process of “frozen raw
shell-on shrimpsÏ includes mechanical sorting according
to size, treatment in a glazing-solution (chemical
treatment), packaging in 1 kg plastic-coated cardboard
boxes, freezing in a plate freezer and storage on board
the trawler at c [25¡C until unloading. After unloading
the shrimps are kept frozen during storage and transportation.
The aim of the present work was to detect possible
seasonal variations of white spot formation and to
determine the inÑuence of the following production and
storage parameters on white spot formation : shrimp
size, production time, chemical treatment, and storage
temperature. The inÑuence of shrimp size, reÑecting the
age of the shrimp, might be considered a biological
parameters as well. The work was conducted on board
shrimp trawlers, and samples were taken from di†erent
sites in the usual production and exposed to the desired
treatments. Unfortunately, it was not always possible to
conduct all planned sampling and treatments of shrimps
on board trawlers. This is the reason for di†erent
number of repeating of treatments.
EXPERIMENTAL
Shrimp samples were taken at di†erent sites at the usual
production line on board Royal Greenland shrimp
trawlers operating in the Davis Strait (between 60¡ and
72¡N and between 60¡W and the coast line of
Greenland) and used for the investigations described
below. After mechanical sorting according to size the
shrimps were treated in a glazing solution for one
minute. Immediately after this chemical treatment the
shrimps were packed in 1 kg low-density polyethylenecoated cardboard boxes and frozen in a plate freezer to
a core temperature of [30¡C or below controlled with
an electronic thermometer with the sensor in the
product. The shrimps were stored in a freezer storage at
c [25¡C on board the trawler until unloading. In each
experiment all samples were from the same catch.
Seasonal variations
Random sampling of raw shrimps from ten trawlers
during 22 months was conducted to study the variation
in white spot occurrence during the year and to study
the dependence of white spot occurrence upon time
from catch to inspection. These samples (in total 2096
1-kg boxes) were taken from the usual production after
freezing and analysed shortly after unloading. As the
trawlers were catching for up to 2 months, samples were
stored for 1È70 days at [25¡C before being unloaded
and analysed.
E†ect of shrimp size
Shrimps of the two most common size categories were
used to investigate the inÑuence of size of the shrimps
on formation of white spots. Eighteen 1-kg boxes of
each shrimp size (small : 9È11 g, and large : 11È14 g)
were taken out from the production line after freezing.
The same number of samples were taken from three
catches and examined after 26, 34, 39, 41, 45 and 48
days of storage, respectively.
E†ect of chemical treatment
Experimental chemical treatments were made in
buckets with shrimps sampled at the production line
prior to the normal chemical treatment : portions of
2È3 kg of shrimps were poured into 6 litres of the
experimental “chemical solutionÏ. After 1 min the solution was poured through a strainer into another bucket,
and the shrimps were drained for a few seconds, packed
in 1-kg boxes and frozen. For each chemical treatment,
6 kg in total were prepared in the solution. Four individual series of chemical treatments were conducted,
each repeated two or three times. All “chemical solutionsÏ (listed in Table 1) were made with sea water.
Glaze-Nole, BL-7P (Shimakyu Chemical Co, Osaka,
Japan) and Oxinon (Cosmo Techno Co, Kanagawa,
Japan) are commercial products for chemical treatment
of shellÐsh, and ingredients of these products are given
in Table 2. Potassium phthalate, potassium dihydrogen
phosphate, disodium diphosphate (Riedel-de HaeŽn,
Seelze, Germany), sodium tetraborate (Merck, Darmstadt, Germany), sodium sulphite, pentasodium triphosphate, and trisodium trimetaphosphate (Sigma, St
Louis, MO, USA) were all of analytical grade. Solution
pH was measured with a pocket pH meter (accuracy
^0É2).
W hite spots in the shell of raw shrimps during frozen storage
435
TABLE 1
E†ect of chemical treatments on white spot formation in shell of raw frozen shrimps investigated in
four experimental series ; “commercial productÏ, “pHÏ “sulphiteÏ, and “phosphatesÏ.
Chemical treatmenta
Solution
pHb
Indexc
E†ect of commercial products
Sea water
Glaze-Nole (10 g litre~1)
BL-7P (15 g litre~1) ] Glaze-Nole (10 g litre~1)d
BL-7P (15 g litre~1)
Oxinon (30 g litre~1)d
Oxinon (30 g litre~1), 2 min
Oxinon (30 g litre~1), 3 min
Oxinon (30 g litre~1), 6 min
Oxinon (60 g litre~1)
7É8
5É9
5É1
4É9
5É7
5É7
5É4
5É7
5É7
2É22a
1É72b
0É15c
0É11c
0É26c
0É30c
0É17c
0É12c
0É18c
E†ect of pH
Sea water
BL-7P (15 g litre~1)
Oxinon (40 g litre~1)d
phthalate (0É02 M)
phthalate (0É02 M)
phosphate (0É02 M)
phosphate (0É02 M)
borax (0É005 M)
borax (0É005 M)
7É7
5É4
5É5
4É5
4É7
5É4
6É1
7É0
7É6
2É98b
1É06c
0É99c
2É72b
2É52b
1É20c
1É38c
3É38a
3É36a
E†ect of sulphite
Sea water
BL-7P (15 g litre~1)
Oxinon (40 g litre~1)d
sulÐte (0É016 M) ] phthalate (0É02 M)
sulÐte (0É032 M)e ] phthalate (0É02 M)
sulÐte (0É048 M) ] phthalate (0É02 M)
7É5
5É3
5É4
5É2
5É1
5É0
3É85a
1É84d
1É86d
3É11b
2É21c
1É51e
E†ect of phosphates
Sea water
BL-7P (15 g litre~1)
Oxinon (40 g litre~1)d
Diphosphate (0É003 M)e
Diphosphate (0É003 M)e ] phthalate (0É02 M)
Tripolyphosphate (0É002 M)e
Tripolyphosphate (0É002 M)e ] phthalate (0É02 M)
Trimetaphosphate (0É001 M)e
Trimetaphosphate (0É001 M)e ] phthalate (0É02 M)
7É3
5É4
5É4
5É3
5É4
7É1
5É5
7É5
5É4
3É79a
1É68c
1É77c
2É92ab
2É63b
2É95ab
3É26ab
3É40ab
3É64a
a All solutions were made with sea water, and all treatments were for 1 minute when not otherwise
indicated.
b pH values are mean values of 2È3 experiments.
c Estimated white spot index, (k ] a ) in equation 3 in the text. Within an experimental series, index
i
values with the same following letters are not signiÐcantly di†erent at the 5% level.
d Common production solution.
e Concentration as in 15 g litre~1 BL-7P solution.
E†ect of production time
The production time consist of two intervals, that is
time from catch to chemical treatment and time from
chemical treatment to freezing. Boxes of shrimps were
randomly sampled at the production line immediately
after chemical treatment and packaging in 1-kg boxes at
the beginning of the production (Time-1 and Time-1a,
see Table 3), and about 1 h later (Time-2). For each of
the three treatments, three 1-kg boxes were used.
A Mikkelsen, B RÏnn, L H Skibsted
436
TABLE 2
Ingredients of commercial products for treatment of shellÐsh according to
product speciÐcations. All three products are produced and allowed for
use in food in Japan.
Product name
Ingredients
Concentration
(g kg~1)
Oxinon
Sodium sulphite
Sodium polyphosphate
Sodium alginate
“Natural substancesÏ
175
75
30
720
BL-7P
L-Tryptophan
Sodium metaphosphate
Sodium D-tartrate
Sodium polyphosphate
Sodium citrate
Sodium hydrogenpyrophosphate
Sodium L-glutamate
Sodium erythorbate
Sodium sulphite
8
24
32
47
33
46
25
113
672
Glaze-Nole
“Natural substancesÏ
Time-1 and Time-2 samples were placed in the freezer
immediately after packaging, while Time-1a samples
were placed in the freezer at the same time as Time-2
samples. The experiment was repeated twice.
E†ect of storage temperature
Samples (312 1-kg boxes) were taken from the production line after freezing. After unloading, the samples
were
placed
in
freezers
at
[16¡C(^2¡C),
[18¡C(^2¡C), [25¡C(^3¡C) and at [29¡C(^2¡C),
respectively. The temperature of each freezer was measured by continuous temperature recordings throughout
the investigation period. Samples were analysed several
times during a storage period of 63 days, the samples
stored at the lower temperatures observed during
1000
longest period (see Fig 4 below). To study the e†ect of a
short storage period at high temperature, simulating
temperature abuse during storage and transportation,
on later development of white spots, samples were
moved from [16¡C and [18¡C, respectively, to
[25¡C at day 6, and stored at [25¡C for the rest of the
storage period. The e†ect of temperature Ñuctuations
were studied by moving samples back and forth
between [29¡C and [25¡C 12 times with minimum
24 h between each movement.
Visual examination
All analyses of white spots in shrimp shell were made by
visual inspection of the shrimps, and for each treatment
three 1-kg boxes were analysed. Each 1-kg box was
TABLE 3
E†ect of production time on white spot formation in shell of raw frozen shrimps treated in
0É40 g litre~1 Oxinon for 1 min.
Production conditions
Time-1
Time-1a
Time-2
Indexa
Chemical treatment at the beginning of the production
and immediately placed in the plate freezer
Chemical treatment at the beginning of the production
and placed in the plate freezer 1 h later
Chemical treatment after 1 h production
and immediately placed in the plate freezer
1É65a
1É54a
2É15b
a Estimated white spot index, (k ] a ) in eqn (3) in the text. Index values with the same following
i
letter (a or b) are not signiÐcantly di†erent at the 5% level
W hite spots in the shell of raw shrimps during frozen storage
thawed under tap water (15¡C ^ 1) for 10 min and
inspected immediately. Depending on the size of white
spots each shrimp were given a score ; 0 \ no white
spots ; 1 \ very small white spots ; 2 \ small white
spots ; 3 \ large white spots and 4 \ splotches of white
spots, and for each 1-kg box a white spot index has
been calculated
index \ ; c N /N
(1)
j j
where c is the score (0È4), N is the number of shrimps
j
j
with score c , and N is the number of shrimps in the
j
box. Thus, “indexÏ, the empirical mean score of a 1-kg
box, accounts for the degree of white spots, including
both the number of shrimps with white spots as well as
the size of the white spots.
Statistics
In the investigation of seasonal variation, the inspection
was made shortly after unloading. White spots had
developed in very few boxes and an assumption of normally distributed observations of the white spot index
would not be reasonable. Therefore, the index was
transformed into a binary response, valued 0 for
index \ 0 and 1 for index [ 0. The transformed
response was analysed by a logistic regression with the
day of catch and the time between catch and inspection
included as Ðxed covariates, and the day of inspection
included as a random e†ect. The model is described by
the formula
ln[p /(1 [ p )] \ a ] b É cos(2n É day /365)
i
i
i
] c É sin(2n É day /365) ] d É time ] C
(2)
i
i
time~i
where p is the probability of box i to contain any
i
shrimps with white spots, and p /(1 [ p ) is odds of
i
i
white spots for box i. Day is the day of catch and time
i
i
is the time between catch and inspection for box i. The
random variable C
is the e†ect of the day of
time~i
inspection and is assumed to be independent and normally distributed. For more details see (McCullagh and
Nelder 1989).
Data from the three series of investigations concerning shrimp size, chemical treatment and production
time were analysed by analysis of variance, with the relevant treatment, ie shrimp size, chemical treatment or
production time, included as a Ðxed e†ect in the model.
A considerable variation between experiments were
observed, and accordingly random e†ects of the experiment as well as interaction between the experiment and
the treatment were included in the model.
index \ k ] a ] E ] (T E) ] e
(3)
ijk
i
j
ij
ijk
where index is the observed index for box k given
ijk
treatment i in experiment j. k is 1È3 as three boxes were
analysed for each treatment. The number of treatments,
437
i, varies between experimental series ; i is 1È3 for the
experimental series of shrimp size, 1È9 for the experimental series of chemical treatment “commercial productsÏ, “phÏ and “phosphatesÏ, 1È6 in the experimental
series of chemical treatment “sulphiteÏ (see Table 1), and
1È3 in the experimental series of production time (see
Table 3). j is the number of repetitions of the experiment. k is the overall mean index, a is the e†ect of
i
treatment i, and E , (T E) are random e†ects of the
j
ij
experiment and interaction between the experiment and
the treatment, respectively. e
is the measurement
ijk
error.
In the shrimp size investigation, the shrimps were
examined several times during the storage period, and
the storage time (time between production and
inspection) was included in the model as a Ðxed e†ect as
well
index \ k ] a ] c É time ] E ] (T E) ] e
(4)
ijk
i
ijk
j
ij
ijk
Before the analysis, the white spot index was transformed by arcsin()index) to achieve a homogeneous
variance.
Data from the experimental series concerning storage
temperature were evaluated using a model where formation and growth of white spots during storage is
described by a polynomium of third degree in the
storage time with a rate depending on the temperature :
arcsin()index ) \ a É (rate
É time ) ]
i
temp
i
b É (rate
É time )2 ] c É (rate
É time )3 ] e (5)
temp
i
temp
i
i
where index is the observed index for box i, time is the
i
i
storage time, and rate
is the rate parameter corretemp
sponding to the storage temperature of box i. a, b and c
are constants, and e is the measurement error. The temi
perature dependence of the rate was evaluated according to an Arrhenius type equation
\ A É exp[[(E /R) É T ~1]
(6)
temp
a
where A is a constant, E is energy of activation, R is
a
the gas constant and T is the absolute temperature.
All statistical calculations were made by the procedures MIXED and NLIN, and the macro %GLIMMIX in SAS version 6.10.
rate
RESULTS
Variation in white spot occurrence during a period of
22 months is shown in Fig 1. Logistic regression
analysis showed a signiÐcant e†ect (P \ 0É003) of the
day of catch on the probability of white spot
occurrence, and shrimps caught in the summer seasons
had markedly increased incidence of white spots. The
statistical analysis also showed a signiÐcant e†ect
(P \ 0É001) of storage time (at c [25¡C) on white spot
formation, as may be seen from Fig 2.
438
A Mikkelsen, B RÏnn, L H Skibsted
Fig 3. Development in white spots during frozen storage of
small (K) and large (=) shrimps. Each data point is a mean of
9 observations (3 experiments with each 3 observations).
Fig 1. White spot occurrence in raw frozen shrimps as function of time of catch. Day of catch \ 0 is 1 January 1994. L,
Observed mean odds of grouped data from random sampling
during 22 months. …, Predicted odds of grouped data (same
mean time of catch as for observed data), corrected for the
groupÏs mean time between time of catch and inspection as for
the observed odds. Full line, predicted odds, for time between
catch and inspection being equal to mean time between catch
and inspection (\24É3) for the entire data set.
Fig 2. White spot occurrence in raw frozen shrimps as function of time (days) between catch and inspection. L, Observed
odds of grouped data from random sampling during 22
months. …, Predicted odds of grouped data (same mean time
between catch and inspection as for observed data). Full line,
predicted odds, for time of catch being equal to mean time of
catch (\335É2) for the entire dataset.
Development in white spots during frozen storage of
small and large shrimps is shown in Fig 3. The data
conÐrm the e†ect of storage time, but no di†erence
between the two sizes was observed (P \ 0É69).
When used in production of raw shrimps, Oxinon is
used at a concentration of 30È40 g litre~1, and BL-7P
and Glaze-Nole are used together at concentrations of
15 g litre~1 and 10 g litre~1, respectively. Treatment
with Glaze-Nole has a minor e†ect only, while treatment with BL-7P or Oxinon strongly reduces white
spot formation (Table 1). A tendency to further
reduction in white spot formation was observed when
Oxinon concentration or the time of treatment was
increased, however, these e†ects were not signiÐcant
(Table 1). The e†ect of pH of the “chemical solutionÏ on
white spot formation was studied for bu†er solutions
with pH in the range 4É5 to 7É6 (Table 1, E†ect of pH).
In general, pH decreased by 0È1 units during treatment
of 6 kg shrimps, most for sea water. No direct e†ect of
solution pH on white spot formation was observed.
Phthalate bu†er treatment (pH 4É5È4É7) had no signiÐcant e†ect, while phosphate bu†er treatment (pH 5É4È
6É1) resulted in less white spot formation compared to
sea water (pH 7É7) and borax bu†er treatment (pH 7É0È
7É6) increased white spot formation. The e†ect of sulphite, the major ingredient of BL-7P and Oxinon, was
investigated for three concentrations (Table 1), and a
strong inÑuence of concentration of sulphite on inhibition of white spot formation was observed. Sulphite
concentration as in the usual BL-7P solution (0É032 M)
has less inhibitory e†ect than BL-7P, whereas 0É048 M
sulphite protects just as well as BL-7P. Sodium diphosphate, sodium tripolyphosphate and sodium trimetaphosphate in concentrations as in the usual BL-7P
solution, with or without addition of phthalate bu†er to
adjust pH to c 5É4, provide no signiÐcant protection
from white spot formation (Table 1).
The time it takes to pass a shrimp catch through the
production process inÑuences white spot formation.
Results in Table 3 show that shrimps that have waited
for 1 h before chemical treatment and freezing (Time-2)
W hite spots in the shell of raw shrimps during frozen storage
439
storage at [25¡C. The white spot formation in this case
was as would be expected for shrimps stored at a constant temperature of c [27¡C. Progress in white spot
formation in shrimps stored for six days at [16¡C and
[18¡C, respectively, followed by storage at [25¡C, is
well described by a combination of the rate at [16¡C
or [18¡C and the rate at [25¡C. The temperature
dependence of white spot formation is well described by
an Arrhenius type equation (Fig 5) with an apparent
energy of activation of 90(^8) kJ mol~1. The high
energy of activation indicates that the rate-limiting step
in the reactions leading to white spot formation is not
enzymatically controlled and not di†usion controlled.
The white spot formation is probably the results of a
sequence of reactions, which may include enzymatic
processes and di†usion of reactants. However, the rate
limiting step is most likely a non-enzymatic hydrolysis.
Fig 4. E†ect of storage temperature on development in white
spot index. Observed index values are shown with symbols (L
and …), and predicted index values are shown as full and
dotted lines. A, storage at [16¡C (…, full line) and [25¡C
(L, dotted line) ; B, storage at [18¡C (…, full line) and
[29¡C (L, dotted line) ; C, storage at [16¡C for 6 days followed by storage at [25¡C (…, full line), and at alternating
[25 and [29¡C (L, dotted line) ; D, storage at [18¡C for 6
days followed by storage at [25¡C (…, full line).
have a higher degree of white spots than shrimps
treated with chemicals immediately after catch (Time-1
and Time-1a). It had no signiÐcant e†ect if the shrimps
were placed in the freezer immediately after packaging
(compare Time-1a with Time-1).
Storage temperature strongly a†ects white spot formation, and the rate of white spot formation increased
with increasing temperature in the range [29¡C to
[16¡C as can be seen from Fig 4. Alternating storage
at [25¡C and [29¡C resulted in increased white spot
formation compared to constant storage at [29¡C, and
decreased white spot formation compared to constant
Fig 5. Arrhenius type plot for white spot formation in the
shell of pink shrimp during frozen storage. Rates are relative
to the rate at [16¡C.
DISCUSSION
Like other crustacean exoskeletons, the shrimp shell
consists of a thin outer epicuticle composed of protein,
lipid and calcium salts, and a thicker procuticle composed of chitin, protein and calcium salts (Stevenson
1985). The cuticle, which also contains water, consists of
several layers, and the white spots are formed within
these layers. We have recently identiÐed the chemical
composition of white spots as calcite and vaterite in
close association with chitin (Mikkelsen et al 1997).
Calcium and carbonate are natural components of the
shrimp shell though no (visible) calcium carbonate crystals are formed in the living shrimp.
From experiments where the development in white
spot formation was followed over a time period (eg in
Figs 2, 3 and 4), it is clear that white spots are initially
formed as very small spots which grow in size, eventually covering the whole animal. This is in agreement
with the identiÐcation of white spots as calcium carbonate, the precipitation of which starts as small crystals
which grow in size. After catch, when the physical conditions as well as physiological conditions of the shrimp
change, a number of factors might initiate crystallization and promote crystal growth in the shrimp
shell.
As a simple model, the aqueous phase of the shell can
be considered as an aqueous solution of calcium and
carbonate ions. When the shrimp is frozen, water forms
ice, resulting in increased activity of solutes. Crystallization of calcium carbonate will occur when the
solubility product constant, K , is exceeded
sp
K \ aCa2` É aCO2~
(7)
sp
3
where aCa2` is activity of calcium ions and aCO2~ is
3
activity of carbonate ions. The solubility product constant, K , varies with temperature, salinity, crystal
sp
440
form, purity and particle size (Simkiss 1976). Besides the
precipitation equilibrium
Ca2` ] CO2~ ¢ CaCO (s)
(8)
3
3
the following gas/solution equilibria must also be taken
into account
CO (g) ¢ CO (aq)
(9)
2
2
CO ] H O ¢ H CO
(10)
2
2
2
3
H CO ¢ HCO~ ] H`
(11)
2
3
3
HCO~ ¢ CO2~ ] H`
(12)
3
3
From eqns (8) and (12), calcium carbonate precipitation
would be followed by a fall in pH, as is also seen in
laboratory experiments (eg Reddy and Nancollas 1973 ;
Reddy 1975 ; Reddy and Wang 1980 ; Koutsoukos and
Kontoyannis 1984). However, during storage of
shrimps, pH increases as the result of other processes
(Bhobe and Pai 1986 ; Riaz and Qadri 1990), the pH
increasing more rapidly at increasing storage temperature (Riaz and Qadri 1990). White spots usually
appear in the carapace (head shell) Ðrst, probably
resulting from a larger increase in carapace pH than in
somite (body shell) pH. During storage for 1 month at
[25¡C, somite pH increased approximately 0É3 units
from initially pH \ 6É9, while carapace pH increased
approximately one unit from initially pH \ 7É9 (Royal
Greenland, unpublished data). The observed increase in
white spot formation with increasing storage temperature (Fig 4) is in agreement with the more rapid rise
in pH at higher storage temperature. From the e†ect of
pH of the chemical solutions on white spot formation
(Table 1) it can be inferred that the chemical solution
does not solely a†ect the reactions of calcium carbonate
precipitation (eqns 8È12) as this would show an increase
in white spot formation with increasing pH of the
chemical solution. The pH of the chemical solution
must a†ect white spot formation indirectly, eg via e†ect
on hydrolytic reactions, protein conformation etc. Alternatively, the observed e†ect of the chemical solutions
may arise from speciÐc inhibiting e†ects of compounds
in the mixture on calcium carbonate crystallisation.
In non-biological systems, calcium carbonate crystal
formation and growth is inhibited by many organic and
inorganic substances (eg Reddy and Nancollas 1973 ;
Reddy 1975 ; Reddy and Nancollas 1976 ; Reddy 1977 ;
Reddy and Wang 1980 ; SoŽhnel and Mullin 1982 ; Koutsoukos and Kontoyannis 1984 ; Meyer 1984 ; Sawada et
al 1990). In the present study, retardation of calcium
carbonate crystal growth by sulphite and phosphate
was demonstrated in shrimp shell. E†ects of di-, tri- or
trimetaphosphate at concentrations equal to their concentrations in commercial products for treatment of
shellÐsh is, however, marginal. The addition of phosphates to commercial products for treatment of shellÐsh
might have other advantages such as increasing water
A Mikkelsen, B RÏnn, L H Skibsted
binding. These compounds may, however, have a synergistic e†ect with other compounds on the inhibition of
calcium carbonate crystallisation. A synergistic e†ect of
compounds in Oxinon and BL-7P is indicated by a
smaller e†ect of 0É032 M sulphite than that of BL-7P
with the same concentration of sulphite (Table 1). The
mechanism of inhibition of calcium carbonate growth is
usually explained as a reduction in the reaction rate due
to surface adsorption of the inhibitor at growth sites on
the crystal surface (Reddy 1977 ; Morse 1990) with different efficiencies depending on di†erent adsorption
affinities which result from di†erent charge, ionic radii
and hydration properties of the inhibitor. Structural differences between SO2~ and CO2~ should be noted. In
3
3
the present investigation we have found that the length
of time from catch to chemical treatment is critical for
white spot formation. Changes in physical and physiological conditions when the shrimps are caught result in
disturbance in the regulation of pH and ion concentrations in the shrimp shell, in e†ect initiating crystal seed
formation and crystal growth of calcite and vaterite. To
be e†ective, inhibition of calcium carbonate crystal
growth should therefore be performed as early as possible in the production process, as clearly indicated
from data in Table 4. The time from chemical treatment
to freezing seems, however, not to be important for later
formation of visible calcite.
To accommodate the growing shrimp, the external
skeleton must be shed and replaced periodically by the
molt process (Gnatzy and Romer 1984 ; Stevenson
1985). The time of molting varies geographically and is
inÑuenced by temperature (Shumway et al 1985), and
might vary slightly from year to year. Before the old
skeleton is shed, parts of it are digested and resorbed. A
new cuticle is formed, followed by shedding of what is
left of the old cuticle. Resorbed ions and other substances are used in formation of the new skeleton.
Movement of ions during the period of molting might
be a condition of easy calcite/vaterite crystal formation
and crystal growth, as observed for two intervals during
a period of 22 months (Fig 1). The data in Fig 1 can not
be correlated to molt period, as this has not been
recorded. However, in the geographical area where the
present work was carried out shrimps molt mainly in
MayÈJune. Another possible explanation of the
increased incidence of white spots in the summer
months might be the increased risk of periodically elevated temperatures during unloading, transportation,
and storage in the warmest period of the year.
The strong temperature dependence (Fig 5) of the rate
limiting step in calcium carbonate crystallisation in
shrimp shell means that careful control of storage temperature is an important tool in avoiding white spot
formation. The high energy of activation (90 kJ mol~1)
of formation of white spots indicates that the process of
white spot formation is neither controlled by di†usion
nor by an enzymatic reaction but more likely by a
W hite spots in the shell of raw shrimps during frozen storage
simple hydrolysis reaction. A very convincing e†ect of
temperature on formation of white spots is also seen
when frozen shrimps, without any white spots present,
are thawed under warm tap water. Within a few
minutes white spots appear in the shell. Therefore, the
thawing process at inspection of the shrimps as well as
by the consumer is very important to the observed incidence of white spots in the product. If shrimps without
white spots are thawed under cold (O15¡C) water and
the shell is dried at room temperature, no white spots
appear upon heating to 100¡C, probably because
calcium and carbonate ions are kept at a distance in the
chitinÈprotein network of the shell during drying at
room temperature. On the other hand, wet heating of
shrimp shell facilitates movement of ions and promotes
precipitation, an e†ect which is further enhanced by the
solubility product of calcite decreasing with increasing
temperature.
CONCLUSIONS
Formation of white spots in the shell of raw shrimps
during frozen storage can be considered a natural phenomenon, as white spots always appear after some time
of storage at [25¡C, if the shrimps are not treated
chemically prior to freezing. Chemical treatment with
sulphite, phthalate and phosphates was found to
decrease calcite crystallisation in shrimp shell, while
treatment with borax promotes crystallisation.
Increased time between catch and chemical treatment as
well as increased storage temperature results in
increased formation of white spots.
As several factors are shown to inÑuence white spot
formation, control of a single factor might not result in
a satisfactory improvement in the visual quality. In contrast, the simultaneous presence of several factors such
as long production time and low concentration of
crystal inhibitors in the chemical bath, followed by high
temperature during unloading, transport and storage,
will certainly cause substantial reduction in the shrimp
quality.
An increased risk of white spot formation is observed
in the summer period. This might result from changes in
ion regulation in the shell during molt, a factor which
should be considered in future investigations.
ACKNOWLEDGEMENTS
This research was a part of the F^TEK programme
sponsored by the Danish Ministry of Research and the
Danish Ministry of Agriculture and Fisheries and conducted as a collaborative project with the company
Royal Greenland (Roslev, Denmark) and LMC-Centre
441
for Advanced Food Studies. Arctic Station (University
of Copenhagen) in Qeqertarsuaq, Greenland, is thanked
for providing laboratory facilities.
REFERENCES
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