A Research Note

Extraction of Carotenoprotein from Shrimp Process Wastes

with the Aid of Trypsin from Atlantic Cod
A. CANO-LOPEZ, B.K. SIMPSON, and N.F. HAARD
ABSTRACT
Atlantic cod trypsin or bovine trypsin were used to aid the extraction
of carotenoprotein from shrimp wastes at 4C. When 25 mg% cod
trypsin was added to extraction medium containing 0.5N ethylene
diaminetetraacetic acid (EDTA) 64% of the astaxanthin and 81% of
the protein of shrimp waste was recovered as carotenoprotein in 24
hr. With 25 mg% bovine trypsin, under otherwise identical conditions,
the carotenoprotein recovered represented 49% of the astaxanthin and
65% of the protein of the waste. Semi-purified cod trypsin was not
as effective as pure trypsin in facilitating recovery of carotenoprotein
from shrimp waste. The recovery of carotenoprotein from shrimp waste,
during extraction at 4C with or without trypsin, was facilitated by
EDTA.
INTRODUCTION
CAROTENOPROTEIN, a by-product from crustacean process
offal, has potential for use as a feed supplement in rations of
farmed fish or as a colorant and flavorant for use in food
products (Simpson and Haard, 1985a; Simpson and Haard,
1985b). When the extraction medium includes 25 mg% bovine
trypsin, 0.5N ethylene diamine tetra acetic acid (EDTA), pH
7.7 at 4C as much as 80% of the protein and astaxanthin of
shrimp waste can be recovered as a stable carotenoprotein com-
plex. At higher temperatures the speed of carotenoprotein ex-
traction is increased and EDTA is not required for high yield;
however the maximum yields are somewhat lower and the odor
and taste of the product is adversely affected.
Given the low habitat temperature of Atlantic cod, its trypsin
is expected to be a more efficient catalyst than bovine trypsin
at low reaction temperatures (Simpson and Haard, 1984a). The
present study was undertaken to compare the efficacy of tryp-
sin from Atlantic cod pyloric ceca with that from bovine pan-
creas in releasing carotenoprotein from shrimp waste at low
reaction temperatures. The utility of semi-purified cod trypsin
isolates to aid recovery of carotenoprotein from shrimp wastes
was also examined.
MATERIALS & METHODS
Materials
Atlantic cod (Gadus morhua), obtained live from holding tanks at
the Marine Sciences Research Laboratory, were stunned with a blow
to the head and the pyloric ceca was removed and used to obtain
trypsin. Raw, frozen shrimp (Pandufus borealis, Kroyer) were do-
nated by Fishery Products International, St. Johns, Nfld. Bovine tryp-
sin, N-bcnzoylarginine-p-nitroanilide (BAPA), soybean ttypsin inhibitor,
EDTA, tris(hydroxymethyl)aminomethane(Tris), cyanogen bromide-
activated Sepharose 4B were obtained from Sigma Chemical Co., St.
Author Haard is with the Institute for Marine Resources, Dept.
of Food Science & Technology, Univ. of California, Davis, CA
95616. Author Cano-Lopez is a technical trainee with Canada-
Mexico Exchange Program at Memorial University of New-
foundland, St. Johns Present address: CIEIAA, Universidad de
Guanajuato, CP 38000, Guanajuato, Mexico. Author Simpsons
present address is Dept. of Food Science & Human Nutrition,
Univ. of Florida, Gainesville, FL 32611.
Louis, MO. Polyethylene lauryl ether (Brij 35) was obtained from
BDH Chemicals, Dartmouth, N.S. All other reagents were of reagent
grade and were obtained from Fisher Scientific, Halifax, N.S.
Isolation of trypsin
Trypsin, from the pyloric ceca of Atlantic cod, was isolated and
purified by a procedure used by us earlier with Greenland cod (Simp-
son and Haard, 1984a). The purified trypsin exhibited one band after
electrophoresis in the presence or absence of SDS as described by
Simpson and Haard (1984a). Trypsin activity was standardized by the
amidase reaction as described by Simpson and Haard (1984a). One
BAPA unit is defined as an absorbancy change (410 nm) of 2.933/
min at 25C, pH 8.2 in a 3 mL reaction mixture.
Extraction of carotenoprotein
Carotenoprotein was extracted and isolated from cooked shrimp
waste as described by Simpson and Haard (1985a) using 25 mg%
trypsin. In certain experiments, when EDTA was not added to the
extraction medium, the pH was maintained at 7.7 by titration with
OSN HCI using a metrohm autotitration unit. In separate experiments,
the Brij fraction or the ammonium sulfate .fraction of semi-
pure trypsin, equivalent to 25 mg% pure trypsin on the basis of its
amidase activity (after activation) with BAPA substrate at 25C, was
used instead of purified cod trypsin.
Total astaxanthin and protein content of carotenoprotein
The lyophilized carotenoprotein was analyzed for total astaxanthin
and protein as described by Simpson and Haard (1985a). The protein
and astaxanthin content of carotenoprotein was expressed as % re-
covered, i.e. the total astaxanthin (or protein) recovered in the caro-
tenoprotein fraction as a per cent of that which was present in the raw
shrimp waste prior to extraction.
RESULTS & DISCUSSION
Amidase activity of Atlantic cod trypsin
At 25C the specific activity of purified cod trypsin was
1.14 BAPA units/mg protein, and was similar to that of bovine
trypsin, 1.13 BAPA unitslmg protein. The temperature coef-
ficients for the amidase reaction catalyzed by cod and bovine
trypsin were 1.76 and 2.08 respectively. Thus at Yc, the spe-
cific activity of cod trypsin (0.37) was almost 50% higher than
that of bovine trypsin (0.26). The relatively low temperature
coefficient for Atlantic cod trypsin is similar to that reported
earlier by us for Greenland cod trypsin, i.e. 1.62 (Simpson
and Haard, 1984a). The specific activities of the Brij and
ammonium sulfate fractions, employed as extraction aids,
were 0.4 and 0.41 BAPA units/mg protein at 25C respec-
tively .
Comparison of bovine and cod trypsin as extraction aids
The recovery of astaxanthin and protein from shrimp waste
when extraction was carried out for various times up to 48 hr
at 4C in 0.5M EDTA, pH 7.7 containing either 25 mg%
bovine trypsin, 25 mg% pure cod trypsin or no enzyme is
shown in Fig. 1. Both sources of trypsin were effective in
Volume 52, No. 2, 1987JOURNAL OF FOOD SCIENCE-503
CAROTENOPROTEIN EXTRACTION FROM SHRIMP WASTES. . .
100 ,
ASTAXANTHIN
0 10 20 30 40
EXTRACTION TIME (h at 4-C)
50
90
90
70
60
50
40
30
20
10
0
PROTEIN
0 10 20 30 40 50
EXTRACTION TIME (h at 4%)
Fig. I-Recovery of astaxanthin and protein from shrimp wastes
as carotenoprotein. Extraction was at 4C with 0.5M EDTA, pH
7.7 containing no added enzyme (01, purified Atlantic cod tryp-
sin @J, orpurified bovine pancreatic trypsin /mJ. The astaxanthin
and protein contents of the shrimp waste employed were 128
mg% and 2.97%, respectively, prior to extraction. Data are the
average of duplicate results for one experiment and are repre-
sentative of results obtained in two other experiments.
aiding the extraction of carotenoprotein compared to the con-
trol which did not contain added enzyme. Cod trypsin was
more effective than bovine trypsin in aiding pigment and pro-
tein recovery. On the basis of the temperature coefficients of
BAPA hydrolysis for the two enzymes, the cod trypsin would
be expected to be almost 1.5 times as active as bovine trypsin
at 4C. Thus Atlantic cod is an advantageous industrial enzyme
for use at low temperature as already shown for Greenland cod
trypsin (Simpson and Haard, 1986b).
Extraction of carotenoprotein at 4C without EDTA
Both cod and bovine trypsin were less effective, and did not
appreciably differ, in aiding extraction of carotenoprotein from
shrimp waste when EDTA was omitted from the extraction
medium. The astaxanthin recoveries after 48 hr extraction with
cod trypsin, bovine trypsin and control were only 36%, 35%
and 18% respectively. The protein recoveries for these samples
were 56%, 54% and 31% respectively. Trypsin aided extrac-
tion at 4C differs from that carried out at 50C since at the
higher temperature EDTA does not have a marked effect on
carotenoprotein extraction (Simpson and Haard, 1985b).
Semi-purified cod trypsin as extraction aids
The AS and Brij fractions of cod trypsin appeared to
be very effective in releasing astaxanthin from shrimp waste
since the chitinous residue obtained after extraction was ren-
Lt 80
8 60
zi
; 40
K
x 20
0
10 20 30 40
EXTRACTION TIME (h at 4%)
80
s 60
8 40
2
8 20
PROTEIN
0
0 10 20 30 40 50
EXTRACTION TIME (.h at 4-C)
Fig. 2-Recovery of astaxanthin andprotein from shrimp wastes
as carotenoprotein. Extraction was at 4C with 0.5 M EDTA, pH
7.7 containing no added enzyme (01, pure cod trypsin @J, Bnj
fraction of cod trypsin (*J or ammonium sulfate fraction of
cod trypsin PJ. Data are the average of duplicate results for one
experiment.
dered colorless. However, the pigment released during extrac-
tion with semi-purified cod trypsin was not recovered, as
completely, in the carotenoprotein fraction compared to pure
cod trypsin (Fig. 2). After making the extracts containing semi-
purified trypsin to 45% saturation with ammonium sulfate and
centrifugation, much of the pigment remained soluble or sep-
arated as an oil. These observations indicate that impurities,
presumably other proteolytic enzymes or lipolytic enzymes which
were not separated from pyloric ceca extracts by the initial
purification steps, act to degrade the carotenoprotein. Previ-
ously we found that technical grade porcine trypsin or Enzeco
protease AP-1 were less effective than pure bovine trypsin in
facilitating release of intact carotenoprotein from shrimp wastes
(Simpson and Haard, 1985a).
CONCLUSIONS
THE PRESENT STUDY indicates Atlantic cod trypsin is a
more effective extraction aid than is bovine trypsin for re-
covering carotenoprotein from shrimp process wastes at 4C
when EDTA is present in the extraction medium. Unfortu-
nately, the semi-purified cod trypsin fractions tested were not
very effective in recovering intact carotenoprotein under the
conditions employed in this study. It is likely that use of pure
trypsin as an extraction aid would be prohibitive for commer-
cial preparation of carotenoprotein because of the expense of
purified enzymes. For this reason, additional study should be
directed toward finding a practical way of obtaining cod trypsin
which does not have residual activity which is autolytic to
carotenoprotein.
-Continued on page 506
504-JOURNAL OF FOOD SCIENCE-Volume 52, No. 2, 1987