Animal Feed Science and Technology, 14 (1986) 291--305 291

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

THE AMINO ACID AND MINERAL COMPOSITION OF WHITE FISH

MEAL CONTAINING ENZYME-DIGESTED OR UNTREATED
STICKWATER SOLIDS

C.G. ZARKADAS
Agriculture Canada's Food Research Centre, St. Hyacinthe, The McGill Nutrition and
Food Science Centre, and the Department o f Agricullural Chemistry and Physics,
Macdonald College, McGill University, Montreal, Quebec H9X 1C0 (Canada)
H.W. HULAN ~ and F.G. PROUDFOOT
Agriculture Canada, Research Station, Kentville, Nova Scotia B4N 1J5 (Canada)
Contribution No. 1836, Kentville Research Station and No. 12, St. Hyacinthe Food
Research Centre
(Received 14 December 1984; accepted for publication 20 August 1985)

ABSTRACT

Zarkadas, C.G., Hulan, H.W. and Proudfoot, F.G., 1986. The amino acid and mineral
composition of white fish meal containing enzyme-digested or untreated stickwater
solids. Anim. Feed Sc£ Technol., 14: 291--305.

Two representative commercial white fish meal samples were used to study the effect
of added enzyme-digested (FMED) or untreated (FMR) stickwater solids on their protein
quality. They contained the same amino acids in approximately similar proportions. The
main advantage of the enzymatic digestion of stickwater solids is that the product be-
comes more soluble and less viscous, thus reducing build up on the interior of the evap-
orators and dryers and that it improves the recovery of fish proteins from the press
liquor. The crude protein content of FMED (64.2%) was comparable to that of FMR
(61.6%), estimated by the Kjeldahl nitrogen method, but these figures were higher than
their true protein contents as determined by quantitative amino acid analysis. Although
the total lipid content (6.0%) of FMED differs from FMR (3.41%), their apparent meta-
bolizable energies were similar. Determination of 5-hydroxylysine as an index of collagen
indicated that FMED and FMR contained 33.5 and 33.3% collagen, respectively.

INTRODUCTION

W h i t e fish m e a l is p r o d u c e d b y c o o k i n g , p r e s s i n g , d r y i n g a n d g r i n d i n g c u l l
fish, s m a l l fish, fish o f f a l a n d o t h e r w a s t e m a t e r i a l s f r o m t h e fish c a n n i n g
a n d f i l l e t i n g p r o c e s s , p r i m a r i l y o f c o d a n d h a d d o c k ( f o r r e v i e w see B a r l o w

' Author to whom correspondence should be addressed.

0377-8401/86/$03.50 © 1986 Elsevier Science Publishers B.V.

292

and Windsor, 1983). In 1978, the world's fish meal production was 4.7
million tonnes (FAO, 1979). About 90% was derived from oily species
such as anchovy, capelin and menhaden. The remaining 10% was white fish
meal produced mainly on the East Coast of North America, Iceland, the
U.K. and South Africa. White fish meal contains between 60 and 70% pro-
tein of high biological value for the diets of animals, poultry and pets (Barlow
and Windsor, 1983; Hulan and Proudfoot, 1981; Al Hassan et al., 1981). It is
rich in the essential amino acids, particularly lysine and the sulfur amino
acids and the digestibilities of the critical amino acids are approximately 94%
for the rat, 89% for the pig and 85% for the chicken (Barlow and Windsor,
1983).
The aqueous portion of the press liquor during fish meal processing,
k n o w n as stickwater, contains dissolved material and fine solids which may
a m o u n t to about 9% by weight (Barlow and Windsor, 1983). Traditionally,
the stickwater has been discarded with the plant effluent creating, in many
instances, a pollution problem. Since the stickwater can contain as much as
20% of the total solids, the present industrial method used for recovering
the protein is by evaporating the stickwater to a thick syrup containing
30--50% solids. The concentrated product, sometimes termed condensed
fish solubles, is usually added back to the press cake and dried with it to
make what is known as whole fish meal. The hot air (500°C) or steam-
heated discs normally employed during the evaporating and drying processes
may cause heat damage or partial denaturation of proteins, which can in-
crease the viscosity of the condensed fish solubles. This frequently results in
a residue coating of the evaporators and dryers and decreases the efficiency
of the heat exchange, increasing fuel oil consumption during fish meal
production by about 1 kg oil per tonne of raw fish materials. To reduce
fuel costs and improve the quality of fish meal production, Jacobsen
and Rasmussen (1983) recommended the use of proteolytic enzymes, such
as Alcalase 0.6 L, which is prepared by submerged fermentation of a selec-
ted strain of Bacillus licheniformis. The major enzyme c o m p o n e n t in com-
mercial Alcalase 0.6 L preparations is subtilisin Carlsberg.
It has been shown that fish meal in poultry diets can lead to increased
meat or egg production and better feed utilization compared with nutrition-
ally balanced diets not containing fish meal {for review see Barlow and
Windsor, 1983). Since little information exists on the nutritive value and
economic potential of fish meal containing enzymatically digested stick-
water solids, the present study was undertaken. The amino acid and mineral
composition of commercially available white fish meal products containing
enzyme-digested (FMED) or untreated (FMR) stickwater solids typical of
those produced in eastern Canada were determined.
 293

MATERIALS AND METHODS

Chemicals and resins

The Beckman type AA-10, 9.0 + 1.0 /~m spherical resin and a type I
standard amino acid calibration mixture were obtained from Beckman
Instruments Inc., Palo Alto, CA. The Durrum type DC-6A 11.0 + 1.0 pm
spherical resin was purchased from Dionex Corporation, Sunnyvale, CA. L-
Tryptophan, D-glucosamine HC1 and DL-ornithine (5-aminonorvaline) were
from Schwarz/Mann, Orangeburg, NY. The diastereoisomer mixture of
5-hydroxy-DL-lysine and allo-5-hydroxy-DL-lysine, D-galactosamine HC1,
4-hydroxy-L-proline and most of the unusual basic amino acids employed
for the preparation of the amino acid standard calibration mixture were
from Calbiochem, La Jolla, CA and were prepared as described previously
(Zarkadas, 1975, 1979). All other chemicals and reagents were of the highest
purity commercially available and were used w i t h o u t further purification.

White fish meal preparation

The two white fish meal (FM) preparations used in these studies were
supplied by National Sea Products Ltd., Lunenburg, Nova Scotia. These
were made up of 40% cod, 30% haddock, 8% pollack, 10% sole and 12%
red fish, except that the stickwater solids normally added back to the FM
cake were either untreated (FMR) or enzymatically digested (FMED) to
yield two different FM products. The procedure of Jacobsen and Rasmussen
(1983) for the continuous enzymatic hydrolysis of the fish press liquor
(stickwater) with a food grade Alcalase 0.6 L preparation (activity 0.6
Anson units; AU per g) supplied by Nova Industri A/S, Novo Alte, Denmark
(Adler-Nissen, 1976) was as follows. The stickwater liquor flowing at 2275
1 h ~ was treated with a diluted Alcalase 0.6 L solution (128 mg ml -~) at
a flow rate of 12.7 1 h -~ (ratio of Alacalase to stickwater fish protein 1:100).
The mixture (pH 8.0) was helu at 60°C in a holding tank for 2 h to com-
plete the digestion. The digest was then flushed into the evaporators to
inactivate the enzyme and to sterilize (121°C) and concentrate the product.
Commercial Alcalase 0.6 L preparations, produced by submerged fermenta-
tion of a selected strain of Bacillus licheniformis, are known to contain
primarily the serine protease subtilisin Carlsberg (mol. wt. 27.3 kdaltons;
EC 3.4.21.14) capable of digesting food proteins into smaller peptide frag-
ments. This procedure renders the concentrated product more soluble,
especially at its isolectric point (Cheftel et al., 1971; Archer et al., 1973),
supposedly w i t h o u t impairing its nutritional value (Adler-Nissen, 1976).

Proximate and elemental analyses

Standard methods for moisture (7.003, 14.002), total ash (14.006),

phosphorus (7.118) and crude protein (2.057; N X 6.25, Kjeldahl method)
294

were followed (Association of Official Analytical Chemists, 1984). Ether-

extractable lipids were determined as described by Crampton (1956). Cal-
cium, zinc, iron, potassium, sodium and magnesium were determined sep-
arately by atomic absorption s p e c t r o p h o t o m e t r y (2.097--2.157) using the
official lanthanum oxide m e t h o d (Association of Offical Analytical Chemists,
1984).
Apparent metabolizable energy (AME) was determined by the method
described by Farrell (1978).

Amino acid analyses

Amino acid analyses were performed with a conventional (Beckman

Spinco Model 120C) or a fully automated amino acid analyzer (Beckman
Spinco Model 121MB) equipped with a Module control (Autolab Spectra-
Physics Gmbh, 61 Darmstadt, F.R.G.) and a companion Autolab system
AA (Beckman methodology bulletins AA-TB-001 to AA-TB-014) for com-
puting peak concentrations. Data processing and precise quantitation of
protein content in fish meal hydrolysates was carried o u t by the method
described by Horstmann (1979).
Triplicate FM samples (0.5 g) were hydrolyzed in Pyrex test tubes (18
× 150 mm) under reduced pressure (< 10 /~m of mercury) with 15 ml of
triple glass-distilled constant-boiling HC1 (6.0 M) at l l 0 ° C for 24, 48, 72
and 96 h (Zarkadas, 1975, 1978). The small amounts of insoluble materials
formed during acid hydrolysis and the fat plug were removed by filtration
(0.22 p m Millipore microfilter). Amino acid analyses of individual filtrates
dissolved in 0.2 M sodium citrate buffer (pH 2.2) were performed in dup-
licate by methods previously described (Moore and Stein, 1963; Zarkadas,
1978, 1979). Similarly, methionine and cysteine were determined in separate
samples (0.1 g) as their oxidation products by the performic acid procedure
of Moore (1963) as described previously (Hidiroglou and Zarkadas, 1976).
4-Hydroxyproline was also determined separately from concentrated hydro-
lysates (equivalent to 0.1 mg protein per column) by the modified method
of Piez and Morris (1960) using a single column (28 X 0.6 cm) packed with
Dionex type DC-6A resin. This column was eluted b y a single buffer contain-
ing 0.20 M sodium citrate buffer, thiodiglycol (10 ml 1-'), isopropanol
(20 ml 1-~), octanoic acid (0.1 ml 1-1) and phenol (1.0 ml 1-') which was
adjusted to pH 2.85 + 0.01 at 25°C. In this system, 4-hydroxyproline and
aspartic acid were completely separated and emerged from the column at
28.5 and 32.7 min, respectively. Recoveries of 4-hydroxyproline were cal-
culated relative to alanine, which elutes at 68.5 min.
Tryptophan in the FM samples (0.1 g) was determined separately after
alkaline hydrolysis (Hugli and Moore, 1972) by an improved chromato-
graphic procedure (Zarkadas et al., 1982). Samples were eluted from a
25 X 0.9 cm column of Dionex DC-6A resin amino acid analyzer (Beckman
model 120C) with 0.21 m sodium citrate buffer (pH 5.40) at 51°C and a
 295

flow rate of 50 ml h - ' (827 kN m-2). Elution time for tryptophan was
88.4 min. The determination of all methylated basic amino acids, the dia-
stereoisomers of 5-hydroxylysine and related c o m p o u n d s were carried o u t
with concentrated hydrolysates (equivalent to 1--5 mg protein) by the
accelerated single-microcolumn (50 X 0.28 cm) system described previously
(Zarkadas, 1979), so that peaks adequate for these components could be
obtained.

Statistical analysis

Data processing and linear regression analysis of the results were carried
o u t by a F O R T R A N c o m p u t e r program developed for this purpose.
Analysis of variance, conducted on the amino acid data for a completely
randomized design, was carried o u t by the SAS (Statistical Analysis System)
general linear m o d e l procedure (SAS, 1982). Differences among sample
means were also tested for significance using Duncan's multiple-range test
(Duncan, 1955).

RESULTS AND DISCUSSION

The proximate and ~lemental composition of Atlantic white FM made

from different species (40% cod, 30% haddock, 8% pollack, 10% sole and
12% red fish) and containing either untreated (FMR) or enzymatically
digested (FMED) stickwater solids is given in Table I. Data obtained by
other investigators for regular white and herring-type FM (quoted from
Barlow and Windsor, 1983) are also included in Table I for comparison.
The crude protein c o n t e n t of FMED (64.2%) was comparable to that of
FMR (61.6%) and FM (65.8%), but is lower than that of herring-type meal.
Since a portion of the Kjeldahl-N in all animal tissues occurs in nitrogenous
c o m p o u n d s other than fish proteins, including purines, pyrimidines, vita-
mins, creatine, etc. (Natl. Acad. Sci. U.S., N.R.C., 1963), the protein con-
tent of both samples was also calculated from their amino acid composition.
From the mean amino acid nitrogen content given in Table II, a new protein
conversion factor o f 5.20 was calculated for white fishmeal. When the
Kjeldahl nitrogen was multiplied by 5.20 (Table I), the protein content of
FMED averaged 534.1 g kg-1 DM compared to 641.9 g kg-' DM reported in
Table I. Similarly, the protein c o n t e n t of FMR averaged 512.5 g kg-1 DM.
Another method used for calculating the protein content of these fish-
meal samples is the procedure described by Horstmann (1979). This m e t h o d
is also based upon knowledge o f the amino acid composition of the protein
or protein mixture and yields accurate estimates of the absolute amount of
protein present. According to this approach, the protein mass of a biologi-
cal sample is calculated by summing the weights of the amino acid residues
of which the proteins are c o m p o s e d and taking into consideration their dif-
ferent contributions to the total weight, as described previously (Nguyen
296
TABLE I

A c o m p a r i s o n ( m e a n values S.D.) o f the p r o x i m a t e and mineral c o m p o s i t i o n (g k g - ' ) ,

a n d a p p a r e n t m e t a b o l i z a b l e energy values (MJ k g - ' ) o f w h i t e fish meals o b t a i n i n g e n z y m e
digested ( F M E D ) and u n t r e a t e d ( F M R ) s t i c k w a t e r solids with h e r r i n g - t y p e meal

No. o f White fish m e a l ' Herring-type

determin- meal 2
ations FMED FMR FM ~

Moisture 12 64 ± 1 53 ± 0.4 70 ± 6 77 ± 13
Total n i t r o g e n 12 103 ± 2 98 ± 2.4
Crude p r o t e i n
(N × 6.25) 642 ± 12 6 1 6 ± 15 658 ± 17 720 ± 20
(N × 5.20) 4 534 512
Totallipid 12 60 t 0.6 34 ± 0.4 34 ± 8 97 ± 16
Total ash 12 228 + 5.8 239 + 2.2 200 101 ± 11
Minerals
Calcium 12 67.0 ± 0.4 61.7 -+ 0.3 80 19.5 +- 5.3
Phosphorus 12 37.5 ± 0.9 33.9 ± 0.0 48 15.0 -+ 3.5
Magnesium 12 1.9 +- 0.0 2.0 ± 0.2 1.5 1.1 ± 0. i
Potassium 12 9.9 + 0. I 9.3 ± 0.i 9.0 12.0 _+ 1.6
Iron 12 0.3 ± 0.0 0. I ± 0.0 0.3 0.1 ± 0.0
Manganese 12 0.02 ± 0.0 0.01 ± 0.0 0.0 0.03 ± 0.0
Zinc 12 0.05 ± 0.0 0.05 ± O.O 0.1 0.1 ,_ O.l
Sodium 12 15.9 .+ 0.1 16.6 ± 0.1 7.7 4.2 ± 1.9
Apparent metabolizable
energy (MJ k g - ' ) 14.6 : 0.0 13.9 +_ 0.2

Mean values and s t a n d a r d deviations (SD) for three samples.

2 F r o m Barlow and W i n d s o r (1983).
Fish meal c o n t a i n i n g no s t i c k w a t e r solids.
4Present study.

et al., 1986). From the weight equivalent (WE) and F constants given in
Table II, the protein content of FMED (49.9%) and FMR (51.7%) samples
were determined by the amino acid analysis procedure described by Horst-
mann (1979). It was found that the crude protein contents of FMED and
FMR estimated by the Kjeldahl nitrogen m e t h o d (N × 6.25) were higher
than their true protein c o n t e n t as determined b y quantitative amino acid
analysis (Table II). These data suggest that a substantial quantity of Kjel-
dahl nitrogen is derived from other nitrogenous c o m p o u n d s present in FM
samples.
FMED, however, has a higher content of lipid material (6.0%) compared
with FMR (3.4%) and is more comparable to South African white FM
(6.3%) and Norwegian herring meal (7.5%). Gunstone et al. (1978) have
shown that stabilized white FM contains high levels of long-chain (C20 and
greater) and polyunsaturated fatty acids which are essential for optimum
chick growth, reproduction and egg production (Menge et al., 1971; Engster
et al., 1975). The higher apparent metabolizable energy of FMED (14.55
MJ kg--') compared with that of FMR (13.96 MJ k g - ' ) can most likely be
attributed to the higher lipid and protein contents of this product. The
 297

T A B L E II

T h e a m i n o acid c o m p o s i t i o n o f w h i t e fish m e a l c o n t a i n i n g u n t r e a t e d ( F M R ) a n d e n z y m e
d i g e s t e d s t i c k w a t e r solids ( F M E D ) (g kg -~ p r o t e i n )

A m i n o acid FMED FMR

M e a n ~ ± S.E.M. C.V. M e a n ~ ± S.E.M. C.V.

A s p a r t i c acid 1 0 2 . 9 ± 0.3 0.62 1 0 2 . 4 ± 0.7 1,40

Threonine 41.5 -+ 0.2 0.89 41.8 ± 0.9 4.36
Serine 53.0 ± 0.2 0.86 54.2 ± 0.8 2.49
G l u t a m i c acid 1 4 7 . 9 ± 0.4 0.49 145.1 ± 1.4 1.95
Proline 57.1 ± 0.6 2.11 63.3 +- 1.5 4.57
Glycine 99.1 ± 0.4 0.86 1 1 0 . 9 ~ 0.8 1.43
Alan±he 66.2 ± 0.2 0.56 69.1 ± 0.6 1.67
Cysteine 13.2 ± 0.0 0.00 12.3 -+ 0.1 1.87
Valine 4 7 . 4 -+ 0.7 2.93 4 6 . 3 ± 0.7 2.96
Methionine 32.6 -+ 0.8 4.73 31.1 +- 0.9 6.30
Isoleucine 4 1 . 4 ± 0.4 1.77 39.1 ± 0.3 1.60
Leucine 70.5 +- 0.5 1.34 68.8 + 0.4 1.17
Tyros±he 31.1 ± 0.4 2.28 31.0 ± 0.9 6.29
Phenylalanine 38.1 ± 0.6 3.25 37.6 ± 0.3 1.88
Lysine 8 5 . 0 ± 0.5 1.15 79.2 ± 0.9 2.31
Histidine 21.9 ± 0.1 1.25 20.8 ± 0.4 3.71
Arginine 7 3 . 2 ± 0.5 1.38 75.1 ± 1.0 2.69
Tryptophan 7.7 ± 0.0 0.55 8 . 3 ± 0.0 1.52
4-Hydroxyproline 23.9 +- 0.0 0.00 20.3 ± 0.1 2.72
5-Hydroxylysine 4.8 ± 0.5 2.55 4.6 -_ 0.0 1.55
Nn-methylhistidine 1.5 z 0.0 1.66 0.4 ± 0.0 1.46
N~-methylhistidine 0.3 -+ 0.0 1.79 0.6 ± 0.0 2.66
T o t a l a m i n o acids 1060.1 ± 1062.5
Ammonia 15.9 +- 0.4 19.7 ± 0.8
Total nitrogen 189.7 194.9
P r o t e i n (g kg -~ D.M.) 498.9 517.3
Pro (40H) 0.29 0.24
(Pro + P r o ( 4 0 H ) )
Lys ( 5 0 H ) 0.05 0.05
(Lys + L y s ( 5 0 H ) )
W e i g h t e q u i v a l e n t = W E (~g)2 0.1039 0.1026
C o n v e r s i o n f a c t o r = F (ag)2 0.1032 0.1032

~Mean values a n d s t a n d a r d error o f m e a s u r e m e n t s (S.E.M.) for 24 d e t e r m i n a t i o n s (3

s a m p l e s ) ; C.V. = c o e f f i c i e n t o f v a r i a t i o n ; D.M. = d r y m a t t e r .
2 T h e WE a n d F c o n s t a n t s were c a l c u l a t e d f r o m t h e a m i n o acid c o m p o s i t i o n f o u n d in
t h e fish meal h y d r o l y s a t e s ( a f t e r H o r s t m a n n , 1 9 7 9 ; N g u y e n et al., 1 9 8 6 ) .

values obtained for essential minerals in FMED and FMR are comparable
to those shown in Table I for regular white FM and are considerably higher
than the values reported for herring-type m e a l b y K i f e r e t al. ( 1 9 6 9 ) . S i m i -
larly, the total ash content o f w h i t e F M ( 2 0 % ) is c o m p a r e d to the values
reported f o r F M E D a n d F M R i n T a b l e I. T h e h i g h e r l e v e l o f a s h i n w h i t e
FM (20.0--24.0%) compared with that of herring meal (10.14%) apparently
reflects their higher contents of calcium and phosphorus.
 ~D
Gt~

T A B L E III

C o m p a r i s o n o f t h e a m i n o acid c o m p o s i t i o n (g p e r 16 g N) o f w h i t e fish m e a l c o n t a i n i n g e n z y m e - d i g e s t e d ( F M E D )
a n d u n t r e a t e d ( F M R ) s t i c k w a t e r solids w i t h c o d b o n e g e l a t i n a n d h e r r i n g - t y p e m e a l

A m i n o acid W h i t e fish m e a l s Cod White ~ Herring- ~

collagen fish m e a l type meal
FMED' FMR ' P> F (gelatin)

Pheuylalanine 3.19 ± 0.10 3.07 ± 0.06 N.S. 1.61 3.29 3.91 ± 0 . 1 7

Tyrosine 2.60 ± 0.06 2.53 ± 0.16 N.S. 0.51 2.60 3.13 ± 0.22
Histidine 1.80 ± 0.02 1.70 ± 0.06 * 0.98 2.01 2.41 ± 0 . 3 1
Isoleucine 3.44 ± 0.06 3.18 ± 0.05 ** 1.27 3.70 4.49:0.27
Leucine 5.90 ± 0.08 5.61 ± 0.06 * 2.51 6.48 7.50 ~ 0 . 3 6
Methionine 2.73 ± 0.13 2.53 ± 0.16 N.S. 1.77 2.60 2 . 8 6 ~0.21
Cysteine 1.11 ± 0.00 1.00 ± 0.00 N.S. n.a. 0.93 0.97 , 0.12
Valine 3.97 ± 0.11 3.77 ± 0.11 N.S. 1.75 4.47 5 . 4 1 _~0 . 3 0
Arginine 6.12 ± 0.10 6.12 ~ 0.16 N.S. 7.30 6.37 5.84 ±0.62
Lysine 7.12 ± 0.08 6.46 ± 0.14 ** 2.88 6.90 7 . 7 3 -+0.52
Threonine 3.48 ± 0.03 3.40 ± 0.15 N.S. 2.32 3.85 4.26 ±0.33
Tryptophan 0.65 ± 0.002 0.68 ± 0.003 N.S. n.a. 0.94 1.15 ± 0 . 1 0
Total essential a m i n o
acids (rag g - ' N ) 2136.89 2014.45 1439.97 2588.25
T A B L E III (continued)

Aspartic acid 8.62 ~ 0.05 8 . 3 5 -+ 0 . 1 1 N.S. 5.74 8.54 9.10 ± 0.48

Glutamicacid 12.38 -+ 0 . 0 6 1 1 . 8 3 -+ 0 . 2 3 N.S. 8.95 12.79 12.77 -+0.68
Serine 4.44 -+ 0 . 0 4 4 . 4 2 +- 0.11 N.S. 5.85 4.75 3.82 -+0.22
Glycine 8.29 -+ 0 . 0 7 9.03 ± 0.13 ** 19.19 9.92 5.97 ±0.37
Alanine 5.54 -+ 0 . 0 3 5 . 6 3 -,. 0 . 0 9 ** 7.30 6.31 6.25 ~:0.21
Proline 4.78 + 0.10 5.16 ± 0.24 ** 9.35 5.34 4.15 ±0.23
4-Hydroxyproline 2.01 -+ 0 . 0 0 1 . 6 6 +_ 0 . 0 0 N.S. 6.38 n.a. n.a.
5-Hydroxyhistidine 0.38 ± 0.009 0 . 3 8 -+ 0 . 0 0 9 N.S. 1.14 n.a. n.a.
N~-methylhistidine 0.029 ± 0.001 0 . 0 4 7 -+ 0 . 0 0 1 N.S. n.a. n.a. n.a.
Total amino acids 89.24 84.05 86.8 91.79 91.72
F (ug) 0.103919 0.102563 n.a. n.a. n.a.
Essential amino
acid i n d e x 4 64.07 61.44 n.a. 69.7 78.53
Protein score 60.56 67.44 n.a. 82.32 83.96
C o l l a g e n (%)s 35.51 33.33

1 M e a n v a l u e s a n d s t a n d a r d d e v i a t i o n s f o r 24 d e t e r m i n a t i o n s (3 s a m p l e s ) ; P > F p r o b a b i l i t y o f a l a r g e r v a l u e o f F , Sig-
n i f i c a n c e d e n o t e d b y : * = P < 0 . 0 5 , * * = P < 0 . 0 1 , N.S. = n o t s i g n i f i c a n t ; n.a. = n o t a v a i l a b l e .
2From Eastoe (1957).
3From Barlow and Windsor (1983).
4From Oser (1951)and Block and Mitchel (1946).
s A m i n o a c i d n i t r o g e n as % t o t a l p r o t e i n N.

t'O
',D
¢.O
 ABSORBANCE
i r w
c
~a
c
g~
3~
f
E o
5¸
v
m~
m m ~,
mr- m I
O~ O
~o ~
mC
00~
 301

The amino acid composition of FMED and FMR expressed as g per kg of

p ro t ein are summarized in Table II and com pared with those o f previous
investigators in Table III. The data represents the average values o f duplicate
d e t e r m i n a t i o n s obtained f r om the triplicate 24-, 48-, 72- and 96-h hydro-
lysates. The means and standard error of the estimates r e p o r t e d for serine,
t h reo n in e and tyrosine (Table II) represent the regression values ext rapol at ed
to zero time o f hydrolysis (Rees, 1946; Sanger and T h o m p s o n , 1963). The
values for valine, leucine, isoleucine and phenylalanine are averages o f data
from 48, 72 and 96 h o f hydrolysis (Blackburn, 1978). The results presented
in Tables II and Ill indicate a narrow range in amino acid composition of
FMR and FMED, as indicated by bot h the m a x i m u m and m i n i m u m values
and the coefficients of variation. Similarly, isoleucine, valine and t r y p t o p h a n
showed narrow ranges o f values and corresponding low coefficients of
variability. A wider range was f ound f or m c t h i o n i n e and tyrosine, although
their coefficients of variability are below the significant level of 5%. FMED
was shown to contain a higher level o f 4 - h y d r o x y p r o l i n e c o m p a r e d with
t h a t o f FMR. Fish collagens are low in 4 - h y d r o x y p r o l i n e c o n t e n t and as
shown in Table III (Eastoe, 1957), 4 - h y d r o x y p r o l i n e is especially low in cod
collagen (53--59 residues per 1000), a recently evolved actinopterygian fish
which inhabits cold waters (Eastoe, 1967). T he low 4 - h y d r o x y p r o l i n e
c o n t e n t o f fish collagens is accompanied by higher levels of serine and
threonine. The proline c o n t e n t of fish collagens is also lower than in mam-
mals. The glycine c o n t e n t is uni f or m l y high in bot h FM samples, while the
balance o f acidic and basic groups appears to be similar in FMED and FMR.
Similarly, Tables II and III list the amounts (g per kg protein) of 5-hydroxy-
lysine, N ~- and N'-methylhistidines present in the acid hydrolysates of
FMED and FMR. The chromatograms illustrated in Fig. I are typical of the
separations o b tained by this m e t h o d of all the unique basic amino acids
likely to be e n c o u n t e r e d in biological systems. Figures l b and l c of both
FMED and FMR reveal the c o m p l e t e separation o f the diastereoisomers
o f 5 - h y d r o x y l y s i n e and small amounts of the m e t h y l a t e d basic amino acids
along with two major (Nos. 17 and 18) and fifteen minor (Nos. 2 - 1 6 ) , as
y e t unidentified, stable com pone nt s . The values obtained for 5-hydroxy-
lysine in FMED are comparable to those reported for FMR and represent
the sum of the values obtained for the diastereoisomers after epimerization
(Zarkadas, 1975). Fish collagens contain slightly more 5-hydroxylysine

Fig. 1. Chromatographic separations of all methylated basic amino acids, the diastereo-
isomers of 5-hydroxylysine and related compounds in white fish meal hydrolysates.
(A) Separation of a synthetic amino acid calibration mixture; (B) A typical chromato-
graphic separation of a 96-h hydrolysate of white fish meal with untreated stickwater
solids (FMR) and (C) Analysis of a 96-h white fish meal hydrolysate containing enzyme-
digested stickwater solids (FMED). Lys (50H), 5-hydroxylysine; aLys (50H), allo-5-
hydroxylysine; Orn, ornithine; Lys (6Me), N~-methyllysine; Lys (6Me2), N~-dimethyl -
lysine; Lys (6Me3), N6-trimethylysine; His (~rMe), Nn-methylhistidine; His (-Me), N ~-
methylhistidine.
302

than mammalian collagens, the value being 6--8 residues per 1000 amino
acids. Eastoe (1967) found that the total number of h y d r o x y groups in the
side chains of fish collagens remains approximately constant at the mam-
malian level (152--162 residues per 1000), despite the reduction in 4-
hydroxyproline. Cod collagen has a particularly high serine level (69 resi-
dues per 1000), as shown in Table III. It should also be noted that the mean
values obtained in the present study for N'-methylhistidine were higher in
FMR compared to those found in FMED (Tables II -- III). These results
are in agreement with those values reported for cod muscle by Rangeley
and Lawrie (1977).
The amino acid profiles (g per 16 g N) of FMED and FMR, as presented
in Table IIi, are very similar. With three exceptions (glycine, alanine and
proline), FMED contained slightly higher levels of each individual amino
acid than FMR and significantly higher levels of the essential amino acids
(EAA) lysine and isoleucine (P < 0.01), histidine and leucine (P < 0.05).
These results differ from the values quoted for white fish and herring-type
meals in Table III (Barlow and Windsor, 1983). Some of these differences
may arise from the fact that the total amino acid composition (g per 16 g
N) of fish meals was based on the Kjeldahl nitrogen (N X 6.25); (Crainpton,
1956) rather than their actual nitrogen values determined by amino acid
analysis. In the present study, the protein content values reported for
FMED and FMR are 13--15% lower than those compiled by Miller (1970).
FMED has a higher content of total EAA (2136.9 mg g-' N) compared with
t h a t of FMR (2014.5 mg g-' N). Herring-type meal, however, has a fairly
high content of total EAA (2588.3 ~g g-' N) and protein score (84%)
compared with the values calculated for FMED (60.6%). This can most
likely be attributed to differences in their t r y p t o p h a n contents (Table III).
The protein quality of the white fish meal (FMED and FMR) was also
assessed by the direct determination of its connective tissue content and
the results are given in Table III. As may be seen in Fig. 1, an accurate
determination of 5-hydroxylysine content can be made from the sum of
the values obtained for its diastereoisomers after epimerization in 6M HC1
at l l 0 ° C for 96 h (Zarkadas, 1975). In this approach, collagen is determined
from the amounts of 5-hydroxylysine f o u n d in white fish meal as follows:
A m o u n t of 5-hydroxylysine-N as % total fish protein-N
× I00
A m o u n t of the same amino acid-N as % total fish collagen-N
The results obtained from the 5-hydroxylysine contents of FMET and
FMR averaged 35.3 and 33.3% collagen, respectively (Table III). Although
collagen has been found in most of the invertebrate phyla (Eastoe, 1967),
only limited biochemical studies have been done on the various collagens
found in the skin (Types I and III), bone (Type II) or the peri- and endo-
mycial fish muscle collagens (Types I, III--V). Eastoe (1957, 1967) provided
some information on the h y d r o x y amino acid content of cod skin gelatin,
 303

a n d his r e s u l t s , s u m m a r i z e d in T a b l e I I I , h a v e b e e n u s e d t o c a l c u l a t e t h e
c o l l a g e n c o n t e n t s o f F M E D a n d F M R . T h e i n c l u s i o n o f w h i t e fish m e a l c o n -
t a i n i n g 3 3 - - 3 6 % c o l l a g e n in b o t h s t a r t e r a n d f i n i s h e r c h i c k d i e t s at t h e r a t e
of 5--10% produced the same or better complementary effects (compared
t o c e r e a l p r o t e i n s ) f o r p r o m o t i n g g r o w t h in b r o i l e r c h i c k e n s ( H u l a n e t al.,
1986).

ACKNOWLEDGEMENTS

T h e a u t h o r s a c k n o w l e d g e N a t i o n a l Sea P r o d u c t s L t d . , L u n e n b u r g , N o v a
S c o t i a f o r p r o v i d i n g t h e w h i t e fish m e a l s u s e d in t h e s e s t u d i e s ; A n a l y t i c a l
C h e m i s t r y Services o f C B R I , A g r i c u l t u r e C a n a d a , f o r p r o x i m a t e a n d ele-
m e n t a l a n a l y s e s a n d f o r use o f t h e a m i n o a c i d a n a l y z e r s , J o h n E m e r y , D.
T u t t e a n d D. N a s h f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e in a m i n o acid
a n a l y s i s . T h e f i n a n c i a l s u p p o r t o f A g r i c u l t u r e C a n a d a in t h e f o r m o f a D S S
c o n t r a c t ( O S V 8 1 - 0 0 5 2 3 ) w i t h M c G i l l U n i v e r s i t y is also g r a t e f u l l y a c k n o w -
ledged.

REFERENCES

Adler-Nissen, J., 1976. Enzymatic hydrolysis of proteins for increased solubility. J.

Agric. Food Chem., 24: 1090--1093.
A1 Hassan, S., McRae, K.B., Hulan, H.W. and Proudfoot, F.G., 1981. Economic analysis
on rapeseed meal and fishmeal as protein supplements in chicken broiler diets. Can.
Farm Econ., 16: 9--15.
Archer, M.C., Ragnarsson, J.O., Tannenbaum, S.R. and Wang, D.I.C., 1973. Enzymatic
solubilization of an insoluble substrate, fish protein concentrate: Process and kinetic
considerations. Biotechnol. Bioeng. 15: 181--196.
Association of Official Analytical Chemists, 1984. Official Methods of Analysis, 14th
edn., The Association, Washington, DC, pp. 16, 20--26, 152 and 249.
Barlow, S.M. and Windsor, M.L., 1983. Fishery by-products. In: M. Rechcigl, Jr. (Editor),
CRC Handbook of Nutritional Supplements, Vol. II. CRC Press, Boca Raton, FL,
pp. 253--272.
Blackburn, S., 1978. Sample preparation and hydrolytic methods. In: S. Blackburn
(Editor), Amino Acid Determination: Methods and Techniques. Dekker, New York,
pp. 7--45.
Block, R.J. and Mitchell, H.H., 1946. The correlation of the amino acid composition of
proteins with their nutritive value. Nutr. Abstr. Rev., 16: 249--278.
Cheftel, C., Ahern, M., Wang, D.I.C. and Tannenbaum, S.R., 1971. Enzymatic solubili-
zation of fish protein concentrate: Batch studies applicable to continuous enzyme
recycling processes. J. Agr. Food Chem., 19: 155--161.
Crampton, E.W., 1956. Applied Animal Nutrition. Freeman, San Francisco, CA, pp. 31--
34 and 47--50.
Duncan, D.B., 1955. The new multiple range and multiple F tests. Biometrics, 11: 1-42.
Eastoe, J.E., 1957. The amino acid composition of mammalian collagen and gelatin.
Biochem. J., 61: 589---600.
Eastoe, J.E., 1967. Composition of collagen and allied products. In: G.N. Ramachan-
dran (Editor), Treatise on Collagen. Vol. 1. Academic Press, New York, pp. 1-72.
Engster, H.M., Caren, J.R. and Foss, D.C., 1975. Effect of herring oil on body weight,
comb size and gonadal development in the chick. Poult. Sci., 54: 2118--2121.
304

FAO, 1979. Yearbook of Fishery Statistics. Vol. 47. Food and Agricultural Organization,
Rome, Italy 1--1 pp. 232.
Farrell, D.J., 1978. Rapid determination of metabolizable energy of foods using cocke-
rels. Br. Poult. Sci., 19: 303--308.
Gunstone, F.D., Wijesundera, R.C. and Serimgeour, C.M., 1978. The component acids
of lipids from marine and freshwater species with special reference to furan-containing
acids. J. Sci. Food Agric., 19: 539--550.
Hidiroglou, M. and Zarkadas, C.G., 1976. The effects of selenium on the metabolism of
methionine in sheep. Can. J. Physiol. Pharmacol., 54: 336--346.
Horstmann, H.J., 1979. A precise method for the quantitation of proteins taking into
account their amino acid composition. Anal. Biochem., 9 6 : 1 3 0 - - 1 3 8 .
IIugli, T.E. and Moore, S., 1972. Determination of the tryptophan content of proteins
by ion-exchange chromatography of alkaline hydrolysates. J. Biol. Chem., 247:
2828--2834.
Hulan, H.W. and Proudfoot, F.G., 1981. Replacement of soybean meal in chicken
broiler diets by rapeseed meal and fishmeal as complementary protein sources. Can. J.
Anim. Sci., 61: 999--1004.
IIulan, H.W., Proudfoot, F.G. and Zarkadas, C.G., 1986. The nutritive value and quality
of white fishmeal containing enzyme digested and untreated stickwater solids for
broiler chickens. Anita. Feed Sci. Technol., in press.
Jacobsen, F. and Rasmussen, O.L., 1983. Energy savings through enzymatic treatment
of stickwater in the fishmeal industry. Proc. Int. Assoc. Fishmeal Manuf., 7--11
April, at Lisbon, Portugal, pp. 1--20.
Kifer, R.R., Payne, W.L., Miller, D. and Ambrose, M.E., 1969. The nutritive content of
menhaden (Brevoorl& tyrannus and pelronus) fishmeal evaluated by chemical meth-
ods. Feedstuffs, 40(20): 36--37.
Menge, H., Miller, E.C. and Denton, C.A., 1971. Effect of an essential fatty acid deficient
diet on the reproductive performance of chickens. Poult. Sci., 42: 1291.
Moore, S., 1963. On the determination of cystine as cysteic acid. J. Biol. Chem., 238:
235--237.
Moore, S., and Stein, W.H., 1963. Chromatographic determination of amino acids by
the use of automatic recording equipment. In: S.P. Colowick and N.D. Kaplan (Edi-
tors), Methods hi Enzymology. Vol. VI. Academic Press, New York, pp. 8:19--831.
National Academy o f Sciences, National Research Council, 1963. Evaluation o f Pro-
tein Quality. National Academy of Sciences, Washington, DC, Publ. 1100, pp. 1--11.
Nguyen, Q., Fanous, M.A., Kamm, L.H., Khalili, A.D., Schuepp, P.H. and Zarkadas,
C.G., 1986. A comparison of the amino acid composition of two commercial porcine
skins (rind). J. Agric. Food Chem., in press.
Oser, B.L., 1951. Method for integrating essential amino acid content in the nutritional
evaluation of Protein. J. Am. Diet. Assoc., 27: 396--402.
Piez, K.A. and Morris, L., 1960. A modified procedure for the automatic analysis of
amino acids. Anal. Biochem., 1: 187--201.
Rangeley, W.R.D. and Lawrie, R.A., 1977. Methylated amino acids as indices in meat
products. II. Further examination of protein sources and the practical application
of methylamino acid titres~in predicting meat content. J. Food Technol., 12: 9--26.
Rees, M.W., 1946. The estimation o f threonine and serine in proteins. Biochem. J., 40:
632--640.
Sanger, F. and Thompson, E.O.P., 1963. Halogenation of tyrosine during acid hydroly-
sis. Biochem. Biophys. Acta, 71: 468--471.
SAS (Statistical Analysis System) User's Guide. Basics. 1982. Ed. SAS Institute, Cary,
NC, pp. 139--199.
Zarkadas, C.G., 1975. A simple chromatographic method for the determination of the
methylated basic amino acids in proteins. Can. J. Biochem., 53: 96--101.
 305

Zarkadas, C.G., 1978. A rapid chromatographic method for the determination of w-N-
methylarginines in protein and muscle tissues. Can. J. Biochem., 56: 952--957.
Zarkadas, C.G., 1979. Accelerated methods for determining basic amino acids in proteins
and muscle tissues and stable crosslinks in elastin and collagens. J. Appl. Biochem.,
1: 148--163.
Zarkadas, C.G., Hulan, H.W. and Proudfoot, F.G., 1982. A comparison of the amino
acid composition of two commercial oat groats. Cereal Chem., 59: 323--327.