1. Characteristics of protein fractions generated from hydrolysed cod
(Gadus morhua) by-products
Rasa Sˇlizˇyte˙a,b
, Egidijus Dauksˇasa
, Eva Falcha,b
, Ivar Storrøa
, Turid Rustadb,*
a
SINTEF Fisheries and Aquaculture, Processing, N-7465 Trondheim, Norway
b
Department of Biotechnology, NTNU, N-7491, Trondheim, Norway
Received 9 June 2004; accepted 1 July 2004
Abstract
The aim of this work was to study how raw material mixtures combined from different separated cod (Gadus morhua) by-products
influenced the composition of the substrate for hydrolysis. The influence of using an endo-peptidase (Flavourzyme) or exo-peptidase
(Neutrase) and the amount of added water on yield, nutritional, physicochemical and functional properties of the hydrolysis products was also
studied. All freeze-dried fish protein hydrolysates (FPH) powders had a light yellow colour and contained 75–92% protein. The dried
insoluble material, sludge, was a grey, greasy powder containing 55–70% protein. Degree of hydrolysis was 18.5–33.7% for FPH and 4.3–
10.9% for sludge. Different ways of combining fish by-products lead to different end products with different properties after hydrolysis. Raw
material containing the highest amount of lipids gave the lowest percentage of solubilised proteins. Addition of water before hydrolysis was
more important than the type of enzyme used for yield, biochemical and functional properties of FPH and sludge. Protein efficiency ratio
(PER) of sludge was generally 1.5 times higher than PER value of FPH. Sludge made up a large part after hydrolysis compared to fish protein
hydrolysate, contained a significant part of the total protein and had good functional properties, in some cases even better than the FPH, which
is often considered the main product of protein hydrolysis.
# 2004 Elsevier Ltd. All rights reserved.
Keywords: Cod; By-products; Enzymatic hydrolysis; Functionality; FPH; Sludge
1. Introduction
Optimal utilisation of fishery by-products is becoming
increasingly important to provide more fish raw material for
various purposes. Seafood processing discards and under-
utilised species of fish serve as sources of raw material for
preparation of protein-based food and feed ingredients [1].
Enzymatic hydrolysis of fish by-products is one of the
approaches for effective protein recovery from the fishery
industry and can be applied to improve and upgrade the
functional and nutritional properties of proteins. Preparation
of protein hydrolysates from fish by-products has received
increasing attention in recent years. Many studies have been
done on the evaluation of the conditions for hydrolysis and
the functional properties of fish protein hydrolysate (FPH)
based on whole fish, fish fillet or muscle. In the most recent
papers dealing with fish by-products: [2–7], neither the
influence of added water nor the amount of added enzyme
was studied. Both of these process parameters are of
economical interest in the hydrolysis process [8]. A
combination of different by-products as substrate for
hydrolysis and impact on the hydrolysis products should
also have scientific and industrial interest.
The nutritive value of a protein depends primarily on its
capacity to satisfy the needs for nitrogen and the essential
amino acids. Since proteins differ in nutritional value,
evaluation of this aspect is important for protein containing
components. A widely used method to evaluate protein
quality is the protein efficiency ratio (PER) test, which
measures protein quality by feeding a diet containing 10% of
the test protein to rats and measuring their weight gain. This
is an expensive and time consuming method. Alsmeyer et al.
[9] showed that the relative quantities of the various amino
www.elsevier.com/locate/procbio
Process Biochemistry 40 (2005) 2021–2033
* Corresponding author. Fax: +47 73 59 3337.
E-mail address: turid.rustad@biotech.ntnu.no (T. Rustad).
0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.07.016

2. acids in the food could be used as reliable estimators of
actual protein quality. Nevertheless, PER evaluation has
some disadvantages [10], the calculated PER values provide
only a rough estimate of protein quality. Comparison of PER
values for cod flesh and cod by-products shows that fish by-
products has a high percentage of essential amino acids and
can be used to produce nutritious products [11]. In addition,
the work of Shahidi et al. [12] on capelin confirmed the
assumption that amino acid profiles of protein hydrolysates
are generally similar to that of raw material except for the
sensitive amino acids such as methionine and tryptophan,
which were present in smaller amounts after hydrolysis and
decolorization of hydrolysate by charcoal. However, except
for the deficit of a few amino acids, hydrolysates have a high
nutritional value.
Extensively hydrolysed proteins also have reduced
immunological reactivities and can be used in formulas
for hyper allergic infants [13]. Furthermore, peptides, being
easily absorbed, may be an optimal nitrogen source in sports
nutrition. In addition peptides with a high biological value
are attractive as a general protein supplement to a wide
variety of diets.
Pour-El [14] defined protein functionality as ‘‘any
property of food or food ingredients except its nutritional
ones that affects its utilisation’’. Degree of hydrolysis
(DH), which indicates the percentage of peptide bonds
cleaved [15], is one of the basic parameters that describes
the properties of the hydrolysates and needs to be
controlled during protein hydrolysis. This is essential
because several properties of protein hydrolysates are
closely related to DH. Hydrolysis of peptide bonds causes
several changes such as an increase of amino and carboxyl
groups, which increase solubility. The molecular weight of
the protein decreases and the tertiary structure is destroyed,
affecting the functional properties of protein [16]. The
functional properties of proteins in a food system depend in
part on the water–protein interaction. Water holding
capacity (WHC) refers to the ability of the protein to
absorb water and retain it against gravitational force within
a protein matrix, such as protein gels or beef and fish
muscle [17]. Kristinsson and Rasco [18] pointed out that
some studies showed that FPH also have good water
holding capacity and thus useful properties for certain food
formulations: addition of 1.5% of fish protein hydrolysate
made from salmon reduced water loss after freezing to 1%
compared with 3% for the control. However a relationship
between degree of hydrolysis and water holding capacity
was not observed.
Proteins are often used as surfactants in emulsion-type
processed foods [16]. Proteins have interfacial properties,
which are important for their application as for example
emulsifiers in sausages or protein concentrates in dressings.
Hydrolysates are also water-soluble and surface active
and promote oil-in-water emulsions, due to their hydro-
philic and hydrophobic functional groups [19]. Proteins
adsorb to the surface of the freshly formed oil droplets
during homogenisation and form a protective membrane
that prevents droplets from coalescing [20]. The emulsify-
ing properties of proteins can also be improved by
controlled hydrolysis. According to Adler-Nissen and
Olsen, emulsifying capacity (EC) could be significantly
increased by gentle hydrolysis to a DH of approximately
5% [21]. Extensive hydrolysis results in a drastic loss of
emulsifying properties [13]: although small peptides
diffuse rapidly and absorb at the interface, they are less
efficient in stabilising emulsions because they cannot
unfold and reorient at the surface like a protein [22].
Mahmoud [13] showed that for DH in the range of 25–67%,
the emulsifying activity of the hydrolysates decreases
linearly with increasing DH. Degree of hydrolysis of
protein hydrolysates also has a significant effect on the
stability of emulsions: as DH increases, emulsion stability
decrease substantially. Generally, the molecular weight of
the hydrolysates has a major influence on the emulsifying
properties. Several reports suggested that there is an
optimum molecular size or chain length for peptides to
provide good emulsifying properties [21,23]. Lee et al.
[23] suggested that peptides should have a minimum chain
length of >20 residues to function as good emulsifiers.
Phospholipids also enhance emulsifying properties in the
system.
The ability of FPH to absorb and hold oil is another
important functional property. It influences not only the taste
of the product but is also an important functional
characteristic especially for the meat industry [18]. The
mechanism of fat absorption is attributed mostly to physical
entrapment of the oil and thus, the higher bulk density of the
proteins, the higher fat absorption [24]. Fat binding capacity
also correlates with surface hydrophobicity [18]. On the
other hand, lipid residues retained in dried FPH after
hydrolysis must be lower than 0.5% to reduce development
of rancid taste during storage [25].
Despite all advantages of the hydrolysis process, active
application of enzymes in the processing of marine raw
material is not extensively used [26]. The amount of raw
material being converted into soluble hydrolysate is a factor
especially important for industrial processes [27]. However,
today not much work has been published on the evaluation
and examination of the non-soluble part after hydrolysis.
The applications of the hydrolysis could be increased by
better description of all fractions after hydrolysis and finding
application for the non-soluble fraction.
The aim of this study was to evaluate how raw material
mixtures combined from different separated cod by-
products influenced the composition of the substrate for
hydrolysis. In addition, the effect of different enzymes and
amount of added water on yield, nutritional, physicochem-
ical and functional properties of the hydrolysis products was
studied. We also wanted to pay more attention to the water
non-soluble part after hydrolysis and compare it with FPH
which is commonly regarded as the main product of the
protein hydrolysis.
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–20332022

3. 2. Materials and methods
2.1. Raw material
Twenty five cod (Gadus morhua) caught in the
Trondheim fjord (Norway) in March 2002 were used for
the experiments. The fish (55 Æ 4 cm in length and 2.05 Æ
0.3 kg in weight) were kept on ice overnight, eviscerated
and hand filleted. The different body parts were separated
and stored on ice for about 1–6 h until mincing. All
procedures were done in a cold room (+4 8C). Four
different groups of raw material mixtures were made.
The different fractions were mixed according to the
proportions found in fish (Table 1): viscera (V), viscera
and backbone (V + BB), viscera without the digestive tract
(V-DT), viscera without digestive tract and backbone (V-DT
+ BB).
The samples were minced twice in a manual mincer with
10 mm holes. Endogenous enzymes were inactivated in a
microwave oven at 900 W (Whirlpool) at 95 Æ 5 8C for
5 min. After cooling the mixtures were vacuum packed and
kept at À40 8C.
2.2. Enzymes and other chemicals
Flavourzyme is a fungal protease/peptidase complex
produced by submerged fermentation of a selected strain of
Aspergillus oryzae which has not been genetically modified
and are used for the hydrolysis of proteins under neutral or
slightly acidic conditions. The optimal working conditions
for Flavourzyme 500 L are reported to be at pH 5.0–7.0 with
an optimal temperature around 50 8C. Flavourzyme 500 L
has a declared activity of 500 L APU/g. Neutrase is a
bacterial endoprotease which can be used in most cases
where proteins have to be broken down either moderately or
more extensively to peptides. The optimal working
conditions for Neutrase 0.8 L are reported to be at pH
5.5–7.5 at a temperature of 45–55 8C. Neutrase 0.8 L has a
declared activity of activity 0.8 AU/g. Both enzymes were
produced by Novozymes A/S (Bagsvaerd, Denmark) and
comply with the recommended purity specifications for
food-grade enzymes given by the Joint FAO/WHO Expert
Committee on Food Additives (JECFA) and the Food
Chemicals Codex (FCC) [28,29].
Casein (Merck, No. 2242) and soy protein isolate
(Vaessen-Schoemaker Chemische Industrie B.V.) were used
as reference samples for evaluation of functional properties
of the hydrolysis products. Methanol, chloroform, hexane,
formaldehyde (all from Merck, Darmstad, Germany) were
used for chemical analysis.
2.3. Hydrolysis process
The minced and frozen fractions were thawed in a
microwave oven. Sample (250 g) was mixed with 250 mL
distilled water and the pH value measured (Philips PW
9420 pH meter, Pye Unicam LTD., England; electrode:
Unikan, Type No. 9436-095-84003). Hydrolysis was
performed in a 4 L closed glass vessel stirred with a
marine impeller (150 rpm). The enzymatic hydrolysis was
started when the temperature of the mixture was 50 8C by
adding either 0.1% (by weight of raw material) Flavour-
zyme 500 L or 0.3% Neutrase 0.8 L. The hydrolysis
proceeded for 60 min followed by enzyme inactivation by
microwave heating for 5 min at 90 8C. The hot hydrolysed
mixtures were centrifuged in 1 L batches at 2250 Â g for
30 min. Four fractions were collected: the sludge (non-
water-soluble part) on the bottom, fish protein hydrolysate
(FPH, water-soluble compounds), the oil fraction on top,
and in some samples an emulsion layer was formed
between FPH and oil fraction. The FPH, emulsion and
sludge fractions were freeze-dried. The pH of the mixtures
after hydrolysis and the inactivation of enzymes were
determined. Experiments were performed in duplicate.
Three controls were included in the processing of the
sample ‘viscera with backbone’ (V + BB). (1) Raw material
and water with no enzymes added (NE). (2) Raw material
and addition of Flavourzyme (FlavNW) or (3) Neutrase
(NeuNW), respectively, were done directly to the 250 g of
mince, without the addition of water.
2.4. Chemical analyses
The moisture content of the dried samples was determined
by infrared drying (Mettler LP16 Infrared Dryer). Measure-
mentswereperformedinduplicate.Ashcontent wasestimated
according to AOAC [30]. Measurements were performed in
triplicate. Total N was determined by CHN-S/N elemental
analyser 1106 (Carlo Erba Instruments s.p.a., Milan, Italy)
and crude protein was estimated by multiplying total N by
6.25. These measurements were performed in quadruplicate.
The extraction of total lipid from the samples was performed
according to the method of Bligh and Dyer [31]. Analysis was
performed in duplicate.
2.5. Degree of hydrolysis
The degree of hydrolysis was evaluated as the proportion
(%) of a-amino nitrogen with respect to the total N in the
sample [32]. Analyses were performed in duplicate.
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–2033 2023
Table 1
Gross composition of raw material used for enzymatic hydrolysis of cod by-
products, g/100 g (mean Æ S.D.)
Composition V-DT + BB V + BB V-DT V
Moisture 64.5 Æ 0.1 70.5 Æ 0.5 60.7 Æ 0.1 60.0 Æ 0.0
Lipids 17.9 Æ 0.6 9.6 Æ 0.2 26.9 Æ 1.3 21.0 Æ 0.5
Proteins 15.4 Æ 0.2 16.1 Æ 0.4 12.6 Æ 1.2 14.9 Æ 2.3
Ash 3.6 Æ 1.4 3.6 Æ 0.2 1.7 Æ 0.2 4.4 Æ 0.3
V-DT + BB: viscera without digestive tract and backbone; V + BB: viscera
and backbone; V-DT: viscera without digestive tract; V: viscera.

4. 2.6. Amount and composition of free amino acids
Amount of free amino acids was determined by high-
pressure liquid chromatography (HPLC). Dry powders were
dissolved in 0.05 M phosphate buffer (pH = 7.0) and
centrifuged for 10 min at 7840 Â g. Reversed phase HPLC
by precolumn fluorescence derivatization with o-phthal-
dialdehyde (SIL-9A Auto Injector, LC-9A Liquid Chroma-
tograph, RF-530 Fluorescence HPLC Monitor, all parts from
Shimadzu Corporation, Japan) was performed using a
NovaPak C18 cartridge (Waters, Milford, MA, USA), using
the method of Lindroth and Mopper [33] as modified by
Flynn [34]. Glycine/arginine and methionine/tryptophane
were determined together, as their peaks merged. This
analysis was performed twice on each sample.
2.7. Amount and composition of total amino acids
The amino acid composition of powdered samples was
determined by digestion in 6 M HCl at 105 8C for 22 h [35]
followed by neutralisation of hydrolysates. After dilution
and filtration amount of 16 amino acids was estimated by
HPLC as described earlier. Hydroxyproline was determined
by a colorimetric method [36]. These tests were performed
in duplicate.
2.8. Gel filtration of proteins
Dry powder were diluted in 0.05 M phosphate buffer (pH =
7.0) and centrifuged at 7840 Â g for 10 min. The separation
was performed using a Superdex1 75 HR 10/30 column, the
flow rate 0.3 mL/min. The standards used were: bovine serum
albumin (Mw = 67000), myoglobin (Mw = 17600), cytochrome
c (Mw = 12270), vitamin B12 (Mw = 1355).
2.9. Calculation of protein efficiency ratio (PER)
Protein efficiency ratio (PER) values of FPH and sludge
were calculated using equations developed by Alsmeyer et
al. [9] and Lee et al. [37]:
PERa
= À0.468 + 0.45[LEU] À 0.105[TYR]
PERb
= À1.816 + 0.435[MET] + 0.780[LEU] + 0.211[HIS]
À 0.944[TYR]
PERc
= 0.08084[
P
AA7] À 0.1094,
where
P
AA7 = threonine + valine +methionine + isoleucine
+ leucine + phenylalanine + lysine.
2.10. TLC lipid classes
Lipid classes were determined by thin-layer chromato-
graphy [38]. Lipid classes were separated and detected by an
Iatroscan thin layer chromatography-flame ionisation
detector system (TLC-FID analyser TH-10 MK-IV, Iatron
Laboratories Inc., Tokyo, Japan). Chromarods SIII were first
scanned twice through the Iatroscan FID immediately before
sample application in order to remove possible contaminants
from the rods.
2.11. Water holding capacity (WHC)
FPH powder was added to fish mince for evaluation of the
ability to influence water holding capacity during frozen
storage. FPH powder (5% of minced muscle mass) was
added to fish mince (minced cod fillet, which were kept in
the freezer and defrosted overnight at 4 8C) and stored at
À24 8C for 1 month. Samples were thawed at room
temperature and a low speed centrifugation method was
used for measuring the WHC. Water holding capacity
(WHC) was determined as described by Eide et al. [39] with
the exception that a centrifugal force of 340 g was used
instead of 1500 g. The WHC is expressed as the percentage
of water retained in the mince. The test was performed in
quadruplicate.
2.12. Fat absorption/oil holding capacity
The ability to bind oil was measured according to the
method of Shahidi et al. [12] with some modifications.
Freeze-dried FPH and sludge powders (0.5 g) were mixed
with 10 mL soybean oil. The mixture was kept at room
temperature for 30 min with stirring every 10 min and then
centrifuged for 25 min at 1360 Â g. Free oil was decanted
and the fat absorbed was determined gravimetrically. This
test was performed in duplicate and fat absorption was
calculated as the mass (g) of fat absorbed by 1 g of proteins
in the powders. Fat adhesion to the walls in the tube was
evaluated in an empty tube.
2.13. Emulsifying properties
Emulsification capacity was measured by mixing 5 mL of
soybean oil with 5 mL of a 5% FPH and sludge solution in
water and homogenising (Ultra-Turrax TP 18/10) at
20000 rpm for 90 s. The emulsion was poured into 10 mL
graded tubes and centrifuged at 2400 Â g for 3 min. The
volume of each fraction (oil, emulsion and water) were
determined and emulsification capacity was expressed as
millilitres of emulsified oil per 1 g of FPH [24]. Emulsion
stability was expressed as the percentage of initial emulsion
remaining after a certain time (1 day at room temperature)
and centrifugation at 2400 Â g for 3 min [40]. Tests were
performed in duplicate.
2.14. Statistical analysis
Depending on the methods, the tests were done in
duplicates-sextuples. The programmes Guideline (Camo
ASA, Oslo, Norway), MatLab (MathWorks Inc., USA) and
Microsoft Excel were employed for data processing and
statistical analysis. Significance level was set at 95%.
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–20332024

5. R.Sˇlizˇyte˙etal./ProcessBiochemistry40(2005)2021–20332025
Table 3
Composition of dried FPH and sludge fractions, g/100 g dry matter (mean Æ S.D.)
Composition Fraction V-DT + BB V + BB V-DT V
Flavourzyme Neutrase Flavourzyme Neutrase Control Flavourzyme Neutrase Flavourzyme Neutrase
NE FlavNW NeuNW
Moisture FPH 5.0 Æ 0.0 6.5 Æ 0.6 4.0 Æ 0.0 3.9 Æ 0.1 7.8 Æ 0.1 9.1 Æ 0.1 8.8 Æ 0.2 4.9 Æ 0.0 5.6 Æ 0.0 4.5 Æ 0.6 3.9 Æ 0.1
Sludge 2.0 Æ 0.1 2.3 Æ 0.1 1.9 Æ 0.1 2.0 Æ 0.1 2.2 Æ 0.0 3.0 Æ 0.2 2.8 Æ 0.4 1.9 Æ 0.0 1.4 Æ 0.1 1.3 Æ 0.1 2.0 Æ 0.4
Proteins FPH 79.9 Æ 1.7 87.2 Æ 1.3 85.6 Æ 1.0 87.7 Æ 0.4 84.8 Æ 0.7 89.5 Æ 1.0 91.6 Æ 0.4 75.0 Æ 1.2 78.2 Æ 0.6 76.5 Æ 1.5 83.5 Æ 1.3
Sludge 63.1 Æ 1.4 58.7 Æ 1.7 60.1Æ 2.5 55.5 Æ 2.3 63.5 Æ 1.8 64.8 Æ 5.7 70.0 Æ 2.1 66.3 Æ 1.8 61.4 Æ 1.9 55.1 Æ 2.5 55.0 Æ 2.6
Lipids FPH 7.7 Æ 0.1 4.1 Æ 0.1 2.3 Æ 0.1 1.8 Æ 0.1 5.4 Æ 0.2 3.5 Æ 0.1 1.4 Æ 0.1 11.5 Æ 0.4 6.9 Æ 0.0 7.8 Æ 0.6 3.0 Æ 0.1
Sludge 23.2 Æ 1.0 29.9 Æ 0.8 25.4 Æ 0.4 31.6Æ1.7 24.6 Æ 0.4 18.0 Æ 0.3 14.9 Æ 1.0 31.4Æ0.5 33.4 Æ 3.9 28.4 Æ 1.3 30.9 Æ 1.5
Ash FPH 10.4 Æ 1.0 9.9 Æ 0.2 10.6 Æ 0.1 9.7 Æ 0.1 12.4 Æ 0.2 11.3 Æ 0.0 10.2 Æ 0.1 13.9 Æ 0.1 11.8 Æ 0.0 13.0 Æ 0.1 12.4 Æ 0.2
Sludge 17.2 Æ 2.6 16.6 Æ 1.5 15.5 Æ 1.2 12.1 Æ 0.3 15.9 Æ 0.2 16.5 Æ 0.4 17.1 Æ 2.9 7.4 Æ 0.0 5.5 Æ 0.2 19.0 Æ 0.7 17.4 Æ 0.0
V-DT + BB: viscera without digestive tract and backbone; V + BB: viscera and backbone; V-DT: viscera without digestive tract; V: viscera; NE: no enzyme added; FlavNW: Flavourzyme, no water added to the
hydrolysis mixture; NeuNW: Neutrase, no water added to the hydrolysis mixture.
Table 2
Yield of dry matter (g/100 g wet weight of raw material) obtained after enzymatic hydrolysis (mean Æ S.D.)
V-DT + BB V + BB V-DT V
Flavourzyme Neutrase Flavourzyme Neutrase Control Flavourzyme Neutrase Flavourzyme Neutrase
NE FlavNW NeuNW
Oil fraction 10.8 Æ 0.1 11.5 Æ 0.1 4.2 Æ 0.1 3.5 Æ 0.2 4.4 Æ 0.2 5.3 Æ 0.2 6.2 Æ 0.4 20.9 Æ 0.7 22.2 Æ 0.1 13.4 Æ 1.0 14.6 Æ 0.3
FPH 6.00 Æ 0.3 7.7 Æ 0.1 7.0 Æ 0.1 8.6 Æ 0.3 6.2 Æ 0.1 2.7 Æ 0.0 2.8 Æ 0.1 4.0 Æ 0.8 6.4 Æ 0.1 5.7 Æ 0.1 7.3 Æ 0.2
Sludge 17.0 Æ 0.4 15.5 Æ 0.3 18.6 Æ 0.2 17.1 Æ 0.8 18.8 Æ 0.3 21.8 Æ 0.3 20.9 Æ 0.9 15.3 Æ 0.2 12.6 Æ 1.2 18.5 Æ 0.7 16.0 Æ 0.6
Emulsion 0.7 Æ 0.1 0.5 Æ 0.1 0.4 Æ 0.1 0.4 Æ 0.1 tr. tr. tr. 2.7 Æ 0.1 2.3 Æ 0.1 1.8 Æ 0.1 1.1 Æ 0.0
V-DT + BB: viscera without digestive tract and backbone; V + BB: viscera and backbone; V-DT: viscera without digestive tract; V: viscera; NE: no enzyme added; FlavNW: Flavourzyme, no water added to the
hydrolysis mixture; NeuNW: Neutrase, no water added to the hydrolysis mixture.

6. 3. Results and discussion
By combining different parts of fish by-products, four
mixtures of raw material were made (Table 1). During
hydrolysis, by-products were converted into yellow-brown-
ish liquid mixtures. In the samples where bones were present
in the raw material, the hydrolysates contained bone
particles. After centrifugation, four fractions were usually
obtained: oil on the top, emulsion, FPH and sludge on the
bottom of the centrifugation vessels. The colour of the oil
varied from yellow to pink, depending on the composition of
the raw material. The FPH was a clear yellow and sticky
liquid and the dried FPH powders had a light yellow colour
and a fishy odour. The sludge was a grey layer and had two
parts: fluffy dust coloured upper part and compact bottom
layer with bone particles. Dried sludge was a grey, greasy
powder. The composition of dried FPH and sludge is given
in Table 3.
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–20332026
Fig. 1. Percentage of soluble protein in fish protein hydrolysates as a function of the amount of lipid in raw material for two different proteases.
Fig. 2. Relationship between amount of free amino acids and degree of hydrolysis of FPH fractions.

7. The yield of dried sludge was significantly higher (p <
0.05) compared to yield of dried FPH for all samples (Table
2). Different enzymes gave different amounts of the
fractions: Flavourzyme produced significantly higher
amount of sludge than Neutrase.
3.1. Degree of hydrolysis
The proteins in the FPH fraction was as expected more
hydrolysed than in the sludge. Degree of hydrolysis varied
between 18.5 and 33.7% for FPH and between 4.3 and
10.9% for sludge [41]. After hydrolysis with addition of
water (29.5 Æ 5.0)% of the protein in the raw material were
obtained in the FPH fraction, while (70.0 Æ 4.7)% of the
protein in raw material were obtained in the sludge. The
percentage of protein calculated on a fat-free basis in the raw
material varied in a very narrow range: 18.2 Æ 0.2%. This
indicates that the amount of proteins available for the
enzymatic reaction was very similar. However, the
percentage of proteins solubilised during hydrolysis with
Flavourzyme varied from 22.7% for V-DT to 34.7% for V +
BB and from 38.8% V-DT to 44.2% for V + BB with
Neutrase. A dependency between amount of lipids and
percentage of solubilised proteins was found (Fig. 1). Raw
material containing the highest amount of lipids gave the
lowest percentage of solubilised proteins. Protein recovery
in the FPH fraction was in average 1.4 times higher for
samples obtained after Neutrase treatment compared to
Flavourzyme treatment. However, after hydrolysis without
dilution of substrate, recovery of proteins in FPH was similar
for both enzymatic treatments and was significantly lower
than recovery of protein after hydrolysis with added water
and enzymes. DH depended slightly on the enzyme used:
Flavourzyme as an exopeptidase on an average gave a higher
degree of hydrolysis for FPH, conversely Neutrase gave
sludge with higher DH. The initial composition of the raw
material also influenced the DH of FPH fractions: raw
material from V had the highest, while raw material from V-
DT + BB had the lowest DH values. The amount of free
amino acids [41] was significantly higher in FPH (15–
97 mg/g dried powder) than in the sludge (3–9 mg/g dried
powder). The relationship between DH and amount of free
amino acids fell into two groups (Fig. 2). For all samples
obtained with Flavourzyme and for most of the samples
obtained with Neutrase, the increase in DH was followed by
a large increase in amount of free amino acids (Fig. 2).
However, for samples hydrolysed with Neutrase and
containing viscera without digestive tract (V-DT), the
increase in free amino acids with increasing DH was
significantly lower. These samples contained proteins and
peptides in the Mw range between approximately 415,000
and 200,000, which were not obtained in other hydrolysates
(Fig. 3). Besides, V-DT samples hydrolysed with Neutrase
had more peptides in the Mw range between approximately
24,000 and 1500 than samples hydrolysed with Flavour-
zyme. The increase in DH for other samples was influenced
by the large amount of smaller peptides (Mw range less than
$1500).
The DH in sludge of the control samples (hydrolysed
without added water) was significantly higher: between 10.2
and 10.9% compared to 5.1 and 5.4% for the sludge samples
with added water. These samples also contained more free
amino acids: 22–32 mg/g in dried sludge powder from
control samples without added water compared to 4–9 mg/g
for the sludge samples with added water. This could
probably be due to increased concentration of free amino
acids in the control hydrolysis compared to the diluted
system. Comparing the treatments with and without added
commercial enzymes, performed under the same conditions,
similar DH values and amount of free amino acids were
found in the FPH fraction: DH was 23.5–24.4 for samples
obtained with commercial enzymes and 23.5 without added
enzymes. The DH of FPH obtained by using Flavourzyme
(Flav/V + BB) and Neutrase (Neu/V + BB) was only 4 and
0.1% higher respectively than the DH of the soluble fraction
obtained using only water (NE/V + BB). Using Flavourzyme
and Neutrase (Flav/V + BB and Neu/V + BB), the increase in
DH in the sludge was 7 and 15%, respectively.
Mohr [42] pointed out that during heating to the
temperature of hydrolysis, proteins in the sarcoplasmic
fraction may denature and precipitate. This is more evident
when the raw material is heated before hydrolysis in order to
inactivate endogenous enzymes. The denatured proteins are
apparently highly resistant to enzymatic breakdown [42],
consequently only a minor part of the denatured proteins will
be solubilised during subsequent enzymatic hydrolysis. In
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–2033 2027
Table 4
Calculated protein efficiency ratio (PER) values of FPH and sludge
Raw material Enzyme Fraction PERa
PERb
PERc
V-DT + BB Flavourzyme FPH 2.53 1.30 1.60
V + BB Flavourzyme FPH 2.50 1.25 1.68
V-DT Flavourzyme FPH 2.54 0.80 1.95
V Flavourzyme FPH 2.58 1.27 1.97
V-DT + BB Neutrase FPH 2.58 1.55 1.78
V + BB Neutrase FPH 2.61 1.53 1.78
V-DT Neutrase FPH 2.98 1.93 2.27
V Neutrase FPH 2.77 1.46 2.08
V-DT + BB Flavourzyme Sludge 3.36 2.18 2.41
V + BB Flavourzyme Sludge 3.49 2.33 2.48
V-DT Flavourzyme Sludge 3.93 2.76 2.80
V Flavourzyme Sludge 3.78 2.84 2.67
V-DT + BB Neutrase Sludge 3.46 2.20 2.50
V + BB Neutrase Sludge 3.64 2.46 2.60
V-DT Neutrase Sludge 4.07 2.89 2.81
V Neutrase Sludge 3.97 2.94 2.79
Cod muscled
2.87 3.24 2.99
a
PER: À0.468 + 0.45[LEU] À 0.105 [TYR].
b
PER: À1.816 + 0.435[MET] + 0.780[LEU] + 0.211[HIS] À
0.944[TYR].
c
PER: 0.08084[
P
AA7] À 0.1094, where
P
AA7 = threonine + valine +
methionine + isoleucine + leucine + phenylalanine + lysine.
d
Data from Shahidi et al. (1991); V-DT + BB: viscera without digestive
tract and backbone; V + BB: viscera and backbone; V-DT: viscera without
digestive tract; V: viscera.

8. addition hydrophobic interactions between peptides or self-
association of larger peptides probably lead to formation of
aggregates which will reduce the susceptibility of the proteins
towards enzymatic breakdown, reducing the yield of FPH
[44]. This assumption was supported by the results in this
experiment.The percentage ofhydrophobic amino acidsinthe
non-soluble fraction was (34.6 Æ 2.2%), which is 1.3–1.5
times higher than in the FPH (24.7 Æ 1.6%). In addition, in the
case when raw material contains relatively high amount of
lipids (10–30%), protein–lipid complexes could be formed.
These complexes might be more resistant to enzymatic
breakdown and extraction of oil and yield of FPH fraction can
be reduced. The relationship between amount of lipids and
percentage of solubilised proteins found in this study supports
this assumption (Fig. 1). The state of the substrate before
hydrolysis may therefore be of great importance. More
attention should therefore be given to a temperature-
programmed hydrolysis, which could give measurably higher
yields of soluble products, as well as better purity and quality
of the oil compared to a conventional enzyme process.
Gel filtration was used for evaluation of the size
distribution of the protein in the FPH powders. The gel
filtration showed that the main part of peptides in the FPH
fraction was smaller than 1355 D (Vitamin B12, Mw = 1355,
used as standard). With the available column, it was not
possible to separate smaller peptides and amino acids. Gel
filtration also indicated that treatment of the same raw
material with Flavourzyme, as expected, gave FPH with
higher molecular weight peptides compared to neutrase (one
more additional peak, more proteins in the range from Mw
200,000 to 13,000) (Fig. 3). Hydrolysis without adding
water gave higher amount of larger peptides. Similarly,
samples obtained without addition of commercial enzymes
had more peptides with higher molecular weight than the
same raw material treated with Flavourzyme and Neutrase.
3.2. Protein efficiency ratio
Calculated PER values (Table 4) showed that the sludge
had significantly (p < 0.05) higher PER value than FPH.
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–20332028
Fig. 3. Gel filtration chromatograms showing the distribution of FPH fractions molecular weight: (a) V-DT/F: viscera without digestive tract after Flavourzyme
hydrolysis (b) V-DT/N: viscera without digestive tract after Neutrase hydrolysis (c) V/F: viscera after Flavourzyme hydrolysis (d) V/N: viscera after Neutrase
hydrolysis.

9. PERc
values of dried powders were found to range from 1.60
to 2.27 for FPH and from 2.41 to 2.81 for sludge. The same
tendency was observed for all calculated PER values. PER
value of sludge was generally 1.5 times higher than PER
value of FPH. Furthermore, PER values of sludge from
different raw materials were very similar or even higher than
PER values for cod muscles calculated by Shahidi [11].
Degree of hydrolysis of protein hydrolysates also has a
significant effect on the nutritional quality of the protein
fractions: with increasing DH, PER values decreased
substantially. This is directly connected to the different
fractions: the sludge fractions were less hydrolysed than
FPH. The sludge had higher PER values and had a higher
content of hydrophobic amino acids, and many of these
amino acids are essential. This confirmed Liaset et al. [45]
data showing that the insoluble fraction produced by
enzymatic hydrolysis from salmon frames was rich in
essential amino acids and could possibly be a dietary protein
supplement to poorly balanced dietary proteins. The
estimation of actual protein quality produced from cod
by-products showed that sludge also had a high nutritional
value. Taking into account that sludge gave significantly
higher dry yield than the FPH fraction, it should be claimed
that by elimination of the non-soluble protein fraction after
hydrolysis huge amounts of nutritionally valuable proteins
are lost.
3.3. Water holding capacity
Several studies have shown that fish protein hydrolysates
have excellent water holding capacity and can increase the
cooking yield when added to minced meat [12,18,46]. The
aim of one part of the experiment was to evaluate how
adding dried powders into comminuted fish muscle and
freezing the mixture influence the WHC of the system after
thawing. The addition of 5% sludge powders to comminuted
fish muscle resulted in an increase of up to 17% in the water
holding capacity after freezing compared to control, but this
was lower than the ability of casein to hold water in the same
system. FPH powders made from raw material without
bones showed 0.5–9% increase, while samples made from
raw material containing bones did not increase the WHC.
Sludge powders had a lower degree of hydrolysis. However,
a relationship between DH and WHC was not observed for
any of the powders. The powders made from viscera without
digestive tract (V-DT) had the best WHC and powders from
V + BB had the lowest. In general FPH powders made from
raw material containing backbones (V-DT + BB and V +
BB) exhibited low WHC. These samples contained 6–12
times more hydroxyproline than samples without back-
bones. A linear relationship between amounts of certain
amino acids and WHC of FPH was observed: decreasing
amounts of glycine/arginine (r = 0.63), alanine (r = 0.62),
hydroxyproline (r = 0.62) and sum of hydrophobic amino
acids increased the WHC of the frozen comminuted fish
muscle (Fig. 4).
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–2033 2029Table5
Fatabsorption(goilpergprotein),waterholdingcapacity,emulsifyingcapacity(goilemulsifiedpergpowder)andemulsionstability(%ofinitialemulsion)
FPHSludge
Fatabsorption,
meanÆS.D.O.M.
Waterholdingcapacity,
meanÆS.D.
Emulsifyingcapacity,
meanÆS.D.O.M.
Emulsionstability,
meanÆS.D.
Fatabsorption,
meanÆS.D.
Waterholdingcapacity,
meanÆS.D.
Emulsifyingcapacity,
meanÆS.D.
Emulsionstability,
meanÆS.D.
V-DT+BB/F4.1Æ0.261.8Æ1.18.5Æ0.636.6Æ0.32.4Æ0.167.6Æ0.96.1Æ0.290.2Æ3.0
V-DT+BB/N3.1Æ0.261.9Æ1.810.5Æ0.933.0Æ5.12.6Æ0.072.2Æ0.310.8Æ0.677.3Æ10.5
V+BB/F2.4Æ0.159.5Æ1.04.3Æ0.339.3Æ15.22.2Æ0.064.4Æ0.20.3Æ0.136.7Æ4.7
V+BB/N2.2Æ0.262.1Æ0.711.8Æ1.236.0Æ0.72.2Æ0.166.0Æ0.30.1Æ0.00.0Æ0.0
V-DT/F5.0Æ0.170.4Æ1.26.0Æ0.719.4Æ7.92.1Æ0.075.1Æ0.97.2Æ0.698.9Æ1.9
V-DT/N4.7Æ0.464.8Æ0.76.4Æ0.333.3Æ3.12.0Æ0.068.0Æ0.411.4Æ0.295.5Æ3.3
V/F3.3Æ0.265.8Æ1.28.6Æ0.941.4Æ5.11.6Æ0.064.2Æ0.80.7Æ0.481.7Æ2.4
V/N2.9Æ0.167.8Æ0.913.2Æ1.530.4Æ8.11.6Æ0.065.6Æ0.81.7Æ0.272.0Æ1.9
V+BB–water6.0Æ0.359.4Æ0.837.0Æ0.732.4Æ4.32.6Æ0.059.1Æ1.0
V+BB/F–water2.1Æ0.062.5Æ1.3118.3Æ1.733.3Æ0.32.8Æ0.159.8Æ0.4
V+BB/N–water1.6Æ0.065.8Æ1.5128.3Æ1.770.6Æ0.22.7Æ0.064.2Æ0.6
Soybeanprotein1.2Æ0.068.4Æ0.2500Æ0.050.0Æ14.1
Casein1.4Æ0.082.4Æ1.617.0Æ0.898.9Æ1.5
Empty(forWHC)64.4Æ0.7
V-DT+BB:viscerawithoutdigestivetractandbackbone;V+BB:visceraandbackbone;V-DT:viscerawithoutdigestivetract;V:viscera;NE:noenzymeadded;FlavNW:Flavourzyme,nowateraddedtothe
hydrolysismixture.NeuNW:Neutrase,nowateraddedtothehydrolysismixture.

10. 3.4. Fat absorption capacity
In contrast to WHC results, FPH powders exhibited
significantly (p < 0.05) higher fat absorption capacity than
sludge powders (Table 5) and had values similar to those
Kristinsson and Rasco [5] observed in their experiment with
FPH powders (15% DH) obtained after Atlantic salmon
muscle hydrolysis. Similar to WHC results FPH powder
made from viscera without digestive tract (V-DT) had the
highest fat absorption ability, while viscera plus backbone
(V + BB) had the lowest. In general, the ability of sludge to
absorb fat was constant (2.3 Æ 0.4 g oil/1 g protein in the
powder) for all samples.
Addition of water before hydrolysis increased the fat
absorption capacity of FPH. The powders obtained without
adding commercial enzymes showed the highest fat
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–20332030
Fig. 5. Relationship between fat absorption and amount of lipids in the FPH powder.
Fig. 4. Relationship between WHC and amount of certain amino acids in the FPH.

11. absorption ability. This might be explained by the presence
of large peptides in the powders, because progressive drop in
fat absorption was observed by Kristinsson and Rasco [5]
with increasing of DH of the samples.
FPH powders containing higher amounts of lipids had
higher fat absorption ability, while sludge exhibited the
opposite tendency: higher amount of lipids in the powder
gave lower fat absorption ability (Fig. 5). A positive
relationship (r2
= 0.90) between fat absorption and amount
of phospholipids was observed in the FPH samples (Fig. 6).
However, this observation did not hold for FPH powder
obtained without addition of commercial enzymes. This
could be due to the conformation of those proteins which
were not hydrolysed by commercial enzymes and had larger
peptides than enzymatically hydrolysed samples. It seems
that the state of proteins in FPH is more important for fat
absorption than amount of phospholipids.
In the sludge it was observed that powders containing
higher amount of charged amino acids, such as aspartic acid,
glutamic acid, lysine and arginine had better fat absorption
ability. A linear relationship was also observed between
amount of alanine (r2
= 0.81), hydroxyproline (r2
= 0.57) and
hydrophobic amino acids (r2
= 0.58) in the sludge and fat
absorption (Fig. 7). In general, FPH and sludge powders
showed good fat absorption properties, significantly better
than casein and soybean proteins, which are both common
food protein ingredients [43,47,48] and were used as
reference.
3.5. Emulsifying properties
FPH powders obtained without adding commercial
enzymes had better EC than samples obtained with added
water and commercial enzymes. The protein structure of the
samples obtained without commercial enzymes was less
hydrolysed than the samples with added enzymes and
therefore play a significant role for the emulsifying
properties.
It was found that the most significant factor influencing
the emulsification capacity of FPH was the amount of added
water before hydrolysis: powders hydrolysed without added
water showed significantly higher emulsification capacity
compared to other samples (Table 5). High emulsifying
capacity of samples obtained without added water could be
due to the plastein reaction, which can start at high
concentration of hydrolysates in the system [18]. In the
plastein reaction, condensation of the peptides occurs
resulting in formation of new polypeptides with new and
different properties.
The amount of proteins and amino acids seems to be
important for emulsification capacity. The EC values
increased with increasing protein content in the FPH
powders. However, a relationship between amount of
proteins and stability of emulsions was not found. These
data are in accordance with Turgeon et al. [22], who
concluded that although hydrolysed proteins and small
peptides diffuse rapidly and adsorb at the interface, they are
less efficient in stabilising emulsions because they cannot
unfold and reorient at the interface like a protein. Treatment
with different enzymes also influenced emulsification
properties: hydrolysates after treatment with neutrase had
significantly (p < 0.05) better emulsification capacity than
samples after hydrolysis with flavourzyme. Reduction of the
emulsifying properties of samples treated with Flavourzyme
can also be explained by higher amount of free amino acids
in the samples. This is in agreement with the results from
Chobert et al. [49], who found that smaller peptides and free
amino acids may have reduced emulsifying properties
R. Sˇlizˇyte˙ et al. / Process Biochemistry 40 (2005) 2021–2033 2031
Fig. 6. Relationship between fat absorption and amount of phospholipids in the FPH powder.

12. compared to larger peptides. The sludge samples with higher
amount of proteins (V-DT + BB and V-DT had more proteins
than V + BB and V samples) also showed better emulsifying
properties. Lower amounts of free amino acids also
increased the emulsifying properties of the sludge.
4. Conclusions
Different ways of combining fish by-products lead to
different end products with different properties after
hydrolysis. Raw material containing the highest amount
of lipids gave the lowest percentage of solubilised proteins.
In general, FPH powders made from raw material containing
backbones contained 6–12 times more hydroxyproline than
samples without backbones and exhibited low WHC. The
powders made from viscera without digestive tract had the
highest fat absorption ability and WHC, while V + BB had
the lowest. The sludge samples with higher amount of
proteins showed better emulsifying properties.
The more important factor affecting the yield, biochem-
ical and functional properties of different fractions was
amount of added water rather than type of enzyme used.
Protein recovery after hydrolysis without addition of water
was more than two times lower than protein recovery after
hydrolysis with added water, while difference in yield for the
use of different enzymes was about 40%. The most
significant factor influencing the emulsification capacity
of FPH was also amount of added water before hydrolysis:
powders hydrolysed without added water showed signifi-
cantly higher emulsification capacity compared to samples
obtained with addition of water. The fat absorption of FPH
and sludge powders was higher than those of soybean
protein and casein. WHC was comparable to that of soybean
protein, but was lower than for casein.
To achieve better utilisation of all fish by-products it is
necessary to pay more attention to the water non-soluble part
after hydrolysis, which constituted a significant part after
hydrolysis and contained 70.0 Æ 4.7% of protein, while FPH
contained 29.5 Æ 5.0% of the protein in the raw material.
PER of sludge was about 1.5 times higher than PER value of
FPH. Sludge also had good functional properties, in some
cases even better than the water-soluble fraction, which is
often considered the main product of protein hydrolysis.
Acknowledgements
Authors wish to thank The Norwegian Research Council
and EU commission (project QLK1-CT2000-01017) for
financial support to carry out experiments. Colleagues at
SINTEF Fisheries and Aquaculture and NTNU are thanked
for their help to prepare this paper.
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