1. Yield and composition of different fractions obtained after enzymatic
hydrolysis of cod (Gadus morhua) by-products
Rasa Sˇlizˇytea,b
, Egidijus Dauksˇasa
, Eva Falcha,b
, Ivar Storrøa,*, Turid Rustadb
a
SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway
b
Department of Biotechnology, Norwegian University of Science and Technology, Norway
Received 22 March 2004; accepted 11 June 2004
Abstract
The aim of this work was to study how different raw material mixtures from cod (Gadus morhua) by-products influenced the composition
of the substrate for hydrolysis, yield and chemical composition of the different fractions after the enzymatic hydrolysis using endo-peptidase
(Flavourzyme) or exo-peptidase (Neutrase).
The most important factor influencing the yield of the different fractions was added water rather than type of enzyme used. The highest
lipid yield was obtained in samples without addition of water resulting in a decreased yield of fish protein hydrolysates (FPH).
Use of Neutrase produced more dry FPH (23–57%) than Flavourzyme. The amount of oil fraction obtained after Neutrase treatment was up
to 10% higher compared to Flavourzyme due to 13–60% smaller emulsion layer and higher degree of hydrolysis in sludge liberating more
lipids from sludge to oil fraction. Emulsion, which is not a desirable fraction after hydrolysis can also be reduced or avoided by reduction or/
and elimination of addition of water into hydrolysis mixture.
# 2004 Elsevier Ltd. All rights reserved.
Keywords: Cod; By-products; Enzymatic hydrolysis; Yield; Recovery; Composition
1. Introduction
Fisheries and fish processing create large amounts of
processing discards and by-products. According to Venu-
gopal and Shahidi [1] approximately 30% of the total
landings can be considered as under-utilised, by-catch,
unconventional or unexploited. Only a small part of these is
used for human consumption. In Norway 110,000 t of cod
by-products were produced in year 2002 and only 41,000 t of
these were used for human consumption [2]. Fish by-
products are dumped, composted as fertiliser, used for fish
meal and oil, for silage or used for production of value added
components [3]. To satisfy the increasing demand for fish
and fish products for human consumption, optimal use of
unexploited or under-utilised resources is important [4]. For
better management of world catches it is therefore a
challenge to utilise the valuable lipid and protein fractions
from the by-products for human consumption and other
value added ingredients [5].
Comparing chemical composition of cod flesh and cod
by-products shows that cod by-products has a high amount
of proteins and can be used to produce different valuable
products [3]. Functional protein powders, kamaboko
products, traditional products (cakes, balls, patties, breaded
products, canned products, etc.), sauces, pasties, flavouring
can be produced with ingredients from under-utilised
species and by-products [4]. High protein products such
as fish meal, surimi, fish protein concentrate and fish protein
hydrolysates (FPH) can be produced from fish by-products
[6]. The production of fish protein hydrolysates can help to
avoid the depletion of the commercially important fish
stocks, reduce environmental pollution from waste of fish
and supply nutritious and functional protein food. Fish
protein hydrolysates have similar amino acid profiles to that
of original material except for the sensitive amino acids such
as methionine and tryptophan that are affected to a relatively
large extent during enzymatic hydrolysis [7].
www.elsevier.com/locate/procbio
Process Biochemistry 40 (2005) 1415–1424
* Corresponding author. Fax: +47 73 59 63 63.
E-mail address: ivar.storro@sintef.no (I. Storrø).
0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.06.033

2. In some countries, production of acid silage is a common
method to hydrolyse proteins and lipids. Acidic treatments
has however been found to deteriorate the lipids by
increasing the level of free fatty acids [8,9]. Hydrolysis
carried out by the addition of enzymes is more reproducible
than the more unspecific autolysis in traditional silage
production. Enzymatic hydrolysis of proteins is an attractive
means of giving better functional and nutritional properties
to food proteins from by-products.
There is an increasing interest in the development of fast
and gentle enzymatic production methods as an alternative
to mechanical or chemical treatments that often damage the
products and reduce product recovery [10]. Furthermore, the
amount of raw material being converted into soluble
hydrolysate is especially important for industrial processes
[11]. However, the yield of soluble material obtained in a
standard batch process using commercial proteases, was low
[12]. Not much work has been done on the evaluation and
examination of the non-soluble part after hydrolysis. The
value of the hydrolysis process could be increased
significantly by performing continuous hydrolysis or/and
finding use and application for the non-hydrolysed fractions.
The aim of this work was to study how raw material
mixtures combined from different separated by-products
influenced the composition of the substrate for hydrolysis,
the yield of different fraction after hydrolysis and the
chemical composition of the different fractions. Both endo-
and exo-peptidases were used for the enzymatic hydrolysis
of different cod (Gadus morhua) by-products. This work
shows the possibility to use selected fractions of fish by-
products in production of protein fractions and fish lipids.
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 À DT), viscera and backbone
without digestive tract (V À 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
temperature 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 is 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
the optimal temperature around 50 8C. Flavourzyme 500 L
has a declared activity of 500 LAPU/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 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) [21,22].
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–14241416
Table 1
Amount of fish by products (g, mean Æ S.D.) and composition of different raw material mixtures (g/100 g)
Parts of fish body Average V À DT + BB V + BB V À DT V
Number of fish in group 25 6 4 10 5
Length, cm 55.4 Æ 4.4
Roe 91.5 Æ 69.9 11.6 26.0 23.9 28.1
Milt 165.8 Æ 48.8 16.5 0.0 26.7 8.0
Liver 94.0 Æ 28.1 24.9 18.2 47.2 30.9
Stomach (with contents) 67.4 Æ 56.3 – 11.4 – 15.4
Gallbladder 2.3 Æ 1.2 0.4 0.4 0.7 0.9
Diverse 3.6 Æ 2.1 1.3 0.7 1.6 0.9
Intestines 28.0 Æ 8.9 – 4.7 – 8.2
Blind shaft 27.0 Æ 7.4 – 4.3 – 7.7
Back bones 173.3 Æ 31.4 45.5 34.3 – –
Chemical composition
Amount of water 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 (6.25N) 15.4 Æ 0.2 16.1 Æ 0.4 12.6 Æ 1.2 14.9 Æ 2.3
Ash 3.6 Æ 0.4 3.6 Æ 0.2 1.7 Æ 0.2 4.4 Æ 0.3

3. Methanol, chloroform, hexane, formaldehyde (all from
Merck, Darmstad, Germany) were used for the chemical
analysis.
2.3. Hydrolysis process
Minced and frozen fractions were thawed in a microwave
oven. An amount of 250 g of sample was mixed with 250 mL
distilled water and the pH values measured (Philips PW 9420
pH meter, Pye Unicam Ltd., England; electrode: Unikan, type
No. 9436-095-84003). The hydrolysis was performed in a 4 L
closed glass vessel stirred with a marine impeller (150 rpm).
The enzymatic hydrolysis was started whenthe temperature of
the mixturewas50 8C byaddingeither 0.1%(byweightofraw
material) Flavourzyme 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, 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. The experiments
were performed in duplicate. Three controls were included in
the processing of the sample ‘viscera with backbone’ (V +
BB).(1) Raw materialand water with noenzymesadded(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). Measurements were performed in duplicate. Ash
content was estimated according to AOAC [13]. Measure-
ments were performed in triplicate. Total N was determined
by CHN-S/N elemental analyser 1106 (Carlo Erba Instru-
ments S.pA., 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 [14]. 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 [15]. Analyses were performed in duplicate.
2.6. Amount and composition of free amino acids
The 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 7850 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 [16] as modified by
Flynn [17]. Glycine/arginine and methionine/tryptophane
were determined together, as their peaks merged. This
analysis was performed twice on each sample.
2.7. Statistical analysis
Dependent upon 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%.
3. Results and discussion
3.1. Yields and properties of the fractions
By combining different parts of fish by-products, four
mixtures of raw material were obtained. The chemical
composition of these samples is presented in Table 1. The
amount of water in the samples varied from 60.0% to
70.5%. The highest amount of water and proteins was
found in the samples with backbones (V À DT + BB and V +
BB), these samples had the lowest amount of lipids. Total
amount of proteins varied from 12.6 to 16.1 g/100 g of wet
raw material. Calculated on lipid free basis, the variation
was only 17.2–18.7 g/100 g of wet raw material. The
highest amount of lipids was found in viscera (V, 21.0 g/
100 g) and in viscera without digestive tract (V À DT,
26.9 g/100 g). V À DT mixture contained up to three times
more lipids than the V + BB mixture. The amount of ash in
the samples depended on the composition of the raw
material mixture. The presence of backbones in the raw
material mixture increased the amount of ash up to 3.6 g/
100 g, however the highest amount of ash (4.4 g/100 g) was
found in all viscera (V) containing samples due to presence
of stones, sand, crustaceans and shells in stomachs and
intestines.
The enzymatic treatments were performed at natural pH
6.7–6.9. These values were within the optimal range (pH
5.5–7.5 for Neutrase and pH 5–7 for Flavourzyme) for both
enzymes. For most samples, pH after hydrolysis was 0.3
units lower than the starting pH. The exception was the
control group (Flav/NW and Neu/NW) without added water
and the viscera (V) group. The control samples had a final
pH in the same range or even higher than the starting pH. In
viscera samples a small decrease in pH was also observed
(0.1–0.2 units).
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–1424 1417

4. Table 2 shows the dry weight obtained of each fraction
after centrifugation, separation and freeze-drying. The
amount of oil fraction obtained after hydrolysis depended
significantly on the amount of lipids in the raw material
(Table 2 and Fig. 1). The yield of oil extracted using
Flavourzyme and Neutrase was 60.3% and 64.5% (V À DT
+ BB), 43.8% and 36.4% (V + BB), 78.0% and 82.8% (V À
DT), 63.6% and 69.4% (V) respectively (Table 2), compared
to amount of oil fraction obtained from the same raw
material by the Bligh and Dyer method (Table 1). The yield
of oil fraction was linearly correlated (y = 1.0109x À6.3946,
R2
= 0.98, Fig. 3) to the amount of lipids (% of wet weight,
without control group) in the raw material. As shown in
Fig. 3, in order to obtain oil after protein hydrolysis, the
raw material must contain more than approximately 6 g
lipids/100 g of wet weight. The calculation on a protein basis
(Fig. 3) showed that the amount of proteins in raw material
can also influence the amount of separated oil fraction.
When amount of proteins in the raw material is more than
17 g/100 g of raw material, the formation of oil fraction can
be prevented. Neutrase gave higher amounts of oil fraction
(6–16%) compared to Flavourzyme (Table 2) from all raw
material mixtures except for the V + BB samples obtained
with enzyme and added water where Neutrase gave 17% less
oil than Flavourzyme.
Using Flavourzyme and Neutrase, respectively 44% and
36% of the lipids present in the raw material could be
separated after hydrolysis. The control incubation without
enzymes (NE/V + BB) showed that 46% of oil can be
obtained without hydrolysis. Running the hydrolysis without
added water, the yield of oil fraction increased even more: to
55% with Flavourzyme (Flav/NW/V+BB) and 64% with
Neutrase (Neu/NW/V + BB). This indicated that the controls
(with enzyme, but without added water, Table 2) gave a
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–14241418
Table 2
Yield of dry matter (g/100 g wet weight of raw material) obtained after enzymatic hydrolysis and drying (mean Æ average relative S.D. of the measurement)
Yield (%) V À DT + BB V + BB V À DT V
Flavourzyme Neutrase Flavourzyme Neutrase Control Flavourzyme Neutrase Flavourzyme Neutrase
NEa
FlavNWb
NeuNWc
Oil fraction, mean Æ 3% 10.8 11.5 4.2 3.5 4.4 5.3 6.2 20.9 22.2 13.4 14.6
FPH, mean Æ 3% 6.0 7.7 7.0 8.6 6.2 2.7 2.8 4.0 6.4 5.7 7.3
Sludge, mean Æ 3% 17.0 15.5 18.6 17.1 18.8 21.8 20.9 15.3 12.6 18.5 16.0
Emulsion, mean Æ 3% 0.7 0.5 0.4 0.4 tr. tr. tr. 2.7 2.3 1.8 1.1
a
Treatment: raw material and water with no enzymes added.
b
Treatment: raw material + Flavourzyme, no added water.
c
Treatment: raw material + Neutrase, no added water.
Fig. 1. Amount of lipids in the raw material and percentage of solubilised proteins by using different enzymes.

5. higher yield of oil fraction (increase of respectively 26% and
57%) compared to Flavourzyme (Flav/V + BB) or Neutrase
(Neu/V + BB) treatment with added water. This can partially
be explained by the absence of the emulsion layer.
Therefore, addition of water before hydrolysis seems to
reduce the yield of oil fraction.
Fish protein hydrolysate (FPH) is the main product of
the protein hydrolysis. Therefore optimal hydrolysis should
give the highest possible amount of FPH. The amount of dry
FPH was influenced by the composition of raw material,
hydrolysis conditions and type of enzyme used (Table 2).
The highest amount of dry FPH was obtained from the raw
material containing the lowest amount of lipid: viscera
with backbones (V + BB) gave 7.0–8.6 g FPH/100 g raw
material depending on the type of enzyme used. Hydrolysis
of V À DT + BB and V produced similar amounts of FPH.
The raw material, containing the highest amount of lipids
(V À DT) produced 4.0–6.4 FPH/100 g raw material. The
percentage of solubilised protein was found to depend on the
amount of lipids in the raw material (Fig. 1). Raw material
containing the highest amount of lipids gave the lowest
percentage of solubilised protein. These investigations
showed that amount of lipids in the raw material is
important for the hydrolysis process.
The presence of backbone and/or digestive tract gave the
largest amount of dry FPH (up to 8.6 g/100 g of raw
material, Neu/V + BB). However, the hydrolysis of the same
raw material under the same conditions without added water
gave only 2.7–2.8 g FPH/100 g raw material (Table 2). This
showed the importance of the added water to obtain more
dried FPH. Mohr [11] stated that during hydrolysis peptide
linkages are being broken in the insoluble and soluble
fraction and high concentrations of soluble peptides in the
reaction mixture markedly reduce both the rate of
hydrolysis, and also the yield of material released into
solution. The strong negative influence on nitrogen recovery
on increasing salmon frames-water ratio was found in the
experiment with salmon frames [18], because the potential
for product inhibition of the enzyme increases with higher
substrate concentrations in the hydrolysis process. Conse-
quently the adding of water to the hydrolysis mixture is
necessary, but because of the expense of removing the water
afterwards by evaporation or drying, optimum raw material–
water ratio and time to add water (before or after hydrolysis)
should be found.
Different enzymes also gave different amounts of the
fractions: Neutrase treatment gave more FPH, and Flavour-
zyme gave a higher amount of sludge. The use of Neutrase
(Neu/V + BB) increased the yield of FPH by almost 38%
compared to the yield of the soluble protein fraction
obtained without using enzymes (NE/V + BB, Table 2).
While using Flavourzyme (Flav/V + BB) the FPH yield was
only 12% higher than the soluble fraction yield of NE/V +
BB. Mohr [12] pointed out that during heating to the
temperature of hydrolysis, the proteins can denature and
precipitate. This is more evident when raw material is
heated before hydrolysis in order to inactivate endogenous
enzymes. The denatured proteins are highly resistant to
enzymatic breakdown [19], which reduces the amount of
proteins which will be solubilised during subsequent
enzymatic hydrolysis. The hydrophobic interactions
between the peptides or self-association of larger peptides
during heat inactivation of enzymes [20] can also reduce the
ability of the enzyme to hydrolyse the proteins and decrease
the yield of FPH. This assumption was supported by the
percentage of hydrophobic amino acids, which was 34.6 Æ
2.2% in the non-soluble fraction, 1.3–1.5 times more than in
the FPH (24.7 Æ 1.6%). In addition, in raw material
containing relatively high amount of oil (10–30%), protein/
lipid complexes could be formed. These complexes also
seem to be more resistant to enzymatic breakdown and
extraction of oil and yield of FPH fraction will be reduced.
The relationship between amount of lipids and percentage of
solubilised proteins confirmed this assumption (Fig. 1). This
indicates that more attention should be given to the state of
the substrate before hydrolysis, because it may have a
considerable significance for the yields of soluble products
compared to a conventional enzyme process.
The crude protein recovery in FPH varied from 22%
(Flav/V À DT) to 44% (Neu/V + BB) of initial amount of
proteins in raw material. The control incubation of raw
material containing viscera and backbones (V + BB) with no
enzyme added (NE/V + BB) showed that 31% of proteins
present in the initial substrate could be found in the FPH
fraction without adding commercial enzymes. By adding
Flavourzyme and Neutrase the yield observed was 7.0 g/
100 g (Flav/V + BB) and 8.6 g/100 g (Neu/V + BB)
respectively, which corresponds to 34% and 44% protein
recovery from initial protein in raw material mixtures.
Running the hydrolysis without added water, protein
recovery was 14% (Flavourzyme) and 15% (Neutrase)
based on raw material. These results showed that addition of
water into the hydrolysis mixture is necessary in order to
increase protein recovery. Water extraction gave more than
two times higher protein recovery in the FPH fraction
compared to enzymatic hydrolysis without addition of water.
The protein in the FPH fraction was as expected more
hydrolysed than in the sludge. Degree of hydrolysis (DH)
varied between 18.5% and 33.7% for FPH and between
4.3% and 10.9% for sludge (Tables 3 and 4). 30 Æ 5% of the
crude protein from the raw material were obtained in the
FPH fraction, while 70 Æ 5% of the crude protein from the
raw material were obtained in the sludge. The amount of
proteins available for the enzymatic reaction was similar in
all mixtures: 18.2 Æ 0.2% on a fat free basis. 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 À DT till 34.7% for V + BB and
from 38.8% V À DT till 44.2% for V + BB with Neutrase.
DH depended on the enzyme used: Flavourzyme as exo-
peptidase on an average gave a higher degree of hydrolysis
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–1424 1419

6. R.Sˇlizˇyteetal./ProcessBiochemistry40(2005)1415–14241420
Table 3
Composition (g/100 g dry matter, mean Æ average relative S.D. of the measurement) degree of hydrolysis (%, mean Æ S.D.) and amount of free amino acids (mg/g dry sample, mean Æ S.D.) of dried FPH fraction
Parameter V À DT+BB V + BB V À DT V
Flavourzyme Neutrase Flavourzyme Neutrase Control Flavourzyme Neutrase Flavourzyme Neutrase
NEa
FlavNWb
NeuNWc
Moisture, mean Æ 3% 5.0 6.5 4.0 3.9 7.8 9.1 8.8 4.9 6.0 4.5 3.9
Proteins, mean Æ 1% 79.9 87.2 85.6 87.7 84.8 89.5 91.6 75.0 78.2 76.5 83.5
Lipids, mean Æ 4% 7.7 4.1 2.3 1.8 5.4 3.5 1.4 11.5 6.9 7.8 3.0
Ash, mean Æ 2% 10.4 9.9 10.6 9.7 12.4 11.3 10.2 13.9 11.8 13.0 12.4
Degree of hydrolysis 19.5 Æ 1.4 18.5 Æ 0.6 24.4 Æ 0.6 23.5 Æ 0.4 23.5 Æ 0.2 27.1 Æ 2.3 28.8 Æ 2.3 27.5 Æ 0.8 25.5 Æ 0.5 33.7 Æ 0.3 25.3 Æ 0.6
Free amino acids 40.8 Æ 2.1 15.2 Æ 0.9 76.4 Æ 9.2 67.9 Æ 8.1 62.4 Æ 6.4 84.8 Æ 0.2 55.1 Æ 0.4 82.8 Æ 7.0 25.2 Æ 0.2 96.6 Æ 2.9 62.4 Æ 3.8
a
Treatment: raw material and water with no enzymes added.
b
Treatment: raw material + Flavourzyme, no added water.
c
Treatment: raw material + Neutrase, no added water.
Table 4
Composition (g/100 g dry matter, mean Æ average relative S.D. of the measurement), degree of hydrolysis (%, mean Æ S.D.) and amount of free amino acids (mg/g dry sample, mean Æ S.D.) of dried sludge
fraction
Parameter V À DT + BB V + BB V À DT V
Flavourzyme Neutrase Flavourzyme Neutrase Control Flavourzyme Neutrase Flavourzyme Neutrase
NEa
FlavNWb
NeuNWc
Moisture, mean Æ 7% 2.0 2.3 1.9 2.0 2.2 3.0 2.8 1.9 1.4 1.3 2.0
Proteins, mean Æ 4% 63.1 58.7 60.1 55.5 63.5 64.8 70.0 66.3 61.4 55.1 55.0
Lipids, mean Æ 4% 23.2 29.9 25.4 31.6 24.6 18.0 14.9 31.4 33.4 28.4 30.9
Ash, mean Æ 6% 17.2 16.6 15.5 12.1 15.9 16.5 17.1 7.4 5.5 19.0 17.4
Degree of hydrolysis 5.3 Æ 0.6 6.9 Æ 0.2 5.1 Æ 0.1 5.4 Æ 0.3 4.7 Æ 0.4 10.9 Æ 0.3 10.2 Æ 0.3 4.3 Æ 0.3 5.6 Æ 0.1 5.0 Æ 0.2 6.3 Æ 0.3
Free amino acids 8.3 Æ 0.0 3.8 Æ 0.3 9.2 Æ 0.5 4.6 Æ 0.1 8.2 Æ 0.7 31.9 Æ 6.2 21.6 Æ 1.7 6.4 Æ 0.2 3.1 Æ 0.2 6.9 Æ 0.1 8.4 Æ 0.5
a
Treatment: raw material and water with no enzymes added.
b
Treatment: raw material + Flavourzyme, no added water.
c
Treatment: raw material + Neutrase, no added water.

7. for FPH, conversely Neutrase gave sludge with higher DH.
Initial composition of the raw material also influenced the
DH of FPH: raw material from V had the highest, while raw
material from V À DT + BB had the lowest DH values.
Consequently the amount of free amino acids (Table 3) was
significantly higher in the FPH than in the sludge.
The insoluble fraction was a grey layer of sludge on the
bottom of the vessels obtained after extraction and centrifuga-
tion. The amount of dry sludge varied between 12.6 and
21.8 g/100 g of raw material (Table 2), the lowest being
obtained after treatment with Neutrase. The presence of
indigestible contents (stones, sand and shells) in the digestive
tract of the fish increased the amount of sludge more than the
presence of backbone (V gave 16.0 and 18.5 g/100 g
compared to 15.5 and 17.0 g/100 g in V À DT + BB). The
largest amount of sludge was found in extractions without
added water (Flav/NW/V + BB and Neu/NW/V + BB). This
shows the need of water to facilitate the enzymatic hydrolysis
and wash out the soluble components. The control extraction
without enzymes gave results close to those of the same raw
material (V + BB) treated with Flavourzyme.
The DH in sludge of the control samples (without added
water) was significantly higher: between 10.2% and 10.9%
compared to 5.1–5.4% 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 was due to increased
concentration of free amino acids in the control hydrolysis
compared to the diluted system. However, the control
‘‘hydrolysis’’ of the same raw material under the same
conditions without added enzymes gave similar DH values
and amount of free amino acids in the FPH fraction: DH for
FPH was 23.5–24.4 for samples obtained with adding
commercial enzymes and 23.5 without adding enzymes. The
DH of FPH obtained using Flavourzyme (Flav/V + BB) and
Neutrase (Neu/V + BB) was only 4% and 0.1% higher than
the DH of soluble fraction obtained using only water (NE/
V+BB). The increase in DH in sludge using Flavourzyme
and Neutrase (Flav/V + BB and Neu/V + BB) was 7% and
15% compared to DH in sludge obtained using only water
(NE/V + BB). This can be due to the higher concentration of
free amino acids in the powders.
Emulsion is not a desired fraction after hydrolysis.
Therefore, reducing or avoiding formation of an emulsion
layer is one of the aims in the modelling of the hydrolysis.
The yield of dry emulsion layer varied between 0.4 and
2.7 g/100 g (Table 2). Flavourzyme treatment resulted in a
greater amount of emulsion (13–60%) relative to Neutrase
because the Neutrase is more efficient in breaking lipid/
protein emulsions (Fig. 2) [21,22]. Moreover Flavourzyme
treatment gave larger peptides, which also took part in an
emulsion formation. The highest yield of emulsion (2.3–
2.7 g/100 g) was measured in samples containing mainly
liver (V À DT), and the lowest (0.4–0.7 g/100 g) in samples
containing backbones. Only trace amounts of emulsion were
found in the control hydrolysis and these were not collected.
The amount of emulsion increase linearly with increasing
amount of lipids in the raw material (R2
= 0.79, Fig. 3) and
were negatively linearly correlated to the amount of proteins
in the raw material (R2
= 0.89, Fig. 3). The minimum amount
of lipids in the raw material should be more than 8.5 g/100 g
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–1424 1421
Fig. 2. The relationship between protein recovery and amount of the lipids in the FPH for both enzymes.

8. in order to form an emulsion. On the other hand, an increase
in amount of proteins in the raw material above 16.5 g/100 g
should decrease emulsion formation (Fig. 3). This effect was
partially observed in the control extraction without added
water, where the highly concentrated FPH fraction probably
prevented formation of emulsion. The lipase in the viscera
was active during sample preparation until heat inactivation.
This lipase could break down the tri-glycerides into mono-,
di-glycerides and free fatty acids, which can take part in
formation of emulsions [23].
Statistical analysisofdataindicated thatthe mostimportant
factor affecting the amount of all fractions was the chemical
composition of the raw material (P < 0.05). The type of
enzyme significantly influenced only the yield and the
composition of FHP and sludge (P < 0.05). Therefore the
type of enzyme used for the hydrolysis did not significantly
influence the amount of emulsion or/and oil fraction.
3.2. Model describing the relationship between yield of
different fractions and chemical composition of raw
material and enzymes used
The relationship between the chemical composition of
the raw material mixtures and yields of the different
fractions was assumed to be linear (Fig. 3). The statistical
model was therefore based on linear regression. The model
was accomplished by using four variables, searching for
coefficients a (lipids), b (proteins), c (ash) and d (moisture),
showing the magnitude of influence of each studied
component for yield of obtained fractions. Numerical
analysis of data showing the relationship between composi-
tion of raw material and type of enzyme used on the amount
of different fractions after hydrolysis is presented in Table 5.
The amount of oil fraction was influenced most by
amount of lipids and proteins in raw material, for instance
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–14241422
Fig. 3. The correlation between raw material and the yield of the fractions.
Table 5
Dependency of yield of hydrolysis products on composition of raw material and type of enzyme
Raw material Oil fraction Emulsion FPH Sludge
Flavourzyme Neutrase Flavourzyme Neutrase Flavourzyme Neutrase Flavourzyme Neutrase
Lipids 0.8745 0.9851 0.1381 0.1028 À0.0662 –0.0281 0.1008 0.0026
Proteins À1.1841 À0.7911 À1.4109 À1.0723 0.2909 0.3365 À1.4872 À0.3752
Ash 0.0199 À0.0687 1.1155 0.6501 0.236 À0.0244 2.5469 1.5432
Moisture 0.2099 0.0998 0.2524 0.2029 0.0292 0.0495 0.4589 0.2483

9. can the yield of oil fraction obtained after Flavourzyme
treatment be expressed by the following equation:
Yieldoil fr:=Flavourzyme
¼ 0:8745ðlipids in the raw materialÞ
þ ðÀ1:1841Þðproteins in the raw materialÞ
þ 0:0199ðash in the raw materialÞ
þ 0:2099ðmoisture in the raw materialÞ
Increased amounts of lipids in raw material gave an
increased amount of oil fraction depending on type of
enzyme (coefficients 0.87 and 0.98). However, increased
amounts of proteins in the raw material may reduce the yield
of oil fraction (coefficients À0.79 and À1.18). This shows
that proteins can entrap lipids and block the formation of an
oil fraction. The ash had a minor influence on the amount of
oil fraction (coefficients 0.02 and 0.07). On the other hand
the moisture of raw material had a positive influence on the
amount of oil fraction and other products after hydrolysis
(coefficients 0.03–0.46). It is probable that the higher
moisture content makes proteins more available for
enzymes, resulting in increased amounts of oil fraction,
emulsion, FPH and sludge.
According to the model, increasing amount of lipids in
the raw material increased the amount of emulsion.
Increasing the amount of proteins decreased the formation
of emulsion (confirming already presented data, Fig. 1).
The amount of FPH increased with increased amount of
proteins in raw material. The model indicated that Neutrase
as a more active enzyme in protein hydrolysis influenced
formation of FPH to a large extent (coefficients 0.2909 and
0.3365). Part of the ash, as mineral compounds, is water-
soluble and appears in the water-soluble part and has
positive influence on the yield of FPH. The increased
amount of lipids in raw material decreased the yield of FPH.
This can be due to interaction between proteins and lipids
before and/or during hydrolysis and movement of proteins to
emulsion or sludge fractions. In addition, higher amount of
fat in the raw material means that the separated oil fraction
will be large, leading to a proportional reduction of FPH
fraction after hydrolysis.
Ash and/or backbones in raw material had significant
influence on the amount of sludge. The relationship between
amount of proteins and formation of sludge was opposite:
more proteins in raw material gave less sludge after
hydrolysis. Amount of lipids in raw material did not
influence the amount of sludge using Neutrase, while it
increased amount of sludge using Flavourzyme.
3.3. Composition of different fractions
Table 3 shows the composition of freeze-dried FPH. The
dried product contained 3.9–6.5% moisture, which is
reported to be in the optimal range (5–7.5%) for storage
of dried FPH [24]. However the control samples contained
higher amount of moisture: 7.8–9.1%. The dried FPH had a
low water activity (0.07–0.21) which is low enough to
prevent microbial growth in the powders [25]. The
exceptions were the control samples, in which the water
activity varied between 0.25 and 0.39.
The residual lipids in FPH were surprisingly high for
some samples: up to 11.5% and increased with increasing
amount of lipids in raw material (R2
= 0.61). The amount of
lipids was dependent on the enzyme used for hydrolysis.
Compared to Neutrase, Flavourzyme produced powders
with 25–158% higher concentration of lipids. Fig. 2 shows
that FPH obtained with Flavourzyme contained more lipids
than the FPH from Neutrase, because Neutrase is more
efficient in breaking lipid/protein emulsions (Fig. 2) [21,22].
The amount of proteins in FPH varied from 75.0 to
91.6 g/100 g and was dependent on the amount of proteins in
raw material. The amount of ash was similar in all FPH and
varied from 9.7 to 13.9 g/100 g in FPH. The Neutrase
samples had from 6% to 25% lower ash content than samples
treated with Flavourzyme.
Freeze-dried sludge contained from 1.3% to 3.0%
moisture (Table 4), which gave water activity from less than
0.03andupto0.09.The proteinswerethe major compoundsin
sludgeandmakeup55–70%ofthe totalamountofsludge.The
largest amounts of proteins were found in the control
treatments followed by Flavourzyme treated samples.
The lipid concentration in dried sludge (except from the
control) varied in the range 23.2–33.4 g/100 g of total
amount of sludge. The lowest amount of lipids was found in
control hydrolysis without adding water (Table 2). However,
comparing concentration of lipids in the sludge with the
initial amount of lipids in raw material, a linear correlation
was found (y = 0.3444x À0.8851, R2
= 0.9443, Fig. 3). The
amount of lipids (Fig. 3, curve ’Lipids in sludge’) appears to
be ’caught and locked’ in the sludge up till approximately
2.6 g lipids/100 g in raw material. This is probably due to
formation of lipid/protein complexes. However, only when
the amount of lipids reached about 6 g/100 g, did the lipids
start to move into the oil fraction. The amount of lipids in
sludge calculated on a protein basis (Fig. 3) showed that the
lower amount of proteins in the raw material gave more
lipids in the sludge. The treatment with Neutrase gave higher
amounts of lipids in sludge, while Flavourzyme left more
lipids in FPH (Table 3). This was due to different efficiency
of enzymes and probably to distribution of different type of
lipid classes during enzymatic treatment [26].
The amount of ash in the sludge (Table 4) varied from 5.5
up to 19.0 g/100 g of sludge and was dependent on the
composition of the raw material. The presence of backbones
and/or digestive tract (containing sand, carapaces and small
stones) increased the amount of ash. The Neutrase treated
sludge also had a relatively lower amount of ash due to a
higher amount of lipids.
Due to negligible amounts of emulsion after hydrolysis
not all emulsions were collected and analysed. The freeze-
dried emulsions contained approximately 1.3 Æ 0.6 g/100 g
R. Sˇlizˇyte et al. / Process Biochemistry 40 (2005) 1415–1424 1423

10. water and had a water activity of 0.11. Amount of ash in the
emulsions varied from 1.2 (V À DT + BB) to 6.0 g/100 g
(V + BB) and was not influenced by type of enzyme used.
4. Conclusions
By combining different parts of fish by-products,
different mixtures of substrate for hydrolysis can be made.
The composition and state of this substrate influences both
the hydrolysis process and the yield and composition of the
hydrolysis products. Raw material containing the highest
amount of lipids gave highest amount of oil fractions and
the lowest percentage of solubilised proteins. A model
describing the relationship between yield of different
fractions and chemical composition of raw material and
enzymes used was developed.
The most important factor influencing the yield of the
different fractions was added water rather than type of enzyme
used. The highest lipid yield was obtained in samples without
addition of water, resulting in a decreased yield of FPH.
Formation of emulsion can be avoided or reduced by
using enzymes, which are more efficient in breaking lipid/
protein emulsion. Moreover reduction of emulsion can be
reached by reduction and/or elimination of addition of water
into hydrolysis mixture.
Comparison of enzymes used showed that the use of
Neutrase gave more FPH (23–57%) than Flavourzyme. Also
the amount of oil fraction obtained after Neutrase treatment
was up to 10% higher (except for the V À DT + BB samples)
compared to Flavourzyme. This was due to 13–60% smaller
layer of emulsion and higher degree of hydrolysis in sludge,
liberating more lipids from sludge to oil fraction.
The state and conditions of the substrate influences the
hydrolysis process, yield and composition of hydrolysis
products. The heat denatured proteins seem to be highly
resistant to enzymatic breakdown, which could reduce the
yield of lipids and increase the presence of lipids in sludge
fraction. More attention should be paid to the temperature
during inactivation and hydrolysis, with the aim of getting
higher yields of FPH and lipids. To achieve better utilisation
of all fish by-products it is also necessary to pay more
attention to the water non-soluble part after hydrolysis. The
sludge contained a relatively high amount of lipids (up to
33.4 g/100 g of dry sludge).
Acknowledgements
Authors wish to thank The Norwegian Research Council
and European Committee (project QLK1-CT-2000-01017)
for financial support to carry out experiments and to prepare
this paper. Anna Ivanova, Ph.D. student at the Centre for
Ships and Ocean Structures, Marine Technology Centre,
NTNU, Norway is thanked for the assistance in the statistical
data treatment.
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