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Process Biochemistry 40 (2005) 2021–2033

Characteristics of protein fractions generated from hydrolysed cod

(Gadus morhua) by-products

Rasa Slizytea,b, Egidijus Dauksasa, Eva Falcha,b, Ivar Storrøa, Turid Rustadb,*

aSINTEF Fisheries and Aquaculture, Processing, N-7465 Trondheim, NorwaybDepartment 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)

* 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

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

R. Slizyte et al. / Process Biochemistry 40 (2005) 2021–20332022

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. Slizyte 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 � S.D.)

Composition V-DT + BB V + BB V-DT V

Moisture 64.5 � 0.1 70.5 � 0.5 60.7 � 0.1 60.0 � 0.0

Lipids 17.9 � 0.6 9.6 � 0.2 26.9 � 1.3 21.0 � 0.5

Proteins 15.4 � 0.2 16.1 � 0.4 12.6 � 1.2 14.9 � 2.3

Ash 3.6 � 1.4 3.6 � 0.2 1.7 � 0.2 4.4 � 0.3

V-DT + BB: viscera without digestive tract and backbone; V + BB: viscera

and backbone; V-DT: viscera without digestive tract; V: viscera.

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 � 4 cm in length and 2.05 �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 � 5 8C for

5 min. After cooling the mixtures were vacuum packed and

kept at �40 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 � 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-

ments were performed in duplicate. Ash content was estimated

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. Slizyte et al. / Process Biochemistry 40 (2005) 2021–20332024

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 � 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 � 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 = �0.468 + 0.45[LEU] � 0.105[TYR]

PERb = �1.816 + 0.435[MET] + 0.780[LEU] + 0.211[HIS]

� 0.944[TYR]

PERc = 0.08084[P

AA7] � 0.1094,

P

where 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

�24 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 � 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 � 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 � 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%.

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Table 3

Composition of dried FPH and sludge fractions, g/100 g dry matter (mean � 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 � 0.0 6.5 � 0.6 4.0 � 0.0 3.9 � 0.1 7.8 � 0.1 9.1 � 0.1 8.8 � 0.2 4.9 � 0.0 5.6 � 0.0 4.5 � 0.6 3.9 � 0.1

Sludge 2.0 � 0.1 2.3 � 0.1 1.9 � 0.1 2.0 � 0.1 2.2 � 0.0 3.0 � 0.2 2.8 � 0.4 1.9 � 0.0 1.4 � 0.1 1.3 � 0.1 2.0 � 0.4

Proteins FPH 79.9 � 1.7 87.2 � 1.3 85.6 � 1.0 87.7 � 0.4 84.8 � 0.7 89.5 � 1.0 91.6 � 0.4 75.0 � 1.2 78.2 � 0.6 76.5 � 1.5 83.5 � 1.3

Sludge 63.1 � 1.4 58.7 � 1.7 60.1� 2.5 55.5 � 2.3 63.5 � 1.8 64.8 � 5.7 70.0 � 2.1 66.3 � 1.8 61.4 � 1.9 55.1 � 2.5 55.0 � 2.6

Lipids FPH 7.7 � 0.1 4.1 � 0.1 2.3 � 0.1 1.8 � 0.1 5.4 � 0.2 3.5 � 0.1 1.4 � 0.1 11.5 � 0.4 6.9 � 0.0 7.8 � 0.6 3.0 � 0.1

Sludge 23.2 � 1.0 29.9 � 0.8 25.4 � 0.4 31.6�1.7 24.6 � 0.4 18.0 � 0.3 14.9 � 1.0 31.4�0.5 33.4 � 3.9 28.4 � 1.3 30.9 � 1.5

Ash FPH 10.4 � 1.0 9.9 � 0.2 10.6 � 0.1 9.7 � 0.1 12.4 � 0.2 11.3 � 0.0 10.2 � 0.1 13.9 � 0.1 11.8 � 0.0 13.0 � 0.1 12.4 � 0.2

Sludge 17.2 � 2.6 16.6 � 1.5 15.5 � 1.2 12.1 � 0.3 15.9 � 0.2 16.5 � 0.4 17.1 � 2.9 7.4 � 0.0 5.5 � 0.2 19.0 � 0.7 17.4 � 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 � 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 � 0.1 11.5 � 0.1 4.2 � 0.1 3.5 � 0.2 4.4 � 0.2 5.3 � 0.2 6.2 � 0.4 20.9 � 0.7 22.2 � 0.1 13.4 � 1.0 14.6 � 0.3

FPH 6.00 � 0.3 7.7 � 0.1 7.0 � 0.1 8.6 � 0.3 6.2 � 0.1 2.7 � 0.0 2.8 � 0.1 4.0 � 0.8 6.4 � 0.1 5.7 � 0.1 7.3 � 0.2

Sludge 17.0 � 0.4 15.5 � 0.3 18.6 � 0.2 17.1 � 0.8 18.8 � 0.3 21.8 � 0.3 20.9 � 0.9 15.3 � 0.2 12.6 � 1.2 18.5 � 0.7 16.0 � 0.6

Emulsion 0.7 � 0.1 0.5 � 0.1 0.4 � 0.1 0.4 � 0.1 tr. tr. tr. 2.7 � 0.1 2.3 � 0.1 1.8 � 0.1 1.1 � 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.

R. Slizyte 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.

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

Fig. 2. Relationship between amount of free amino a

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.

cids and degree of hydrolysis of FPH fractions.

R. Slizyte 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: �0.468 + 0.45[LEU] � 0.105 [TYR].b PER: �1.816 + 0.435[MET] + 0.780[LEU] + 0.211[HIS] �

0.944[TYR].c PER: 0.08084[

PAA7] � 0.1094, where

PAA7 = 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.

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 � 5.0)% of the protein in the raw material were

obtained in the FPH fraction, while (70.0 � 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 � 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. Slizyte 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.

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 of hydrophobic amino acids in the

non-soluble fraction was (34.6 � 2.2%), which is 1.3–1.5

times higher than in the FPH (24.7� 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. Slizyte et al. / Process Biochemistry 40 (2005) 2021–2033 2029T

able

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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. Slizyte et al. / Process Biochemistry 40 (2005) 2021–20332030

Fig. 4. Relationship between WHC and amount of certain amino acids in the FPH.

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

Fig. 5. Relationship between fat absorption an

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 � 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

d amount of lipids in the FPH powder.

R. Slizyte et al. / Process Biochemistry 40 (2005) 2021–2033 2031

Fig. 6. Relationship between fat absorption and amount of phospholipids in the FPH powder.

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. Slizyte et al. / Process Biochemistry 40 (2005) 2021–20332032

Fig. 7. Relationship between fat absorption and amount of certain amino acids in the sludge.

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 � 4.7% of protein, while FPH

contained 29.5 � 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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