Journal of Aquatic Food Product Technology

ISSN: 1049-8850 (Print) 1547-0636 (Online) Journal homepage: http://www.tandfonline.com/loi/wafp20

Bitterness in Fish Protein Hydrolysates and

Methods for Removal

Egidijus Dauksas , Rasa Slizyte , Turid Rustad & Ivar Storro

To cite this article: Egidijus Dauksas , Rasa Slizyte , Turid Rustad & Ivar Storro (2004) Bitterness
in Fish Protein Hydrolysates and Methods for Removal, Journal of Aquatic Food Product
Technology, 13:2, 101-114, DOI: 10.1300/J030v13n02_09

To link to this article: http://dx.doi.org/10.1300/J030v13n02_09

Published online: 11 Oct 2008.

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Bitterness in Fish Protein Hydrolysates

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and Methods for Removal

Egidijus Dauk as
Rasa li yte
Turid Rustad
Ivar Storrø

ABSTRACT. Enzymatic hydrolysis is a processing method for recover-

ing protein from under utilized fish biomass and fish by-products. How-
ever, the hydrolysis process often creates bitter taste in the product. The
bitterness restricts the practical uses of these hydrolysates. The presence
of bile in whole fish and fish viscera is shown to cause bitterness in fish
protein hydrolysates. The fat and ash content could also cause bitter
taste. The content of total amino acids and hydrophobic amino acids did
not correlate with bitterness.
Three different methods were used to eliminate or reduce bitterness
from FPHs after enzymatic hydrolysis with commercial enzymes:
(1) treatment with endopeptidases (Flavourzyme®), (2) extraction with
butanol and (3) treatment with cholestyramine resin. Flavourzyme® did

Dr. Egidijus Dauk as is Researcher, SINTEF, Fisheries and Aquaculture, Norway

(E-mail: Egidijus.Dauksas@sintef.no).
Rasa li yte is a PhD Student, Department of Biotechnology, Norwegian Univer-
sity of Science and Technology, Norway (E-mail: Rasa.Slizyte@sintef.no).
Dr. Turid Rustad is Associate Professor, Department of Biotechnology, Norwegian
University of Science and Technology, Norway (E-mail: turid.rustad@biotech.ntnu.no).
Dr. Ivar Storrø, Senior Researcher, SINTEF, Fisheries and Aquaculture, Norway
(E-mail: Ivar.Storro@sintef.no).
Address correspondence to: Dr. Egidijus Dauk as, SINTEF, Fisheries and Aqua-
culture, N-7465 Trondheim, Norway.
The authors thank The Norwegian Research Council (project 134407/140) and The
European Commission (project QLK1-CT-2000-01017) for financial support.
Journal of Aquatic Food Product Technology, Vol. 13(2) 2004
http://www.haworthpress.com/web/JAFPT
 2004 by The Haworth Press, Inc. All rights reserved.
Digital Object Identifier: 10.1300/J030v13n02_09 101
 102 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY

not reduce the bitterness. The use of butanol and cholestyramine resin
separately or in combination reduced the bitter taste from FPH to levels
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barely discernible by our sensory panel in 1% concentration. [Article cop-

ies available for a fee from The Haworth Document Delivery Service:
1-800-HAWORTH. E-mail address: <docdelivery@haworthpress.com> Web-
site: <http://www.HaworthPress.com>  2004 by The Haworth Press, Inc. All
rights reserved.]

KEYWORDS. Bitterness, hydrolysis, bile, viscera, cod, Gadus morhua,

by-products

INTRODUCTION

Biologically processed fish have long traditions in South-East Asia where

fish sauce is the major product. Fermented fish were also known in ancient
Greece and Rome (Corcoran, 1963). Shortly after the Second World War, Ca-
nadian researchers developed methods for the enzymatic preparation of pro-
tein hydrolysates from fish meat (Tarr, 1948).
One of the shortcomings for the wide application of the protein hydroly-
sates is their bitter taste. The possible sources of bitter taste may be the compo-
sition of starting material or the hydrolysis process itself. Hydrolysates of
whole fish and fish viscera will contain the bile from the gall bladder. The bile
contains bitter components and may influence the bitterness of the fish protein
hydrolysate. The bitter tasting components in bile are cholic and taurocholic
acid. Cholic acid is slightly soluble in water, but is soluble in alcohol (JECFA,
1973) like the hydrophobic amino acids and peptides. The most common way
to remove bile compounds is to complex the bile acids to compounds that can
be easily removed from the mixture. Kahlon and Woodruff (2002) used soy
protein, pinto beans, black beans or wheat gluten to bind bile acids in vitro.
They found that black beans had the highest binding ability. Cholestyramine
resin is also known to bind bile acids due to its cationic properties.
In the hydrolysis process peptides are formed. The chain length of the pep-
tides is of special interest in relation to the organoleptic and functional charac-
teristics, because properties such as solubility, emulsifying capacity and
bitterness depend, at least in part, on molecular size. Bitterness is also related
to the average hydrophobicity of the peptide (Mohr, 1980). Shahidi (1994)
claimed that the taste of products obtained by proteolytic modification of pro-
teins depends on degree of hydrolysis. Adler-Nissen (1984) showed that a low
bitterness could usually be ascertained by restricting the degree of hydrolysis
(DH) to values of 3-5%. Alternatively, a high DH indicating a complete hy-
drolysis to free amino acids decreases the bitterness, because hydrophobic
 Daukšas et al. 103

peptides are considerably more bitter than the corresponding mixture of free
amino acids (Belitz and Wieser, 1976). The highest risk of bitterness is when
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DH is between 4 and 40%.

The bitterness of small peptides is highest when the hydrophobic amino ac-
ids are blocked at both ends through a peptide bond. The bitterness is lower
when hydrophobic amino acids are in the C- or N-terminal position and lowest
when the amino acids are free (Pedersen, 1994). If bitter peptides are already
present, debittering may be achieved by application of specific peptidases
(Sugiyama et al., 1991). By applying Flavourzyme® with its combination of
exopeptidase and endopeptidase activity, the risk of bitterness is minimized
(Novozymes, 1999). The exopeptidase activity is able to remove the bitterness
of a bitter peptide by removing hydrophobic terminal amino acids. A reduced
bitterness can also be obtained by iso-electric precipitation of bitter peptides
(Adler-Nissen, 1984). Suh et al. (2000) described a procedure for the debittering
of protein hydrolysates with active carbon. However, the removal of bitter
peptides resulted in reduced yield and reduced nutritional quality of the hy-
drolysates (Stevenson et al., 1998).
The bitter taste of autolytic extracts can also be enhanced by shaking treat-
ment enhancing oxygen transfer leading to increased lipid oxidation (Liu et
al., 2000).
The objective of this work was to determine how the presence of bile in the
starting material influences the bitterness in the FPH, and to compare the influ-
ence of the bile on the bitter taste relative to the other bitter compounds in the
fish protein hydrolysates. If bitter compounds from bile are present in the start-
ing material, it may not be enough to prevent formation of bitter peptides dur-
ing hydrolysis, but removal of bile acids may also be necessary. Three
different methods were used to eliminate or reduce bitterness from FPHs after
enzymatic hydrolysis: (1) secondary treatment with endopeptidases (Flavour-
zyme®), (2) treatment with butanol and (3) treatment with cholestyramine resin.

MATERIALS AND METHODS

Farmed cod (Gadus morhua) were slaughtered in November 2002. The di-
gestive tract and the gall bladder from 31 fish (length 59 ± 4 cm and weight
3600 ± 500 g) were used for the experiments. The fish were kept on ice over-
night, eviscerated and hand filleted in a cold room (+4°C). Stomach, caecum,
intestines and gall bladder were separated and the weight of each fraction re-
corded for each fish. The fractions were stored on ice for 1-6 hours before
mincing. The fractions were minced separately twice in a manual mincer with
10 mm holes and homogenized in the kitchen homogenizer (Siemens) for 5
min, vacuum packed and kept at ⫺80°C.
 104 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY

Other materials and reagents: Flavourzyme® 500 L (protease/peptidase,

activity 500 LAPU/g), Alcalase® 2.4 L (protease, activity 2.4 AU/g) from
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Novozymes A/S (Denmark), n-butanol (HPLC grade), cholestyramine resin

(copolymer of styrene and divinylbenzene with quaternary ammonium func-
tionality, CAR), methanol, chloroform and caffeine (99%) were from Sigma-
Aldrich.
Hydrolysis. After thawing (~0°C), the different parts of fish viscera were
mixed in proportions similar to the average of the proportion in the live fish
(Table 1) and 50 g of sample were mixed with 50 ml distilled water. The hy-
drolysis was performed in a closed 1L plastic vessel at 50°C in a shaking water
bath. When the temperature of the mixture had reached 50°C, the enzymatic
hydrolysis was initiated by adding 0.1% (by weight of starting material)
Flavourzyme® or Alcalase®. The experiment was executed in three steps (Fig-
ure 1):

A. Enzymatic hydrolysis by Flavourzyme® or Alcalase® followed by inac-

tivation of enzymes by microwave heating (850 W, 95 ± 5°C, 5 min) and
rapid chilling. This procedure gave level A-mixtures: A1, A2, A7 and A8.
B. Two samples hydrolyzed by Alcalase® were not inactivated but addi-
tionally hydrolyzed by Flavourzyme® (0.1% by weight of starting mate-
rial) for 60 min, followed by microwave inactivation leading to mixture
B11 and B13. All four A-level mixtures were treated with n-butanol
(BuOH) or cholestyramine resin (CAR) producing level B-mixtures
(B3, B5, B9 and B12) and (B4, B6, B10 and B14), respectively.
BuOH 100 ml was added (1:1) and the vessels placed in a 50°C shak-
ing water bath for 30 min. After centrifugation at 2250 ⫻ g for 30 min
the BuOH layer was removed. The extraction procedure was repeated
twice and the BuOH fractions pooled.
Cholestyramine resin (5 g) was added to level A-mixtures and the ex-
traction procedure performed as described for butanol treatment but not
repeated.
C. Some of the B-mixtures were treated a second time by BuOH or CAR
producing the C-mixtures (C15 to C18). The performed procedures
were as described for level B-mixtures.

All mixtures were centrifuged as described. The aqueous phase was sepa-
rated, and freeze dried in a Hetosicc freeze dryer (CD 13-2, Heto AB Equip-
ment A/S, Denmark) and stored at ⫺18°C until evaluation of the bitter taste
and chemical analysis.
Evaluation of bitterness. A group of 12 people evaluated the bitter taste of
the hydrolysates. The participants in the sensory panel were selected among
students and university staff on the basis of their threshold level for bitter taste
 Daukšas et al. 105

TABLE 1. The mixture of starting material, %.

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Body part With gallbladder Without gallbladder

Stomach (with content) 40 43
Gallbladder 5 -
Intestines 25 26
Pyloric (Caecum) 30 31
Total 100 100

FIGURE 1. The experimental design.

Starting material

Gall- No gall- Gall- No gall-

bladder bladder bladder bladder

Flavour-
zyme® Alcalase®

A1 A2 A7 A8

Flavour- Flavour-
Butanol Butanol CAR Butanol CAR
CAR zyme® Butanol zyme® CAR

B3 B4 B5 B6 B9 B10 B11 B12 B13 B14

Butanol Butanol CAR Butanol

C15 C16 C17 C18

and trained using caffeine solutions (0%, 0.006%, 0.014% and 0.027%). A
concentration of 1% of fish protein hydrolysate (FPH) in water was presented
for evaluation. A ranking test was used and the bitterness was evaluated in
range from 0 (no presence of bitterness) up to 5 (very bitter). The relative stan-
dard deviation of the mean (RSDOM) of bitter taste was 2.8%. One or two ref-
erence samples were included in each sensory evaluation.
 106 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY

Chemical analysis. Total N was determined using CHN-S/N elemental ana-

lyzer 1106 (Carlo Erba Instruments S.pA., Milan, Italy) and amount of crude
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protein was estimated by multiplying total N by 6.25. The total lipid content in
the samples was determined according to the method of Bligh and Dyer
(1959). Ash content was estimated according to AOAC (1990). The degree of
hydrolysis was evaluated as the proportion (%) of α-amino nitrogen with re-
spect to the total N in the sample (Taylor, 1957). Amount of free amino acids
was determined by reversed phase HPLC by pre-column fluorescence der-
ivatization with o-phthaldialdehyde, using a NovaPak C18 cartridge (Waters,
Milford, MA, USA) and standard reference solution (The Sigma Amino Acid
standard nr. AA-S-18 together with Asn, Gln and Aba) according to the
method of Lindroth and Mopper (1979) as modified by Flynn (1988). Glycine/
arginine and methionine/tryptophane were determined together, as their peaks
merged. The pH of hydrolysates was measured by Philips PW 9420 pH meter
(Pye Unicam LTD, England), electrode: Unicom (type No. 9436-095-84003).
Statistical analysis. Depending on the method, the tests were done in dupli-
cates-sextuples. The statistical programme Guideline (Camo ASA, Norway)
and Microsoft Excel were employed for data processing and statistical analy-
sis. Significance level was set at 95%.

RESULTS AND DISCUSSION

Bitterness

The effect of gallbladder on bitterness. The sensory evaluation of mixtures

(A1, A2, A7 and A8, Figure 2) showed the influence of bile on bitterness. The
hydrolysates free from bile (A2 and A8) have a relatively low value of bitter-
ness (‘2.1’ and ‘2.7’) compared to hydrolysates obtained from starting mate-
rial with gall bladder (A1 and A7 with values of ‘4.0’ and ‘4.2’). These results
and statistical evaluation (Table 2) show that presence of bile in the starting
material clearly influence the bitter taste of the fish protein hydrolysate made
from fish viscera.
Effect of type of enzyme on bitterness. The hydrolysates obtained by use of
Alcalase® were more bitter than hydrolysates obtained using Flavourzyme®
(A1, A7 and A2, A8, Figure 2). The type of enzyme had a significant influence
(Table 2, p = 0.0018) on bitterness. However, the difference in bitterness was
smaller in hydrolysates produced from bile containing starting material com-
pared to difference in hydrolysates from starting material without bile. This in-
dicates that bitterness from bile had more influence on the bitter taste in these
hydrolysates than the enzymes used.
 Daukšas et al. 107

FIGURE 2. Bitterness of FPH as a function of dry yield of FPH, RSDOM for bit-
terness 2.8%, RSDOM for yield 0.93%.
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4.5
A7
4.0 A1 B11

3.5

3.0
B13
A8
Bitterness

2.5 B9

B12 A2
2.0
B3 C16
C18
1.5
C15 B5
1.0
C17 y = 0.5385x⫺4.6072
B4 B10 2
0.5 B14 R = 0.8314
B6
0.0
9 10 11 12 13 14 15 16
Yield of FPH, g/100 g of wet starting material

TABLE 2. Main and interaction effect (p-value) of different factors for bitterness
and yield measurement.

Compound/Procedure Bitterness Yield

Raw material (± gallbladder) 0.0000 0.0011
Enzymes (Alcalase®/Flavourzyme®) 0.0018 0.0027
Treatment (BuOH/CAR/Flavourzyme®) 0.0000 0.0000
Raw material/Enzymes 0.1622 0.6045
Raw material/Treatment 0.0000 0.1721
Enzymes/Treatment 0.0074 0.0743

Reduction of bitterness by BuOH treatment. The extraction with BuOH re-

duced bitterness in all treated mixtures. The reduction in bitterness from sam-
ples containing bile (A1-B3 = 2.1 and A7-B9 = 1.7) was higher than for samples
without gallbladder (A2-B5 = 0.9 and A8-B12 = 0.7). This indicates that BuOH
reduces bitterness more in samples containing bile acids, than in samples with-
out bile. This is probably due to extraction of bile compounds. The level of bit-
terness after the first BuOH treatment (B-mixtures) was ‘1.9,’ ‘1.2,’ ‘2.5,’ and
‘2.0’ showing variable bitterness dependent upon starting material.
 108 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY

Removal of bitterness by cholestyramine resin. The extraction with CAR

reduced bitterness in all treated mixtures. The reduction in bitterness from
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samples containing bile (A1-B4 = 3.3 and A7-B10 = 3.3) was higher than for
samples without gall bladder (A2-B6 = 1.7 and A8-B14 = 2.1). This indicates
that CAR reduces bitterness more in samples containing bile acids, than in
samples without bile. The reduction of bitterness is also larger using CAR than
using extraction with BuOH. The level of bitterness after the first CAR treat-
ment (B-mixtures) was ‘0.7,’ ‘0.4,’ ‘0.9,’ ‘0.6’ showing very low and fairly
constant bitterness for all B mixtures with minimal dependence upon starting
material.
Removal of bitterness by Flavourzyme®. Using Flavourzyme® after hydro-
lysis with Alcalase®, a reduction in bitterness was expected, due to the ability
of Flavourzyme® to remove terminal hydrophobic amino acids. The bitterness
in hydrolysates was reduced by 0.3 conditional units (from A7 to B11) or in-
creased by 0.1 (from A8 to B13) when Alcalase® and Flavourzyme® worked
together the last hour of hydrolysis. The use of Flavourzyme® in cooperation
with Alcalase® did not reduce the bitterness in the samples tested.
Secondary treatment by BuOH and cholestyramine resin. This secondary
treatment was tried in order to remove bitter tasting compounds left from the
first treatment, assuming the different treatments were working selectively.
The secondary treatment by BuOH (C16 and C18) shows that it is possible to
use BuOH after Flavourzyme® treatment to decrease bitterness. This treat-
ment reduced bitter taste from ‘3.9’ to ‘2.0’ and from ‘2.8’ to ‘1.9’ for starting
material with and without bile. CAR treatment reduced mixture C17 (Alcalase®
hydrolysed and primary Flavourzyme® treated) to even lower bitterness (‘0.9’).
However, the BuOH-extraction was not effective to remove bitterness after
prior treatment with CAR (C15).
The statistical analysis of bitterness (Table 2) shows that chemical treat-
ment had a significant influence on bitterness. However, there was no signifi-
cant interaction effect between starting material and enzymes, except for the
interaction between starting material and treatment (p = 0).
Removal of bitterness from hydrolysates can be achieved in several differ-
ent ways. The easiest way to reduce bitter taste in hydrolysates of fish viscera
is to remove the gall bladder. By using BuOH and CAR it was possible to im-
prove the taste of hydrolysates. A negative effect of using BuOH was the pres-
ence of ‘plastic’ taste in dried hydrolysates, and powders produced from
starting material with gall bladder had a dirty green color. The CAR treated
samples either had a reduced or no detectable bitter taste and the powders had
a light yellow color. Endopeptidases (Flavourzyme®) did not reduced bitter-
ness in hydrolysates produced by Alcalase® in these experiments.
 Daukšas et al. 109

Yield
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The effect of gallbladder on yield. The bile is an isotonic solution contain-

ing about 90% of water. However, the presence of bile in starting material (5%
w/w) increased the yield of dry hydrolysates by 7.7-11% (A1, A7-A2, A8, cal-
culation on the bile free starting material) significantly (Table 2). This is par-
tially due to the increased emulsification of lipids, increasing the lipid content
of the water phase (about 50-60% of the increase) and increased amount of
mineral compounds in hydrolysates (about 10% of the increase). No signifi-
cant increase in the amounts of free and total amino acids that could explain
the increase in yield was found.
The effect of type of enzyme on the yield (A samples). The hydrolysates with
the lowest bitterness also had lower yields of FPH (Figure 2). The yields are
presented as gram dry weight hydrolysate per 100 g of starting material
(RSDOM = 0.93%). It was found that by using Alcalase® the yield (A7, A8,
Figure 2) was slightly (up to 6%) but significantly (p = 0.0027, Table 2) higher
compared to yields using Flavourzyme® (A1, A2). This may be explained by
the higher hydrolyzing power of Alcalase®.
Effect of primary treatment on yield (B samples). The hydrolysis with
Alcalase® for 60 min and additional treatment with Flavourzyme® for 60 min
(together with Alcalase®, B11, B13) gave a 6.9-9.9% increase in yield com-
pared to only Alcalase® or Flavourzyme® hydrolyzed samples (A1, A2, A7,
A8). This shows that the hydrolysis was close to completion after 60 min with
Alcalase® alone, this is also supported by the high degree of hydrolysis (Ta-
ble 3).
Effect of secondary treatment on yield (C samples). All chemical treatments
were found to reduce yield of hydrolysates. The average reduction in yield af-
ter treatment by CAR was 30-31%, while the reduction using BuOH varied be-
tween 16-18%. The statistical analysis (Table 2) shows that the secondary
treatment had the largest influence on the yield (p = 0.0000). However, no sig-
nificant interaction effects were found on yield.

Bitter Compounds

A linear correlation was found between bitterness and yield of hydrolysate

(r = 0.91, Figure 2). This implies that the bitter taste was probably caused by a
compound or compounds proportional to the yield of FPH.
The amount of crude proteins in the hydrolysates varied from 7.51 to 12.13
g/100 g of starting material (RSDOM = 0.22%, Figure 3). The lowest amounts
were found in hydrolysates produced from bile-containing starting material
(B6, B14). The chemical treatment of these hydrolysates decreased the amount
of peptides due to extraction or binding of some peptides and amino acids. The
treatment by Flavourzyme® increased the amount of crude proteins and re-
 Downloaded by [University of Auckland Library] at 15:35 13 November 2017

110
TABLE 3. Degree of hydrolysis and pH in FPH.

Sample A1 A2 B3 B4 B5 B6 A7 A8 B9 B10 B11 B12 B13 B14 C15 C16 C17 C18
DH, % 45.50 43.49 40.14 45.54 42.71 46.83 40.67 42.23 38.46 48.00 46.90 38.97 47.46 44.88 37.24 41.91 45.47 45.46
pH 6.40 6.42 6.72 6.14 6.67 6.30 6.38 6.41 6.71 6.23 6.38 6.77 6.37 6.28 6.34 6.85 6.22 6.81
 Daukšas et al. 111

FIGURE 3. Relationship between bitterness and amount of crude proteins in

FPH and, RSDOM for bitterness 2.8%, RSDOM for yield 0.17%.
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4.5
A7
4.0 B11
A1

3.5

3.0
A8 B13
Bitterness

2.5 B9

B12 A2
2.0 C16
B3 C18 y = 0.7146x⫺4.8373
2
R = 0.7521
1.5
B5
C15
1.0 C17
B4 B10
0.5 B14
B6

0.0
7 8 9 10 11 12 13
Crude proteins, g/100 g of wet starting material

sulted in a small increase in bitter taste. A linear correlation was found be-
tween the amount of proteins in hydrolysates and bitterness (r = 0.87) and the
bitterness increased with increasing amount of crude proteins in the hydroly-
sates.
The amount of fat (RSDOM = 1.37%) and ash (RSDOM = 0.25%) are pre-
sented in Figure 4. The highest concentrations of fat were found in hydroly-
sates produced from starting material with gall bladder and without treatment
(0.64-0.71 g/100 g, A1, A7, B11) probably due to emulsification of fat in the
FPH fraction. Hydrolysates produced using Alcalase® had more fat (0.27-0.29
g/100 g, A8, B13) compared to Flavourzyme® produced hydrolysates (0.12 g/
100 g, A2) from the same bile free starting materials. As expected, the BuOH
and CAR treated samples had reduced amount of fat (down to 0.01-0.07 g/
100 g). CAR (a hydrophobic resin) will probably also bind non-specific hy-
drophobic compounds. The bitterness was found to increase linearly with in-
creasing amount of fat in hydrolysates, and the correlation coefficient was r =
0.88.
The amount of ash in hydrolysate powders varied between 0.78 and 1.10 g/
100 g. The highest amounts were obtained in hydrolysates that had not been
chemically treated. A negative correlation (r = 0.82) was found between
amount of ash and bitterness.
 112 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY

FIGURE 4. Relationship between amounts of fat (䊏) and ash (♦) in FPH and bit-
terness, RSDOM for bitterness 2.8%, RSDOM for yield of fat 0.01%, RSDOM
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for yield of ash 0.02%.

4.5
A7
A7
4.0 A1 A1
y = 4.4123x + 1.1859 B11
2 B11
R = 0.7761
3.5

3.0
B13 A8
A8 B13
Bitterness

2.5 B9 B9
A2
C16 B12 A2
2.0 C16
C18 B12 B3
B3 C18
1.5
B5 B5
C15 C17
1.0 B10 C15
C17B4 y = 10.187x⫺7.6467 B10
2
R = 0.6566 B4
0.5 B14 B14
B6 B6
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Yield, g/100 g of wet starting material

The composition, pH and degree of hydrolysis and properties of obtained

hydrolysates are presented in Table 3. The degree of hydrolysis (DH) varied
from 37 to 48% (RSDOM = 0.29%). A higher DH was found in hydrolysates
produced by Flavourzyme® (A1, A2) compared to hydrolysates produced by
Alcalase®. BuOH treatment reduced the DH by 13% (B3, B5, B9, B12, C15,
C16, C17), but CAR treatment increased the DH by up to 18% (B4, B6, B10,
B14, C17) due to selective removal of amino acids or other nitrogen-contain-
ing peptide compounds. No correlation was found between DH and bitter-
ness.
The pH (RSDOM = 0.13%) was measured in each hydrolysate. The pH was
slightly lower than neutral (6.2-6.8) and was not influenced by the type of en-
zymes used or by the presence of bile in starting material (pH 6.4). The treat-
ment with CAR reduced the pH value to 6.1-6.3 while the treatment by BuOH
increased the pH value to 6.7-6.9. Even secondary treatment by BuOH (No.
15) after CAR increased the pH. No correlation was, however, found between
pH value and bitterness.
The compositions of total amino acids (52-92% of the total crude proteins)
and free amino acids (28-62% of the total amino acids) were dependent on
starting material, hydrolysis and treatment (data not presented). However, no
 Daukšas et al. 113

correlation between single or group (hydrophobic) of amino acids and bitter-

ness was found. Phenylalanine is known as a bitter amino acid (Liu, 2002), but
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no relationship between the amount of free phenylalanine and bitterness was

found. It was expected to find correlation between content of peptides (total
amino acids minus free amino acids) and bitterness, however, the correlation
was not strong (r = 0.62).

CONCLUSIONS

The results summarized in this paper illustrate the possibilities to reduce the
bitterness by chemical treatment and thereby probably increase the use of fish
protein hydrolysates for human food after taste improvement. The powders of
FPH produced from fish viscera with gall bladder had a more bitter taste than
powders produced without gallbladder. The bitterness of fish protein hydroly-
sates was also influenced by the use of Alcalase® or Flavourzyme® but to a
minor extent. Both treatments with BuOH and cholestyramine resin reduced
the bitter taste of fish protein hydrolysates (in primary treatment). The largest
reduction of bitterness was obtained after treatment with CAR. BuOH reduced
bitterness of the FPH but left the powders with ‘plastic’ taste. The bitterness
was not reduced by secondary treatment with Flavourzyme®. The largest dis-
advantage of chemical treatments was the reduced yield of FPH.
Although the degree of hydrolysis was higher than 40%, the fish protein hy-
drolysates had a marked bitter taste and no correlation was found between bit-
terness and free amino acids or bitter amino acids was found.
The observed bitterness is not fully explained by the presence of bile or bit-
ter amino acids/peptides. More research should be focused on bitterness of
fish protein hydrolysates to be used for human food applications.

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