Small-Scale Productlon Of Fish Protein Hydrolysate
SMALL-SCALE PRODUCTlON OF FISH PROTEIN HYDROLYSATE
P u ~ w oARBIANTO
Bandung Institute o f Technology, Bandung, Indonesia
ABSTRACT
Biotechnology and the biotechnology industry open up new horizons for human activity, and
especially for industry in the near future. Starting from science and from a range of technologies,
biotechnoiogy does not apply to one area of industrial activity alone but to a whole range: the
agrofood industry, pharmaceuticals, the energy sector and some aspects of agriculture as well.
These new technologies must be adapted to suit the specific cultural as well as ecological
circumstances. Thus i t is vitally important that developing countries should have their own pool of
scientists and engineers working in biotechnology, that is, people fully conversant with the
problems of their own country or region and who are skilled in using their own domestic resources.
The Center for Environmental Studies and the Biotechnology Team of the Bandung lnstitute of
Technology, has been carrying out a project in Ciamis, West Java for nearly four years. The project
is an attempt at rural ecosystem development and is known as the "Ciamis Project". This rural
ecosystem development at the grass-roots level forms the building blocks, or the microstructure,
on which biotechnology industry development rests. The superstructure, or the meso and macro
structure, is already there and may contain powerful forces detrimental or sympathetic, alien or
neutral t o the microstructure. The project role is to help build up the microstructure and to be a
mediator in the process of integration of micro -, meso - and macrostructures. This paper concerns
about the development of a process designed for village level industry.
Fresh water and sea fish constitute an important source of food for large segments of the
populations of South East Asia. However, outside the main urban centers of these countries,
consumption of fresh or processed fish may actually have declined. The lack of appropriate landing
and transport facilities puts a limit to the amount of fish which can be marketed before spoilage
takes place. Indeed, small-scale fisheries, which are more appropriate for the supply of fresh on
cured fish for consumption by low-income groups than large scale fisheries, generally tend to be
neglected.
In an effort to overcome the problem posed by the inadequate landing transport facilities and
marketing channels, a simple processing method is developed in the form of a Fish-Protein
Hydrolysate or Fish Sauce. Fish sauces are basically water extracted solutions of fully fermented
fish and are used in a similar mener to soya bean sauce. Fermentation involves the hydrolysis or
breakdown of protein into their constituents peptides and amino acids Putrefaction is prevented by
the addition of salt. In some circumstances, carbohydrates may be added which result in the
formation of acids which further help to impart a characteristic flavor and odour as well as
provifing a further degree of preservation.
In the method developed the fish protein was hydrolysed by bromelain entrapped in the fibers
of the core of pineapples. The flavor was then developed through fermentation of the fish hydrolysate.
INTRODUCTION
Fresh water and sea fish constitute an important source of food for large
segments of the populations of South-east Asia. However, outside the main
urban centers of these countries, consumption of fresh or prosessed fish
by low-income groups, has not progressed and may actually have declined. The
lack of appropriate landing and transport facilities puts a limit t o the amount of
fish which can be marketed before spoilage takes place. Indeed, small-scale
fisheries, which are more appropriate for the supply of fresh or cured fish for
consumption by low income groups than large scale fisheries, generally tend t o
be neglected.
In an effort t o overcome the problem posed by the inadequate landing and
transport facilities and marketing channels, a simple processing method was
developed in the form of a fish-protein hydrolysate or plain fish-sauce.
Fish sauces are basically water extracted solutions of fully fermented fish
and are used in a similar manner t o soy-bean sauce. Fermentation involves the
hydrolysis or breakdown of protein into their constituent peptides and amino
acids. Putrefaction is prevented by the addition of salt. In some eircurnstances,
carbohydrates may be added which result in the formation of acids which
further help t o impart a characteristic Flavor and odor as well as providing a
further degree of preservation.
Fresh fish
washed
Arranged in layers with salt in
between, add water such as t o cover
all the fish
(2-3 days), the fermentation
takes place for 1-2 years.
filtered
KECAP IKAN
first quality
Mixed and left-overs of
the former batch and dried
for 2 - 4 days.
filtrated
NAM-PLA
Residue, add water and
fermented for 3 months
filtered
KECAP lKAN
2nd quality
In Vietnam, fish sauce nuoc-mam, provides up t o 20% of the daily protein
intake. Pattis in the Philippines is similar t o nuoc-mam. Nam-pla is made in
Thailand, while i n Malaysia, a sauce t o known as budu is made from small
anchovies. In Indonesia the souce is called kecap ikan. I n the traditional
method, the fish and salt are kept i n a container for some time. Most of the fish
protein will dissolve due t o the action of autolytic enzymes and Microbes.
The longer the fermentation takes place, the higher the concentration of
the peptides and amino acids in the resulting clear liquid. To fasten the
process, in this study, an enzyme found in pine-apples were used i n the
following manner :
Mesh of core of pine-apples
suspension
( 3 parts)
Fish protein
suspension
(1 part)
Incubated overnight at 4 0 C.
Protein hydrolysate
+ NaCl2%, glucose 5%, starch 1 0 %
Inoculated with Str. thermophillus
L . bulgaricus
Bacillus natto.
Hydrolysate
(pH 4.5)
+
NaCl 2 % add Sach. rouxii
Rhodotorula Pilimonde
Fish sauce.
MATERIALS AND METHODS
All chemicals are of reagent grade.
Carbohydrates were determined according t o Luff-Schoori.
Lipids by ether extraction, and
Protein-through Kjeldahl and Lowry.
Bromelein iSolation :
The core of pine-apple fruits were collected, pressed t o obtain about 100
ml of juice. This juice was cooled and cold aceton (-15OC added slowly until a
total volume of 200 ml was reached. The mixture was centrifuged for 30
minutes at 0-4OC and 2000 g. The residue was collected and the supernatant were treated with aceton as above. This was repeated three times and
the residues were combined and lyophilized.
RESULTS AND DISCUSSION
Bromelein is a simple protein w i t h a molecular weight of 31 000 and an
iso-electric point of 4.6 (Vamada, 197 1 ). Bromelein extract has a pH optimum
of 6.5 while the isolate showed an optimum of 7.8. More important. More
important, unlike when using the pure enzyme, the bromelein extract caused
no bitter flavor, due t o certain types of peptides (Fujimaki, 197 1).
Thus, in the procedure used, bromelein is not isolated, but a suspension of
the core of the pine apple fruits was made by using a blender. This greatly
simplify the enzyme preparation aspect. The ratio of pine-apple core suspension and the fish protein suspension for optimal conditions were determined.
Table 1. Ratio of pine-apple core suspension and fish protein suspension for optimal hydrolysis
(pH 6.5, 50°C).
Ratio of suspension fish proteinlpine-apple core
Mg proteinlrnl
After standing overnight a clear yellowish protein hydrolysate was
. obtained after filtration. The fish hydrolysate still smells fishy and still has the
taste of pine-apples. The following fermentation step is designed t o impart the
taste similar t o soy-sauce.
Fish contain only small amounts of fermentable carbohydrates, thus
glucose and starch was added. Although lactic acid bacteria are widespread in
nature, they are not found in large numbers in fish microflora. It is therefore
essential t o add a starter culture of lactobacilli for successful fermentation. In
our case Lactobacillus bulgaricus and Streptococcus thermophilus were
selected. In addition Bacillus natto was also added t o help hydrolyze the starch
into maltose and glucose. The three bacteria have an optimal temperature of
about 40°C. L. bulgaricus and Sir. ithermophillus are both heterofermentative.
They have the ability t o change glucose into lactic acid, alcohol and GO,. The
difference is that Str, thermophillus is still active at pH 5 whereas L. bulgaricus
is not. The result of this step can be seen at the following table.
Table 2. The Lactic acid Fermentation (pH 7, temperature 40°C).
lnocullum
L. bulgaricus
Str. thermophillus
+
-
-
+
+
+
-
-
+
+
8.natto
Final product
-
smell of fish & pine-apple
+
+
smell
smell
smell
smell
of fish & pine-apple
of yogurt
of fish & pine-apple
resembles soy-sauce
The final step resembles brine fermentation. Here L. bulgaricus, Str.
thermophillus and B. natto does not grow anymore but are still active in
producing lactic acid and esters characteristic for soy-sauce. Salt is added in a
way as not t o cause a salting-out of the fish protein. In addition Saccharornyces rouxii and Rhodoterulla pilimonde were added t o complete the
hydrolisis of starch still available in the liquid.
Table 3. The Brine Fermentation Stage (pH 4.5, temperature 40°C).
Salt concentration %
0
10
15
Results after 2 days
The smell of alcohol is dominant.
Microbial growth on the surface.
Alcohol small less dominant.
No surface growth.
No alcohol smell, no surface growth, salting out of protein.
The addition of 1 0 % salt also ensures a long shelf-life.
CONCLUSIONS
Since the fish used could be any kind of fish, even waste from fish
industries, this process offers a method for bioconversion of waste products.
The short time required and the simplicity of the process makes it suitable for
primary industries a t the village level. The taste and flavor developed could be
adjusted t o peoples food habit. In our case, the flavor and taste of soy-sauce
were tried.
REFERENCES
BENDER
A.E. AND MUNCKR. 1970. Evaluation of Novel Protein Products. Pergamon press, page 55.
FUJIMAKIM., KOTOH., ARAIS., YAMASKITA
M. 197 1. J. Appl. Biol. 34, 1 19.
HEVIAP.,OLCOTTM.S.1977.J. AgricFoodChem., 25,4.
LOWRYO.H. AND RESEBRDUGH
ETAL. 1951. J. Biol. Chern. 1 93, 26 r.
OHTAS., HORIEK., MEVACHIT. 19 72. J. Biol. Chem. 7 1, 8 1 7-830.
P u ~ w A,,
o RUKMA.1964. Laporan Proyek lkan Asin.
SHIBASAKI
I., INOUE
Y., TAKANO
M. 1980. JSPS-NRCT Seminar.
YAMADAF., TAKASHIN., HAGNUOT.1976. J. BiolChem. 79, 1223-1234.
B.P. K. 19 78. lsolasi bromelein dari nenas Departernen Perindustrian Pusat Penelitian dan Pengernbangan, Aneka lndustri dan Kerajinan, September.
P u ~ w oARBIANTO
Bandung Institute o f Technology, Bandung, Indonesia
ABSTRACT
Biotechnology and the biotechnology industry open up new horizons for human activity, and
especially for industry in the near future. Starting from science and from a range of technologies,
biotechnoiogy does not apply to one area of industrial activity alone but to a whole range: the
agrofood industry, pharmaceuticals, the energy sector and some aspects of agriculture as well.
These new technologies must be adapted to suit the specific cultural as well as ecological
circumstances. Thus i t is vitally important that developing countries should have their own pool of
scientists and engineers working in biotechnology, that is, people fully conversant with the
problems of their own country or region and who are skilled in using their own domestic resources.
The Center for Environmental Studies and the Biotechnology Team of the Bandung lnstitute of
Technology, has been carrying out a project in Ciamis, West Java for nearly four years. The project
is an attempt at rural ecosystem development and is known as the "Ciamis Project". This rural
ecosystem development at the grass-roots level forms the building blocks, or the microstructure,
on which biotechnology industry development rests. The superstructure, or the meso and macro
structure, is already there and may contain powerful forces detrimental or sympathetic, alien or
neutral t o the microstructure. The project role is to help build up the microstructure and to be a
mediator in the process of integration of micro -, meso - and macrostructures. This paper concerns
about the development of a process designed for village level industry.
Fresh water and sea fish constitute an important source of food for large segments of the
populations of South East Asia. However, outside the main urban centers of these countries,
consumption of fresh or processed fish may actually have declined. The lack of appropriate landing
and transport facilities puts a limit to the amount of fish which can be marketed before spoilage
takes place. Indeed, small-scale fisheries, which are more appropriate for the supply of fresh on
cured fish for consumption by low-income groups than large scale fisheries, generally tend to be
neglected.
In an effort to overcome the problem posed by the inadequate landing transport facilities and
marketing channels, a simple processing method is developed in the form of a Fish-Protein
Hydrolysate or Fish Sauce. Fish sauces are basically water extracted solutions of fully fermented
fish and are used in a similar mener to soya bean sauce. Fermentation involves the hydrolysis or
breakdown of protein into their constituents peptides and amino acids Putrefaction is prevented by
the addition of salt. In some circumstances, carbohydrates may be added which result in the
formation of acids which further help to impart a characteristic flavor and odour as well as
provifing a further degree of preservation.
In the method developed the fish protein was hydrolysed by bromelain entrapped in the fibers
of the core of pineapples. The flavor was then developed through fermentation of the fish hydrolysate.
INTRODUCTION
Fresh water and sea fish constitute an important source of food for large
segments of the populations of South-east Asia. However, outside the main
urban centers of these countries, consumption of fresh or prosessed fish
by low-income groups, has not progressed and may actually have declined. The
lack of appropriate landing and transport facilities puts a limit t o the amount of
fish which can be marketed before spoilage takes place. Indeed, small-scale
fisheries, which are more appropriate for the supply of fresh or cured fish for
consumption by low income groups than large scale fisheries, generally tend t o
be neglected.
In an effort t o overcome the problem posed by the inadequate landing and
transport facilities and marketing channels, a simple processing method was
developed in the form of a fish-protein hydrolysate or plain fish-sauce.
Fish sauces are basically water extracted solutions of fully fermented fish
and are used in a similar manner t o soy-bean sauce. Fermentation involves the
hydrolysis or breakdown of protein into their constituent peptides and amino
acids. Putrefaction is prevented by the addition of salt. In some eircurnstances,
carbohydrates may be added which result in the formation of acids which
further help t o impart a characteristic Flavor and odor as well as providing a
further degree of preservation.
Fresh fish
washed
Arranged in layers with salt in
between, add water such as t o cover
all the fish
(2-3 days), the fermentation
takes place for 1-2 years.
filtered
KECAP IKAN
first quality
Mixed and left-overs of
the former batch and dried
for 2 - 4 days.
filtrated
NAM-PLA
Residue, add water and
fermented for 3 months
filtered
KECAP lKAN
2nd quality
In Vietnam, fish sauce nuoc-mam, provides up t o 20% of the daily protein
intake. Pattis in the Philippines is similar t o nuoc-mam. Nam-pla is made in
Thailand, while i n Malaysia, a sauce t o known as budu is made from small
anchovies. In Indonesia the souce is called kecap ikan. I n the traditional
method, the fish and salt are kept i n a container for some time. Most of the fish
protein will dissolve due t o the action of autolytic enzymes and Microbes.
The longer the fermentation takes place, the higher the concentration of
the peptides and amino acids in the resulting clear liquid. To fasten the
process, in this study, an enzyme found in pine-apples were used i n the
following manner :
Mesh of core of pine-apples
suspension
( 3 parts)
Fish protein
suspension
(1 part)
Incubated overnight at 4 0 C.
Protein hydrolysate
+ NaCl2%, glucose 5%, starch 1 0 %
Inoculated with Str. thermophillus
L . bulgaricus
Bacillus natto.
Hydrolysate
(pH 4.5)
+
NaCl 2 % add Sach. rouxii
Rhodotorula Pilimonde
Fish sauce.
MATERIALS AND METHODS
All chemicals are of reagent grade.
Carbohydrates were determined according t o Luff-Schoori.
Lipids by ether extraction, and
Protein-through Kjeldahl and Lowry.
Bromelein iSolation :
The core of pine-apple fruits were collected, pressed t o obtain about 100
ml of juice. This juice was cooled and cold aceton (-15OC added slowly until a
total volume of 200 ml was reached. The mixture was centrifuged for 30
minutes at 0-4OC and 2000 g. The residue was collected and the supernatant were treated with aceton as above. This was repeated three times and
the residues were combined and lyophilized.
RESULTS AND DISCUSSION
Bromelein is a simple protein w i t h a molecular weight of 31 000 and an
iso-electric point of 4.6 (Vamada, 197 1 ). Bromelein extract has a pH optimum
of 6.5 while the isolate showed an optimum of 7.8. More important. More
important, unlike when using the pure enzyme, the bromelein extract caused
no bitter flavor, due t o certain types of peptides (Fujimaki, 197 1).
Thus, in the procedure used, bromelein is not isolated, but a suspension of
the core of the pine apple fruits was made by using a blender. This greatly
simplify the enzyme preparation aspect. The ratio of pine-apple core suspension and the fish protein suspension for optimal conditions were determined.
Table 1. Ratio of pine-apple core suspension and fish protein suspension for optimal hydrolysis
(pH 6.5, 50°C).
Ratio of suspension fish proteinlpine-apple core
Mg proteinlrnl
After standing overnight a clear yellowish protein hydrolysate was
. obtained after filtration. The fish hydrolysate still smells fishy and still has the
taste of pine-apples. The following fermentation step is designed t o impart the
taste similar t o soy-sauce.
Fish contain only small amounts of fermentable carbohydrates, thus
glucose and starch was added. Although lactic acid bacteria are widespread in
nature, they are not found in large numbers in fish microflora. It is therefore
essential t o add a starter culture of lactobacilli for successful fermentation. In
our case Lactobacillus bulgaricus and Streptococcus thermophilus were
selected. In addition Bacillus natto was also added t o help hydrolyze the starch
into maltose and glucose. The three bacteria have an optimal temperature of
about 40°C. L. bulgaricus and Sir. ithermophillus are both heterofermentative.
They have the ability t o change glucose into lactic acid, alcohol and GO,. The
difference is that Str, thermophillus is still active at pH 5 whereas L. bulgaricus
is not. The result of this step can be seen at the following table.
Table 2. The Lactic acid Fermentation (pH 7, temperature 40°C).
lnocullum
L. bulgaricus
Str. thermophillus
+
-
-
+
+
+
-
-
+
+
8.natto
Final product
-
smell of fish & pine-apple
+
+
smell
smell
smell
smell
of fish & pine-apple
of yogurt
of fish & pine-apple
resembles soy-sauce
The final step resembles brine fermentation. Here L. bulgaricus, Str.
thermophillus and B. natto does not grow anymore but are still active in
producing lactic acid and esters characteristic for soy-sauce. Salt is added in a
way as not t o cause a salting-out of the fish protein. In addition Saccharornyces rouxii and Rhodoterulla pilimonde were added t o complete the
hydrolisis of starch still available in the liquid.
Table 3. The Brine Fermentation Stage (pH 4.5, temperature 40°C).
Salt concentration %
0
10
15
Results after 2 days
The smell of alcohol is dominant.
Microbial growth on the surface.
Alcohol small less dominant.
No surface growth.
No alcohol smell, no surface growth, salting out of protein.
The addition of 1 0 % salt also ensures a long shelf-life.
CONCLUSIONS
Since the fish used could be any kind of fish, even waste from fish
industries, this process offers a method for bioconversion of waste products.
The short time required and the simplicity of the process makes it suitable for
primary industries a t the village level. The taste and flavor developed could be
adjusted t o peoples food habit. In our case, the flavor and taste of soy-sauce
were tried.
REFERENCES
BENDER
A.E. AND MUNCKR. 1970. Evaluation of Novel Protein Products. Pergamon press, page 55.
FUJIMAKIM., KOTOH., ARAIS., YAMASKITA
M. 197 1. J. Appl. Biol. 34, 1 19.
HEVIAP.,OLCOTTM.S.1977.J. AgricFoodChem., 25,4.
LOWRYO.H. AND RESEBRDUGH
ETAL. 1951. J. Biol. Chern. 1 93, 26 r.
OHTAS., HORIEK., MEVACHIT. 19 72. J. Biol. Chem. 7 1, 8 1 7-830.
P u ~ w A,,
o RUKMA.1964. Laporan Proyek lkan Asin.
SHIBASAKI
I., INOUE
Y., TAKANO
M. 1980. JSPS-NRCT Seminar.
YAMADAF., TAKASHIN., HAGNUOT.1976. J. BiolChem. 79, 1223-1234.
B.P. K. 19 78. lsolasi bromelein dari nenas Departernen Perindustrian Pusat Penelitian dan Pengernbangan, Aneka lndustri dan Kerajinan, September.