use of koji and protease in fish sauce...

Singapore J Pri Ind 32: 19-29 2005/06

* Marine Fisheries Research Department, 2 Perahu Road, Singapore 718915

19

USE OF KOJI AND PROTEASE IN FISH

SAUCE FERMENTATION

Hariono I*, Yeap S E*, Kok T N* and Ang G T*

ABSTRACT

Traditionally, in Southeast Asia, fish sauce is

produced by fermenting fish mixed with salt. Usually,

the traditional fermentation process may take up to a

year or two to complete. A study was conducted to

investigate the possibility of the use of soya sauce koji

(starter mash) and commercial protease enzyme to

accelerate the fermentation process in the processing

of fish sauce from pelagic fish species without

affecting the characteristic flavour and nutritional

quality of the fish sauce as compared to that produced

by the traditional fermentation process. Parameters

such as pH, formol-N, total soluble solids, acidity I

and acidity II were monitored regularly during the

fermentation process. Yield of fish sauce, sodium

chloride, volatile basic nitrogen (VBN), total-N and

amino acid composition were measured at the end of

fermentation. The accelerated process was able to

produce fish sauce of comparable quality (according

to the Thai fish sauce, Nam-pla standard) to that of

the traditional process but in about 10 weeks.

INTRODUCTION

Fish is a major source of animal protein in many

countries in Southeast Asia. However, due to its

perishable nature, fish undergoes rapid spoilage,

especially under tropical conditions of high

temperature and high humidity. Traditional fish

fermentation is one of the low-cost preservation

methods, used widely in Southeast Asia. There are a

wide variety of fermented fish products, of which fish

sauce is probably one of the most well known. In

Southeast Asia, fish sauce is made from various types

of fish, from both freshwater and marine fish species,

by various methods. Each has its unique taste and

characteristic. Fish sauce is known as ‘nam pla’ in

Thailand, ‘patis’ in Philippines, ‘kecap ikan’ in

Indonesia, ‘budu’ in Malaysia, ‘nuoc nam’ in Vietnam

and Cambodia and ‘ngan pya ye’ in Myanmar (Putro,

1993; Ismail, 1977; Baens-Arcega, 1977; Phithakpol

et al., 1995; Tyn, 1993; Tran, 2002). The processing

method for fish sauce can be different in each country,

but the basic principle is quite similar. The main

ingredients of fish sauce are fish and salt. The fish is

washed and mixed with salt with the ratio ranges from

1:1 to 1:5. The mixture is left at room temperature for

a period varying from 5 to 24 months. The liquid will

be skimmed off the top or drained from the bottom of

the container. During this fermentation process, the

fish will undergo hydrolysis, both microbial and

enzymatic. The term ‘fish sauce’ itself, according to

Amano (1962) refers to ‘clear, brown liquid

hydrolysate from salted fish’.

The worldwide popularity of fish sauce has been

rising with the growing popularity of ethnic foods,

such as Vietnamese and Thai dishes. According to

FAO, the worldwide export of fermented fish

products, including fish sauce in year 2000 was

336,213 metric tonnes, valued at more than US$ 800

million. This quantity has been growing from 311,006

metric tonnes in 1998 and 312,995 metric tonnes in

1999, and it is expected to increase further in future.

As the traditional process of fish fermentation can

take up to 24 months to complete, there have been

some attempts to accelerate the fermentation process


20

without affecting the characteristic flavour and

nutritional quality of the fish sauce, for example, by

increasing the fermentation temperature (Mabesa et

al, 1989), adding the antibacterial agent instead of salt

(Amano, 1962), using plant protease such as

bromelain, papain or ficin (Ooshira et al, 1981,

Beddows and Ardeshir, 1977), or even using soya

sauce koji (Chae et al, 1989)

Koji is an essential ingredient in the fermentation

process in soy sauce production. The term ‘koji’

derives from the Chinese characters, meaning mouldy

grain. During fermentation, koji serves as a source of

a variety of enzymes which catalyse the degradation

of solid raw materials to soluble products that provide

fermentable substrates for yeast and bacteria in the

subsequent fermentation stage (Mheen, 1972).

Pelagic fish catch is the backbone of fishery in

Southeast Asia. Small-size pelagic fish contribute to

between 25 and 40% of total fish landing in this

region. However, due to its small size and high fat

content, pelagic fish tend to deteriorate more rapidly

if not properly handled and stored. In some Southeast

Asian countries, as much as 30% of the fish caught is

utilised as low-value fish meal or fertilizer.

It would be advantageous for the production of fish

sauce, (a) if the fermentation period can be shortened

to reduce the capital costs and to increase the

throughput, (b) if fish that is underutilised and has

low value can be used. The present study was

conducted to investigate the use of soya sauce koji

inoculated with Aspergillus oryzae and commercial

protease for the acceleration of fish sauce production

from pelagic fish species without affecting the

characteristic flavour and quality of the traditionally

produced fish sauce.

MATERIALS AND METHODS

Materials

Koji

Dried soybean and wheat flour were purchased from

local supermarket. Aspergillus oryzae culture was

obtained from Konno Moyashi Co. Ltd.

Fish Sauce

Pelagic fish, round scad (Decapterus macrosoma) and

hard-tail (Megalaspis cordyla) were purchased from

Geylang Serai market. Table salt was obtained from

local supermarket. Protease, derived from Aspergillus

oryzae was obtained from Nagase Enzyme Co. Ltd.

Food grade lactic acid was purchased from Merck

(Darmstadt, Germany).

Methods

Preparation of Koji

The soybean was washed and steamed for 30 minutes.

At the same time, the wheat flour was roasted at

150ºC for one hour. After cooling the ingredients to

less than 40ºC, the cooked beans and roasted wheat

were mixed in the ratio of 1:1 (w/w) and inoculated

with Aspergillus oryzae seed culture, added in the

ratio of 1:16,000. The mixture was then kneaded to a

dough, placed in plastic trays and incubated in a

temperature-humidity chamber (Hotpack, Model

435314). The temperature was adjusted, according to

the stage of growth of the Aspergillus oryzae culture,

that is:

1. Germination of Aspergillus, occurred at optimum

temperature of 35ºC

2. Initial growth of Aspergillus

During this phase, the growth of Aspergilus

would generate heat. The increase of dough

temperature would be observed. However,

enzyme would be deactivated if the temperature

rose too high. Hence, during this stage, it is

necessary to reduce the temperature of the

chamber to 30ºC and to spread the koji to around


21

30 cm thickness. The surface of the koji should

be covered with a wet cloth to maintain a

saturated humidity of 100%. Rewetting of cloth

should be done frequently as the cloth would dry

up by the heat generated.

3. Logarithmic growth stage of mould

By this stage, the surface of koji would be

covered by white mycelium. Wet cloth should be

removed and the koji should be broken to small

pieces and re-spread. The temperature of the

chamber should be maintained at 30ºC

4. Final stage

Koji was maintained at a humidity of 95% for 18

to 20 hours. At this stage, the temperature of the

chamber should be maintained to 25ºC to prevent

deactivation of peptidase

The flow chart for koji preparation is shown in Fig 1.

Preparation of Fish Sauce

The composition of materials for the preparation of

fish sauce is shown in Table 1. The raw round scad or

hard-tail was washed with chilled water. The fish was

cut into two or three pieces, depending on the size of

the fish. The fish was then cooked in boiling water for

around five minutes until the flesh changed colour.

This step was necessary to remove the lipid

component to prevent rancidity in the final product.

The boiled fish was then drained to remove excess

water. After the fish was cooled, protease and water

were added. The pH of mixture was then adjusted to

6.5 by adding lactic acid. The mixture was incubated

at 45ºC for four hours and then cooled to 28ºC. After

that, koji and salt were added to the fish mixture,

which was then allowed to ferment at room

temperature (28ºC) for 2.5 months. During

fermentation, the mixture needed to be stirred daily to

dissolve salt evenly. The stirring was done daily for

the first five days of the fermentation process and

subsequently, once every three days.

At the end of fermentation, the whole fermentation

mixture was filtered with cheesecloth and the liquid

was collected and heated to 80ºC for 15 minutes, then

filtered with Whatman filter paper No. 35 to obtain

the liquid (fish sauce).

Fig 1: Flow chart for koji preparatioin

Fig 2: Flow diagram of preparation of fish sauce


22

Monitoring of Fermentation Process

A series of chemical tests were carried out to monitor

the fermentation process. A small amount of the

liquid from the fermentation mixture was collected

regularly for chemical analysis. Acidity I, acidity II,

formol-N and total soluble solids tests were carried

out weekly while pH measurements were done

fortnightly. At the end of fermentation, the yield of

fish sauce was determined and analyses of total

nitrogen (Total-N), volatile basic nitrogen (VBN),

sodium chloride and amino acid composition of the

fish sauce were conducted.

The pH was measured by Istek pH meter, Model

725P. Acidity I and Acidity II were determined by the

analytical method for Japanese soy sauce (Okuhara,

1985). The content of amino-type nitrogen were

determined by the Formol-N method (Official Method

of Miso Analysis, 1968). Total soluble solids was

measured by using hand refractometer (Atago, Type

N1 (0 – 32%) and Type N2 (28 – 62%)).

The yield of fish sauce was measured by filtration of

the whole fermentation mixture at the end of the

fermentation period. The weight of liquid obtained

from the filtration would be calculated against the fish

weight. Sodium chloride content measurement was

done by potentiometric titration with 0.1 N silver

nitrate (AOAC, 1980). Total-N was determined by

Kjeldahl method and VBN was determined by

Conway microdiffusion method (Conway, 1950).

Amino acid composition was determined using high

pressure liquid chromatography by the Food and

Nutrition Branch, Veterinary Public Health Division,

Agri-Food and Veterinary Authority of Singapore.

RESULTS AND DISCUSSION

Changes in Chemical Composition during

Fermentation Process

pH, Acidity I

The pH decreased from 5.51 and 5.67 respectively for

hard-tail and round scad at the beginning of

fermentation to 5.15 and 5.06 at the end of

fermentation (Fig 3). Note that in fish sauce

processing, most of the fermentation occurring is

lactic acid fermentation. This was shown further by

the measurement of acidity I, which is correlated with

the amount of lactic acid during fish sauce

fermentation (Fig 4). Acidity I was used to roughly

indicate the adequacy of fermentation, i.e.

fermentation process could cease once the acidity I

reached 10 ml NaOH.

Acidity II, Formol-N

Acidity II measures the total amino acid level and

formol-N test measures the amino-type nitrogen. As

fermentation progresses, protein is broken down to

amino acids, affecting the flavour of the product. The

greater the amino acid content in the product, the

more flavourful the product will be, though the

overall amino acid composition still plays an

important part in determining overall flavour balance.

As fermentation proceeds, the acidity II and formol-N

values should increase until a certain extent, then

stabilize.

TABLE 1

COMPOSITION OF MATERIALS FOR PREPARATION OF FISH SAUCE

Sample

number

Fish type Protease (%

of fish weight)

Water (% of

fish weight)

Koji (% of fish-

water mixture

weight)

Salt (% of fish-

water mixture

weight)

RS Round scad 1.0 30.0 25.0 16.0

HT Hard-tail 1.0 30.0 25.0 16.0


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0

5

10

15

20

25

0 1 2 3 4 6 7 8 9 10Ferm entation period (weeks)

0.1

N N

aO

H (

ml)

HT RS

Linear (RS) Linear (HT)

0

5

10

15

20

25

0 1 2 3 4 6 7 8 9 10

Fermentation period (weeks)

0.1

N N

aO

H (

ml)

HT RS


Fig 3: Changes in pH in fish sauce fermentation Fig 4: Changes in acidity I in fish sauce fermentation

0

5

10

15

20

25

0 1 2 3 4 6 7 8 9 10


0.1

N N

aO

H (

ml)

HT RS

Linear (HT) Linear (RS)

0.00

0.50

1.00

1.50

2.00

0 1 2 3 4 6 7 8 9 10


Co

nc

en

tra

tio

n (

Ng

/10

0m

l)

HT RSLinear (HT) Linear (RS)

Fig 5: Changes in acidity II in fish sauce fermentation Fig 6: Changes in Formol-N in fish sauce fermentation

20

30

40

50

1 2 3 4 6 8 9 10

Fermentation period (Week)

Bri

x

HT RS


Fig 7: Changes in total soluble solids in fish sauce fermentation


23


0.1N

NaO

H (m

l)

Fig 3: Changes in pH in fish sauce fermentation Fig 4: Changes in acidity I in fish sauce fermentation


0.1N

NaO

H (m

l)


Con

cent

ratio

n (N

g/10

0ml)

Fig 5: Changes in acidity II in fish sauce fermentation Fig 6: Changes in Formol-N in fish sauce fermentation


Brix

Fig 7: Changes in total soluble solids in fish sauce fermentation


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Acidity II values at the beginning and the end of

fermentation was 11 ml NaOH and 13.8 ml NaOH

respectively for hard-tail and 19.6 ml NaOH and 21.3

ml NaOH for round scad (Fig 5). The formol-N values

also increased from 0.840 to 1.540 g/100 ml for hard-

tail and 1.092 to 1.596 g/100 ml for round scad (Fig

6).

Total Soluble Solids

The total soluble solids content increased from 39.0%

and 38.2% respectively for the fish sauce prepared

from hard-tail and round scad, reaching 49.0% and

43.7%, respectively, when the products were

harvested (Fig 7). The measurement of total soluble

solids by refractometer could be used to estimate the

degree of protein hydrolysis during fermentation. The

refractive index of the solution is dependent on the

amount of free amino acids and small peptides

released through protein hydrolysis.

Chemical Composition of Lab-produced and

Commercial Thai Fish Sauce

The yield of both hard-tail and round scad fish sauce

is shown in Table 1. The chemical composition of

both lab-produced fish sauce and commercial Thai

fish sauce are shown in Table 2.

Both of the lab-produced fish sauce had higher total

soluble solids, acidity I and acidity II, formol-N and

total- N values than commercial Thai fish sauce. Lab-

produced fish sauce also had lower salt content than

commercial fish sauce. Commercial fish sauce had a

higher ratio of formol-N/ total-N and lower VBN

content.

The pH of the two lab-produced fish sauces were

almost similar to that of commercial fish sauce. It is

worth noting that most nam pla is seasoned during

ripening with food grade additives such as citric acid

and sorbic acid to lower pH and adjust colour

(Mabesa et al, 1972)

Both lab produced fish sauces had similar yield, based

on 4,000 g of fish used. Yield of fish sauce depends

on the digestible portion of fish, namely flesh and

viscera.

Amino Acid Composition

In traditional fish sauce fermentation, proteolysis of

fish tissues by fish gut enzymes and microbial

enzymes gives rise to amino acids and small

molecular peptides. Saisithi (1994) stated that the

amount of amino acids produced during fermentation

of nam pla increased gradually from 2.9 to 12.5 g/

100 ml at the end of 12 months of fermentation. These

amino acids are responsible for the flavour

components of the fish sauce.

TABLE 2

CHEMICAL COMPONENTS OF LAB-PRODUCED AND COMMERCIAL THAI FISH SAUCE

(NAM PLA)

Fish

sauce

Yield (ml)*

Total

Soluble

Solids (%)

Salt (%)

pH Acidity I (ml of

NaOH)

Acidity II (ml of

NaOH)

Formol-

N (g/100 ml)

Total N (g/100 ml)

Formol-N/

Total-N

VBN (mg/

100ml)

RS 1,800 43.7 20.00 5.06 12.30 21.20 1.60 2.60 0.64 317.84

HT 1,800 49.0 24.40 5.15 15.60 19.90 1.54 2.73 0.56 301.20

Comm NA 41.5 25.00 5.11 7.15 12.80 1.37 1.95 0.70 139.03

RS: Round scad HT: Hard-tail Comm: Commercial Thai fish sauce, Tra Chang Brand NA: Not available *based on 4,000 g fish used


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TABLE 3

AMINO ACID COMPOSITION OF LAB-PRODUCED AND COMMERCIAL THAI FISH SAUCE

(NAM PLA)

Round scad fish sauce

(g/100ml)

Hard-tail fish sauce

(g/100ml)

Commercial Thai fish

sauce

(g/100ml)

Aspartic acid 1.21 1.22 1.03

Glutamic acid 2.10 1.83 1.69

Serine 0.37 0.57 0.40

Glycine 0.60 0.73 0.63

Threonine 0.55 0.54 0.51

Alanine 0.73 0.76 0.72

Proline 0.52 0.31 0.15

Histidine 0.52 0.26 0.04

Arginine 0.62 0.89 0.17

Tyrosine 0.13 0.15 0.06

Cystine 0.18 0.02 0.08

Valine 0.67 0.64 0.58

Methionine 0.30 0.32 0.23

Phenylalanine 0.42 0.43 0.32

Isoleucine 0.48 0.48 0.34

Leucine 0.60 0.61 0.46

Lysine 1.14 0.82 0.82

Total amino acid 11.14 10.58 8.23

Sum (g/total)

Umami 3.31 3.05 2.72

Sweet 2.77 2.91 2.41

Bitter 5.06 4.62 3.1

Sum (% of total amino acid)

Umami 29.71 28.83 33.05

Sweet 24.87 27.50 29.28

Bitter 45.42 43.67 37.67


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The free amino acid composition of the two lab-

produced and commercial fish sauce are shown in

Table 3. The total free amino acid content of lab-

produced fish sauces is higher than commercial Thai

fish sauce (10.58 and 11.14 g/100 ml for hard-tail and

round scad sauce vs 8.23 g/100 ml). Among all the

amino acids, glutamic acid, the amino acid

accountable for umami taste, was the most abundant

in all fish sauces analysed. It accounted for 17 to 21%

of the total amino acid content of the fish sauce.

The amino acids that contribute to umami taste are

glutamic acid and aspartic acid. Serine, glycine,

alanine, threonine and proline are associated with

TABLE 4

THAI FOOD AND DRUG ADMINISTRATION STANDARD FOR FISH SAUCE (NAM PLA)*

No. Requirement Type of Nam Pla

Naturally processed Artificially processed Blended

1. Sodium chloride (%) >20 >20 >20

2. Total-N (g/100 ml) >0.9 >0.9 >0.4

3. Amino acid nitrogen (%) [(amino acid-N/ Total-N) x 100]

40 – 60 40 – 60 -

4. Glutamic acid/ Total-N (G/N) 0.4 – 0.6 0.4 – 0.6 0.4 – 1.3 *Source: Virulhakul, 2000

TABLE 5

THAI INDUSTRIAL STANDARD INSTITUTE STANDARD FOR FISH SAUCE (NAM PLA)*

(REFERENCE NO. TIS 3-1983)

No. Requirement Grade 1 Grade 2

1. Relative density at 27ºC 1.2 1.2

2. pH 5.0 – 6.0 5.0 – 6.0

3. Sodium chloride (g/l), not less than 230 230

4. Total-N (g/100 ml), not less than 20 20

5. Glutamic acid/ Total-N (G/N) 0.4 – 0.6 0.4 – 0.6

6. Amino acid nitrogen (g/l) 10 7.5 *Source: Virulhakul, 2000

TABLE 6

COMPARISON OF LAB-PRODUCED AND COMMERCIAL FISH SAUCE WITH STANDARDS

SET BY THAI FDA AND TISI

Sodium

chloride

(%)

Total-N

(g/100 ml)

Amino acid

nitrogen (%)

[(amino acid-N/

Total-N) x 100]

Glutamic acid/

Total-N (G/N)

Relative

density at

27ºC

pH

Thai

FDA

>20 >0.9 40 – 60 0.4 – 0.6 - -

TISI >23 >2.0 40 – 60 0.4 – 0.6 1.2 5.0 – 6.0

HT 24.40 2.73 NA 0.77 NA 5.15

RS 20.00 2.60 NA 0.70 NA 5.06

Comm 25.00 1.95 NA 0.87 NA 5.11 RS: Round scad Comm: Commercial Thai Fish sauce, Tra Chang Brand HT: Hard-tail NA: Not available


27

sweet taste. Histidine, arginine, tyrosine, cystine,

valine, methionine, phenylalanine, isoleucine, leucine

and lysine are related to bitter taste (Funatsu, 2001).

Though commercial fish sauce has the lowest total

amino acid content, it has the highest umami amino

acid proportion (33%) and the lowest bitter amino

acid proportion (37%) as compared to the lab-

produced fish sauce (the proportion of umami and

bitter proportion were 29.71% and 45.42% in round

scad sauce and 28.83% and 43.67% in hard-tail

sauce).

The overall flavour of fish sauce depends on the

balance of all amino acids, that is those that account

for umami, sweet and bitter taste. As taste is

subjective, there is no standard formula of a

“balanced” amino acid composition. However, it is

worthy to note that taste contributed by amino acids in

these oligopeptides can mask the saltiness of fish

sauce (Funatsu, 2001).

Comparison with Thai Standards

Thailand is the world’s largest fish sauce producer,

contributing more than 20% of total world fish sauce

production. The production of Thai fish sauce, nam

pla, is regulated by two standards established by the

Thai Food and Drug Administration (FDA) and Thai

Industrial Standard Institute (TISI). The former is

mandatory whereas the latter is voluntary. The

requirements of both standards are shown in Table 4

and 5.

The comparison of lab-produced and commercial fish

sauce with requirements as set by the Thai FDA and

TISI is shown in Table 6.

In general, both lab-produced fish sauces are able to

meet the standards as set by the Thai FDA and TISI.

CONCLUSION

It is possible to make a good quality fish sauce from

underutilised pelagic fish by the accelerated method

using koji and protease. The lab-produced fish sauce

have comparable amounts of essential amino acids

with the commercial Thai fish sauce produced by

traditional method and are able to meet the standards

set by the Thai FDA and TISI. The accelerated

method also took only ten weeks as compared to one

to two years for the traditional method, thus reducing

overall production cost and increasing productivity in

fish sauce processing.

ACKNOWLEDGEMENTS

This study has been greatly assisted by the Food and

Nutrition Branch, Veterinary Public Health Division,

Agri-food and Veterinary Authority, for which the

authors feel deeply grateful.

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