CHAPTER 10

Production of fish protein

hydrolyzates by
• •
mIcroorganIsms
V. Venugopal

10.1 INTRODUCTION

Of the current world fish landing of around 100 million tonnes, con-
sumer preference is restricted to only a few selected items. A significant
proportion of total available fish forms the by-catch of shrimp trawling,
and includes non-conventional species which are not commercially
exploited. Nevertheless, these fish are sources of nutritive proteins
which could be utilized for satisfying human protein requirements. The
necessity of exploitation of under-utilized fish species as well as reduc-
tion of postharvest fishery losses have been pointed out by several
authors (James, 1986; Morrissey, 1988; Burt, Hardy and Whittle, 1990;
Sikorski, 1990).

10.1.1 Mince from under-utilized fish

Under-utilized fish could be used to develop several value-added pro-
ducts. The first step in utilization invariably consists of hygienic collec-
tion of the meat by a mechanical deboning operation. The meat mince
obtained could be used as a raw material for the development of a
number of products such as fermented items, fabricated foods and
surimi-based products (Grantham, 1981; Regenstein, 1986; Venugopal,
1992; Venugopal, Ghadi and Nair, 1992). Fish protein hydrolyzate (FPH)
is one product, currently used as a protein supplement for animal foods,
which also has potential as a human food item (Kilara, 1985). At present
FPHs are produced by digestion of fish meat by proteolytic enzymes

A. M. Martin (ed.), Fisheries Processing

© Chapman & Hall 1994
224 Production of fish protein hydrolyzates
from vegetable as well as animal sources. This article will discuss the
scope of applying biotechnological methods for the production of FPH.
At the outset, some aspects on the properties of fish mince, microbial
extracellular proteases and immobilization of microorganisms will be
discussed.

10.1.2 Properties of the mince

Fish mince, as compared with intact fish muscle, is more unstable and
is prone to rapid deteriorative changes during storage (Grantham, 1981).
Mechanical deboning causes disruption of the tissue and exposure of
the meat to air which accelerates several deteriorative processes, such
as lipid oxidation, bacterial proliferation and resulting protein changes.
The higher temperature associated with deboning also affects the stabil-
ity of the mince. The mince contains about 20% soluble components
belonging to the sarcoplasmic fraction and about 2-3% connective tissue
(although this may be as high as 10% in the case of elasmobranch
species). The rest is composed of myofibrillar proteins. Washing of the
mince in chilled water removes soluble compounds, significant amounts
of lipids, blood and pigments to give concentrated myofibrillar proteins,
having a bland taste (Lee, 1986). Fatty fish like mackerel or sardine may
require washing with bicarbonate to remove oil (Putro, 1989). The mince
may be stored frozen in the presence of cryoprotectants, e.g. polyphos-
phates, which can control protein denaturation during storage.
The washed myofibrillar proteins from most fish species are capable
of turning into gel either on prolonged incubation at low temperature
or by heating to 40-S0°C (Lanier, 1986). Gelation results from cross-
linking of randomly dispersed polymer chains in solution to give matri-
ces which immobilize water in the interstitial structure. The process
occurs in two stages consisting of initial unfolding of the protein chains
followed by protein-protein interactions which help entrapment of the
water. The stability of the gel depends upon the extent of such interac-
tions. The presence of salts, low pH and mild heat can bring about
unfolding of the proteins leading to gelation (Lanier, 1986; Foegiding,
1988; Zeigler and Foegeding, 1990). The washed mince could be the
ideal raw material for the development of several foods (Grantham 1981;
Lee, 1986). After gelation, the washed mince could be the ideal starting
material for the preparation of hydrolyzates, as will be discussed later.

10.2 SOLUBILIZATION OF FISH MINCE

10.2.1 Conventional methods to make FPH

FPHs are distinguished from fermented foods by their high solubility in
water, low fat and ash contents as well as high protein contents. FPHs
 Solubilization of fish mince 225
Whole fish

~
I...------J
Mince
Water, equal volume;
~ L Enzyme, 0.15-0.3%

Incubation, 45-50·C, 4 h

~
Heat to 90·C to inactivate enzyme

~
Separation (oil, residue)

~
Liquefied fish protein

~
Concentrate

~
Spray dry

~
FPH powder

Figure 10.1 Flow diagram for production of FPH.

are produced by conventional methods by proteolytic digestion of fish

and fish waste at optimal conditions of pH and temperature required
by the enzymes. Liquefaction of the proteins takes place within a few
hours. The hydrolyzate is then decanted and centrifuged to remove
scales and bones. The soluble fraction is concentrated by evaporation
and is suitably dried. The production of FPH has been described by
several authors (Adler-Nissen, 1977, 1985; Kinumaki, 1978; Mackie,
1982a, b; Kilara, 1985; Qaglia and Orban, 1987a, b). The process is
depicted in Figure 10.1.
By using different fish species, enzymes and digestion conditions, a
wide range of hydrolyzates can be produced. The enzymes can be from
plant (papain, ficin, etc.), animal (trypsin, pancreatin, etc.) or microbial
(pronase, alcalase, etc.) sources. Uncontrolled or prolonged proteolysis
results in the formation of smaller and highly soluble peptides comple-
tely lacking the functional properties of native proteins, such as water-
holding capacity, emulsification capacity and foaming ability. By careful
control of hydrolysis, it is possible to suitably modify the functional
properties which are useful in food formulations (Mackie, 1982a). The
degree of hydrolysis, which is expressed as the percentage of (X-amino
nitrogen in the soluble fraction (Monti and Jost, 1979), is important in
optimizing the process parameters in the product development. Quaglia
226 Production of fish protein hydrolyzates
and Orban (1987b) showed higher solubility of sardine meat accompa-
nied by a decrease in the molecular weight of protein fragments with
increasing degree of hydrolysis. The feasibility of employing 23 different
proteolytic enzymes was examined by Hale (1969), who observed that
pancreatin, papain and pepsin were suitable for the process. Sen et al.
(1962) defined digestion conditions using papain and found that at pH
7.0 maximum solubilization occurred in the first few hours of treatment.
The authors advocated 40°C instead of 65°C for obtaining longer pepti-
des during digestion. Venugopal and Lewis (1981) reported maximum
solubilization of low cost fish using pepsin. Hevia, Whitaker and Olcott
(1976) examined fish meat hydrolysis using bromelain, ficin and pro-
nase, and showed that pronase gave a product with less bitterness. Lean
fish species are ideal for the process since fatty species give products
containing significant amounts of lipids. Such hydrolyzates may require
centrifugation in order to remove the oil. Antioxidants may also have to
be used to control rancidity development during hydrolysis. Ritchie and
Mackie (1982) hydrolyzed several fish including both fatty and lean
varieties using papain added at 5% of the weight of fish meat. The
authors faced fewer problems in the preparation of spray dried FPH
from lean fish varieties as compared with fatty species. The dry powder
had 92% protein, 1.7% lipid and 6.4% ash. The yield of the powder
varied from 8 to 16% of the initial raw material (Merritt, 1982). A process
to make FPH from a low cost fish, i.e. dhoma, consisted of treatment
of the mechanically deboned meat with papain at 55°C for 2 h. The
powder obtained after spray drying of the hydrolyzate had a creamy
color and 90% protein content. Polyphosphate improved the functional
properties of the powder (Ghadi, Warrier and Ninjoor, 1987). Partial
deodorization of the mince prior to enzyme treatment could be done by
boiling it in water for a few minutes followed by screw pressing of the
cooked meat (Venugopal and Lewis, 1981). Preparation of FPH from
Antarctic krill, one of the major sources of non-conventional fish, has
been described by Sikorski and Naczk (1981). The process consisted of
initial low speed centrifugation to remove the gut of the fish followed
by mechanical deboning. The mince obtained was treated with either
neutrase or alcalase at 50°C for 2 h. The enzyme was inactivated by
steaming and the hydrolyzate was spray dried.

10.2.2 Properties and uses of FPH

The protein hydrolyzates have better functional properties as compared
with whole fish meat. However, bitterness is a commom problem of
protein hydrolyzates, which has been attributed to the formation of
peptides containing bulky hydrophobic groups towards the C-terminal
end (Roy, 1992; Kilara, 1985). Bitterness is more intense if extensive
hydrolysis of the protein has occurred. Several method have been
 Microbial proteases 227

suggested to mask the bitterness which include incorporation in the

FPH of glutamic acid or glutamyl-rich peptides, polyphosphates, gelatin
or glycine (Roy, 1992; Kilara, 1985). Another method is through the
plastein reaction, which is brought about by the addition of certain
proteases to the concentrated hydrolyzates, resulting in the synthesis of
a new polypeptide. Raghunath and McCurdy (1991) observed signifi-
cant amounts of plastein synthesis in autolyzed fish waste by pepsin at
pH values ranging from 5 to 8. The plastein reaction also improves the
nutritional quality and physical properties of the hydrolyzates (Satter-
lee, 1981). A protease derived from Aspergillus has been recognized to
be beneficial for the purpose (Kilara, 1985). Biotechnologically, the
bitterness score of a tryptic hydrolyzate of casein was decreased when
the product was incubated with immobilized cells of Erwinia ananas
(Watanabe, Shimizu and Arai, 1990).
The commercial aspects of FPH production have been discussed by
Kinumaki (1978), Ritchie and Mackie (1982) and Merritt (1982). Piggott,
Bucone and Ostrander (1978) studied the engineering aspects of a plant
for FPH production. The spray drying costs could be reduced if the fish
meat is directly treated with the enzyme, without addition of water,
which gave higher solid contents in the hydrolyzate (Rebecca, Pefta-
Vera and Diaz-Castaneda, 1991). However, the production cost of FPH,
based on a yield of 14% of the raw material, was more than that of fish
meal. The economic advantage of FPH as a milk replacement has to be
considered in this respect (Merritt, 1982).
Protein hydrolyzates are generally used for the modification of func-
tional properties of foods and in dietetic foods as a source of small
peptides and amino acids. As a result of its high solubility and amino
acid balance, FPH has obvious advantages over dried products like fish
protein concentrate or even human grade fishmeal, in these respects.
Its high dispersability makes it suitable as a replacement for milk
proteins. Although, at present, it is used as a milk replacer for calves in
France and the US (Kilara, 1985), its potential has been pointed out as
a protein supplement to cereal foods (Yanez, Ballester and Monckeberg,
1976), soups and bread (Kinumaki, 1978), and crackers (Yu and Tan,
1990). The development of products involving fish hydrolysis has been
reviewed by Owens and Mendoza (1985).

10.3 MICROBIAL PROTEASES

10.3.1 Characteristics of microbial extracellular proteases

Microbial proteases can be either extracellular or intracellular. Extracel-
lular enzymes are those secreted by the organisms and comprise mostly
hydrolytic enzymes including proteases. Several microorganisms sec-
228 Production of fish protein hydrolyzates
rete a variety of proteases, the yields of which depend upon the
nutritional composition of the growth media and the growth conditions.
For practical applications, proteases are produced from high-yielding
microbial strains by fermentations under controlled conditions. These
enzymes act on proteins at specific sites and, depending upon the site
of cleavage, they can be divided into exoproteases or endoproteases.
Exoproteases cleave the protein substrate at the N-terminal (aminopepti-
dases) or at the C-terminal (carboxy peptidases) end. On the other hand,
endoproteases (or proteinases) act on the protein molecule at sites away
from the terminal amino acid residues. All proteinases which are of
industrial importance are endoproteases rather than exoproteases. Mic-
robial proteases have broad specificities as indicated by the require-
ments for specific amino acid residues near the splitting point. Apart
from their sites of action on the protein, the enzymes are classified as
acidic, neutral or alkaline, depending upon pH requirements for optimal
activities. Acid proteases are widely distributed in molds and yeasts, but
are seldom found in bacteria, which generally secrete neutral or alkaline
proteases. Hartley (1960) classified the proteases based on their mechan-
ism of action and sensitivities to various types of inhibitors. These
classes include serine, thiol, metallic and acid (aspartic) proteases,
which have serine, thiol, metal ion or aspartic acid, respectively, in their
active sites. Several authors have summarized the information on micro-
bial proteases relating to their production, extraction, classification,
properties and uses (Glenn, 1976; Priest, 1983; Ward, 1985; Loffler,
1986). The role of microbial extracellular proteases in the spoilage of fish
has been discussed by Venugopal (1990).
Serine proteinases have both serine and histidine in their active sites.
They include trypsin-like proteinases and alkaline proteinases (Mori-
hara, 1974). Trypsin-like proteinases have an optimum pH of 8 and may
also possess esterase and amidase activities. Serine alkaline proteinases
are produced by a number of bacteria, yeast and molds including
Aspergillus orizae. These enzymes are commercially important since they
are used in detergents. Thiol proteinases are activated by reducing
compounds like cysteine and are inhibited by oxidizing agents. They are
sensitive to thiol reagents such as parachloro mercurybenzoate. Acid
proteinases show specificity towards aromatic or bulky amino acid
residues at both sides. They have an optimal pH of 3-4, and are
insensitive to inhibitors like difluorophosphate, parachloro mercuryben-
zoate and ethylenediamine tetraacetic acid. These proteinases find seve-
ral industrial applications. The metalloproteinases, which involve a
metal ion in their catalytic mechanisms, can be either neutral or alkaline.
Several bacterial and fungal proteinases belong to this category. Some of
the prominent microbial proteinases in terms of their structural or
catalytic nature are grouped as follows:
 Microbial proteases 229
Serine proteinases:
Bacillus subtilisin
Pseudomonas serine proteinase
Pseudomonas elastase
Aspergillus alkaline protease
Streptomyces serine proteinase
Staphylococcus serine proteinase
Saccharomyces alkaline proteinase
Thiol proteinases:
Clostridium histolyticum (clostripain)
Yeast proteinase B
Streptococcal proteinase
Staphylococcal thiol proteinase
Acid proteinases:
Aspergillus oryzae carboxy proteinase
Aspergillus saitoi carboxy proteinase
Aspergillus niger carboxy proteinase
Mucor pusillus carboxy proteinase
Rhizopus carboxy proteinase
Saccharomyces cerevisiae carboxy proteinase
Most of these enzymes have molecular weights ranging from 20 to 50
kDa.
Synthesis of proteinases by microorganisms occurs at minimal levels
during exponential growth. Enzyme secretion increases with a decrease
in nutrients and reaches a maximum towards the late exponential or
early stationary phase (Eveleigh and Montencourt, 1979; Glenn, 1976).
Several regulatory mechanisms, including catabolite repression, end
product inhibition and induction, operate in the syntheses and activities
of proteases. Although certain amino acids and other low molecular
weight compounds could inhibit enzyme synthesis, peptides and pro-
teins are capable of inducing protease production. Keil-Dlouhia, Mur-
shain and Keil (1976) reported that collagenase and a neutral protease
of Achromobacter iophagus were induced by high molecular weight subs-
trates such as collagen and ~-casein. Similarly, trypsinized casein,
neopeptone, gelatin and certain pep tides were found to be potent
inducers of some Aeromonas proteases (Litchfield and Prescott, 1970),
while peptone induced collagenase synthesis in Vibrio alginolyticus
(Reid, Woods and Rebb, 1980). Protease synthesis by Serratia marcescens
was enhanced by bovine serum albumin and peptone (Braun and
Schmitz, 1980). Washed cells of Aeromonas hydrophila (Alur, Nerkar and
Venugopal, 1988) and Bacillus subtilis (May and Elliott, 1968) secreted
proteases in the presence of myosin or casein hydrolysate, respectively.
Similarly, protease secretion by Erwinia chrysanthemi was enhanced by
230 Production of fish protein hydrolyzates
hydrolyzed casein and not by whole casein (Wandersman, Ando and
Bertheau, 1986). These data suggest the requirement of a certain short
chain peptide to trigger the synthesis of the enzyme at the optimal level.
It is now accepted that a basal level of constitutive exoenzyme degrades
to the exogenous protein substrate and the resultant low molecular
weight peptides enter the cells and induce further protease synthesis.
Whooley, D'Callaghan and McLoughlin (1983) demonstrated growth-
linked production of exoprotease by Pseudomonas aeruginosa in complex
substrates such as proteins. They observed that the ability to synthesize
protease provides the organism with a mechanism to survive in muscle
foods.
The mechanism of enzyme secretion has been explained through the
signal hypothesis (Blobel and Dobberstein, 1975). According to this,
enzyme synthesis proceeds on the membrane-bound ribosomes of the
organism. The primary precursors of the enzymes contain a hydropho-
bic amino acid sequence at the membrane receptors facilitating secretion
of the growing peptides through the membrane. The signal sequence is
cleaved proteolytically on the external side of the membrane and the
nascent pep tides acquires a stable conformation and activity.

10.3.2 Uses of microbial proteases in the food industry

As compared with animal and plant enzymes, microbial proteases have
general advantages in food applications. These include their compara-
tively lower cost of production, less time required for enzyme isolation,
possibilities for mass production and amenability of the organisms to
genetic manipulations to improve the enzyme yields. The applications
include production of oriental foods, bread-making, cheese ripening,
meat tenderization and chill-proofing of beer. A number of microbial
proteases also find applications in the detergent industry as well as for
diagnostic purposes. Some of the organisms which are sources of
commercial proteases include Aspergillus oryzae, Aspergillus miehi, Mucor
pusillus and Streptomyces griseus (Taylor and Richardson, 1979; Ward,
1985; Loffler, 1986). Many of the proteases from these sources have been
affirmed by the US Food and Drug Administration as 'generally recog-
nized as safe' (GRAS) (Loffler, 1986).

10.3.3 Uses of microbial proteases in fish hydrolysis

A number of microbial enzymes have been found to be superior to plant
enzymes in their ability to cause solubilization of proteins, including
those from fish. For example, bacterial neutral and alkaline proteases
were about 140% more efficient as compared with papain (Kilara, 1985).
Hale (1969) showed that hydrolysis of raw fish with Bacillus subtilis
protease at pH 8.5 gave a high yield of soluble product having an
 Biotechnological approaches to solubilization 231

excellent balance of amino acids. Similarly, pronase, a bacterial protease,

had twice the activity at 50°C as compared with that of ficin at 40°C or
bromelain at 50°C. The product obtained using pronase was also less
bitter (Hevia, Whitaker and Olcott, 1976). Liquefied fish protein having
negligible bitterness could also be obtained using protease from Aspergil-
lus niger (Kinumaki, 1978). Rebecca, Pena-Vera and Diaz-Castaneda
(1991) examined the efficacies of several microbial proteases for the
production ofFPH from mullet (Mugil cephalus). The fish protein was
hydrolyzed as such without the addition of water. An alkaline protease
from Bacillus subtilis (Pescalase 560) was found to be more efficient than
two neutral proteases from the same bacterium, giving more than 80%
nitrogen solubilization. The bacterial proteases showed higher activities
than fungal (Aspergillus oryzae) enzymes. The FPH had 70-85% available
lysine and could be used for fortification of bakery products, as a milk
substitute for calves as well as in fermentations as a highly concentrated
soluble nitrogen source. Archer et al. (1973) examined the kinetics of
solubilization of fish protein concentrate by Bacillus subtilis protease
(monozyme). The authors showed that the enzyme was adsorbed on
the surface of the substrate and the initial rate of reaction was propor-
tional to the surface area of the substrate exposed to the aqueous phase.
The overall kinetics were described by a sequence of two first-order
processes - an initial fast reaction in which loosely bound polypeptide
chains were cleaved from an insoluble protein particle and a second,
slower reaction in which more compact core protein was digested. These
results suggest that successful solubilization of fish meat could be
achieved by employing certain microbial proteases. It is, however,
essential that such microorganisms should be safe for food applications.

10.4 BIOTECHNOLOGICAL APPROACHES TO FISH MEAT

SOLUBILIZATION

10.4.1 Immobilized enzymes and microbial cells

Recent advances in biotechnology have shown potential for a variety of
food processing operations. Applications of microorganisms and micro-
bial enzymes are some major areas of biotechnology (Nga and Lee,
1990). Immobilized microbial enzymes and microorganisms offer scope
for the modification of food ingredients. This section will deal briefly
with some of these techniques and their applications in food processing.
Biocatalysts (enzymes and whole cells) whose mobilities are restricted
by chemical or physical means are called immobilized biocatalysts and
can be used for bioconversions. The different techniques of immobiliza-
tion have been described by a number of authors (D'Souza and Nad-
karni, 1981; Klibanov, 1983; Klein and Wagner, 1983; Klein and Vorlop,
Table 10.1 Some examples of biocatalysts immobilized on different carriers

Biocatalyst Method of Carrier Reference

immobilization
Acetobacter aceti adsorption cellulose Bar, Gainer and Kirwan (1986)
Clostridium thermocellum adsorption cellulose Weigel and Dykatra (1986)
Pseudomonas spp. adsorption activated charcoal Eharhardt and Rehm (1984)
Saccharomyces cerevisiae adsorption glass Haecht, BoIipombo and Rouxhet (1985)
Saccharomyces cerevisiae adsorption polyster Black et al. (1984)
Azotobacter vinclamdii ionic binding cellulose Diluccio and Kirwan (1984)
Saccharomyces cerevisiae covalent binding kieselguhr Navarro and Durand (1977)
Escherichia coli entrapment carrageenan Umemura et al. (1984)
Saccharomyces cerevisiae entrapment calcium-alginate Quereshi and Tamhane (1985)
 Biotechnological approaches to solubilization 233

1985; Hartmeier, 1986; Brodelius and Vandamme, 1987; Scott, 1987).

Applications of immobilized cells have also been discussed by Chibata
and Tosa, (1981), Linko and Linko, (1984) and Tanaka and Nakajima
(1990).
Immobilization can be achieved by binding biocatalysts to one another
or to carrier substances, by entrapping in the network of a polymer
matrix or by means of membrane confinement. Binding to carriers could
be through ionic adsorption, covalent binding or cross-linking. In
adsorption, biocatalysts are held to the surface of a carrier by physical
forces. The adsorbents could be selected from a wide variety of inorganic
as well as organic substances. A disadvantage of the technique is the
relative weakness of adsorptive binding which causes desorption by
temperature fluctuation, ionic concentration, etc. Ionic binding is based
on electrostatic attraction between oppositely charged groups of ion
exchange resins such as DEAE-cellulose. The binding is, however,
weaker than covalent binding. Covalent binding is generally employed
for coupling enzymes but not whole cells. In cross-linking, the indi-
vidual biocatalysts are formed with the help of multi-functional reagents
such as glutaraldehyde. However, since the particles obtained are gelati-
nous and access to substrate becomes limited, they are not ideal for
packed bed reactors.
Immobilization through matrix entrapment is widely used for whole
cell systems. The network gives good cell retention and porosity for
substrate and product transport. Hydrocolloids such as alginate and
carrageenan are frequently used for the purpose. The network has to
be formed in the presence of the cells to be entrapped and therefore the
network forming reactions have to be adapted to the physiological
requirements of stability and activity of the enzymes or cells. The
process is referred to as ionotropic gelation since the water-soluble
polyelectrolyte (alginate or carrageenan) is mixed with appropriate
counter ions when solidification by polysalt formation occurs. The
biocatalysts are formed by forcing the mixture of the hydrocolloid and
the microorganism into an aqueous solution of the gelling material. The
droplets falling into the solution are converted into beads upon gelation
which contain the entrapped organism. The stability of the beads may
be improved by drying (Scott, 1987). The well-known example is the
calcium alginate gel which is obtained by gelation of a sodium alginate
solution in CaCl2 solution (Klein and Vorlop, 1985).
Table 10.1 gives some of the examples of the different techniques used
for the immobilization of whole microbial cells.

10.4.2 Immobilised proteolytic enzymes

Immobilization of proteolytic enzymes has been reported by several
authors. Cross-linking of pepsin and trypsin using glutaraldehyde has
Table 10.2 Some applications of immobilized proteases and proteolytic microorganisms

Process Bioreactor Reference

Casein hydrolysis protease bioreactor Haque and Mozaffar (1992)

Soy protein hydrolysis pepsin on glass Mason et al. (1975)
Milk coagulation pepsin/calf rennet on glass Cheryan et al. (1975)
Fish hydrolysis Bacillus megaterium in Venugopal, Alur and Nerkar (1989)
calcium-alginate
Protein hydrolysis protease on fibre Deeslie and Cheryan (1981)
Protein hydrolysis immobilized pronase Lasch, Koelsch and Krelschmer (1987)
Meat fermentation lactic acid bacteria Kearney, Upreon and McLoughlin (1990)
in calcium alginate
Debittering of casein Erwinia ananas in Watanabe, Shimizu and Aral (1990)
hydrolysate calcium alginate
 Biotechnological approaches to solubilization 235

been developed by Khan and Siddiqui (1985). Proteolytic enzymes have

also been immobilized on polyethylene terephthalate (Blassberger, Free-
man and Goldstein, 1978), glass (Mason, Detar and Weetal 1975) and
chitosan (Leuba and Widmer, 1979). Such systems have been proposed
for food applications (Taylor and Richardson, 1979; Hultin, 1983). Lasch,
Koelsch and Krelschmer (1987) demonstrated continuous hydrolysis of
several proteins using immobilized protease reactors such as the stirred
tank, packed bed, fluidized bed and bubbled columns. The last men-
tioned reactor gave maximum efficiency. Other applications include
milk coagulation and cheese ripening (Table 10.2) (Deeslie and Cheryan,
1981; Loffler, 1986; Haque and Mozaffar, 1992). Commercial applications
of these processes, however, have not yet been achieved. Some of the
problems that may be encountered in these systems include autodiges-
tion of immobilized proteases, problems relating to mass transfer and
diffusion when working with viscous liquids (see Table 10.2).

10.4.3 Advantages of immobilized whole cells

As compared with immobilized enzymes, immobilized whole cells offer

several advantages. These include the following:

1. processes for extraction and purification of enzymes are not neces-

sary;
2. the yield of enzyme activity from immobilized cells is generally high;
3. the operational stability of immobilized cells is high;
4. the volume required for continuous reactors is low as compared with
batch reactors or conventional fermentations (for review, see Der-
vakos and Webb, 1991).

Immobilization of proteolytic microorganisms could result in enhanced

protease secretion by entrapped cells as compared with free cells (Klein
and Vorlop, 1985; Hartmeier, 1986; Scott, 1987).
Klein and Vorlop (1985) pointed out the need for consideration of
three factors in the development of an immobilized cell reactor. These
are activity, efficiency and stability. The relative activity of a reactor is
the ratio of activity of the immobilized system to that of free cells; while
absolute activity is the rate of reaction based on the unit weight or
volume of whole cells. High relative activity can be obtained using
natural polymers such as alginate or carrageenan for cell entrapment.
Efficiency is defined as the ratio of the actual reaction rate to the
maximum possible reaction rate. Loss of efficiency is caused by trans-
port limitation of a rate controlling substrate. Stability is the parameter
used to characterize the kinetic performance of immobilized systems.
Stability relates to both operational as well as storage stability of the
236 Production of fish protein hydrolyzates
system. Operational stability should be considerably higher for immobil-
ized systems than for whole cells.
Several types of reactors have been designed for the use of immobil-
ized whole cells. These consist of both batch-type and continuous
reactors. In continuous reactors the efficiency is higher since problems
of enzyme inhibition by products formed are minimal. Most bioreactor
systems now being used for immobilized cells are of the continuous
type, such as packed bed or fluidized bed systems (Scott, 1987). The
reader is also referred to information provided by Hartmeier (1986).
According to Scott (1987), immobilization of cells is well developed and,
therefore, the emphasis should now be on process developments,
including pilot plants, for the increased use of such systems in industry.
The efficiency of proteolysis using immobilized organisms could be
improved using recombinant DNA technology, as pointed out by Loffler
(1986). Such techniques would result not only in enhanced rates of
enzyme secretion by the cells but also in improvement of pH as well as
thermal stabilities of the secreted enzymes.

10.5 SOLUBILIZAnON OF FISH MEAT BY IMMOBILIZED

MICROBIAL CELLS

Only limited studies have been reported on the solubilization of fish

meat using immobilized microbial cells. The work done in the author's
laboratory (Venugopal, Alur and Nerkar, 1989) examined the efficacy of
three immobilized bacteria in causing hydrolysis of meat obtained from
a typical low cost fish, i.e. Johnius dissumeri. The mechanically deboned
fish meat was partially deodorized by boiling in excess water for 15 min
at low pH. The boiled meat was pressed in a screw press to remove
water and the odor-bearing compounds. The fish meat (4 g) was sus-
pended in 50 ml of water (pH adjusted to 8.0) and incubated with
calcium alginate beads containing about 1011 cells/g. The extent of
solubilization was determined by measuring tyrosine as well as the
nitrogen content in the supernatants. Figure 10.2 shows the extent of
solubilization of the fish meat by some immobilized microbial cells. It
can be seen that the degree of solubilization, measured in terms of total
nitrogen in the supernatant, was proportional to the incubation time
and reached a maximum at 24 h. The other two bacterial cells were less
efficient in causing solubilization as compared with Bacillus megaterium.
The optimal pH for solubilization was about 9.0, although significant
solubilization was observed in the pH range 7-9. Since the pH of the
suspension decreased due to the liberation of acidic amino acid residues,
pH was intermittently monitored using a pH wand. The calcium algin-
ate beads containing Bacillus megaterium cells had a storage life of about
1 month. A bead-holding assembly was used for treatment of the fish
 Future prospects 237

r----------------------,,--------------------.30
(a) (b)

Figure 10.2 Reaction progress curves for solubilization of fish meat by immobil-
ized bacterial cells. The fish meat (4 g) in 50 ml water was incubated with 4 g of
immobilized bacterial cells. The degree of solubilization was expressed as
percent of total nitrogen in the supernatant. •, B. megaterium; 1:::., A. hydrophila;
0, P. marinoglutinosa; x, mixture of beads of above cells. From Venugopal et al.
(1989). Copyright 1989, reprinted by permission of John Wiley & Sons, Inc.

meat in batch operations (Figure 10.3). The bead holder consisted of a

perforated acrylic cylinder, 5 cm in length and 3.5 cm in diameter. The
beads were distributed on disks inside the cylinder. The perforations on
the cylinder helped in the contact of fish slurry with the beads. Move-
ment of the slurry was attained by slow rotation of the assembly through
its attachment to a motor. An acrylic piece attached to the bottom of the
assembly facilitated stirring of the slurry during treatment. The equip-
ment helped in the recovery of the beads after use.

10.6 FUTURE PROSPECTS

The foregoing sections were intended mainly to point out the possibili-
ties of fish meat solubilization through biotechnological means.
Although only limited studies have been carried out in this respect,
scope exists for the use of microorganisms for the preparation of FPHs.
Since the raw material is non-commercial fishery resources, the method
for solubilization should be economically viable. Therefore, proteolyti-
238 Production of fish protein hydrolyzates

Figure 10.3 Bead-holding assembly for the solubilization of meat. From Venu-
gopal et al. (1989). Copyright 1989, reprinted by permission of John Wiley &
Sons, Inc.

cally active immobilized microbial cells appear to be ideal for the pur-
pose.
Instead of the batch process (Venugopal et al., 1989), the possibility
exists for development of continuous reactors for meat solubilization.
Continuous reactors for food processing involving hydrolysis have been
recently reported. Sims and Cheryan (1992) observed the feasibility of
using a membrane reactor for the hydrolysis of liquefied corn starch.
Enzyme bioreactors for continuous production of casein hydrolyzate
have been reported by Haque and Mozaffar (1992) and Mason (1975).
Development of a continuous system f;)r fish solubilization necessitates
that the meat should be in a free-flowing state, so that the substrate
could be run through a reactor containing immobilized microbial cells.
Recent studies have shown that washed meat mince from low cost fish,
 References 239
such as threadfin bream, Atlantic mackerel, capelin and shark, could be
readily converted into a gel (Venugopal and Shahidi, 1994; Venugopal,
Doke and Nair, 1994). The washing step, which is accomplished by
repeated treatment of the mince with chilled water, removes soluble
nitrogenous compounds and pigments as well as significant proportions
of lipids, and helps gelation (Lanier, 1986). The free-flowing characteris-
tic of the gel helps continuous hydrolysis in a suitable reactor. The
washed mince is highly deodorized and can give protein hydrolysates
having significantly less odor. The treated proteins could be concen-
trated by appropriate means such as spray drying. Studies in our
laboratory have already shown the possibility of preparing highly func-
tional, spray dried powders from protein gels of some low cost fish
items.
Immobilized microbial cells having proteolytic activity could also be
used to improve the drying properties of fish stick water, a by-product
from the fishmeal industry. Drying of the stick water often poses
problems due to a viscosity increase accompanying the concentration of
proteins. Conventionally, viscosity during drying operations is reduced
by the addition of proteases such as alcalase (Jacobsen and Rasmussen,
1984). It is likely that the stick water could be treated with immobilized
proteolytic microorganisms prior to dehydration. A third possible area
of application is for the reduction in the fermentation time required for
fish sauce production, which is usually prepared by the action of
autolytic enzymes on fish muscle (Greig and Estrella, 1988). As observed
recently by Dervakos and Webb (1991), while several advantages could
be claimed for viable cell immobilization, the technique may also intro-
duce certain problems relating to cell over-growth and leakage as well
as mechanical stability of the matrix, depending upon individual appli-
cation. Successful industrial applications of the process for fish protein
processing would therefore depend upon careful evaluation of the
advantages of the process for protein hydrolysis.

ACKNOWLEDGMENT

I thank Dr M.D. AIur for his valuable help in the experimental work
described in this chapter.

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