Fish Protein Hydrolysates: Production, Biochemical, and Functional

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=bfsn20

Download by: [Texas A&M University Libraries] Date: 12 January 2016, At: 11:28

Critical Reviews in Food Science and Nutrition

ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: http://www.tandfonline.com/loi/bfsn20

Fish Protein Hydrolysates: Production,

Biochemical, and Functional Properties

Hordur G. Kristinsson & Barbara A. Rasco

To cite this article: Hordur G. Kristinsson & Barbara A. Rasco (2000) Fish Protein Hydrolysates:

Production, Biochemical, and Functional Properties, Critical Reviews in Food Science and

Nutrition, 40:1, 43-81, DOI: 10.1080/10408690091189266

To link to this article: http://dx.doi.org/10.1080/10408690091189266

Published online: 03 Jun 2010.

Submit your article to this journal

Article views: 1946

View related articles

Citing articles: 262 View citing articles

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

Critical Reviews in Food Science and Nutrition, 40(1):43–81 (2000)

I. INTRODUCTION

At this time there are huge amounts of pro-

tein-rich byproduct materials from seafood pro-

cessing plants discarded without any attempt of

recovery. At the same time many processors are

no longer allowed to discard their offal directly

into the ocean, resulting in a very high cost of

refining the material before it is discarded. To

meet the need of the seafood processing industry,

an alternative to discarding these byproducts

should be developed. Recovery and alteration of

the fish muscle proteins present in the byproduct

material and using these as functional ingredients

in food systems is a very exciting and promising

alternative. However, for the industry to develop

processes for byproduct recovery and utilization

it has to be more economically feasible than dis-

carding the byproducts.

Every year over 91 million tons of fish are

harvested, of which 29.5% is transformed into

fishmeal.

1,2

Possibly more than 50% of the re-

maining fish tissue is considered to be processing

waste and not used as food.

With a dramatically

increasing world population and a world catch of

fish presently on the verge of exceeding the esti-

mated sustainable limits of the suggested 100

million tons/year, there is obviously an increased

need to utilize our sea resources with more intel-

ligence and foresight. By applying enzyme tech-

nology for protein recovery in fish processing, it

may be possible to produce a broad spectrum of

Fish Protein Hydrolysates:

Production, Biochemical, and Functional

Properties

Hordur G. Kristinsson* and Barbara A. Rasco**

Institute for Food Science and Technology, The School of Fisheries, University of Washington, Seattle,

Washington 98105

Referee: Dr. George M. Pigott, President, Sea Resources Engineering Inc., 4525 105 Avenue, N.W., Kirkland, WA, 98033

* Corresponding author: Present address: Department of Food Science, University of Massachusetts at Amherst, Marine Foods

Laboratory, Marine Station, Gloucester, Massachusetts 01930; Fax: (978) 281-2618; E-mail: [email protected]

** Present address: Department of Food Science and Human Nutrition, Washington State University, P.O. Box 646376, Pullman,

Washington 99164

ABSTRACT: Considerable amounts of fish processing byproducts are discarded each year. By developing

enzyme technologies for protein recovery and modification, production of a broad spectrum of food ingredients

and industrial products may be possible. Hydrolyzed vegetable and milk proteins are widely used food ingredients.

There are few hydrolyzed fish protein foods with the exception of East Asian condiments and sauces. This review

describes various manufacturing techniques for fish protein hydrolysates using acid, base, endogenous enzymes,

and added bacterial or digestive proteases. The chemical and biochemical characteristics of hydrolyzed fish

proteins are discussed. In addition, functional properties of fish protein hydrolysates are described, including

solubility, water-holding capacity, emulsification, and foam-forming ability. Possible applications of fish protein

hydrolysates in food systems are provided, and comparison with other food protein hydrolysates where pertinent.

KEY WORDS: fish protein hydrolysates, fish protein, functional properties, chemical hydrolysis of fish protein,

enzymatic hydrolysis of fish protein, protein functionality, fish byproducts.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

food ingredients or industrial products for a wide

range of applications. This would utilize both

fishery byproducts or secondary raw materials

and, in addition, underutilized species that would

otherwise be discarded.

Enzymatic modification of proteins using

selected proteolytic enzyme preparations to cleave

specific peptide bonds is widely used in the food

industry.

Hydrolysis of food proteins has a long

history, mainly for vegetable and milk proteins;

these proteins are widely used in the food indus-

try. Most work on the hydrolysis of fish proteins

was conducted in the 1960s. Some fish protein

hydrolysate (FPH) preparations at that time were

quite successful.

During the 1960s, research was

directed to the production of cheap nutritious pro-

tein sources for rapidly growing developing coun-

tries, or toward animal feed production, primarily

through production of fish protein concentrates

(FPC). Little work has been done recently on

FPH, but some research has been directed into the

potential of using powdered hydrolysates in food

formulations. Many studies have resulted in fish

protein hydrolysates with excellent functional

properties. However, taste defects, specifically

bitterness, and process economics are still major

limiting factors for FPH applications.

This review gives an overview of the differ-

ent techniques for production of fish protein hy-

drolysates, past and present research on their prop-

erties, and various methods to study the extent of

hydrolysis and product functionality.

II. THE BIOCHEMICAL

CHARACTERISTICS OF FISH MUSCLE

PROTEIN

In foods, a protein is traditionally categorized

as a fibrous or globular protein based on its ter-

tiary conformation. Each type of food protein has

a unique molecular structure that determines its

functional properties, in addition to a range of

environmental conditions over which it exhibits

such properties.

These factors and their effect on

functionality are discussed in more detail later in

this review.

The functional and structural properties of

food proteins thus vary tremendously, and to fully

understand the process of protein hydrolysis it is

crucial to have a good understanding of the nature

of the protein substrate and the hydrolyzing agent.

During protein hydrolysate manufacture, the pro-

tein substrate is hydrolyzed by either a proteolytic

enzyme or an acid or base.

Our diet contains a wide variety of proteins

from different sources. It is generally accepted

that the relative concentration of dietary essential

amino acids is the major factor determining the

nutritional value of food protein.

Proteins de-

rived from animal sources are considered to be

nutritionally superior to those from plants be-

cause they contain a better balance of the dietary

essential amino acids. Of these egg and milk pro-

teins (casein) are frequently used as reference

proteins for evaluating protein quality. Proteins

derived from meat and poultry muscle are also of

very high quality and fish muscle proteins are

equally nutritious.

Fish muscle contains an ex-

cellent amino acid composition and is an excel-

lent source of nutritive and easily digestible pro-

teins.

9,10

However, because fish is extremely

perishable and because chemical composition can

vary, the utilization of fish as a basic raw food

material presents unique food processing prob-

lems.

The muscle of different animals is very simi-

lar, containing similar protein and similar amino

acid profiles. There are slight differences between

fish muscle and the muscle of land animals. These

are mainly associated with the differences in

muscle structure required for swimming and buoy-

ancy. Fish are supported by a mass of water, thus

the muscle fibers require less structural support

than those in land animals. Because of this, fish

muscle tends to have less connective tissue than

muscles from terrestrial animals, resulting in more

tender texture. Also, because of the unique move-

ment of fish, the structural arrangement of muscle

fibers is quite different from terrestrial animals. A

large fraction of commercially utilized fish stocks

are cold adapted or poikilothermic, and because

of this their muscle proteins have different bio-

chemical properties compared with those of en-

dothermic animals.

Poikilothermic characteris-

tics of fish proteins make them more heat sensitive

than mammalian muscle proteins, with a greater

tendency to denature at elevated temperatures.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

Fish muscle proteins from cold water species are

more prone to denaturation than those from tropi-

cal waters.

13,14

The T-50 values (temperature re-

quired for 50% denaturation) of fish muscle pro-

teins are also influenced by pH and were reported

to be 29 to 35°C at pH 7.0 and 11 to 27°C at pH

5.5.

Protein composition in muscles varies by

muscle type. Of the three types (striated, smooth,

and cardiac muscle) of muscles, the striated

muscles are the predominant form in fish. Striated

muscle tissue is arranged into muscle fibers that

are bound together by a connective tissue to make

a fiber bundle. Fish muscle has “white” and “dark”

meat.

The white meat is generally more abun-

dant, contains less lipids than the dark meat, and

is the most widely consumed type of muscle tis-

sue. It is composed of about 18 to 23% of protein,

depending on the species and time of harvesting.

Fish proteins can be divided further into different

groups based on their solubility. About 70 to 80%

of fish muscle are structural proteins. These struc-

tural protein are soluble in cold neutral salt solu-

tions of fairly high ionic strength. The remaining

20 to 30% contain sarcoplasmic proteins that are

soluble in water and dilute buffers, and a final

part of the structural protein, 2 to 3%, being in-

soluble connective tissue proteins.

Recent stud-

ies, however, challenge these generally accepted

solubility data, showing that the muscle protein

components can be highly soluble at low ionic

strenghts.

16,17

Myofibrillar proteins are the primary food

proteins of fish, comprising 66 to 77% of the total

protein in fish meat. The myofibril protein com-

plexes contain myosin and actin. These are the

main components of the thick filament, and thin

filament, respectively. Myosin comprises 50 to

60% of the myofibrillar contractile proteins, and

actin only 15 to 30%.

12,15

Myosin is the most

abundant of the single muscle proteins, making

up around 38% of the total, and is a large mol-

ecule containing two identical heavy chains

(223 kDa) and two light chains (22 and 18 kDa).

The molecule has two identical globular head

regions that incorporate the light chains and a

significant fraction of the heavy chains. The tails

of the heavy chains form very long α-helices that

wrap around each other

(Figure 1). Myosin can

be cleaved by proteases at two sites on the mol-

ecule, one recognized by both trypsin and chy-

motrypsin and the other by papain. Papain cleaves

near the head region, releasing the head from the

tail. Trypsin and chymotrypsin cleave further from

the head, dividing the molecule into two compo-

nents called the heavy meromyosin (with the head

region) and the light meromyosin, both with dif-

ferent functional properties.

Myosin molecules are connected via their head

region to the polymerized actin molecules in the

thin filaments due to the ATPase activity of the

head molecules. This complex is called actomyo-

sin and is responsible for muscle contraction and

relaxation. Actomyosin plays a major role in de-

termining the quality of fish meat because it is

quite labile and easily affected during processing

and storage. For example, during frozen storage

the actomyosin becomes progressively less soluble

and the flesh becomes increasingly tougher.

The thin filament is a complex of actin mol-

ecules making a double helix. Tropomyosin sits

within the grooves of the thin filaments and two

FIGURE 1. Fish myosine molecule.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

troponin molecules bind the actin filament at each

helical repeat. Actin is the most prominent pro-

tein of the three protein in the thin filaments,

making up about 13% of the total muscle pro-

teins. Actin occurs in two forms, G-actin, a spheri-

cal monomer, and F-actin, a large polymer that

connects to myosin. The thin filaments play a

very important role by regulating muscle contrac-

tion. From the point of view of muscle biochem-

istry, thin filaments are very important, however,

their content is low in meat and their role with

respect to food processing has not been studied

completely. Other contractile proteins of interest

are C-protein, α-, and β-actinin, connectin and

paramyosin; however, they are of limited interest

as food proteins. With respect to protein hydroly-

sis, the myofibrillar protein myosin, actin, or ac-

tomyosin are subject to enzymic cleavage and are

the greatest focus here.

III. PROTEIN HYDROLYSIS

Proteolytic modification of food proteins to

improve palatability and storage stability of the

available protein resources is an ancient technol-

ogy.

Hydrolysates can be defined as proteins

that are chemically or enzymatically broken down

into peptides of varying sizes.

Protein hydroly-

sates are produced for a wide variety of uses in

the food industry, including milk replacers, pro-

tein supplements, stabilizers in beverages and fla-

vor enhancers in confectionery products. The

benefits of hydrolyzing food proteins to make

functional protein ingredients and nutritional

supplements is a more recent technology, with the

first commercially available protein hydrolysates

appearing only around the late 1940s. Although

production is massive worldwide, the proper con-

trol of the process and the exact mechanism be-

hind protein hydrolysis is in most cases not fully

understood. Recent advances have given research-

ers insight into the connection between the pro-

cess/extent of hydrolysis and the physicochemi-

cal mechanisms responsible for specific functional

properties of the hydrolyzed protein. Recent re-

search on enzyme catalysis has also aided with

the proper selection of enzyme catalysts and pro-

cessing conditions to obtain better control over

the reaction and characteristics of the final prod-

uct.

Chemical and biological methods are the most

widely used for protein hydrolysis with chemical

hydrolysis used more commonly in industrial prac-

tices. Biological processes using added enzymes

are employed more frequently, and enzyme hy-

drolysis holds the most promise for the future

because it results in products of high functionality

and nutritive value. The chemical and biological

hydrolysis are discussed in more detail below,

with an emphasis on hydrolysis with added en-

zymes. In addition, there are many potential tech-

niques for extracting protein from animal tissue.

These include the use of aqueous and organic

solvents; the conventional processes of cooking,

pressing, drying, and hot oil extraction.

The

extraction of protein by means of solvent is also

worth mentioning due to its industrial and histori-

cal importance for fish protein recovery.

A. Chemical Methods for Protein

Hydrolysis

1. Chemical Extraction: The Making of

Fish Protein Concentrate

The extraction methods mentioned above,

other than the chemical and biological hydrolysis

methods, do not hydrolyze protein. They are used

primarily to concentrate intact protein by the re-

moval of water and oil from the substrate. The

method of solvent extraction has been frequently

employed when producing fish protein concen-

trate (FPC). The development of FPC was one of

the earliest attempts to recover fish protein from

processing waste and to produce a protein ingre-

dient from underutilized species. FPC was the

precursor to the field of enzyme hydrolysis of fish

proteins. A small but extensive research program

on the large-scale production of FPC by the Bu-

reau of Commercial Fisheries, now the National

Marine Fisheries Service (NMFS) of the Depart-

ment of the Commerce, began in 1961. The gen-

eral aim of the program was to study the manufac-

ture and use of FPC as a solution to global protein

malnutrition and as a potential economic stimulus

to the American fishing industry.

Solvent-ex-

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

tracted FPC (type A FPC) is produced by using

primarily isopropanol or azeotropic extraction with

ethylene dichloride, although other solvents such

as ethanol have been used successfully as well. A

standard process presented by Sikorski and

Naczk

shown in Figure 2 is to grind a whole or

eviscerated fish, extract it with isopropanol at a

low temperature (20 to 30°C) for 50 min, then

collect the supernatant and extract it twice again,

first at 75°C for 90 min in isopropanol and then at

75°C for 70 min with azeotropic isopropanol. The

final supernatant fraction is collected, dried, milled,

and screened to separate out bone particles. The

final product has a high biological value and is

colorless and odorless, with less than 1% lipids.

The problem with type A FPC is that it is not

readily soluble or dispersible in foods and has

poor emulsification properties.

9,25,26

Dubrow et al.

FIGURE 2. A production scheme for fish protein concentrate. (Adapted from Ref. 24.)

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

reported that FPC produced at higher tempera-

tures (50°C) compared with lower temperatures

(20°C) had significantly lower emulsifying prop-

erties, but both had very poor solubility. General

poor functionality, off-flavors the high cost of

production, and traces of solvent in the final prod-

uct made solvent extracted FPC commercially

unsuccessful despite concerted efforts.

3,28

Al-

though FPC lacks solubility, it reportedly has

good foaming properties over a wide range of pH

(pH 2 to 11), making strong, stable foams.

29–31

Despite problems with protein functionality, sol-

vent extraction is the method of choice for the

abundant fatty pelagic fish species such as sar-

dine, herring, and capelin because the protein is

effectively separated from the lipids, thereby re-

ducing stability problems normally associated with

residual oxidizable lipid. For fatty fish, isopro-

panol was a slightly more efficient solvent than

ethanol considering the residual amounts of lip-

ids, but absolute ethanol produced FPC of lighter

color and a neutral flavor.

Many studies with FPC have also been con-

ducted with solvent-extracted FPC as a substrate

for enzyme hydrolysis, both to defat the substrate

and to make it more accessible to enzymatic hy-

drolysis, with excellent functional and nutritional

properties.

5,25,33,34,35,36

However, enzymatic hy-

drolysis using FPC as a starting substrate resulted

in loss of some functional properties because of

excessive protein breakdown but increased nitro-

gen solubility.

Taste and odor problems are

generally minimized with a FPC starting mate-

rial.

Recent studies with solvent-extracted FPC

have produced FPC with better protein function-

ality. For example, recently Vareltzis et al.

stud-

ied the addition of ethanol-extracted FPC made

from sardine to hamburger patties and found that

the overall functional properties (water binding

and cooking yield) and the penetration depth and

shear force value of the hamburger increased with

the addition of FPC. However the hamburgers

had a slightly unfavorable fishy flavor. Hoyle and

Merritt

found that herring protein extracted with

ethanol in a similar manner and then hydrolyzed

with either Alcalase or papain produced a hy-

drolysate with a markedly reduced bitterness and

less fishy odor.

2. The Chemical Hydrolysis Process

Chemical hydrolysis of proteins is achieved

by cleaving peptide bonds with either acid or

base. Several processes have been proposed for

the acid or alkaline hydrolysis of fish.

This has

been the method of choice in the past for the

industry primarily because it is relatively inex-

pensive and quite simple to conduct. There are,

however, many limitations to food ingredients

using this method. Chemical hydrolysis tends to

be a difficult process to control and almost invari-

ably leads to products with variable chemical

composition and functional properties.

21,38

Pro-

tein hydrolysis with strong chemicals and sol-

vents is performed at extreme temperatures and

pH and generally yield products with reduced

nutritional qualities, poor functionality, and re-

stricted to use as flavor enhancers.

39,40

a. Acid Hydrolysis

Acid hydrolysis of proteins is used more com-

monly than hydrolysis under alkaline conditions.

A vast majority of hydrolyzed proteins consumed

in the U.S. are prepared by acid hydrolysis, mostly

from inexpensive vegetable protein sources that

otherwise would have poor nutritive and little

functional value in foods. Although the process is

harsh and hard to control, it is still the preferred

method for hydrolyzed vegetable proteins. Hy-

drolyzed vegetable protein, which are widely used

for flavor and taste enhancement properties, re-

quire extensive acid hydrolysis.

Applications of

hydrolyzed vegetable proteins are primarily as

flavoring agents in processed meat, crackers, and

soup mixes. Acid hydrolysis of fish protein has

usually involved reacting fish proteins with hy-

drochloric acid, or in some cases sulfuric acid,

and the proteins are completely hydrolyzed at

high temperature, and often high pressure. The

hydrolysate is then neutralized to pH 6.0 to 7.0

and concentrated to either a paste or further dried.

Because the product is hydrolyzed extensively,

its primary functional property is high solubility.

Total hydrolysis of fish protein substrate can be

achieved in 18 h at 118°C in 6N hydrochloric

acid.

In addition, following the neutralization of

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

the digest, the hydrolysate contains large amount

of salt (NaCl), which can make the product unpal-

atable and interferes with functionality in food

systems. Another drawback of acid hydrolysis is

the destruction of tryptophan, which is an essen-

tial amino acid. Orlova et al.

proposed an acid

hydrolysis process of whole fish, where steam

distillation is used to remove aromatic substances

followed by filtration then concentration. The

concentrate was used in dehydrated soup cubes

and as a microbial media.

The acid hydrolysis is

also widely utilized to convert underutilized and

secondary raw material from fish into fertilizer

due to the low production cost and resulting ex-

tensive hydrolysis.

b. Alkali Hydrolysis

The use of alkali reactants, primarily sodium

hydroxide, to hydrolyze protein often results in

poor functionality and more importantly can ad-

versely affect the nutritive value of the hydroly-

sate. Despite this, limited alkali treatment is used

in the food industry to recover and solubilize a

broad range of proteins. For example, mechani-

cally deboned turkey residue (MDTR) includes a

significant proportion of alkali-soluble proteins

that can be recovered by alkali treatment and used

in food applications. Fonkwe and Singh

dis-

cussed the use of alkali extraction to recover

MDTR with an alkaline sodium chloride solution

but found it to be unsuitable due to low recovery.

Alkaline hydrolysis of fish proteins has primarily

used FPC as the starting substrate. During alka-

line hydrolysis of fish protein, rapid cleavage to

large water-soluble polypeptides takes place, fol-

lowed by further degradation at a slower rate.

Alkali treatment can aid in modifying the proper-

ties of insoluble FPC.

Tannenbaum et al.

45,46

have studied the alkaline process for hydrolyzing

insoluble FPC and its applications. They devel-

oped a small-scale batch process that utilizes high

pH (12.5) and 95°C for 20 min. The product

consisted of large peptides, some relatively in-

soluble at the isoelectric point, but with an overall

improvement in functionality with respect to the

original FPC. Use of the solubilized FPC as a

milk substitute gave a product far superior to that

obtained with FPC starting material, which had

poor solubility and dispersibility.

Several deleterious reactions occur in alka-

line solutions during hydrolysis. These are initi-

ated by hydrogen abstraction from the alpha car-

bon of an amino acid and include racemization of

L-amino acids, which produces D-amino acids,

which are not absorbed by humans. Also, disul-

fide bonds are split with loss of cysteine, serine,

and threonine via β-elimination reactions and

formations of lysinoalanine, ornithinoalanine,

lanthionine, and β-amino alanine can also oc-

cur.

Some of these elimination and addition re-

actions may lead to the formation of toxic sub-

stances (e.g., lysinoalanine) that are highly

undesirable in foods.

47,48

Alkaline hydrolysis re-

action products have an inhibiting effect on pro-

teolytic enzymes, reducing the rate of hydroly-

sis.

Some of the possible reaction products that

may form during alkali hydrolysis are shown in

Figure 3.

High collagen solubility is also ob-

served with alkali treatment.

B. Biochemical Methods for Fish Protein

Hydrolysis

Biochemical hydrolysis to produce fish or

other food protein hydrolysates is performed by

utilizing enzymes to hydrolyze peptide bonds.

This can be done via proteolytic enzymes already

present in the fish viscera and muscle (endog-

enous proteases), or by adding enzymes from other

sources. To understand the process of enzymatic

hydrolysis, it is very important to understand the

nature and activity of proteolytic enzymes.

1. Proteolytic Enzymes

Enzymes are biochemical catalysts vital for

living beings, because they accelerate chemical

reactions between organic constituents within the

cells that otherwise would take an extremely long

time to complete. In food science and technology

enzymes are exploited to perform desired func-

tions in processing and analysis and to facilitate

the conversions of raw materials into higher-qual-

ity, more desirable foodstuffs.

Enzymes make

this possible because the active site of an enzyme

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

FIGURE 3. Possible chemicals that may form in the alkali-treated proteins.

is highly specific for certain substrates. Enzymes

catalyze only one specific reaction and function

by forming a complex with the substrate whose

transformation they catalyze.

Enzymes used by the food industry and in

food research are predominantly hydrolases, most

of which are carbohydrases, followed by pro-

teases and lipases. Proteases are among the best

characterized enzymes. Proteolytic enzyme prepa-

rations are economically the most important group

of enzymes, and their use is well established in

the food industry.

Proteases are categorized ac-

cording to the specificity of the peptide bonds

they attack (hydrolyze) and the mechanism by

which they act.

Four major classes of proteases

are known. They are designated by the principal

functional group in their active site: serine, thiol,

carboxyl and metallo.

Proteases are character-

ized further by their hydrolyzing mechanism into

endoproteinases or exopeptidases. The endo-

proteinases cleave/hydrolyze the peptide bonds

within protein molecules, usually at specific resi-

dues to produce relatively large peptides. The

exopeptidases systematically remove amino acids

from either the N terminus, called aminopepti-

dases, or the C terminus, called carboxypepti-

dases, by hydrolyzing the terminal peptide bonds.

In food protein hydrolysis, endoproteinases are

always used, but occasionally endoproteinases are

combined with exopeptidases to achieve a more

complete degradation.

Although the four classes of proteases men-

tioned above have different catalytic mechanisms,

they all share a common transition state (interme-

diate) during catalysis. To discuss enzyme kinet-

ics in any detail would require a separate review,

and this is not intended here. However, it is im-

portant to know the basic steps in enzyme cataly-

sis and to understand the mechanism of protein

hydrolysis. Some proteases preferentially cata-

lyze the hydrolysis of bonds adjacent to a particu-

lar amino acid residue, while others are less spe-

cific. The catalysis by proteases occurs primarily

as three consecutive reactions: (1) the formation

of the Michaelis complex between the original

peptide chain and the enzyme, (2) cleavage of the

peptide bond to liberate one of the two peptides,

and (3) a nucleophilic attack on the remains of the

complex to split off the other peptide and to re-

constitute the free enzyme.

20,54

The hydrolysis of

peptide bonds leads to an increase in the numbers

of ionizable groups (NH

and COO

), with a con-

comitant increase in hydrophobicity and net

charge, a decrease in molecular size of the polypep-

tide chain, and an alteration of the molecular struc-

ture leading to the exposure of the buried hydro-

phobic interior to the aqueous environment.

55–58

Giving the substrate the symbol S, the enzyme E

and the peptides in the reaction P, the overall

mechanism can be presented as:

E S ES EP H P

EOHPHP

+←→→+−

′

→+−+−

′

–

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

This enzyme-substrate complex may dissoci-

ate back to reactant substrate and free enzyme, or

to free enzyme and product molecules. In other

words, classic Michaelis-Menten kinetics apply.

The generally accepted mechanism for proteases

indicates that the second step is the rate-determin-

ing step, thus k

primarily determines the overall

reaction speed, and K

is more or less equal to the

true dissociation constant. This simple mecha-

nism does not, however, deal with the detailed

question of how the enzyme and substrate are

bound or what molecular configurations lead to

product formation. To fully understand the ca-

talysis, a fairly detailed explanation is in order,

which is not the purpose of this review.

Enzymatic hydrolysis of proteins is a complex

process because of several peptide bonds and their

specific accessibility to enzymatic reactions.

The

specificity of enzymes is not the only factor that

affects the peptide profile of the final product.

Environmental factors such as temperature and pH

play an important role. Both temperature and pH

can greatly affect the enzyme reaction kinetics, and

the effect of these factors is different for each

enzyme. Generally, there is an optimum combina-

tion of both pH and T, where an enzyme is the most

active. Temperature and pH extremes deactivate

the enzymes by denaturing them.

2. Autolytic Hydrolysis

Biochemical production of fish protein hy-

drolysates may be carried out by employing an

autolytic process. An autolytic process depends

on the action of the digestive enzymes of the fish

itself. There are no enzyme costs involved, and it

is a simple operation.

The end product of au-

tolytic hydrolysis is generally a fairly viscous

liquid rich in free amino acids and small peptides.

The digestive enzymes in question are primarily

the serine proteases trypsin and chymotrypsin,

and the thiol protease pepsin, all major enzymes

of fish viscera and digestive tract. Lysosomal

proteases, or catheptic enzymes, present in fish

muscle cells also contribute to proteolytic break-

down to some extent.

The endogenous enzymes in autolytic hydroly-

sis are a very complex mixture of enzymes, all

with different activity requirements, which result

in end products of different molecular profiles.

Another complication is that the presence of cer-

tain digestive enzymes and their concentration

may be highly seasonal, gender and age specific,

and can vary tremendously within a species as

well as between species. These variations make it

very hard to control the hydrolytic process, and

direct the production of hydrolysates with spe-

cific molecular properties. Autolytic methods such

as chemical methods often result in a final prod-

uct with bad functionality. Despite these prob-

lems, endogenous proteolytic enzymes are used

to produce hydrolyzed products, specifically fish

sauces and fish silage.

The production of fish sauce preceded fish

silage production and is the major fermented fish

product presently consumed in the world. Its pro-

duction has thousands of years of tradition in

Asia, and it is also known to have been produced

in Mediterranean countries in ancient times. Pres-

ently, fish sauce is used mainly as a condiment on

rice dishes like the popular Nuoc-Nam produced

in Vietnam, and the annual production in South-

east Asia is about 250,000 metric tons.

The

production of fish sauce does not require elabo-

rate processing equipment. The substrate is usu-

ally fish from one or more pelagic species, such

as anchovies or sardines, or minced whole fish of

low commercial value. The substrate is immersed

in a solution containing high concentrations of

salt (20 to 40%) and at relatively high tempera-

tures, preferably ambient tropical temperatures.

In the case of whole fish, the visceral proteolytic

enzymes start by hydrolyzing the stomach con-

tents, then work their way through the stomach

wall, and finally reach the muscle tissue, where

they join with catheptic enzymes to hydrolyze

fish muscle proteins. The pH of the fish sauce

process is usually neutral, because no base or acid

is added to the reaction mixture. The acid-depen-

dent proteolytic enzymes, such as pepsin and the

catheptic enzyme, contribute little to fish sauce

production. The endogenous serine proteases very

slowly breakdown the fish muscle for 6 to 12

months (Nuoc-Mam) under anaerobic conditions.

This slow but extensive breakdown results in a

liquefied fish sauce composed predominantly of

free amino acids, with up to 50% nitrogen recov-

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

ery. The high concentration of salt is responsible

for the slow hydrolysis because this slows the

activity of the proteolytic enzymes. More impor-

tantly, the high salt concentration and anaerobio-

sis totally inhibits growth of spoilage microor-

ganisms once the salt has fully penetrated the

tissue.

Lower concentrations of salt, however,

result in sauces with higher yield, lower levels of

volatile acids, and better balanced composition of

amino acids.

During autolysis to produce fish

sauce, a lipid phase, an aqueous soluble phase

that contains much protein but little lipid, and

insoluble sediment of protein and lipid is formed.

After the hydrolysis is completed, the liquid pro-

tein hydrolysate is tapped, filtered, and bottled,

with the final product containing up to 10% free

amino acids and low-molecular-weight peptides,

and 25% salt.

26,60

Although the production of fish

sauce does not improve the nutritive value of the

protein, the keeping quality is greatly increased

and organoleptic characteristics are generally

improved.

The process of manufacturing fish silage is

different, but the product shares many character-

istics with fish sauce. The production of fish si-

lage was not started until the middle of this cen-

tury, and it is far from being as widely employed

as fish sauce production. The application of fish

silage is primarily for animal feed production

instead of food applications. The process is rapid

and enzymes involved are very effective in pro-

ducing oil and proteins fractions that are readily

separated. The substrate is usually secondary raw

material from fish processing or underutilized fish

species. The substrate is mixed with strong min-

eral acids or organic acids such as formic acid to

acidify the mixture below pH 4. At this pH the

serine proteases are generally inactive, but pepsin

and the catheptic enzymes are highly active. The

pepsin content can be very high in the visceral

portion of fish, and the enzyme is primarily re-

sponsible for fish silage production. Under acidic

conditions the active endogenous enzymes par-

tially hydrolyze the fish over several weeks to

produce a slurry containing up to 12% amino

acids and peptides. Usually about 80% of the

protein in acid fish silage become solubilized af-

ter 1 week at temperatures around 23 to 30°C.

The rate is primarily dependent on conditions

such as ambient temperature and relative amounts

of the visceral organs present. The processing

time is much shorter than for fish sauce because

no salt is added.

26,60

The production of fish silage

using lactic acid bacteria as the hydrolyzing agent

has been reported.

66,67

The bacterial fermentation

is initiated by mixing minced or chopped fish

with a fermentable sugar that favors the growth of

lactic acid bacteria, which is advantageous be-

cause the bacteria produces acid and antimicro-

bial factors that inhibit competing bacteria.

The feed applications of fish silage are prima-

rily limited to young animals due to the extensive

hydrolysis of the proteins. For fish silage to be

incorporated successfully in animal feeds, it has

to contain the majority of the nitrogen fraction as

intact proteins or peptides rather than as free amino

acids, which are less well absorbed. A shorter

processing time and added commercial proteases

may be useful in such instances. Also, problems

connected with the development of bitterness in

the hydrolyzed silage can make the product highly

unpalatable not only for humans but also animals

fed feeds rich in fish silage. The utilization of fish

silage as a primary protein source in fish feeds has

been neither fully investigated nor commercially

successful; however, it is incorporated into diets

for pigs, poultry, and mink.

Research on fish hydrolysates made with en-

dogenous enzymes for human food applications

has been very limited. Fish sauce is almost the

only autolytically produced food of aquatic ori-

gin. The main reason for this is fairly straightfor-

ward. To produce a functional protein hydroly-

sate with specific properties, a good knowledge

of the enzymes involved is crucial. Endogenous

enzymes in fish are a complex and highly variable

mixture, and thus the properties of functional pro-

tein hydrolysates so prepared may vary greatly

under the same reaction conditions. Also, in the

U.S. the sale of processed fish foods containing

visceral material of any kind is prohibited by the

FDA. Despite this, some work on developing fish

silage for human consumption has been conducted.

In 1972, Malcolm B. Hale and a group of scien-

tists at NMFS and the University of Maryland

conducted a comprehensive study on making func-

tional FPH by enzymatic hydrolysis for human

consumption.

This study involved autolysis of

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

red hake (Urophycis chuss), a relatively lean fish,

and alewife (Alosa pseudoharengus), a very fatty

fish. The autolysis of raw hake was conducted at

optimal conditions for the native enzymes, 50°C

and pH 7.0, for 24 h where fish was 50% of the

total slurry. The resulting hydrolysate was either

spray dried or tested as a concentrate. The aver-

age yield of dry solids for hake was lower than for

any other proteolytic enzyme employed, 10.0%

(dry solubles/wet fish), compared with the high of

14.3% from using Alcalase. Similarly, the chemi-

cal score attained for soluble hydrolysate were

quite low due to low tryptophan content that is

correlated with a low recovery. The protein effi-

ciency ratios for both red hake and alewife hy-

drolysates were essentially equivalent to that of

casein. Also, the inclusion of insoluble solids in

the final product resulted in very high fat content.

The results for lipid pressed alewife were similar.

However, by lowering the reaction period to 4 h

and raising the temperature to 55°C, a satisfactory

product with good nutritional value was prepared.

Both the red hake and alewife were only 50 to

70% soluble, requiring substantial additions of

commercial enzymes, at uneconomic levels, to

become fully soluble. The alewife hydrolysate

also suffered from very fishy taste; however, the

red hake hydrolysate had a less fishy taste and

odor. Food applications of the products obtained

by Hale and co-workers were limited and could

be used primarily as a protein supplement in cul-

tures where its taste would be acceptable and the

caloric value of the lipid desirable.

Shahidi et al.

hydrolyzed ground capelin

(Mallotus villosus) by endogenous enzymes and

found that it enhanced the overall extraction of

the fish protein at both acid and alkaline pH, as

both acid and alkaline proteases are present in

fish muscle and viscera. The protein recovery of

hydrolysates produced autolytically was, however,

considerably lower compared with commercial

enzymes, 22.9% compared with 70.6% with

Alcalase. A recent study by Cui

with chum

salmon (Oncorhynchus keta) mince and visceral

content showed a surprisingly extensive and rapid

hydrolysis at an acid pH and 37°C. The hydroly-

sate also showed a marked difference in the mo-

lecular weight distribution of peptides when com-

pared with a commercial pepsin hydrolysis. The

native acid enzymes resulted in a product with the

majority of peptides of lower molecular weight

than the pepsin hydrolysate under the same ex-

perimental conditions. This indicates that protein

hydrolysates can be obtained through autolysis

very efficiently at relatively mild temperatures.

The functional properties of the product were,

however, not investigated.

The main limitation of work performed on

autolytic hydrolysis of food proteins is the lack of

research on functional properties. Studies have

shown that protein recovery can be adequate and

that nutritional requirements are good, but infor-

mation on functional properties of the resulting

hydrolysate is very important to successfully evalu-

ate its use in formulated foods.

3. Enzymatic Hydrolysis of Fish Muscle

Proteins with Added Enzymes

Using added enzymes to hydrolyze food pro-

teins is a process of considerable importance used

to improve or modify the physicochemical, func-

tional, and sensory properties of the native pro-

tein without jeopardizing its nutritive value, and

often protein absorption is improved. These en-

zyme-based processes occur under mild condi-

tions over a series of stages and do not produce

hydrolytic degradation products via racemization

reactions observed with both acid and alkaline

hydrolysis.

The process of using added enzymes

instead of chemicals or endogenous enzymes of-

fers many advantages because it allows good con-

trol of the hydrolysis and thereby the properties of

the resulting products.

Processes can be designed

to take advantage of substrate specificity and the

relative reaction rates of different enzymes under

the reaction conditions employed. The physico-

chemical and functional properties of hydrolyzed

fish proteins are discussed separately in a later

section.

Enzymatic hydrolysis has been employed on

a variety of different proteins derived from live-

stock and poultry meat,

39,44,47,70,71

milk,

57,72–76

and

plants.

77,78

Hydrolysis of fish and other aquatic

foods is also being seen more frequently in the

literature. Several different aquatic protein sources

have been investigated for the production of func-

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

tional fish protein hydrolysates. These include

Rastelliger canagurta and Barbus carnaticus

both Indian tropical fishes,

79,80

hake (Urophycis

chuss),

10,25,33

shark (Isurus oxyrinchus),

81,82

sardine (Sardina pilchardus),

35,36,83,84

herring

(Clupus harengus),

crayfish,

lobster (Panulirus

spp.),

pollack (Theragra chalcogramma),

cape-

lin (Mallotus villosus),

dogfish (Squalus

acanthias),

chum salmon (Oncorhynchus keta),

Pacific whiting (Merluccius productus),

and

Atlantic salmon (Salmo salar).

It can readily be

seen from the list above that the majority of the

sources represent underutilized species or are

connected to utilization of processing wastes.

Enzymatic hydrolysis of fish protein has been

employed primarily as an alternative approach for

converting underutilized fish biomass, which is

commonly used in making feed or even fertilizer

into edible protein products.

15,88

More recently,

fish processing waste, or, more appropriately,

secondary raw material, has been connected to

FPH studies. In many cases this is due to strict

government waste regulations. Many processors

are no longer allowed to discard their offal di-

rectly to sea, resulting in a very high cost of

refining the material before discarding. Second-

ary raw material is the material remaining after

fillets are removed, and if viscera is included, this

can represent something on the order of 64% of

the weight of whitefish, the protein content of this

waste being about 10%.

Hydrolysis of fish pro-

tein with selected proteolytic enzymes provides

the possibility of controlling cleavage degree of

protein in the substrate. Using suitable enzyme/

substrate ratios and reaction times, this permits

the production of hydrolysates with different

molecular structures and different functional prop-

erties that could find applications in various food

formulations.

The hydrolytic process and reaction condi-

tions differ between different substrates and en-

zymes used and also depend on the properties

desired for the hydrolysate. Most of the described

processes are conducted under research condi-

tions and may have limited applications in the

industry. Commercial production of fish protein

hydrolysates is still limited on a worldwide basis,

but has reached a significant level in a few coun-

tries, including France, Japan, and Southeast

Asia.

Commercial batch protein hydrolysis has

several disadvantages such as (1) high cost of

using large quantities of enzymes, (2) difficulty in

controlling the extent of reaction that can result in

nonhomogenous products consisting of fractions

of varying molecular weight, (3) low yields, and

(4) the need to inactivate enzymes by pH or heat

treatment at the end of the reaction, which adds to

the processing costs.

Also, the enzymes em-

ployed in the process cannot be reused.

Figure 4

outlines a fairly typical process for producing fish

protein hydrolysates. Each step is given a detailed

discussion below.

a. The Substrate and Its Preparation

Lean species, or material derived from them,

is the substrate of choice for enzymatic hydroly-

sis as problems with lipid oxidation can be re-

duced. From an economic standpoint, however,

the abundant underutilized pelagic fish would be

preferred. The small pelagics comprise 23% of

the world’s catch,

of which only 42% is used as

human food. These are mostly fatty species such

as herring, sardines, anchovies, and mackerel, and

FPH prepared from them would contain high

amounts of lipid, which would require additional

treatments such as centrifugation to remove ex-

cess fat.

The fewer steps that are involved in the

production, the more economically viable the

operation becomes. If a whole fish is used, it is

eviscerated and washed, then ground in a meat

grinder, usually mixed with an equal amount of

water and homogenized in a blender until a vis-

cous homologous mixture is achieved. In some

instances a buffer solution is added to the minced

fish, for example, phosphate buffer

1,88

and boric

acid-NaOH buffer.

The presence of buffer salts

may affect the final properties of the hydroly-

sates. In a study of whey protein hydrolysis,

Kuehler and Stine

decided not to buffer the

solutions because of the influence buffer salts

might have on foaming or emulsifying properties.

Processes for fatty and lean species are differ-

ent. If the FPH contains more than 1% fish fat, the

fat must either be removed by solvent extraction

or stabilized by antioxidants

3,60

such as butylated

hydroxytoluene, butylated hydroxyanisol,

propylgallate.

A fish protein hydrolysate with

high lipid content may darken. The formation of

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

brown pigments may result from aldol condensa-

tion of carbonyls produced from lipid oxidation

after reaction with basic groups in proteins.

Re-

searchers have developed many means of mini-

mizing the lipid content in FPH. To obtain a

product of a lipid content not exceeding 0.5% by

weight, as established by the Protein Advisory

Groups of FAO for a fish protein hydrolysate

suitable for human consumption,

Quaglia and

Orban

36,84

defatted ground sardines by extraction

with isopropanol three times (solvent: substrate

ratio 1:1) at 46°C for 30 min, and then homog-

enized the mixture with water. Hoyle and Merritt

used an ethanol (90%) extraction directly on

minced herring at the fish/ethanol ratio of 1:2 at

70°C for 30 min, then mixed with equal volume

of water, hydrolyzed the mixture, then spray dried

it. Through this procedure the lipid content was

reduced to 0.9 from 4.0% of raw herring. Also

before placing the treated substrate in the reaction

vessel, chemical agents such as NaCl, sorbic acid,

or ethanol are occasionally added to the minced

fish to minimize bacterial degradation,

espe-

cially if reaction conditions are at neutral or alka-

line pH. However, adding NaCl can reduce the

rate of hydrolysis, increasing reaction time. Etha-

nol can also adversely affect the reaction process

in too high concentration by inhibiting protease

activity, although sorbic acid has not been found

to affect hydrolysis in concentrations up to 0.5%.

b. The Choice of Enzyme

The water mince mixture is added to a reac-

tion vessel where the hydrolysis takes place. Of-

FIGURE 4. A flow sheet for the enzymatic hydrolysis of fish protein to make fish protein hydrolysate

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

ten a flask, ranging from 0.5 to 3 L with a close-

fitting multisocket lid, has been used. The sockets

in the lid usually carry: a stirrer, driven by a

overhead variable speed motor to ensure adequate

mixing of the system, a thermometer to monitor

temperature, a pH electrode to monitor pH, and a

“pH-stat” device, where acid or base is added to

maintain a constant pH (Figure 5). The tempera-

ture of the reaction vessel is controlled. After the

required temperature is achieved, the pH of the

slurry is adjusted to the desired value. It is impor-

tant that the mixture is well mixed and consis-

tently stirred when the pH is added to allow for

uniform distribution of the added acid or base.

Processing temperature and pH is normally se-

lected to optimize the kinetics of the selected

enzyme or enzyme mixture.

A commercial pro-

tease is added in varying concentrations depend-

ing on the rate of hydrolysis needed. Given a

particular enzyme and a particular substrate, any

hydrolysis process involves at least five indepen-

dent variables. These are S (protein substrate con-

centration: %N × 6.25), E/S (enzyme-substrate

ratio in % or in activity units per kg N × 6.25), pH,

T (temperature), and t (time).

A wide variety of commercial enzymes exist

that have been used successfully to hydrolyze fish

and other food proteins. Proteolytic enzymes from

plants and microorganisms are most suitable to

prepare fish protein hydrolysates.

Enzymes used

to hydrolyze fish protein have at least one com-

mon characteristic: they have to be food grade,

and, if they are of microbial origin, the producing

organism has to be non-pathogenic.

The choice

of enzyme(s) is usually determined by a combina-

tion of efficacy and economics.

The screening for a suitable enzyme in a pro-

cess or experiment is very important if the prod-

uct is to have predetermined properties. The

screening process can be conducted in a variety of

ways, and there is no standard methodology for

this selection, leaving it primarily up to the indi-

vidual researcher what method is most appropri-

ate. Good examples of selection procedures are

found in studies by Hale,

Cheftel et al.,

Arzu

et al.,

Rebeca et al.,

Baek and Cadwallader,

and Kristinsson and Rasco.

In the comprehen-

sive study by Hale,

the relative activities of

more than 20 commercially available proteolytic

enzymes were measured for the hydrolysis of a

washed and freeze-dried fish protein substrate

from haddock. Preliminary tests at 1 h, 40°C, and

pH 7 resulted in the plant enzyme ficin to be most

active, but with papain, also a plant enzyme, hav-

ing a much higher relative ranking. When the

enzymes were tested at 24 h the picture changed,

60% digestion (the set limit) was achieved fastest

by Pronase, which exhibited the greatest activity

per unit weight. The enzymes pepsin, papain, and

pancreatin were most suitable if the lowest cost

per unit of proteolytic activity was to be followed.

These cost estimates are less valid today due to

the commercial availability of bacterial enzymes.

Although many would prefer to use acid pro-

teases so microbial growth could be more easily

limited, they usually yield a product with low

protein yield and too excessive hydrolysis for

food use.

39,84,86

Therefore, milder enzymes at neu-

FIGURE 5. A typical enzymatic hydrolysis reaction system in the laboratory. (Adapted from Ref. 20.)

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

tral and slightly alkaline conditions have been

used more frequently in recent years. In some

cases, a high level of solubilization is desired.

The acid enzyme pepsin has been most successful

in solubilizing fish protein. Liu and Pigott

100

pro-

duced a high-quality, fluffy, water-soluble fish

protein hydrolysate by pepsin hydrolysis of rock-

fish fillets. Tarky et al.

101

used pepsin at 37°C and

pH 2.0 to hydrolyze the entire fish waste resulting

from filleted English sole. The final product, after

ultrafiltration and spray drying, was a creamy

white, nonhygroscopic, water-soluble hydrolysate

with low lipid content but very poor nutritional

value.

Alcalase, an alkaline enzyme produced from

Bacillus licheniformis and developed by Novo

Nordisk (Bagsvaerd, Denmark) for the detergent

industry, has been proven repeatedly by many

researchers to be one of the best enzyme used to

prepare functional FPH and other protein hydroly-

sates.

20,36,59,84,88,89

Shahidi et al.

successfully used

Alcalase to optimize processing conditions to

produce capelin protein hydrolysates. Alcalase-

treated hydrolysates exhibited superior protein

recovery (70.6%) compared with the alkaline pro-

tease Neutrase and papain. Alcalase-treated hy-

drolysates also had the lowest lipid content (0.18%)

and excellent functional properties. Quaglia and

Orban

studied the same three enzymes at opti-

mal conditions on enzymatic solubilization of

sardine proteins. Hydrolysates produced using

Alcalase and papain were almost identical in ni-

trogen recovery, which increased with increasing

enzyme concentration (70% recovery at a en-

zyme/substrate ratio of 4%). Neutrase-treated

hydrolysates at the same ratio had only over 20%

nitrogen recovery. Hydrolysates from Alcalase

and papain also exhibited better functional prop-

erties and high nutritional value than those from

Neutrase. Improved nitrogen recovery of fish pro-

tein hydrolysates with increase of protease con-

centration has been reported elsewhere.

1,79

Alcalase

and Neutrase were studied further recently by

Benjakul and Morrissey

on Pacific whiting solid

waste at pH 9.5, 60°C and pH 7.0, 55°C, respec-

tively. Alcalase had a considerably higher activ-

ity than Neutrase and led to a more efficient hy-

drolysis. Optimum conditions for Alcalase were

20 Anson Units (AU)/kg, 1 h reaction time, and

waste:buffer ratio of 1:1 (w/v) at 60°C and pH

9.5. The resulting hydrolysate had a high protein

content with excellent nitrogen yield (up to 70%)

and an amino acid composition comparable to

fish muscle. Further, Alcalase was found to be

the most cost-effective enzyme out of five en-

zyme preparations tested to hydrolyzed salmon

muscle proteins.

Other new enzyme prepara-

tions have shown excellent potential for hydro-

lyzing fish proteins to make highly functional

FPH, including Flavourzyme 1000L (Novo

Nordisk, Bagsvaerd, Denmark), Corolase 7089

(Rohm Enzymes; Somerset, NJ) and Corolase

PN-L (Rohm Enzymes, Somerset, NJ).

In an extensive paper,

Hale reported the

effects of various processing conditions and com-

mercially available proteolytic enzymes on yield

and composition of water-soluble fish protein

hydrolysates. He concluded that the hydrolysis of

raw hake (Urophycis chuss) with Alcalase at pH

8.5 or above gave the best balance of essential

amino acids and a high yield of soluble product,

followed by pancreatin (a mixture of serine pro-

teases). One of the first studies on added enzyme

hydrolysis of fish protein was with papain, due to

its favorable properties of pH and temperature

optima for activity.

79,80

Two fish species were

used as substrate, one freshwater, Barbus

carnaticus, and the other marine, Rastrelliger

canagurta, and studied at 40 and 55°C, and pH 5

and 7. Total solids and nitrogen recovery for both

species was high, with pH 7 having the highest

total solids and nitrogen recovery (69.7% at 55°C

for freshwater species) compared with pH 5, pos-

sibly attributed to better hydration at pH 7.

To properly compare enzyme activity on the

same substrate, it is necessary to determine the

general proteolytic activity units at specific reac-

tion conditions. Unfortunately, very few research-

ers have done this, and most compare enzyme

activity on a weight basis of enzymes used in the

reaction mixture. Adding enzymes on the basis of

weight is meaningless if relative enzyme activity

is to be compared, because enzyme activity per

weight is different for each enzyme under experi-

mental conditions used. However, there exist few

studies that use and compare enzymes on the

basis of their proteolytic activity. Gonzalez-Tello

et al.

69,102

studied three proteases on whey protein

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

substrate by adding them to the system based on

Anson Units (AU). Unfortunately, they did not

use the three enzymes at the same AU. In addi-

tion, the activity units were obtained from the

manufacturer and not assayed by the researchers.

Also, reaction conditions used in the hydrolysis

experiments were different from the conditions

used in the enzyme assays, thus making the activ-

ity units very unreliable.

Benjakul and Morrissey

studied the hydroly-

sis of Neutrase and Alcalase on Pacific whiting

solid wastes by adding the enzymes to the system

based on AU units. This study has the same limi-

tation as the studies by Gonzalez-Tello et al.

69,102

by using reaction conditions different than those

used to assay the enzyme for proteolytic activity

and relying on enzyme units provided by the sup-

plier instead of assaying the enzymes themselves.

Other studies on fish protein hydrolysis add en-

zymes according to AU units and also suffer from

these same limitations.

59,82

A study by Beddows

and Ardeshir

103

is the most carefully conducted

study in the literature with respect to using stan-

dardized relative enzyme activity. They assayed

three proteases, bromelain, ficin, and papain, by

using BApNA (Benzoyl-Arg-para-Nitroanilide) to

obtain some indication of the relative proteolytic

activities of these enzymes. They then added the

enzymes to a system of minced Ikanbilis

(Stolephorus sp.), a tropical fish, at the same ac-

tivity units as based on their assay. The assay,

however, involved different reaction conditions

than the hydrolysis experiment, which limited its

reliability. Assaying enzymes with BApNA also

only estimates the trypsin activity of the enzyme

preparation, not their general proteolytic activity.

Using BapNA, therefore, gives a less accurate

and and probably underestimated value of en-

zyme activity. In a research conducted by

Kristinsson and Rasco,

where salmon muscle

proteins were hydrolyzed, the enzymes were as-

sayed under the same reaction conditions as they

were used in the hydrolysis experiment. A syn-

thetic protein substrate for proteolytic activity,

Azocoll, was used to obtain a uniform level of

proteolytic activity for all enzymes used. Azocoll

is an insoluble cowhide preparation consisting

largely of collagen. The method is based on dye

release from the insoluble substrate Azocoll when

a proteolytic enzyme cleaves the peptide linkages

in it. The rate at which the dye is released can be

used to quantitatively measure the amount of pro-

teolytic enzyme(s) activity working in a solution

by measuring the absorbance at 520 nm. The

enzymes were then used to hydrolyze the sub-

strate, all at the same activity unit according to the

Azocoll assay, thus comparing them at the same

activity level on the same substrate. No reports in

the literature have taken this approach; however,

the use of Azocoll in fish protein hydrolysis ex-

periments has been reported by Ferreira and

Hultin.

104

c. The Mechanism of Enzymatic Hydrolysis

The enzymatic hydrolysis of fish muscle pro-

teins is characterized by an initial rapid phase,

during which a large number of peptide bonds are

hydrolyzed, after this rate of enzymatic hydroly-

sis decreases and reaches a stationary phase where

no apparent hydrolysis takes place

(Figure 6).

The shape of the hydrolysis curve has been asso-

ciated with enzyme inactivation, product inhibi-

tion by hydrolysis products formed at high de-

grees of hydrolysis, a low K

value for the soluble

peptides that act as effective substrate competi-

tors to the unhydrolyzed fish protein,

and possi-

bly autodigestion of the enzyme.

105

Shahidi et

al.

found that a high concentration of soluble

fish peptides in the reaction mixture, released

during the initial phase of hydrolysis, reduced

both the rate of hydrolysis and the recovery of

soluble proteins. Thus, removal of hydrolysate

from the reaction mixture should enhance the

hydrolysis rate and the protein recovery.

By increasing the protease concentration, and

thereby increasing the extent of hydrolysis, re-

covery of soluble nitrogen increases,

1,71,103

al-

though increasing enzyme concentrations may not

be cost effective. Substrate concentration has also

negative effects on protein recovery. Linder et

al.

found that more than 8% protein concentra-

tion in the system, regardless of enzyme concen-

tration, seemed to have an inhibiting effect on

protein recovery. Baek and Cadwallader

reported

using Optimase to hydrolyze crayfish processing

byproducts the %DH increased as substrate con-

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

centration decreased to 45% (w/v), suggesting

that high %DH did not coincide with a high amount

of hydrolysate. Similarly, Surowka and Fik,

who

measured the production of protein hydrolysate

with Neutrase from chicken heads, reported that

hydrolysis increased as substrate concentration

decreased. Ferreira and Hultin,

104

using Newlase

A to hydrolyze cod (Gadus morhua) frames, found

that enzyme autolysis can be reduced at higher

substrate concentrations.

Because fish tissue is a very complex sub-

strate and also contains large amounts of protein-

ase inhibitors, it is impossible to explain the mecha-

nisms of protein hydrolysis in detail for this

system.

A kinetic study of the process is also

quite complicated due to the various types of

peptide bonds involved and their differing vulner-

ability to attack by enzymes during the hydrolytic

process.

Very few studies on kinetics of fish and

food protein enzymatic hydrolysis are reported.

Sakai et al.

conducted a kinetic study on the

hydrolysis of pollack surimi protein using a acid

protease derived from Aspergillus niger, deter-

mining the effects of temperature, pH, initial sub-

strate concentration, and enzyme concentration

on the kinetics of protein solubilization. Their

experimental data followed Michaelis-Menten

type kinetics. Michaelis-Menten kinetics has also

been observed with whey protein hydrolysis.

Kristinsson and Rasco

studied the kinetics of

five different enzyme preparations during the

hydrolysis of salmon muscle proteins and found

that the initial rate of the reaction for all enzymes

showed a linear relationship to enzyme activity.

These experiments indicated that the initial rate

constants for each enzyme tested were in the same

order. This confirmed previous studies by Hevia

et al.

106

and Cheftel et al.

on menhaden fish

protein as a substrate.

A kinetic model of whey protein hydrolysis

with Alcalase has been proposed, where the hy-

drolytic reaction is zero-order for substrate. The

enzyme denatures simultaneously via a second-

order reaction due to free enzymes attacking the

enzyme bound to the substrate.

Moreno and Cuadrado

107

hydrolyzed vegetable

proteins with Alcalase and found reaction mecha-

nism consistent with substrate inhibition and a

second-order deactivation with respect to the en-

zyme concentration. Enzyme autolysis was de-

FIGURE 6. Hydrolysis curve for salmon muscle mince with Corolase 7089 at three different activity units (AzU =

Azocoll Units).

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

pendent on the substrate concentration. Cheftel et

al.

used Pronase to hydrolyze fish protein con-

centrate and found that the rate constant decreased

with time, and that proteolysis did not follow

first-order kinetics with respect to concentration

of the peptide bonds. Cheftel et al.

suspected

this to be due to the multitude of possible sub-

strates in FPC, the number of different proteolytic

enzymes present, the inhibitory effects of sub-

strate or self-digestion, as well as the different

specificities that are known to be present in Pro-

nase. Archer et al.

108

also studied the kinetics of

the enzymatic hydrolysis of FPC. Their research

found that the enzyme is initially adsorbed to the

surface of the protein, with the initial rate of

reaction being proportional to the surface area of

substrate exposed to the aqueous phase. The over-

all kinetics were described by a sequence of two

first-order processes, an initial, fast reaction in

which loosely bound polypeptide chains are

cleaved from an insoluble protein particle, and a

second, slower reaction in which a more com-

pacted core protein is digested.

108

Langmyhr

109

studied the kinetics of the hydrolytic breakdown

of cod muscle with acetyltrypsin. The results in-

dicated that enzyme molecules are rapidly and

firmly bound to fish proteins and that the maxi-

mum binding occurred under the optimum pH

and T conditions for the enzyme. Results also

suggested that muscle proteins are hydrolyzed in

the same way and to the same extent whether they

occur separately or are integrated into intact

muscle.

d. Measuring the Extent of Enzymatic

Hydrolysis

To follow the reaction kinetics and get a

measure for the extent of the hydrolytic degrada-

tion, a parameter named degree of hydrolysis

(%DH) is employed. In principle, there are sev-

eral control methods, but under practical indus-

trial conditions there are few.

The degree of the

hydrolysis is most commonly used to describe

hydrolysis of food proteins. The advantage of

using %DH as a process parameter is that %DH

appears to determine unambiguously the proper-

ties of a protein hydrolysate for a given protein-

enzyme system.

The degree of hydrolysis dem-

onstrates both theoretically and empirically that

four processing variables, S, E/S, T, and t, can be

left uncontrolled, provided that %DH is con-

trolled.

From this, it is obvious that %DH is a

very simple and rapid method of measuring the

extent of protein breakdown.

Two methods for measuring %DH have been

studied thoroughly and shown to be satisfactory,

the pH-stat technique and osmometer technique.

The pH-stat method is more commonly used and

more useful for industrial applications. The prin-

ciple behind the pH-stat method is relatively simple

and is based on maintaining a constant pH during

the reaction. By pH-stat, the %DH is calculated

from the volume and molarity of base or acid used

to maintain a constant pH. The degree of hydroly-

sis is defined as the percent ratio of the numbers

of peptide bonds broken (h) to the total numbers

of bonds per unit weight (h

tot

; meq/kg protein,

calculated from the amino acid composition of

the substrate): %DH = (h/h

tot

) × 100.

%DH can also be expanded to:

%DH

hMP

tot

⋅

⋅⋅

⋅

100

where B = base consumption in ml (or acid in

case of acid proteases), N

= normality of the base

(or acid), α = average degree of dissociation of

the –NH groups or COOH groups, MP = mass of

protein in grams (%N × 6.25).

The degree of

dissociation is found by the following equation:

α=

110

pH pK

–

This equation for %DH is valid when hydrolysis

is conducted at a pH above the pK of the α-NH

group (for hydrolysis at neutral or alkaline pH).

Under these conditions, the reaction will result in

a net release of protons (H

) when peptide bonds

are cleaved and the base consumption is propor-

tional to the number of peptide bonds split.

110

Above pH 6.5, the dissociation of the protonated

α-NH group becomes significant. Therefore, it is

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

important to know the pK values of the α-NH

groups, both to determine the reaction pH and to

calculate the dissociation constant correctly. The

pK value varies significantly with temperature.

The pK values at different temperatures can be

calculated according to Steinhardt and Beychok:

111

⋅

⋅78

298

2400.

–

where T = temperature in Kelvin.

If samples are drawn during the experiment,

it is crucial to correct for the actual value of base

consumed. Actual base consumption (B′) will be

smaller than the theoretical base consumption (B)

if this is the procedure. To correct for this, B can

be calculated from the following formula:

∆∆TK C

=⋅⋅ω

where K

is 1.86 K/mol for water, ω the osmotic

coefficient, and C the osmolality of solution. From

the increase in osmolality, ∆C, %DH can be cal-

culated according to the following formula:

Sf wh

osm tot

⋅

⋅⋅













⋅

∆ 11

100

where ∆C = depression of freezing point mea-

sured in milli-osmol, S% × f

osm

= g protein/1000

ml H

0, 1/w = calibration factor for the osmom-

eter: 1.04, h

tot

= total number of peptide bonds in

the substrate.

The f

osm

factor is calculated by knowing the

percent substrate dry matter, D%, present in the

reaction mixture:

osm

1000

100 – %

Although the osmometer technique is a very

good method to determine the degree of hydroly-

sis, it is being less employed frequently. The de-

gree of hydrolysis is now being more commonly

determined by using either the trichloroacetic acid

(TCA) method or the trinitrobenzenesulfonic acid

(TNBS) method. Both of these methods are very

useful when working within the pH 3 to 7 range,

where the pH-stat is unusable. Several variations

of these methods exist. The most commonly used

TCA method is based on determining the ap-

proximate degree of hydrolysis (%DH) of protein

hydrolysates by the ratio of percent 10% TCA

soluble nitrogen in the hydrolysate compared to

total amount of protein in sample. This is gener-

ally done by removing aliquots at selected inter-

vals and mixing with 20% TCA to create 10%

TCA-soluble and TCA-insoluble fractions. These

mixtures are then centrifuged and the supernatant

analyzed for nitrogen. The degree of hydrolysis is

thus calculated from the following equation:

%DH = (10% TCA-soluble N in sample/Total N

in sample) × 100

where B′

, B′

, etc. are the actual base consumption

at the drawing of samples number 1, 2, etc. The

value n is the sample number and m is the sample

size (ml).

The pH-stat method, however, has some

limitations. Use of pH-stat as a means of control

is only practical outside the approximate pH in-

terval 3 to 7.

This has to be taken into consider-

ation when deciding on pH values for the hy-

drolysis reaction. Within this pH range, process

control may depend on other methods. Therefore,

the pH-stat method for %DH determination is

useful mainly under alkaline conditions.

The other commonly used method to measure

hydrolysis, the osmometer technique, is more

universally applicable. This technique measures

the freezing point of samples drawn at regular

intervals during hydrolysis to construct a hydroly-

sis curve. For this method, the freezing point

depression (∆T) is measured and converted to

milli-osmol (∆C) as follows:

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

This method has been used successfully in fish

and other food protein hydrolysis.

44,70

The TNBS

method is based on the concentration of primary

amino groups in the hydrolysate. It is a spectro-

photometric assay of the chromophore formed by

the reaction of TNBS with liberated amino groups

(420 nm), at slightly alkaline conditions.

112

The

degree of hydrolysis can then be calculated as

presented by Baek and Cadwallader,

and

Benjakul and Morrissey,

for crayfish hydrolysis

and whiting hydrolysis, respectively:

–

max

=⋅

100

where L

= the amount of a specific liberated

amino acid at time t, L

= the amount of the

specific amino acid in original substrate (blank),

max

= the maximum amount of the specific amino

acid in the substrate obtained after hydrolysis.

New methods and modifications of previous

methods for determining the degree of hydrolysis

are being developed. In the special case of fish, a

simple method for monitoring the enzymatic hy-

drolysis of fish protein was developed by Ukeda

et al.

113

The degree of hydrolysis was monitored

by an amino group determination method with

gluteraldehyde (GA). The method is based on the

consumption of dissolved oxygen during the re-

action between gluteraldehyde and the liberated

amino groups of the protein substrate. The results

by the GA method were in agreement with the

TNBS method previously described, with a corre-

lation coefficient of r = 0.992.

e. Termination of Enzymatic Reaction

When a desired %DH is attained, it is neces-

sary to terminate the enzymatic reaction. This is

very important as otherwise the enzymes would

remain active in the substrate and further hydro-

lyze the protein and peptides. Deactivation of

enzymes is achieved either by chemical or ther-

mal means. Usually the slurry of hydrolysate and

enzymes are transferred to a heat bath, where the

enzymes are deactivated by exposing them to

temperatures ranging from 75 to 100°C for 5 to

30 min, depending on the type of enzyme. For

example, papain is very heat tolerant, and has

been reported to need at least 90°C for 30 min to

be fully inactivated.

Terminating the reaction by

thermal means is undesirable

114

because of the

effects of heat denaturation on the protein that

leads to exposure of hydrophobic residues and

subsequently protein aggregation. Diniz and Mar-

tin

suggest that this form of heating can have the

advantage of being very effective in the separa-

tion of oil from the fish protein substrate, al-

though Webster et al.

found that some protein-

fat interaction occurred at elevated temperatures

that prevented their separation, when using bo-

vine lungs as the protein substrate.

The temperatures at which various proteins

denature and unfold vary enormously. Because

fish muscle proteins are adapted to function at

lower temperatures, they may not be particularly

heat stable. Denaturation is usually undesirable

because it results in altered physicochemical prop-

erties, particularly a loss in protein solubility and

functionality.

Spinelli et al.

are among few

researchers that have recognized the adverse ef-

fect elevated temperatures may have on fish pro-

tein hydrolysates. To both terminate the enzy-

matic reaction and quantitatively recover the

protein fractions, they reacted enzymatically hy-

drolyzed muscle proteins from rockfish with 5%

sodium hexametaphosphate (HP), following slight

acidification to form an insoluble protein-phos-

phate complex. By isoelectric precipitation, they

recovered up to 90% of the protein. In addition,

the complex could be washed free of occluded

nonprotein nitrogen components with no loss of

protein nitrogen.

Chemical inactivation would be to either lower

or raise the pH of the slurry to a point where the

enzyme deactivates. Some enzymes are more sen-

sitive to pH changes than they are to temperature

changes. Alcalase is a relatively thermostable

enzyme, but it is very sensitive to acid pH. Com-

plete inactivation of Alcalase therefore is obtained

by lowering the pH to 4.0.

20,59

Neutralizing the

slurry to pH 7.0 would inactivate pepsin and most

other acid proteases. Extremes of pH can also,

like elevated temperatures, have detrimental ef-

fects on protein and peptides. Many proteins un-

fold at pH values less than about 5 or greater than

10. Unfolding at such extremes of pH usually

occurs because the folded protein (or oligopeptide)

has groups buried in nonionized form that can

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

ionize only after unfolding.

Stabilizing salt-

bridges between ionizing groups can also be dis-

rupted by extreme pH values. The termination of

the enzymatic reaction thus is a formidable ob-

stacle in any FPH operation. The choice of inac-

tivation method should be carefully made and

based up on what the enzyme under study is

sensitive to, whether it is heat or pH. In some

cases a combination of elevating temperatures

and lowering pH have been used.

f. Protein Hydrolysate Concentration

Commonly, the slurry is desludged by cen-

trifugation, which results in several fractions (Fig-

ure 7): sludge in the bottom, aqueous layer in the

middle, lipid-protein fraction between aqueous

layer and sludge, aqueous and oil layers and oil

layer on the top.

A single centrifugation step can

eliminate the vast majority of the lipids present.

The oil layer overlying the aqueous layer is then

removed and the soluble fraction collected. Be-

cause lipid in the final hydrolysate is a major

concern for FPH, it is important to remove it.

Lipid residues in FPH must be lower than 0.5% to

prevent alteration of the lipid fraction during stor-

age.

More than one centrifugation step is often

required to separate the soluble proteins from the

lipids and insoluble solids. The second centrifu-

gation step would be performed only on the soluble

fraction. Other separation methods for fish pro-

tein hydrolysates have been reported such as suc-

tion filtration of the sludge

and filtering the

slurry by passing it through a 2-mm mesh screen.

The removal of colored and odiferous matter has

also been reported by treating the first soluble

fraction obtained by centrifugation with 1% w/v

charcoal at 55°C and 30 min before the next

separation step.

In a commercial operation, the final soluble

fraction is generally spray dried to convert the

hydrolysate to a powdered form, which can be

incorporated into food formulations. The insoluble

fraction or the sludge precipitated during cen-

trifugation may be used as animal feed. Spray

drying of the soluble fraction is one of the most

energy consuming and expensive steps in the pro-

duction of protein hydrolysates. In industrial pro-

duction a compromise must be made between the

hydrolysate yield and the amount of water that

must be removed to obtain a dry product.

suspension of equal amounts of water and fish

substrate appears to be convenient from an eco-

nomic point of view. Rebeca et al.

performed a

study where eviscerated mullet (Mugil cephalus)

was enzymatically hydrolyzed without the addi-

tion of water, to lower the cost of spray drying.

Interestingly, the experiment resulted in higher

dry matter content, with high protein recovery, of

the soluble fraction and the cost of drying was

reduced. Generally, in the laboratory, hydroly-

FIGURE 7. Different fractions obtained when recovering soluble fish protein hydroysates.

LIGHT LIPID-PROTEIN

AQUEOUS

SLUDGE

CLEAR OIL

LIPID-PROTEIN SMEAR

HEAVY LIPID-PROTEIN

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

sates are neutralized and freeze dried. By neutral-

ization the final product can have a fairly high salt

content, which is undesirable (HCl + NaOH →

O + NaCl). This can be mostly avoided if the

soluble fraction is passed through an ion exchange

column before freeze-drying. Dialysis to desalt

the soluble fraction can also be a useful process.

Increasingly, ultrafiltration membranes have

been introduced into the production process for

hydrolysates. The hydrolysate after enzyme inac-

tivation is filtered directly through a membrane

with a specific molecular weight cut-off value.

The ultrafiltration process can contain more than

one filter, yielding several molecular weight frac-

tions depending on the product desired. A major

advantage for such a process is the control of

molecular size of selected peptides so that a uni-

form product is possible,

as the molecular size

of the hydrolyzed proteins is a key factor in deter-

mining functional properties of hydrolysates.

115

The method has, however, not found its way into

fish protein hydrolysate production. The reason

probably being that in order to successfully use

the ultrafiltration, the sample has to be very pure

and free of lipids. It is, however, possible that the

method could be applied to highly purified and

defatted FPH powders. The method has found to

be very useful in soy,

115

whey,

75,116

and casein

protein hydrolysis.

In the case of whey protein,

large-molecular-weight fractions are primarily

responsible for allergic reactions. However, small

peptides and free amino acids are less well ab-

sorbed by humans. Therefore, protein hydrolysis

should not be more extensive than the minimum

required to eliminate allergic responses. By ultra-

filtration therefore it is possible to exclude both

the too large and too small peptides and collect

the molecular weight in between by passing the

hydrolysate solution through two filters, the first

with a higher cut-off value than the second.

116

Applying this technique to FPH would be an inter-

esting task to undertake.

IV. PHYSICOCHEMICAL AND

FUNCTIONAL PROPERTIES OF FISH

PROTEIN HYDROLYSATES

As previously mentioned, one of the major

advantages and goals of enzymatically hydrolyz-

ing fish proteins is to modify and improve their

functional properties. The functional properties

of fish protein hydrolysates are important, par-

ticularly if they are used as ingredients in food

products.

Enzymatic hydrolysis of fish proteins

generates a mixture of free amino acids, di-, tri-,

and oligopeptides, increases the number of polar

groups and the solubility of the hydrolysate, and

therefore modifies functional characteristics of

the proteins, improving their functional quality

and bioavailability. The choice of substrate and

proteases employed and the degree to which the

protein is hydrolyzed affect the physicochemical

properties of the resulting hydrolysates.

105

En-

zyme specificity is important to peptide function-

ality because it strongly influences the molecular

size and hydrophobicity of the hydrolysate.

117

Thus, the peptides obtained have different mo-

lecular profiles, and the surface energy of the

hydrolysate is different, depending on the en-

zyme used; these variations have a bearing on the

functionality of the mixture.

114,118

The more nar-

row the specificity, the are the larger peptides

produced; the broader the specificity, the smaller

are the peptides generated. As the range of enzy-

matic activities within commercial preparations

is increased, the hydrolysate becomes more com-

plex.

The chain length of peptides or breaking of

linkage is also dependent on the extent of hy-

drolysis; conditions of hydrolysis; concentration

of enzyme and the type of protein to be hydro-

lyzed.

Due to the complex peptide profile, it is

often useful to calculate the average peptide chain

length (PCL) introduced by Adler-Nissen and

Olsen

119

to express the composition of the protein

hydrolysate. PCL is calculated from the degree of

hydrolysis in the following way:

PCL

100

This approximation is acceptable in nearly all

practical cases.

The relationship between PCL

and %DH is shown in Figure 8.

Manipulating the reaction conditions during

enzymatic hydrolysis of food proteins produces

hydrolysates with different solubility and emulsi-

fying characteristics, foaming properties, or taste

characteristics.

The control of the enzymatic re-

action is very important, as previously discussed.

Uncontrolled or prolonged hydrolysis of fish pro-

teins may result in the formation of highly soluble

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

peptides, completely lacking the functional prop-

erties of the native proteins. This may promote

the formation of undesirable bitter peptides. By

controlled hydrolysis it is possible to eliminate

these drawbacks and to obtain hydrolysates with

different physicochemical and functional proper-

ties, in some cases even better than the originals.

The physical and chemical properties that govern

protein functionality include size, shape, amino

acid composition and sequence, net charge and

distribution of charges, hydrophobicity/hydrophi-

licity ratio, peptide structures, molecular flexibil-

ity/rigidity, and the ability to interact/react with

other components.

120

Functionality of food pro-

teins has been defined as: “those functional and

chemical properties which affect the behavior of

proteins in food systems during processing, stor-

age, preparation and consumption”.

The follow-

ing sections discuss the main functional proper-

ties that fish protein hydrolysates exhibit, that is,

solubility, water holding, emulsifying, foaming

and sensory properties, and research aimed to-

ward evaluating these properties for various FPH

preparations.

A. Solubility

Solubility is probably the most important of

protein and protein hydrolysate functional prop-

erties. Many of the other functional properties,

such as emulsification and foaming, are affected

by solubility,

121

and therefore it is an excellent

indicator of the protein hydrolysate functionality,

and its potential (and limitations of) applica-

tions.

31,58

Hydrophobic and ionic interactions are

the major factors that influence the solubility char-

acteristics of proteins. Hydrophobic interactions

promote protein-protein interactions and result in

decreased solubility, whereas ionic interactions

promote protein-water interactions and result in

increased solubility. Ionic residues on the surface

of peptides and proteins introduce electrostatic

repulsion between protein molecules and repul-

sion between hydration shells around ionic groups,

both major contributors to increased solubility of

proteins. Solubility of protein and protein hy-

drolysates is generally measured by employing

the nitrogen solubility index (NSI), a standard-

ized method developed by AOCS and later modi-

fied by Morr et al.

122

The NSI is determined by

suspending a protein hydrolysate sample in water

then stirring and centrifuging the mixture. The

supernatant is then analyzed for nitrogen content

by the Kjeldahl procedure, and the NSI calculated

as a percentage of the soluble nitrogen to the

percentage of total nitrogen in the sample.

Intact fish myofibrillar proteins have the prob-

lem of the lack of solubility in water over a wide

range of pH,

13,123

and enzymatic hydrolysis is very

important in increasing the solubility of these

FIGURE 8. The relationship between average peptide chain length (PCL) and degrees of hydrolysis (%DH).

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

proteins. The effects of enzymatic hydrolysis on

fish protein are straightforward. Enzymatic break-

down of the protein involves a major structural

change in that the protein is gradually cleaved

into smaller peptide units, and as the degree of

enzymatic hydrolysis increases the solubility of

fish proteins increases. The enhanced solubility

of the hydrolysates is due to their smaller molecu-

lar size compared with the intact protein, and the

newly exposed ionizable amino and carboxyl

groups of the amino acids, that increase the hy-

drolysate hydrophilicity.

58,117

To effectively bind

to water molecules, the peptides have to have the

ability to form hydrogen bonds between its hy-

drophilic polar amino acid side groups and the

water molecules. Hydrolysis exposes some of the

hydrophobic groups to the surface, but at the

same time converts even more hydrophobic groups

to hydrophilic groups by generating two end car-

bonyl and amino groups. Thus, the smaller pep-

tides from myofibrillar protein hydrolysis have

proportionally more polar residues, with the in-

creased ability to form hydrogen bonds with wa-

ter. This increases protein solubility to that of the

intact protein. In addition, ion-dipole interactions

between water and simple ions such as Na

and

are also important in the interactions between

the polar or charged groups on biomolecules and

water.

Biomolecules tend to be very soluble at

favorable NaCl conditions. The increased solubil-

ity is partly through the formation of sodium salts

of carboxyl groups of proteins, COONa.

This is

true for fish myofibrillar proteins, but the hydro-

phobicity of globular proteins, such as milk pro-

teins, may increase with exposure of apolar amino

acid residues after hydrolysis.

117

Although in-

creased solubility has a positive relationship to

the extent of hydrolysis, care has to be taken that

the substrate is not too extensively hydrolyzed. A

very high degree of hydrolysis may lead to high

solubility, but this can have very negative effects

on the rest of the functional properties. To main-

tain or improve functionality, generally low de-

grees of hydrolysis are necessary.

Sugiyama et al.

studied enzymatic hydroly-

sis with several alkaline, neutral, and acid pro-

teases on defatted sardine meal. The result showed

that all the alkaline proteases had higher ability to

produce highly soluble protein hydrolysates com-

pared with the neutral and acid ones. Gel chroma-

tography showed that hydrolysates prepared with

the alkaline proteases had a lower average mo-

lecular weight. The increased hydrophilicity of

these preparations could be due to their larger

charge to size ratios compared with the longer

proteins such as those found in nonhydrolyzed

proteins from a similar source.

Interestingly,

pepsin had lower solubilized protein ratio than all

of the alkaline proteases, but pepsin is generally

considered one of the best enzymes to solubilize

fish protein.

35,36,68,97,100

Quaglia and Orban

35,36

also

studied the properties of hydrolysates produced

from sardine and concluded that Alcalase and

papain, at optimum pH and temperature, both

gave hydrolysates characterized by high solubil-

ity. Hydrolysates made with Alcalase at higher

degrees of hydrolysis showed a decrease in high-

molecular-weight fractions, and increased solu-

bility.

This connection between %DH and solu-

bility is also reported for other food protein.

124

significant increase in solubility was observed

with an increase in %DH from 2.5 to 15% for an

enzymatically hydrolyzed single cell microbial

protein (“Pruteen”), from less than 10% solubility

to over 90%.

124

Similar results were observed

with soy protein hydrolysates from 1 to 8.3%

DH.

Yu and Fazidah

125

hydrolyzed Aristichthys

noblis, a Chinese freshwater fish, with protease P

“Amano” 3, and reported excellent solubility at

15% DH after a 3-h digestion.

Hoyle and Merritt

found that enzymatically

hydrolyzed ethanol-extracted herring FPH had

highest solubility compared with nonextracted

enzymatically hydrolyzed FPH. This finding sug-

gests that the lower lipid content of ethanol-ex-

tracted herring FPH may have resulted in less

competitive water binding of proteins compared

with hydrolysates with higher lipid contents. Vieira

et al.

studied the functional properties of hy-

drolysates from lobster processing wastes and

found these to be highly soluble, pepsin yielding

a fraction with higher solubility than papain or a

fungal protease. The highest nitrogen solubility

index (NSI) for these hydrolysates was found at

extremes of pH. The solubilities of all the prod-

ucts were low at pH 5, approximately correspond-

ing to the isoelectric point of protein, at which it

precipitates. Shahidi et al.

also found that cape-

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

lin protein hydrolysates (CPH) prepared by dif-

ferent enzymes had different solubility profiles at

different pH conditions. However, in contrast to

the findings of Vieira et al.,

Shahidi et al.

found lower solubility at pH 7 to 8, and with close

to 100% solubility for Alcalase at pH 5. The

lowest solubility value at pH 7 to 8 was, however,

84%, thus CPH had excellent solubility over the

range of pH 2 to 11. Hydrolyzed salmon muscle

proteins exhibited excellent solubility at 5 to 15%

DH, between 92 to 100%, with over 96% solubil-

ity in the pH range of 2 to 11.

The solubility of enzymatically hydrolyzed

fish protein is generally much higher than for

chemically produced fish protein concentrate

(FPC). A FPC type-B air-dried fish powder had

extremely poor solubility at a wide range of NaCl

molarity and pH,

126

and alkaline hydrolysis of red

hake exhibited lower than 30% solubility at pH 1

to 7.

45,46

Pure fish myofibrillar protein are very in-

soluble close to their isoelectric point; therefore,

high solubility of FPH over a wide range of pH is

a very useful property for many food applica-

tions, including beverage applications. The good

solubility and good nutritive value of enzymati-

cally hydrolyzed fish protein hydrolysates also

makes them well suited to produce milk replacers

for weanling animals.

1,10,124

Presently, this is be-

ing done in Japan and France. In Japan, one com-

pany is making “bio-fish flour” by enzymatic

digestion of sardines that is used in feeds as a

milk replacer for calves and piglets.

60,126

Menha-

den hydrolysate produced by pancreatine was

found to be an excellent milk replacer with high

PER value with the process cost half as much as

dried skimmed milk.

127

Soluble FPH is an excel-

lent amino acid source for supplementing cereal

proteins

and to be used in bakery products, soups,

and infant formulas.

Pasta enriched up to 3.5% of

fish protein hydrolysate increased the protein

content by 5%, with a dramatic increase in the

dietary essential amino acids lysine (37.5%), va-

line (31%), and threonine (18%).

128

Soluble FPH

from extensive hydrolysis is an excellent source

of nitrogen for microbial growth. Extensive hy-

drolysis results in a product of free amino acids

and low-molecular-weight peptides, and there-

fore has found to be very promising for microbial

peptone production.

129

In the U.S., the main appli-

cation of FPH is not for foods but as weanling

feed for piglets, and increasingly as a pellet coat-

ing for pet food.

B. Water-Holding Capacity

Water-holding capacity refers to the ability

of the protein to imbibe water and retain it against

gravitational force within a protein matrix, such

as protein gels or beef and fish muscle, and it is

positively correlated with water-binding capac-

ity.

120

Water-holding capacity of proteins added

to muscle tissue is of great importance to the food

industry because retaining water in a food system

often improves texture. The functional properties

of proteins in a food system depend in part on the

water-protein interaction, and the final outcome

greatly depends on how well the protein binds

and holds water in a food system. However, a

successful specific application of a functional

protein ingredient or additive for enhancing a

single specific functional property may not be

transferable to other food systems. Fish protein

hydrolysates are highly hygroscopic, and this has

to be considered when producing them. Proper

packaging and low relative humidity of air during

processing is an important consideration. The

presence of polar groups such as COOH and NH

that increase during enzymatic hydrolysis have a

substantial effect on the amount of adsorbed wa-

ter and moisture sorption isotherm for these ma-

terials. The recommended maximum water con-

tent of FPH for storage is 0.075 g/g at less than

15% RH.

130

Fish protein hydrolysates have excellent wa-

ter-holding capacity, and thus useful properties

for certain food formulations. Kristinsson

made

salmon FPH with several enzymes to 5, 10, and

15% DH and added these to minced salmon pat-

ties at 1.5%. Water loss after freezing (48 days)

and thawing was reduced to 1% compared with

3% for the control. In this study there was no

connection observed between %DH and water

loss, FPH made using Alcalase had the best wa-

ter-holding properties in salmon mince patties,

but all FPH exhibited better water-holding prop-

erties than egg albumin and soy protein concen-

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

trate. Onodenalore and Shahidi

increased cook-

ing yield of comminuted pork by 2.4 to 9.3% by

adding 0.5 to 3.0% of a shark protein hydrolysate.

Cooking yield increased with increasing addition,

and the hydrolysates obtained via an Alcalase-

assisted process from the washed myofibrillar

shark proteins were more effective than hydroly-

sates made from untreated shark muscles. Shahidi

et al.

found similar results with capelin protein

hydrolysates (CPH) when added at a 3% level to

communited pork, CPH increased cooking yield

of approximately 4%. At even lower addition lev-

els there was a large reduction in the amount of

drip loss, indicating that CPH has strong water-

binding capacity. In addition, CPH at 0.5 to 3.0%

level inhibited the formation of TBARS by 17.7

to 60.4%, suggesting that CPH may have antioxi-

dant properties, perhaps due to chelation effects.

Hatate et al.

131

also found that sardine myofibril

protein hydrolysates exhibited antioxidant activ-

ity. More importantly, hydrolysates appeared to

act synergistically with several commercial anti-

oxidants. These antioxidant effects are highly

dependent on the amino acid composition and

molecular size of the hydrolysate peptides.

Fish protein concentrate (FPC) produced from

sardine with an ethanol process has also been

studied in a meat model system with respect to

water holding. FPC was added to a hamburger-

type product made from beef at 20, 40, and 60%

addition, and increased percentage of FPC in the

formulations significantly improved the cooking

yields of the products.

C. Emulsifying Properties

The emulsifying properties of FPH are di-

rectly connected to their surface properties, or

how effectively the hydrolysate lowers the inter-

facial tension between the hydrophobic and hy-

drophilic components in food. Proteins adsorb to

the surface of freshly formed oil droplets during

homogenization and form a protective membrane

that prevents droplets from coalescing.

Hydroly-

sates are surface active and promote oil-in-water

emulsions because they have hydrophilic and hy-

drophobic functional groups and are water

soluble.

121

Hydrolysates orient their hydrophobic

loops in the apolar oil phase, while the polar

segments extend into the aqueous phase. Desir-

able surface active proteins and protein hydroly-

sates have three major attributes: (1) ability to

rapidly absorb to an interface, (2) ability to rap-

idly unfold and reorient at an interface, and (3) an

ability, once at the interface, to interact with the

neighboring molecules and form a strong cohe-

sive, viscoelastic film that can withstand thermal

and mechanical motions.

120,132

Emulsifying capacity and emulsifying stabil-

ity are two methods generally used to measure the

ability of protein hydrolysates to form and stabi-

lize emulsions. Emulsifying capacity is usually

defined as the volume of oil (ml) that can be

emulsified by the protein hydrolysate (g), before

phase inversion or collapse of emulsion occurs.

Because FPH produces emulsions of low viscos-

ity, the most accurate way to measure this is to

use an oil titration method,

133

and measure the

electrical resistance during oil titration. A sudden

increase in resistance is observed when the maxi-

mum emulsifying capacity is reached. Emulsion

stability refers to the ability of an emulsion to

resist changes in its properties over time

134

and

can be determined simply using gravitational

methods. Measurement involves blending the

hydrolysate with oil and water, centrifuging, and

measuring total volume of emulsion, its stability

expressed as the difference between total volume

of an emulsion and the aqueous volume to total

volume.

135,136

The emulsifying properties of hydrolyzed

protein are improved by carefully controlling the

extent of hydrolysis. Extensive hydrolysis results

in a drastic loss of emulsifying properties.

Al-

though small peptides are highly stable and dif-

fuse rapidly and adsorb at the interface, they are

less efficient in reducing the interfacial tension

because they cannot unfold and reorient at the

interface, like proteins with higher molecular

weight.

137

Solubility seems to play an important

role in emulsification because rapid migration to

and adsorption at the interface are critical.

138

However, complete solubility is not an absolute

requirement. Solubility and emulsifying proper-

ties have been found to correlate up to 25% pro-

tein solubility.

120

Casein hydrolyzed with pancre-

atin showed a linear decrease in emulsifying

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

activity with increase in %DH. Casein hydroly-

sate at 67% DH consisted solely of amino acids,

di-, and tripeptides with drastically reduced emul-

sifying activity.

Smaller and smaller peptides

are formed as the enzymatic hydrolysis progresses

and this impacts emulsifying properties.

138

Pro-

tease specificity also plays a key role in the emul-

sifying properties of protein hydrolysates because

this strongly influences the molecular size and

hydrophobicity of the resulting peptides. Kuhler

and Stine

found that whey protein hydrolyzed

with Prolase yield large-molecular-weight pep-

tides with excellent emulsifying stability and ac-

tivity. In contrast, Pronase, which has a broad

specificity, produced much smaller peptides and

yielded hydrolysates with very poor emulsifica-

tion properties. Hence, a careful choice of en-

zymes and low degrees of hydrolysis are recom-

mended if good emulsifying properties are desired.

There is a relationship between %DH and

emulsifying properties for fish protein hydroly-

sates. Enzymatic hydrolysis had a negative influ-

ence on the capacity to form and stabilize emul-

sions as degree of hydrolysis increased for sardine

protein hydrolysates

and salmon protein hydroly-

sates.

Different molecular weight distributions

of the hydrolysates show that a higher content of

high-molecular-weight protein fractions plays an

important role in stabilizing emulsions. Hydroly-

sates with lower degrees of hydrolysis have higher

surface hydrophobicity and sardine hydrolysates

at low %DH have better emulsifying capacity

than commercial sodium caseinate.

Cui

ob-

tained similar results for enzymatically hydro-

lyzed chum salmon muscle.

Hydrophobicity plays an important positive

role in determining emulsifying properties. Kato

and Nakai

139

reported that effective hydrophobic-

ity determined fluorometrically showed signifi-

cant correlation with interfacial tension and emul-

sifying activity of a wide variety of proteins

studied. Li-Chan et al.

140

also found that surface

hydrophobicity predicted the emulsifying proper-

ties of meat proteins. A positive correlation be-

tween surface activity and peptide length has been

found,

and it has since been generally accepted

that a peptide should have a minimum length of

>20 residues to possess good emulsifying and

interfacial properties.

141

Various emulsification properties have been

found for hydrolyzed seafood protein products.

Hydrolyzed processing wastes from lobster using

papain and an undefined fungal protease had a

rather poor capacity for oil emulsification.

Also,

relatively poor emulsifying capacity and stability

were reported for enzymatically produced capelin

protein hydrolysate

and shark protein hydroly-

sates;

however, neither of these studied defined

the %DH of the products. Enzymatically hydro-

lyzed myofibrillar proteins from rockfish fillets

showed an increase in emulsifying capacity and

stability with hydrolysis compared to the intact

protein.

34,123

Emulsifying capacity reached a point

at which there was no change with %DH.

Hy-

drolysates from rockfish muscle made with bro-

melain had relatively poor emulsifying capacity

and yielded unstable emulsions.

136

Removing lip-

ids from rockfish protein hydrolysates resulted in

a dramatic loss in emulsifying capacity; the loss

increased with increasing extraction temperature.

Fish protein derivatives with good emulsify-

ing properties can also be prepared by acetyla-

tion.

Acylating agents such as acetic or succinic

anhydrides can react with the amino groups of

proteins in an alkaline environment, forming amide

groups.

24,142

The acetyl residues participate in the

formation of additional hydrogen bonds or else

can contribute to hydrophobic adherences, thus

increasing emulsifying properties. Groninger and

Miller

136,142

are among researchers that reported

an increase in emulsifying properties of fish pro-

tein by acetylation. Miller and Groninger

136

found

that emulsifying properties of FPH prepared by

bromelain increased until 43 to 59% of the free

amino groups were acetylated, with further acety-

lation having no effect.

Emulsifying properties of fish protein con-

centrates have also been reported.

Fish protein

concentrate produced by isopropanol extraction

had a decreased emulsifying capacity with in-

creased solvent extraction. This loss of emulsifi-

cation capacity was believed to be tied to a loss in

solubility.

The emulsifying ability of other food pro-

teins, primarily milk protein, has been studied

extensively. Enzymatically hydrolyzed whey pro-

tein had excellent emulsifying capacity and sta-

bility when made with the protease Prolase (~3.0 g

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

oil/mg protein).

Pepsin and Pronase hydrolyzed

whey protein yielded emulsions of less capacity

and stability, both decreasing with time of hy-

drolysis and %DH.

The degree of hydrolysis

was, however, not specified. Each of these en-

zymes have different substrate specificities and

action patterns. Pepsin and Pronase hydrolysates

had higher %DH values than Prolase hydroly-

sates. Enzymatic hydrolysis progressively de-

creased the emulsifying activity index of casein,

with a more than twofold reduction after hydroly-

sis to a %DH of 58%, with an additional small

decrease at 67% DH.

A drop in emulsifying

capacity was highly correlated with a drop in

hydrophobicity, and reduced molecular size, and

thus in agreement with our findings.

Trypsin

hydrolysis was found to greatly improve the emul-

sifying properties of casein and whey protein and

was highly influenced by pH.

138

Chicken head hydrolysates

70,71

and turkey

waste hydrolysates

have also been tested for

emulsifying capacity, and both had poor emulsi-

fication ability. A significant drop in emulsifying

properties as hydrolysis progressed was observed

when alpha-chymotrypsin was used to hydrolyze

peanut protein fractions, reaching a stationary

phase after a certain time.

There are many different factors that may

account for the difference observed between hy-

drolysates in both the ability to form an emulsion

and emulsion stability. Peptides molecular char-

acteristics and peptide chain length are the major

reason for the different emulsification ability of

hydrolysates. Environmental conditions such as

pH, ionic strength, temperature, etc. also have an

effect on the emulsification properties. Also, some

synergistic effects of peptides on emulsifying

properties have been noted.

117

In short, the fore-

going indicates that peptide behavior is complex

and not easy to explain.

D. Foaming Properties

The chemistry underlying foaming properties

of protein and protein hydrolysates have many

things in common with emulsifying properties.

Both rely on the surface properties of protein.

Food foams consist of air droplets dispersed in

and enveloped by a liquid containing a soluble

surfactant lowering the surface and interfacial

tension of the liquid.

The amphiphilic nature of

proteins makes this possible; the hydrophobic

portion of the protein extends into the air and the

hydrophilic portion into the aqueous phase.

Townsend and Nakai

143

showed that total hydro-

phobicity, or the hydrophobicity of exposed or

unfolded protein, have a significant correlation to

foaming formation. The surface hydrophobicity,

which is important for emulsification, does not

correlate with foam formation.

Many different methods have been developed

to measure foaming properties of proteins and

protein hydrolysates. Most, if not all, of these

methods have the drawback of poor experimental

reproducibility, thus making it hard to compare

findings between laboratories. Foaming proper-

ties are usually expressed as foam formation and

foam stability. Phillips et al.

144

developed a method

where the ability of protein to form foams is

described as overrun. Overrun is the percentage

of excess volume produced by whipping a protein

containing liquid compared with the initial vol-

ume of the liquid. Foam stability is measured by

whipping the protein solution and measuring the

time required to decrease half of the volume.

Foam stability is a highly empirical method, but

is simple to perform and has found useful appli-

cations with FPH.

Very few studies have been performed on

foaming properties of fish protein hydrolysates.

Shahidi et al.

and Onodenalore and Shahidi

studied the foam formation, or whippability, and

foam stability of shark and capelin protein hy-

drolysates, respectively. In both processes Alcalase

was the protease used for hydrolysis. The capelin

protein hydrolysate had good whippability, 90%,

at a 12% DH. Shark protein hydrolysates from

shark meat had higher whippability, 106%, and

hydrolysates from washed shark myofibrillar pro-

teins had whippability of 130%. Capelin hydroly-

sates had higher foam stability after 30 s that

dropped significantly compared with hydrolyzed

shark myofibrillar protein. Hydrolyzed whey pro-

tein isolates prepared using Alcalase had excel-

lent foaming formation and stability at a 3% DH.

145

The foaming ability for whey was much higher

than for shark and capelin protein hydrolysates.

There is a connection between the degree of

hydrolysis and foaming properties. Kuehler and

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

Stine

concluded that whey proteins hydrolyzed

to a limited degree had a increased foaming abil-

ity but reduced foam stability. This was attributed

to more air being incorporated into solution of

smaller peptides, but the smaller polypeptides do

not have the strength required to give a stable

foam. This was seen in the initial 30 min of

hydrolysis with further hydrolysis resulting in

peptides that lack the ability to stabilize the air

cells in the foam. Weak foams are commonly

observed when food proteins are hydrolyzed.

However, the advantage of using hydrolyzed pro-

teins as foaming agents is their insensitivity to

change in pH. The pH of the dispersing medium

markedly affects foaming, particularly foam sta-

bility, with foaming properties being highest close

to the isoelectric point of the protein.

146

Fish pro-

tein hydrolysates and especially fish protein con-

centrates (FPC) have the unusual property of hav-

ing good foaming properties, and of making strong,

stable foams over a wide pH range.

29-31

The foam

formation of FPC is provided by the soluble pro-

teins, only 1% of total FPC, while the remaining

denatured proteins act as foam stabilizers.

Foaming properties of fish protein hydroly-

sates can be improved by acylation. Groninger

and Miller

147

succinylated hydrolysates produced

by hydrolyzing rockfish fillets with bromelain.

These foams were significantly more stable com-

pared with foams from modified soy protein and

egg white; however, they had smaller foam vol-

ume. The succinylated protein foams were more

stable over a wide range of pH (3 to 9) compared

with the other protein. The succinylated FPH was

incorporated into a dessert topping, a soufflé, and

both a chilled and a frozen dessert, and the prod-

ucts were as acceptable to a taste panel as similar

foods containing no fish protein. Ostrander et

al.

148

incorporated succinylated FPH, as prepared

by Groninger and Miller,

147

into whipped gelatin

desserts. The succinylated FPH formed a stable

foam with smooth texture, highly acceptable with

respect to flavor and mouthfeel.

E. Fat Absoption

Various methods for the measurement of fat

absorption have been described. Commonly, hy-

drolysates are mixed with a specified amount of

excess fat for a particular time and then centri-

fuged at a low centrifugal force and the fat ad-

sorption expressed as the millilter of fat bound by

1 g protein hydrolysate

59,90

for FPH. The mecha-

nism of fat absorption, as assessed by this method,

is attributed mostly to physical entrapment of the

oil, and thus the higher bulk density of the protein

the more fat absorption.

Fat-binding capacity of

proteins correlates with surface hydrophobicity.

Fat absorption of salmon protein hydroly-

sates produced using different enzymes to 5, 10,

and 15% DH was studied by Kristinsson.

The

FPH at all %DH exhibited excellent fat absorp-

tion, greater than both egg albumin and soy pro-

tein concentrate. The hydrolysates at 5% DH had

significantly higher fat absorption (5.98 to 7.07 ml

oil/g FPH) than 10% DH (3.22 to5.12 ml oil/g

FPH) and 15% DH (2.86 to 3.86 ml oil/g FPH)

due to the larger peptide sizes. Only two other

publications dealing with fat absorption for FPH

could be found, for capelin protein hydrolysates

and shark protein hydrolysates.

Both studies fail

to define the units used to express fat absorption,

and thus it is impossible to compare them to other

studies.

Other protein hydrolysates have been studied

with respect to fat absorption. Casein hydroly-

sates with excellent oil-holding capacity, made

with papain, were dissociated by SDS, indicating

that hydrophobic interactions were primarily re-

sponsible. The substrate specificity of enzymes

also seemed to play a major role in this.

114

This

was further confirmed by Kristinsson,

who

showed that different enzymes used to hydrolyze

salmon muscle protein had different fat absorp-

tion ability. The capacity of a hydrolysate to ab-

sorb fat/oil is an important attribute that not only

influences the taste of the product, but it is also an

important functional characteristic that is required

especially for the meat and confectionery indus-

try. Fish protein hydrolysates therefore could very

well be used in such applications.

F. Sensory Properties

Although enzymatic hydrolysis of proteins

develops desirable functional properties, it has

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

the disadvantage of generating bitterness. This is

a common problem with fish protein hydroly-

sates, and the major reason for its slow accep-

tance as a food ingredient. The mechanism of

bitterness is not very clear, but it is widely ac-

cepted that hydrophobic amino acids of peptides

are a major factor. In fact, the hydrophobicity of

peptides can be measured, and it has been found

that most bitter peptides have values over 1400

kcal/mol, whereas nonbitter peptides have values

below 1300 kcal/mol. This principle is, however,

only valid for molecular weights below 6000 D.

149

Peptides with a molecular weight from 1000 to

6000 D and with hydrophobic characteristics have

shown most likely to be bitter.

102

Hydrolysis of

protein results in exposing buried hydrophobic

peptides, which will be readily able to interact

with the taste buds resulting in detection of bitter

taste. An extensive hydrolysis to free amino ac-

ids, however, decreases the bitterness of these

bitter peptides because hydrophobic peptides are

far more bitter compared with a mixture of free

amino acids. Free amino acids, are however, un-

desirable from a functional standpoint. Strict con-

trol of any hydrolysis experiment and termination

at low %DH values therefore is desirable to pre-

vent the development of a bitter taste and the

retention of functional properties.

Common features found in the structure of

bitter peptides are, for example, N, or C, terminal

basic amino acid residues, C, or N, terminal hy-

drophobic peptide fragments and peptide chain

folding due to proline residues within the peptide

structure.

150

Bitterness is due to particular arrange-

ments of certain chemical groups in the peptides.

Two functional groups are necessary to produce

bitterness, such as a pair of hydrophobic groups

or a hydrophobic or a basic group.

151

The sensa-

tion of bitterness may also be exerted by chemical

compounds having a hydrophobic region and one

hydrophilic group spaced 0.3 nm apart.

Because

enzymes have different preferences for amino

acids, choosing the most appropriate enzyme

preparation for hydrolysis can control the bitter-

ness. Enzymes with a high preference for hydro-

phobic amino acids such as Alcalase are often

preferred and frequently yield products of low

bitterness.

20,96

The use of exopeptidases, as op-

posed to endoproteinases, can also be helpful in

overcoming the bitterness in fish protein hydroly-

sates, particularly exopeptidases that split off

hydrophobic amino acids from bitter peptides.

However, for an enzyme preparation to be effec-

tive for hydrolyzing protein, both exopeptidases

and endoproteinases are required. Many studies

have shown that proteolytic preparations contain-

ing exopeptidases and endoproteinases produce

less bitter peptides than single proteases.

152-155

Trials have shown that by the additional action of

exopeptidases in proteolytic processes, it is pos-

sible to move the bitterness point up to higher

degree of hydrolysis.

156

Despite the problem with bitterness of fish

protein hydrolysates, little research has been con-

ducted. While FPH is highly nutritious and safe

and functional properties are good, the sensory

properties are extremely important for the suc-

cessful adaptation and acceptance by the food

industry and the consumer. Yu and Fazidah

125

found a connection between the degree of hy-

drolysis and the intensity of bitterness when hy-

drolyzing Aristichthys nobilis with a commer-

cial protease. As the %DH increased so did the

bitterness, and after a 5-h hydrolysis samples

were described as “very bitter”. Interestingly, no

bitterness was observed until after 4 h of hy-

drolysis. Papain-hydrolyzed herring had higher

bitterness scores than Alcalase-hydrolyzed her-

ring.

Alcalase hydrolyzed samples at higher

degree of hydrolysis were less bitter than pa-

pain-hydrolyzed samples at the same %DH. The

greater hydrophobic specificity of Alcalase com-

pared with papain could result in a reduced bitter-

ness. Enzymatically hydrolyzed ethanol-extracted

herring, with either Alcalase or papain, had very

low bitterness, suggesting that reduced bitter-

ness could be due in part to lower lipid content.

Lalasidis et al.

157

hydrolyzed cod filleting offal

with Alcalase, which produced a product with a

bitter taste. This bitter taste was eliminated when

the hydrolysate was treated further with

pancreatine, which is rich in exopeptidase activ-

ity.

Fish protein hydrolysates have been in-

corporated into food systems to evaluate their

acceptability, and sometimes with satisfactory

results.

147,148

Proteins from Oreochromis

mossambicus, a freshwater fish, were hydrolyzed

with Alcalase to produce a soluble hydrolysate

that was incorporated into crackers.

158

Sensory

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

evaluation showed the fried crackers with up to

10% addition of FPH were highly acceptable.

Vieira et al.

hydrolyzed lobster processing

waste for 5 h with several commercial enzymes

and found no difference in taste between the hy-

drolysates and a control sample, and detected no

bitterness. This is perhaps due to the extensive

hydrolysis. The hydrolysate possessed a distinct,

pleasant smell, and it was proposed for use in

various formulated food products such as flavor

enhancers. Is has long been known that proteases

can be used for recovery of flavor.

159

Baek and

Cadwallader

attempted to fully utilize crayfish

processing byproducts as flavorings by hydrolyz-

ing them with Optimase at optimal conditions.

They found that it is possible that the hydroly-

sates could serve as feedstocks for the production

of value-added seafood flavor extracts. Fujimaki

et al.

160

reported that a fish protein concentrate

treated with Pronase had a flavor potentiating

activity like that of monosodium glutamate (MSG)

but was accompanied by an unfavorable bitter

flavor. A low molecular acidic peptide fraction

contributed significantly to this MSG-like flavor.

The acidic peptides isolated later from the same

fish protein hydrolysate showed that at least four

dipeptides (Glu-Asp, Glu-Glu, Glu-Ser and Thr-

Ser) and five tripeptides (Asp-Glu-Ser, Glu-Asp-

Glu, Glu-Gln-Glu, Glu-Gly-Ser, and Ser-Glu-Glu)

had a flavor quantitatively resembling that of

MSG, but with weaker intensities.

161

Hevia and

Olcott

162

analyzed basic tripeptides that were re-

sponsible for bitter taste in fish protein hydroly-

sates. The tripeptides contained asparagine and

lysine as the second and C-terminal residues, re-

spectively, with the N-terminal residue leucine or

glycine.

Many techniques have been suggested to re-

duce or mask bitterness in hydrolysates, but few

of them applied to FPH. These include treating

hydrolysates with activated carbon that partly

removes bitter peptides with absorption,

59,163

ex-

tracting bitter peptides with solvents,

163

applica-

tion of further enzyme treatment with exopepti-

dases,

152

and by using the so-called plastein

reaction.

164

Chakrabarti

165

reported success in

debittering fish protein hydrolysate using ethyl

alcohol. Higher concentrations of alcohol low-

ered the bitter fraction in the hydrolysate.

The plastein reaction is among few that have

been studied with respect to fish protein hydroly-

sates. Figure 9

166

outlines the steps involved in the

plastein reaction. Heck

164

investigated the plas-

tein reaction in FPH from peptic hydrolysis and

found that it reduced the bitterness but with an

accompanied loss of FPH solubility. Some suc-

cess using the plastein reaction has been reported

with fish waste

167

and sardines.

168

Plastein can be defined as a protein substrate

that may be prepared by reversing hydrolysis with

a protease such as pepsin or papain in a concen-

trated protein hydrolysate.

60,169

The plasteins are a

mixture of peptides of varied composition and

molecular weight between 1000 and 500.000

24,166

Three factors are necessary for completion

of the plastein reaction: (1) a low-molecular-weight

protein hydrolysate as a substrate, (2) a high sub-

strate concentration, and (3) a reaction environ-

ment of pH 4 to 6 regardless of the enzyme in-

volved.

164

During plastein synthesis, a high

concentration of hydrolysate (30 to 50%) is incu-

bated with an enzyme resulting in a condensation

of the peptides. As new polypeptides are formed,

they aggregate via hydrophobic associations. The

final product has a low lipid content and special

hydrophobic functional properties.

31,41,60

Tanimoto

et al.

170

have studied the mechanism of plastein

synthesis with α-chymotrypsin and found that it

proceeds in two stages. The first stage is charac-

terized by a reaction of the peptide with the hy-

droxyl group of Ser 195 that produces a peptidyl-

α-chymotrypsin complex that then undergoes

aminolysis as a result of a nucleophilic attack of

a second peptide. The plastein reaction has also

been found useful for protein recovery from ex-

tensively autolytically produced fish silage.

171

is strongly recommended that this process be stud-

ied further for fish protein hydrolysis.

V. FUTURE DEVELOPMENTS AND

POTENTIAL APPLICATIONS

The use of FPH as a functional food ingredi-

ent still has a long way to go until it becomes

economically feasible and accepted by industry

and consumers. However, there is a wonderful

opportunity for this to happen, due to regulations

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

FIGURE 9. The plastein reaction. (Adapted from Ref. 166.)

of processing waste and the abundance of small

pelagic underutilized species that will be the fron-

tiers of fisheries in this century. These factors

should stimulate the conversion of low value fish

materials into more valuable and palatable prod-

ucts. Other applications outside the food industry

may also be a feasible option for FPH. Soluble

FPH from extensive hydrolysis is an excellent

source of nitrogen for maintaining the growth of

different microorganisms. Previously, it was noted

that extensive hydrolysis of fish protein results in

a product containing free amino acids and low-

molecular-weight peptides, which is very promis-

ing for microbial peptone production.

129

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

The beneficial chemical composition of FPH

and FPC has also led to using these materials as

fertilizer with good results. The FPH might be too

expensive for this purpose, as added enzymes are

used, and the product is usually dried. However,

Ferreira and Hultin

104

developed an enzymatic

process using the acid protease Newlase A to

extensively hydrolyze cod frames. This process is

currently being used. The use of FPC as fertilizer

is more common, either from chemical hydrolysis

or autolysis. A successful process using second-

ary raw material from processing plants (frames

and viscera) and using the acid endogenous en-

zymes to extensively hydrolyze the material is

being operated in Washington State. The final

product is sold in bulk to local farmers, mainly

cranberry growers, and with superior results com-

pared with other commercially available fertiliz-

ers.

172

The mechanism behind the effect of the

FPH and FPC to stimulate growth and develop-

ment better than synthetic fertilizers need to be

studied further. Novel applications taking advan-

tage of plant growth-stimulating effect of FPH

and FPC could possibly be developed. An exten-

sive research program supervised by Dr. Kalidas

Shetty at the University of Massachusetts at

Amherst examines the effect of FPH on plants. In

one study the effects of using FPH to stimulate

somatic embryogenesis in Anise (Pimpinella

anisum) when compared with proline, a known

stimulator, was examined.

173

The conclusion was

that FPH could well become a proline and amino

acid substitute in plant tissue culture applications.

The positive effects of FPH due to proline and its

precursor glutamate on plant growth was con-

firmed in a recent study by the same group, where

melon (Cucumis melo L.) shoot organogenesis

was stimulated.

174

Proline and glutamate can be

obtained from FPH and potentially can be used

for value-added applications in the plant propaga-

tion industry,

174

a new arena of application for

FPH and possibly a very valuable one.

Using FPH and FPC for animal feed applica-

tions due to its good amino acid balance and high

protein content could be quite feasible. As men-

tioned previously, fish silage is used primarily for

this purpose and is essentially limited to young

animals, due to the extensive hydrolysis of the

proteins.

Another little-studied property of FPH is its

antioxidant potential. Studies have found power-

ful antioxidant activity in intact fish protein

131

and

in hydrolysate.

These effects are probably highly

dependent on the amino acid composition and

molecular size of the FPH peptides and deserve

further study.

VI. CONCLUSION

There have been several attempts to make

functional fish protein hydrolysates with enzymes,

some successful and some not. Most of these

studies are rather crude, perhaps due to the ap-

plied nature of the field, and have many short-

comings, specifically failing to compare enzymes

at the same activity level, controlling the %DH

properly and characterizing the chemical and func-

tional properties of the final products. There is

potential for these products to be produced and

sold as functional food ingredients, but at present

other applications such as plant nutrients, fertiliz-

ers, and animal feeds might be more feasible.

Standardized procedures to examine the functional

properties are needed, as well as more studies on

using endogenous enzymes to make functional

FPH. With more basic research on the molecular

level the future of FPH could be bright, especially

in light of the environmental problems facing

fisheries. It is time for FPH research to take a

new, fresher, and more scientific direction, be-

cause the field has been more or less stagnant for

the last 20 years or so.

REFERENCES

1. Rebeca, B. D., Pena-Vera, M. T., and Diaz-

Castaneda, M., Production of fish protein hydroly-

sates with bacterial proteases; Yield and nutritional

value, J. Food. Sci., 56, 309, 1991.

2. FAO, Food and Agricultural Organization Yearbook,

Fishery Statistics, Rome, Italy, 1986.

3. Mackie, I. M., Fish protein hydrolysates, Proc.

Biochem., 17(1), 26, 1982.

4. Mullally, M. M., O’Callaghan, D. M., FitzGerald,

R. J., Donnelly, W. J., and Dalton, J. P., Proteolytic

and peptidolytic activities in commercial pancreatin

protease preparations and their relationship to some

whey protein hydrolysate characteristics, J. Agric. Food

Chem., 42, 2973, 1994.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

5. Hoyle, N. and Merritt, J.H., Quality of fish protein

hydrolysates from herring (Clupea harengus), J. Food

Sci., 59, 76, 1994.

6. Demetriades, K., Coupland, J. N., and McClements,

D. J., Physical properties of whey protein stabilized

emulsions as related to pH and NaCl, J. Food Sci.,

62(2), 342, 1997.

7. Shiau, S-Y., Seafood Protein in human and animal

nutrition, in Seafood Proteins, ed., Sikorski, Z. E.,

Pan, B. S., and Shahidi, F., Eds., Chapman & Hall,

New York, 1994.

8. Friedman, K., Nutritional value of proteins from dif-

ferent food sources: a review, J. Agric. Food Chem.,

44(1), 6, 1996.

9. Venugopal, V., Chawla, S. P., and Nair, P. M.,

Spray dried protein powder from threadfin beam:

preparation, properties and comparison with FPC type-

B, J. Muscle Foods, 7, 55, 1996.

10. Yanez, E., Ballester, D., and Monckeberg, F., En-

zymatic fish protein hydrolyzate: chemical composi-

tion, nutritive value and use as a supplement to cereal

protein, J. Food Sci., 41, 1289, 1976.

11. Spinelli, J. and Dassow, J. A., Fish proteins: their

modification and potential uses in the food industry,

in Chemistry and Biochemistry of Marine Food Prod-

ucts, AVI Publishing Company, Westport, CT, 1982.

12. Hultin, H. O., Characteristics of Muscle Tissue, in

Food Chemistry, 2nd ed., Fennema, O. R., Ed., Marcel

Dekker Inc., New York, 1985.

13. Venugopal, V. and Shahidi, F., Thermostable water

dispersions of myofibrillar proteins from Atlantic

mackerel (Scomber scombrus), J. Food Sci., 59(2),

256, 1994.

14. Sikorski, Z. E., The myofibrillar proteins in seafood,

in Seafood Proteins, Sikorski, Z. E., Pan, B. S., and

Shahidi, F., Eds., Chapman & Hall, New York, 1994.

15. Suzuki, T., Fish and Krill Protein: Processing Tech-

nology, Applied Science, London, UK, 1981.

16. Stefansson, G. and Hultin, H. O., On the solubility

of cod muscle protein in water, J. Agric. Food. Chem.,

42, 2656, 1994.

17. Feng, Y. and Hultin, H. O., Solubility of the proteins

of mackerel light muscle at low ionic strength, J.

Food Biochem., 21, 479, 1997.

18. Zubay, G., Biochemistry, William C. Brown Publish-

ers, Dubuque, IA, 1993.

19. DeMan, J. M., Principles of Food Chemistry, Van

Nostrand Reinhold, New York, 1990.

20. Adler-Nissen, J., Enzymic Hydrolysis of Food Pro-

teins, Elsevier Applied Science Publishers, Barking,

UK, 1986.

21. Skanderby, M., Protein hydrolysates: their function-

ality and applications, Food Technol. Int. Eur., 10,

141, 1994.

22. Pigott, G. M., Enzyme modifications of fishery by-

products, in Chemistry and Biochemistry of Marine

Food Products, AVI Publishing Company, Westport,

CT, 1982.

23. Snyder, D. G., Bureau of commercial fisheries pro-

gram, Food Technol., 21(9), 70, 1967.

24. Sikorski, Z. E. and Naczk, M., Modification of tech-

nological properties of fish protein concentrates, Crit.

Rev. Food Sci. Nutr., 4, 201, 1981.

25. Cheftel, C., Ahern, M., Wang, D. I. C., and

Tannenbaum, S. R., Enzymatic solubilization of fish

protein concentrate: Batch studies applicable to con-

tinuous enzyme recycling processes, J. Agric. Food

Chem., 19, 155, 1971.

26. Mackie, I. M., Proteolytic enzymes in recovery of

proteins from fish waste, Process Biochem., 12, 12,

1974.

27. Dubrow, D. L., Kramer, A., and McPhee, A. D.,

Effects of temperature on lipid extraction and func-

tional properties of fish protein concentrate (FPC) J.

Food Sci., 38, 1012, 1973.

28. Finch, R., Whatever happened to fish protein concen-

trate?, Food Technol., 31(5), 44, 1977.

29. Sheustone, F. S., Foaming of fish protein concen-

trate, Food Preserv. Q., 13, 45, 1953.

30. Hermansson, A. M., Sivik, B., and Skjoldebrand,

C., Factors affecting solubility, foaming and swelling

of fish protein concentrate, Lebensm. Wiss. Technol.,

4, 201, 1971.

31. Kinsella, J. E., Functional properties of proteins in

foods: a survey, Crit. Rev. Food Sci. Nutr., 8(4), 219,

1976.

32. Moorjani, M. N., Nair, R. B., and Lahiry, N. L.,

Quality of fish protein concentrate prepared by direct

extraction of fish with various solvents, Food Technol.,

22(12), 1557, 1968.

33. Hale, M. B., Making fish protein concentrate by en-

zymatic hydrolysis, NOAA Technical Report NMFS

SSRF-675, US Department of Commerce, Seattle, WA,

pp. 1–31, 1972.

34. Spinelli, J., Koury, B., and Miller, R., Approaches

to the utilization of fish for the preparation of protein

isolates; enzymic modifications of myofibrillar fish

proteins, J. Food Sci., 37, 604, 1972b.

35. Quaglia, G. B. and Orban, E., Enzymic solubilisation

of proteins of sardine (Sardina pilchardus) by com-

mercial proteases, J. Sci. Food Agric., 38, 263, 1987a.

36. Quaglia, G. B. and Orban, E., Influence of the de-

gree of hydrolysis on the solubility of the protein

hydrolsyates from sardine (Sardina pilchardus), J.

Sci. Food Agric., 38, 271, 1987b.

37. Vareltzis, K., Soultos, N., Zetou, F., and Tsiaras, I.,

Proximate composition and quality of a hamburger

type product made from minced beef and fish protein

concentrate, Lebensm. Wiss. u. Technol., 23(2), 112,

1990.

38. Blenford, D. E., Protein hydrolysates: functionalities

and uses in nutritional products, Int. Food Ingr., 3, 45,

1994.

39. Webster, J. D., Ledward, D. A., and Lawrie, R. A.,

Protein hydrolysates from meat industry by-products,

Meat Sci., 7, 147, 1982.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

40. Loffler, A., Proteolytic enzymes: sources and appli-

cations, Food Technol., 40(1), 63, 1986.

41. Thakar, P. N., Patel, J. R., and Joshi, N. S., Protein

hydrolysates: a review, Indian J. Dairy Sci., 44(9),

557, 1991.

42. Thomas, D. and Loffler, F., Improved protein

functionalities by enzymatic treatment, Food Market.

Technol., 2, 1994.

43. Orlova, T. A., Nelichik, N. N., and Fleider, K. A.,

[Edible protein concentrate from fish raw material],

Rybnoe Khozyaistvo, 10, 59, 1979.

44. Fonkwe, L. G. and Singh, R. K., Protein recovery

from enzymatically deboned turkey residue by enzy-

mic hydrolysis, Process Biochem., 31(6), 605, 1996.

45. Tannenbaum, S. R., Ahern, M., and Bates, R. P.,

Solubilization of fish protein concentrate. I. An alka-

line process, Food Technol., 24(5), 604, 1970a.

46. Tannenbaum, S. R., Ahern, M., and Bates, R. P.,

Solubilization of fish protein concentrate. II. Utiliza-

tion of the alkaline-process product, Food Tecnol.,

24(5), 607, 1970b.

47. Linder, M., Fanni, J., Parmentier, M., Sergent, M.,

and Phan-Than-Luu, R., Protein recovery from veal

bones by enzymatic hydrolysis, J. Food Sci., 60(5),

949, 1995.

48. Lahl, W. J. and Braun, S. D., Enzymatic production

of protein hydrolysates for food use, Food Technol.,

58(10), 68, 1994.

49. Krause, W. and Schmidt, K., [Studies on the enzy-

matic hydrolysis of alkali-treated milk proteins],

Untersuchungen zur enzymatischen hydrolyse von

alkalibehandelten milchproteinen, Nahrung, 18(8),

833, 1974.

50. Ledward, D. A. and Lawrie, R. A., Recovery and

utilization of by-product proteins of the meat industry,

J. Chem. Tech. Biotechnol., 34B, 223, 1984.

51. Richardson, T. and Hyslop, D. B., Enzymes, in Food

Chemistry, 2nd ed., Fennema, O. R., Ed., Marcel

Dekker Inc., New York, 1984.

52. Godfrey, T. and Reichelt, J., Eds., Industrial Enzy-

mology. The Application of Enzymes in Industry,

MacMillan, London, UK, 1983.

53. Chreighton, T. E., Proteins: Structures and Molecu-

lar Properties, W.H. Freeman & Co., New York,

1993.

54. Svendsen, I., Chemical modifications of the subtilisin

with special reference to the binding of large sub-

strates. A review, Carlsberg Res. Commun., 41, 237,

1976.

55. Phillips, R. D. and Beuchat, L. R., Enzyme modifi-

cation of proteins, in Protein Functionality in Foods,

Cherry, J. P., Ed., American Chemical Society, Sym-

posium Series 147, Washington, DC, 1981.

56. Kester, J. J. and Richardson, T., Modification of

whey protein by trypsin, J. Dairy Sci., 67, 2757, 1984.

57. Mahmoud, M. I., Malone, W. T., and Cordle, C. T.,

Enzymatic hydrolysis of casein: effect of degree of

hydrolysis on antigenicity and physical properties, J.

Food Sci., 57(5), 1223, 1992.

58. Mahmoud, M. I., Physicochemical and functional

properties of protein hydrolysates in nutritional prod-

ucts, Food Technol., 58(10), 89, 1994.

59. Shahidi, F., Han, X-Q., and Synowiecki, J., Produc-

tion and characteristics of protein hydrolysates from

capelin (Mallotus villosus), Food Chem., 53, 285,

1995.

60. Gildberg, A., Enzymic processing of marine raw

materials, Process Biochem., 28, 1, 1993.

61. Owens, J. D. and Mendoza, L. S., Enzymatically

hydrolysed and bacterially fermented fishery prod-

ucts, J. Fd. Technol., 20, 273, 1985.

62. Gildberg, A., Espejo-Hermes, J., and Magno-

Orejana, F., Acceleration of autolysis during fish

sauce fermentation by adding acid and reducing the

salt content, J. Sci. Food Agric., 35(12), 1363, 1984.

63. Raa, J. and Gildberg, A., Autolysis and proteolytic

activity of cod viscera, J. Fd. Technol., 11, 619, 1976.

64. van Veen, A. G. and Steinkraus, K. H., Nutritive

value and wholesomeness of fermented foods, J. Agric.

Food Chem., 18(4), 576, 1970.

65. Raa, J. and Gildberg, A., Fish silage: a review, CRC

Crit. Rev. Food Sci. Nutr., 14, 383, 1982.

66. van Wyk, H. J. and Heydenrych, C. M. S., The

production of naturally fermented fish silage using

various lactobacilli and different carbohydrate sources,

J. Sci. Food Agric., 36, 1093, 1985.

67. Dong, F. M., Fairgrieve, W. T., Skonberg, D. I.,

and Rasco, B. A., Preparation and nutrient analyses

of lactic acid bacteria ensiled salmon viscera, Aquac-

ulture, 109, 351, 1993.

68. Cui, H. The Influence of Degree of Hydrolysis on the

Structural and Functional Properties of Fish Protein

Peptic Hydrolysates, Masters thesis, University of

Washington, Seattle, WA, 1996.

69. Gonzalez-Tello, P., Camacho, F., Jurado, E., Paez,

M. P., and Guadix, E. M., Enzymatic hydrolysis of

whey proteins. I. Kinetic models, Biotechnol. Bioeng.,

44, 523, 1994a.

70. Surowka, K. and Fik, M., Studies on the recovery of

proteinaceous substances from chicken heads. I. An

application of neutrase to the production of protein

hydrolysate, Int. J. Food Sci. Technol., 27, 9, 1992.

71. Surowka, K. and Fik, M., Studies on the recovery of

proteinaceous substances from chicken heads. II.

Application of pepsin to the production of protein

hydrolysates, J. Sci. Food Agric., 65, 289, 1994.

72. Kuehler, C. A. and Stine, C. M., Effect of enzymatic

hydrolysis on some functional properties of whey

protein, J. Food Sci., 39, 379, 1974.

73. Jost, R. and Monti, J. C., Partial enzymatic hydroly-

sis of whey protein by trypsin, J. Dairy Sci., 60, 1387,

1977.

74. Vegarud, G. E., Svenning, C., Molland, T., and

Langsrud, T., Enzymatic hydrolysis of milk proteins

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

improved functional prperties, Med. Fac. Landbouww.

Rijksuniv. Gent, 56(4a), 1649, 1991.

75. Mutilangi, W. A. M., Panyam, D., and Kilara, A.,

Hydrolysates from proteolysis of heat-denatured whey

proteins, J. Food Sci., 60(5), 1104, 1995.

76. Lin, S-B., Chiang, W-E., Cordle, C. T., and

Thomas, R. L., Functional and immunological prop-

erties of casein hydrolysate produced from a two-

stage membrane system., J. Food Sci., 62(3), 480,

1997.

77. Parrado, J., Millan, F., Hernandez-Pinzon, I.,

Bautista, J., and Machado, A., Characterization of

enzymatic sunflower protein hydrolysates, J. Agric.

Food Chem., 41(11), 1821, 1993.

78. Monteiro, P. V. and Prakash, V., Alteration of func-

tional properties of peanut (Arachis hypogaea L.) pro-

tein fractions by chemical and enzymatic modifica-

tions, J. Food Sci. Technol., 33(1), 19, 1996.

79. Sen, D. P., Sripathy, N. V., Lahiry, N. L.,

Sreenivasan, A., and Subrahmanyan, V., Fish hy-

drolysates. I. Rate of hydrolysis of fish flesh with

papain, Food Technol., 5, 1962.

80. Sen, D. P., Sripathy, N. V., Lahiry, N. L.,

Sreenivasan, A., and Subrahmanyan, V., Fish hy-

drolysates. II. Standardization of digestion conditions

for preparation of hydrolysates rich in peptones and

proteoses, Food Technol., 5, 1962.

81. Limonta, B. A., Behnke, U., and Ruttloff, H., [Manu-

facture of fish protein hydrolysate] Herstellung von

fischproteinhydrolysate, German Democratic Repub-

lic Patent no. 148437, 1981.

82. Onodenalore, A. C. and Shahidi, F., Protein disper-

sions and hydrolysates from shark (Isurus oxyrinchus),

J. Aquat. Food Prod. Technol., 5, 43, 1996.

83. Quaglia, G. B. and Orban, E., Influence of enzy-

matic hydrolysis on structure and emulsifying proper-

ties of sardine (Sardina pilchardus) protein hydroly-

sates, J. Food Sci., 55(6), 1571, 1990.

84. Sugiyama, K., Egawa, M., Onzuka, H., and Oba,

K., Characteristics of sardine muscle hydrolysates

prepared by various enzymic treatments, Nippon Suisan

Gakkaishi, 57(3), 475, 1991.

85. Baek, H. H. and Cadwallader, K. R., Enzymatic

hydrolysis of crayfish processing by-products, J. Food

Sci., 60, 929, 1995.

86. Viera, G. H. F., Martin, A. M., Saker-Sampaiao,

S., Omar, S., and Goncalves, R. C. F., Studies on the

enzymatic hydrolysis of Brazilian lobster (Panulirus

spp.) processing wastes, J. Sci. Food Agric., 69, 61,

1995.

87. Sakai, N., Noyori, M., Matsunaga, H., and

Hanzawa, T., A kinetic study on the hydrolysis of

fish protein by acid protease, Nippon Shokuhin Kogaku

Kaishi, 42(5), 301, 1995.

88. Diniz, F. M. and Martin, A. M., Use of response

surface methodology to describe the combined effects

of pH, temperature and E/S ratio on the hydrolysis of

dogfish (Squalus acanthias) muscle, Int. J. Food Sci.

Technol., 31, 419, 1996.

89. Benjakul, B. and Morrissey, M. T., Protein hydroly-

sates from Pacific whiting solid wastes, J. Agric. Food

Chem., 45, 3424, 1997.

90. Kristinsson, H. G., Reaction Kinetics, Biochemical

and Functional Properties of Salmon Muscle Proteins

Hydrolyzed by Different Alkaline Proteases, Masters

thesis, University of Washington, Seattle, WA,

1998.

91. Deeslie, W. D. and Cheryan, M., Functional proper-

ties of soy protein hydrolsyates from a continous ul-

trafiltration reactor, J. Agric. Food Chem., 36(1), 26,

1988.

92. Unido, Sectoral Studies Branch, Industrial develop-

ment strategies for fishery systems in developing coun-

tries, Food Rev. Int., 6, 1, 1990.

93. Ritchie, A. H. and Mackie, I. M., Preparation of fish

protein hydrolysates, Anim. Feed Sci. Technol., 7(2),

125, 1982.

94. FAO, Protein Advisory Group, Bulletin 12, 1971.

95. Adler-Nissen, J., Control of the proteolytic reaction

and the level of bitterness in protein hydrolysis pro-

cesses, J. Chem. Technol. Biotechnol., 34B, 215, 1984.

96. Petersen, B. R., Removing bitterness from protein

hydrolysates, Food Technol., 58(10), 96, 1994.

97. Hale, M. B., Relative activities of commercially-avail-

able enzymes in the hydrolysis of fish proteins, Food

Technol., 23, 107, 1969.

98. Arzu, A., Mayorga, H., Gonzales, J., and Rolz, C.,

Enzymatic hydrolysis of cottonseed protein, J. Agric.

Food Chem., 20, 805, 1972.

99. Kristinsson, H. G. and Rasco, B. A., Kinetics of

enzymatic hydrolysis of Atlantic salmon (Salmo salar)

muscle proteins by alkaline proteases and a visceral

serine protease mixture, Proc. Biochem., submitted,

2000.

100. Liu, L. L. and Pigott, G. M., Preparation and use of

inexpensive crude pepsin for enzyme hydrolysis of

fish, J. Food Sci., 46, 1569, 1981.

101. Tarky, W., Agarwala, O. P., and Pigott, G. M.,

Protein hydrolysate from fish waste, J. Food Sci., 38,

917, 1973.

102. Gonzalez-Tello, P., Camacho, F., Jurado, E., Paez,

M. P., and Guadix, E. M., Enzymatic hydrolysis of

whey proteins. II. Molecular-weight range, Biotechnol.

Bioeng., 44, 529, 1994b.

103. Beddows C. G. and Ardeshir, A. G., The production

of soluble fish protein solution for use in fish sauce

manufacture, J. Fd. Technol., 14, 603, 1979

104. Ferreira, N. G. and Hultin, H. O., Liquefying cod

fish frames under acidic conditions with a fungal en-

zyme, J. Food Proc. Pres., 18, 87, 1994.

105. Mullally, M. M., O’Callaghan, D. M., FitzGerald,

R. J., Donnelly, W. J., and Dalton, J. P., Zymogen

activation in pancreatic endoproteolytic preparations

and influence on some whey protein characteristics, J.

Food Sci., 60(2), 227, 1995.

106. Hevia, P., Whiteker, J. R., and Olcott, H. S., Solu-

bilization of a fish protein concetrate with proteolytic

enzymes, J. Agric. Food Chem., 24(2), 383, 1976.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

107. Moreno, M., M., C. and Cuadrado, V. F., Enzymic

hydrolysis of vegetable proteins: mechanism and ki-

netics, Process Biochem., 28, 481, 1993.

108. Archer, M. C., Ragnarsson, J. O., Tannenbaum, S.

R., and Wang, D. I. C., Enzymatic solubilization of

an insoluble substrate, fish protein concentrate: pro-

cess and kinetic considerations, Biotechnol. Bioeng.,

15, 181, 1973.

109. Langmyhr, E., Enzymatic hydrolysis of fish protein,

Dissertation Abstracts International, 43(4), 758, 1981.

110. Adler-Nissen, J., Enzymatic hydrolysis of food pro-

teins, Process Biochem., 12(6), 18, 1977.

111. Steinhardt, H. and Beychok, S., Interaction of pro-

tein with hydrogen ions and other small ions and

molecules, in The Proteins, Vol. 2, Neurath, H., Ed.,

Academic Press, New York, 1964.

112. Adler-Nissen, J., Determination of the degree of

hydrolysis of food protein hydrolysates by trinitro-

benzenesulfonic acid, J. Agric. Food Chem., 27(6),

1256, 1979.

113. Ukeda, H., Matsumoto, K., Katsuki, I., and

Osajima, Y., A simple method for monitoring the

enzymatic hydrolysis of fish meat: determination of

amino groups using gluteraldehyde, Nippon

Nogeikagaku Kaishi, 65(8), 1229, 1991.

114. Haque, Z. U., Influence of milk peptides in determin-

ing the functionality of milk proteins: a review, J.

Dairy Sci., 76(1), 311, 1993.

115. Deeslie, W. D. and Cheryan, M., Fractionation of

soy protein hydrolysates using ultrafiltration mem-

branes, J. Food Sci., 57(2), 411, 1991.

116. Van Beresteijn, E. C. H, Peeters, R. A., Kaper, J.,

Meijer, R. J. G. M., Robben, A. J. P. M., and

Schmidt, D. G., Molecular mass distribution, immu-

nological properties and nutritive value of whey pro-

tein hydrolysates, J. Food Protection, 57(7), 619, 1994.

117. Gauthier, S. F., Paquin, P., Pouliot, Y., and

Turgeon, S., Surface activity and related functional

properties of peptides obtained from whey proteins, J.

Dairy Sci., 76(1), 321, 1993.

118. Haque, Z. U., Importance of peptides for food protein

functionality, in Food Polymers, Gels and Colloids,

Royalty Society of Chemistry London, England,

1991.

119. Adler-Nissen, J. and Olsen, H., The influence of

peptide chain length on taste and functional properties

of enzymatically modified soy protein, in Food Chem-

istry, Pour-El, A., Ed., American Chemical Society,

Washington, D.C., 1979.

120. Damodaran, S., Amino acids, peptides, and proteins,

in Food Chemistry, 3rd ed., Fennema, O. R., Ed.,

Marcel Dekker Inc., New York, 1996.

121. Wilding, P., Lilliford, P. J., and Regenstein, J. M.,

Functional properties of proteins in foods, J. Chem.

Technol. Biotechnol., 34B, 182, 1984.

122. Morr, V., German, B., and Kinsella, J. E.,

Regenstein, J. M., Van Buren, J. P., Kilara, A.,

Lewis, B. A., and Mangino, M. E., A collaborative

study to develop a standardized food protein solubil-

ity procedure, J. Food Sci., 50, 1715, 1985.

123. Spinelli, J., Koury, B., and Miller, R., Approaches

to the utilization of fish for the preparation of protein

isolates: Isolation and properties of myofibrillar and

sarcoplasmic fish protein, J. Food Sci., 37, 599,

1972a.

124. McNairney, J., Modification of a novel protein prod-

uct, J. Chem. Technol. Biotecnol., 34B, 206, 1984.

125. Yu, S. Y. and Fazidah, S., Enzymic hydrolysis of

proteins from Aristichthys noblis by protease P

“Amano” 3, Trop. Sci., 34, 381, 1994.

126. Venugopal, V. and Shahidi, F., Value-added prod-

ucts from underutilized fish species, Crit. Rev. Food

Sci. Nutr., 35(5), 431, 1995.

127. Hale, M. B. and Bauersfeld, Jr., P. E., Preparation

of a menhaden hydrolysate for possible use in a milk

replacer, Mar. Fish. Rev., 40(8), 14, 1978.

128. Mynov, V. A. and Kim, L. V., [Enrichment of pasta

with fish protein hydrolysates], Khlebopekarnaya I

Konditerskaya Promyshlennost, 5, 43, 1980.

129. Skorupa, K. and Sikorski, Z. E., Mozliwosci

wykorzystania mniej cennych surowcow rybnych do

otrzymywania peptonow mivrobiologicznych,

Przemysl spozywczy, 6, 159, 1993.

130. Buinov, A. A., Ginzburg, A. S., and Syroedov, V. I.,

[Hygroscopic properties of fish protein hydrolysates,

dried as a foam], Izvestiya Vysshikh Uchebnykh

Zavedenii, Pishchevaya Technologiya, 3, 110, 1977.

131. Hatate, H., Numata, Y., and Kochi, M., Synergistic

effect of sardine myofibril protein hydrolyzates with

antioxidant, Nippon Suisan Gakkaishi, 56(6), 1011,

1990.

132. Phillips, M. C., Protein conformation at liquid inter-

faces and its role in stabilizing emulsions and foams,

Food Technol., 35, 50, 1981.

133. Webb, N. B., Ivey, F. J., and Craig, H. B., The

measurement of emulsifying capacity by electric re-

sistance, J. Food Sci., 35, 501, 1970.

134. McClements, D. J., Food Emulsions: Principles,

Practice and Techniques, CRC Press, Boca Raton,

FL, 1999.

135. Yatsumatsu, K., Sawada, K., and Moritaka, S.,

Whipping and emulsifying properties of soybean prod-

ucts, Agric. Biol. Chem.(Japan), 36(5), 719, 1972.

136. Miller, R. and Groninger, H. S., Functional proper-

ties of enzyme-modified acylated fish protein deriva-

tives, J. Food Sci., 41, 268, 1976.

137. Turgeon, S. L., Gauthier, S. F., and Paquin, P.,

Interfacial and emulsifying properties of whey pep-

tide fractions obtained with a two step ultrafiltration

process, J. Agric. Food Chem., 39, 637, 1991.

138. Chobert, J-M., Bertrand-Harb, C., and Nicolas,

M-G., Solubility and emulsifying properties of caseins

and whey proteins modified enzymatically by trypsin,

J. Agric. Food Chem., 36(5), 883, 1988.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

139. Kato A. and Nakai, S., Hydrophobicity determined

by a fluorescence probe method and its correlation

with surface properties of proteins, Biochim. Biophys.

Acta, 13, 624, 1980.

140. Li-Chan, E., Nakai, S., and Wood, D. F., Hydropho-

bicity and solubility of meat proteins and their rela-

tionship to emulsifying properties, J. Food Sci., 49,

354, 1984.

141. Lee, S. W., Shimizu, M., Kaminogawa, S., and

Yamaguchi, K., Emulsifying properties of a mixture

of peptides derived from the enzymatic hydrolysates

of β-casein, Agric. Biol. Chem., 51, 161, 1987.

142. Groninger, H. S. and Miller, R., Some chemical and

nutritional properties of acylated fish proteins, J. Agric.

Food Chem., 27(5), 949, 1979.

143. Townsend, A. A. and Nakai, S., Relationships be-

tween hydrophobicity and foaming characteristics of

food proteins, J. Food Sci., 48, 588, 1983.

144. Phillips, L. G., Haque, Z., and Kinsella, J. E., A

method for the measurement of foam formation and

stability, J. Food Sci., 52, 1074, 1987.

145. Althouse, P. J., Dinakar, P., and Kilara, A., Screen-

ing of proteolytic enzymes to enhance foaming of

whey protein isolates, J. Food Sci., 60(5), 1110,

1995.

146. Kinsella, J. E., Functional properties of proteins:

possible relationships between structure and function

in foams, Food Chem., 7, 273, 1981.

147. Groninger, H. S. and Miller, R., Preparation and

aeration properties of an enzyme-modified succinylated

fish protein, J. Food Sci., 40, 327, 1975.

148. Ostrander, J. G., Nystrom, P. J., and Martinsen,

C. S., Utilization of a fish protein isolate in whipped

gelatine desserts, J. Food Sci., 42(2), 559, 1977.

149. Petersen, B. R., The impact of the enzymatic hy-

drolysis process on recovery and use of proteins, in

Enzymes and Food Processing, Elsevier Applied Sci-

ence Publishers, London, UK, 1981, 149–175.

150. Yeom, H. W., Kim, K. S., and Rhee, J. S., Soy

protein hydrolysate debittering by lysine-acetylation,

J. Food Sci., 59(5), 1123, 1994.

151. Tamura, M., Mori, N., Miyoshi, T., Koyama, S.,

Kohri, H., and Okai, H., Practical debittering using

model peptides and related compounds, Agric.

Biol.Chem., 54(1), 41, 1990.

152. Clegg, K. M. and McMillan, A. D., Dietary enzymic

hydrolysates of protein with reduced bitterness, J.

Food Technol., 9, 21, 1974.

153. Cogan, V., Moshe, M., and Mokady, S., Debittering

and nutritional upgrading of enzymic casein hydroly-

sates, J. Sci. Food. Agric., 32, 459, 1981.

154. Vegarud, G. E. and Langsrud, The level of bitter-

ness and solubility of hydrolysates produced by con-

trolled hydrolysis of caseins, J. Dairy Res., 56, 375,

1989.

155. Moll, D., Manufacturing protein hydrolysates without

giving use to a bitter taste, in Food Ingredients Eu-

rope Conference, Expoconsult Publishers, Maarssen,

The Netherlands, 1990, 257–260.

156. Przybyla, A. E., Enzymes used for protein hydroly-

sate debittering; Enzymatic hydrolysis of proteins to

produce flavorings, dietary supplements and other

products that can result in a bitter taste, Food Eng., 9,

51, 1989.

157. Lalasidis, G., Bostrom, S., and Sjoberg, L.-B., Low-

molecular-weight enzymatic fish protein hydrolysates:

Chemical composition and nutritive value, J. Agric.

Food Chem., 26(3), 751, 1978.

158. Yu, S. Y. and Tan, L. K., Acceptability of crackers

(‘Keropok’) with fish protein hydrolsyates, Int. J. Food

Sci. Technol., 25(2), 204, 1990.

159. Haard, N. F., A review of proteolytic enzymes from

marine organisms and their application in the food

industry, J. Aquat. Food Prod. Technol., 1(1),

1992.

160. Fujimaki, M., Arai, S., Yamashita, M., Kato, H.,

and Nogushi, M., Taste peptide fractionation from a

fish protein hydrolysate, Agric. Biol. Chem. (Japan),

37, 2891, 1973.

161. Noguchi, M., Arai, S., Yamashita, M., Kato, H.,

and Fujimaki, M., Isolation and identification of

acidic oligopeptides in a flavor potentiating fraction

from a fish protein hydrolysate, J. Agric. Food Chem.,

23(1), 49, 1975.

162. Hevia, P. and Olcott, H. S., Flavor of enzyme-solu-

bilized fish protein concentrate fractions, J. Agric.

Food Chem., 25(4), 772, 1977.

163. Lalasidis, G., Four new methods of debittering

protein hydrolysates and a fraction of hydrolysates

with high content of essential amino acids, Annales

de la Nutrition et de l’Alimentation, 32(2/3), 709,

1978.

164. Heck, N. E., Characterization of Fish Protein Hy-

drolysate Plastein and the Identification of Glutamyl-

Lysine in the Plastein Material, Ph.D. disseration,

University of Washington, Seattle, WA, 1983.

165. Chakrabarti, R., A method of debittering fish pro-

tein hydrolysate, J. Food Sci. Technol., 20(4), 154,

1983.

166. Arai, S., Yamashita, M., and Fujimaki, M., Plastein

reaction and its application, Cereal Foods World, 20(2),

1975.

167. Onoue, M. and Riddle, L. M., Use of plastein reac-

tion in recovering protein from fish waste, J. Fish.

Res. Board (Canada), 30, 1745, 1973.

168. Kim, S. K. and Lee, E. H., Enzymatic modification

of sardine protein concentrate, J. Korean Agric. Chem.

Soc., 30, 234, 1987

169. Montecalvo, J., Jr., Constantinides, S. M., and

Yang, C. S. T., Enzymatic modification of fish frame

protein isolate, J. Food Sci., 49, 1305, 1984.

170. Tanimoto, S., Yamashita, M., Arai, and Fujimaki,

M., Probes for catalytic action of α-chymotrypsin in

plastein synthesis, Agric. Biol. Chem. (Japan), 36,

1595, 1972.

171. Raghunath, M. R. and McCurdy, A. R., Synthesis

of plastein from fish silage, J. Sci. Food Agric., 54,

655, 1991.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016

2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without

the consent of the publisher is prohibited.

172. Pigott, G. M., University of Washington at Seattle,

personal communication, 1998.

173. Eguschi, Y., Bela, J. S., and Shetty, K., Simulation

of somatic embryogenesis in Anise (Pimpinella

anisium) using fish protein hydrolysates and proline,

J. Herbs and Spices, 5(3), 61, 1997.

174. Milazzo, M. C., Zheng, Z., Kellett, G., Haynesworth,

K., and Shetty, K., Stimulation of benzyladenine-

induced in vitro shoot organogenesis and endogenous

proline in melon (Cucumis melo L.) by fish protein

hydrolysates in combination with proline analogues,

J. Agric. Food Chem., 47(4), 1771, 1999.

Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016