Biochemical, and Functional Properties Fish Protein ......Enzymatic modification of proteins using selected proteolytic enzyme preparations to cleave specific peptide bonds is widely

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Critical Reviews in Food Science and Nutrition

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

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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 ofrecovery. At the same time many processors areno longer allowed to discard their offal directlyinto the ocean, resulting in a very high cost ofrefining the material before it is discarded. Tomeet the need of the seafood processing industry,an alternative to discarding these byproductsshould be developed. Recovery and alteration ofthe fish muscle proteins present in the byproductmaterial and using these as functional ingredientsin food systems is a very exciting and promisingalternative. However, for the industry to develop

processes for byproduct recovery and utilizationit has to be more economically feasible than dis-carding the byproducts.

Every year over 91 million tons of fish areharvested, of which 29.5% is transformed intofishmeal.1,2 Possibly more than 50% of the re-maining fish tissue is considered to be processingwaste and not used as food.3 With a dramaticallyincreasing world population and a world catch offish presently on the verge of exceeding the esti-mated sustainable limits of the suggested 100million tons/year, there is obviously an increasedneed to utilize our sea resources with more intel-ligence and foresight. By applying enzyme tech-nology for protein recovery in fish processing, itmay be possible to produce a broad spectrum of

Fish Protein Hydrolysates:Production, Biochemical, and FunctionalProperties

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 FoodsLaboratory, 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 developingenzyme technologies for protein recovery and modification, production of a broad spectrum of food ingredientsand 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 reviewdescribes 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 fishproteins are discussed. In addition, functional properties of fish protein hydrolysates are described, includingsolubility, water-holding capacity, emulsification, and foam-forming ability. Possible applications of fish proteinhydrolysates 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.

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food ingredients or industrial products for a widerange of applications. This would utilize bothfishery byproducts or secondary raw materialsand, in addition, underutilized species that wouldotherwise be discarded.

Enzymatic modification of proteins usingselected proteolytic enzyme preparations to cleavespecific peptide bonds is widely used in the foodindustry.4 Hydrolysis of food proteins has a longhistory, mainly for vegetable and milk proteins;these proteins are widely used in the food indus-try. Most work on the hydrolysis of fish proteinswas conducted in the 1960s. Some fish proteinhydrolysate (FPH) preparations at that time werequite successful.5 During the 1960s, research wasdirected to the production of cheap nutritious pro-tein sources for rapidly growing developing coun-tries, or toward animal feed production, primarilythrough production of fish protein concentrates(FPC). Little work has been done recently onFPH, but some research has been directed into thepotential of using powdered hydrolysates in foodformulations. Many studies have resulted in fishprotein hydrolysates with excellent functionalproperties. However, taste defects, specificallybitterness, and process economics are still majorlimiting 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 ofhydrolysis and product functionality.

II. THE BIOCHEMICALCHARACTERISTICS OF FISH MUSCLEPROTEIN

In foods, a protein is traditionally categorizedas a fibrous or globular protein based on its ter-tiary conformation. Each type of food protein hasa unique molecular structure that determines itsfunctional properties, in addition to a range ofenvironmental conditions over which it exhibitssuch properties.6 These factors and their effect onfunctionality are discussed in more detail later inthis review.

The functional and structural properties offood proteins thus vary tremendously, and to fully

understand the process of protein hydrolysis it iscrucial to have a good understanding of the natureof the protein substrate and the hydrolyzing agent.During protein hydrolysate manufacture, the pro-tein substrate is hydrolyzed by either a proteolyticenzyme or an acid or base.

Our diet contains a wide variety of proteinsfrom different sources. It is generally acceptedthat the relative concentration of dietary essentialamino acids is the major factor determining thenutritional value of food protein.7 Proteins de-rived from animal sources are considered to benutritionally superior to those from plants be-cause they contain a better balance of the dietaryessential amino acids. Of these egg and milk pro-teins (casein) are frequently used as referenceproteins for evaluating protein quality. Proteinsderived from meat and poultry muscle are also ofvery high quality and fish muscle proteins areequally nutritious.8 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 extremelyperishable and because chemical composition canvary, the utilization of fish as a basic raw foodmaterial presents unique food processing prob-lems.11

The muscle of different animals is very simi-lar, containing similar protein and similar aminoacid profiles. There are slight differences betweenfish muscle and the muscle of land animals. Theseare mainly associated with the differences inmuscle structure required for swimming and buoy-ancy. Fish are supported by a mass of water, thusthe muscle fibers require less structural supportthan those in land animals. Because of this, fishmuscle tends to have less connective tissue thanmuscles from terrestrial animals, resulting in moretender texture. Also, because of the unique move-ment of fish, the structural arrangement of musclefibers is quite different from terrestrial animals. Alarge fraction of commercially utilized fish stocksare cold adapted or poikilothermic, and becauseof this their muscle proteins have different bio-chemical properties compared with those of en-dothermic animals.12 Poikilothermic characteris-tics of fish proteins make them more heat sensitivethan mammalian muscle proteins, with a greatertendency to denature at elevated temperatures.

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Fish muscle proteins from cold water species aremore 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 reportedto be 29 to 35°C at pH 7.0 and 11 to 27°C at pH5.5.15

Protein composition in muscles varies bymuscle type. Of the three types (striated, smooth,and cardiac muscle) of muscles, the striatedmuscles are the predominant form in fish. Striatedmuscle tissue is arranged into muscle fibers thatare bound together by a connective tissue to makea fiber bundle. Fish muscle has “white” and “dark”meat.15 The white meat is generally more abun-dant, contains less lipids than the dark meat, andis 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 differentgroups 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 remaining20 to 30% contain sarcoplasmic proteins that aresoluble in water and dilute buffers, and a finalpart of the structural protein, 2 to 3%, being in-soluble connective tissue proteins.11 Recent stud-ies, however, challenge these generally acceptedsolubility data, showing that the muscle proteincomponents can be highly soluble at low ionicstrenghts.16,17

Myofibrillar proteins are the primary foodproteins of fish, comprising 66 to 77% of the totalprotein in fish meat. The myofibril protein com-plexes contain myosin and actin. These are the

main components of the thick filament, and thinfilament, respectively. Myosin comprises 50 to60% of the myofibrillar contractile proteins, andactin only 15 to 30%.12,15 Myosin is the mostabundant of the single muscle proteins, makingup 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 headregions that incorporate the light chains and asignificant fraction of the heavy chains. The tailsof the heavy chains form very long α-helices thatwrap around each other18 (Figure 1). Myosin canbe cleaved by proteases at two sites on the mol-ecule, one recognized by both trypsin and chy-motrypsin and the other by papain. Papain cleavesnear the head region, releasing the head from thetail. Trypsin and chymotrypsin cleave further fromthe head, dividing the molecule into two compo-nents called the heavy meromyosin (with the headregion) and the light meromyosin, both with dif-ferent functional properties.

Myosin molecules are connected via their headregion to the polymerized actin molecules in thethin filaments due to the ATPase activity of thehead molecules. This complex is called actomyo-sin and is responsible for muscle contraction andrelaxation. Actomyosin plays a major role in de-termining the quality of fish meat because it isquite labile and easily affected during processingand storage. For example, during frozen storagethe actomyosin becomes progressively less solubleand the flesh becomes increasingly tougher.19

The thin filament is a complex of actin mol-ecules making a double helix. Tropomyosin sitswithin the grooves of the thin filaments and two

FIGURE 1. Fish myosine molecule.

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troponin molecules bind the actin filament at eachhelical 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 thatconnects to myosin. The thin filaments play avery 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 withrespect to food processing has not been studiedcompletely. Other contractile proteins of interestare C-protein, α-, and β-actinin, connectin andparamyosin; however, they are of limited interestas food proteins. With respect to protein hydroly-sis, the myofibrillar protein myosin, actin, or ac-tomyosin are subject to enzymic cleavage and arethe greatest focus here.

III. PROTEIN HYDROLYSIS

Proteolytic modification of food proteins toimprove palatability and storage stability of theavailable protein resources is an ancient technol-ogy.20 Hydrolysates can be defined as proteinsthat are chemically or enzymatically broken downinto peptides of varying sizes.21 Protein hydroly-sates are produced for a wide variety of uses inthe food industry, including milk replacers, pro-tein supplements, stabilizers in beverages and fla-vor enhancers in confectionery products. Thebenefits of hydrolyzing food proteins to makefunctional protein ingredients and nutritionalsupplements is a more recent technology, with thefirst commercially available protein hydrolysatesappearing only around the late 1940s. Althoughproduction is massive worldwide, the proper con-trol of the process and the exact mechanism be-hind protein hydrolysis is in most cases not fullyunderstood. 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 functionalproperties of the hydrolyzed protein. Recent re-search on enzyme catalysis has also aided withthe 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 mostwidely used for protein hydrolysis with chemicalhydrolysis used more commonly in industrial prac-tices. Biological processes using added enzymesare employed more frequently, and enzyme hy-drolysis holds the most promise for the futurebecause it results in products of high functionalityand nutritive value. The chemical and biologicalhydrolysis 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 organicsolvents; the conventional processes of cooking,pressing, drying, and hot oil extraction.22 Theextraction of protein by means of solvent is alsoworth mentioning due to its industrial and histori-cal importance for fish protein recovery.

A. Chemical Methods for ProteinHydrolysis

1. Chemical Extraction: The Making ofFish Protein Concentrate

The extraction methods mentioned above,other than the chemical and biological hydrolysismethods, do not hydrolyze protein. They are usedprimarily to concentrate intact protein by the re-moval of water and oil from the substrate. Themethod of solvent extraction has been frequentlyemployed when producing fish protein concen-trate (FPC). The development of FPC was one ofthe earliest attempts to recover fish protein fromprocessing waste and to produce a protein ingre-dient from underutilized species. FPC was theprecursor to the field of enzyme hydrolysis of fishproteins. A small but extensive research programon the large-scale production of FPC by the Bu-reau of Commercial Fisheries, now the NationalMarine 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 proteinmalnutrition and as a potential economic stimulusto the American fishing industry.23 Solvent-ex-

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tracted FPC (type A FPC) is produced by usingprimarily isopropanol or azeotropic extraction withethylene dichloride, although other solvents suchas ethanol have been used successfully as well. Astandard process presented by Sikorski andNaczk24 shown in Figure 2 is to grind a whole oreviscerated fish, extract it with isopropanol at alow temperature (20 to 30°C) for 50 min, thencollect the supernatant and extract it twice again,

first at 75°C for 90 min in isopropanol and then at75°C for 70 min with azeotropic isopropanol. Thefinal supernatant fraction is collected, dried, milled,and screened to separate out bone particles. Thefinal product has a high biological value and iscolorless and odorless, with less than 1% lipids.The problem with type A FPC is that it is notreadily soluble or dispersible in foods and haspoor emulsification properties.9,25,26 Dubrow et al.27

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

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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. Generalpoor functionality, off-flavors the high cost ofproduction, and traces of solvent in the final prod-uct made solvent extracted FPC commerciallyunsuccessful despite concerted efforts.3,28 Al-though FPC lacks solubility, it reportedly hasgood 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 theabundant fatty pelagic fish species such as sar-dine, herring, and capelin because the protein iseffectively separated from the lipids, thereby re-ducing stability problems normally associated withresidual oxidizable lipid. For fatty fish, isopro-panol was a slightly more efficient solvent thanethanol considering the residual amounts of lip-ids, but absolute ethanol produced FPC of lightercolor and a neutral flavor.32

Many studies with FPC have also been con-ducted with solvent-extracted FPC as a substratefor enzyme hydrolysis, both to defat the substrateand to make it more accessible to enzymatic hy-drolysis, with excellent functional and nutritionalproperties.5,25,33,34,35,36 However, enzymatic hy-drolysis using FPC as a starting substrate resultedin loss of some functional properties because ofexcessive protein breakdown but increased nitro-gen solubility.30 Taste and odor problems aregenerally minimized with a FPC starting mate-rial.33

Recent studies with solvent-extracted FPChave produced FPC with better protein function-ality. For example, recently Vareltzis et al.37 stud-ied the addition of ethanol-extracted FPC madefrom sardine to hamburger patties and found thatthe overall functional properties (water bindingand cooking yield) and the penetration depth andshear force value of the hamburger increased withthe addition of FPC. However the hamburgershad a slightly unfavorable fishy flavor. Hoyle andMerritt5 found that herring protein extracted withethanol in a similar manner and then hydrolyzedwith either Alcalase or papain produced a hy-drolysate with a markedly reduced bitterness andless fishy odor.

2. The Chemical Hydrolysis Process

Chemical hydrolysis of proteins is achievedby cleaving peptide bonds with either acid orbase. Several processes have been proposed forthe acid or alkaline hydrolysis of fish.33 This hasbeen the method of choice in the past for theindustry primarily because it is relatively inex-pensive and quite simple to conduct. There are,however, many limitations to food ingredientsusing this method. Chemical hydrolysis tends tobe a difficult process to control and almost invari-ably leads to products with variable chemicalcomposition and functional properties.21,38 Pro-tein hydrolysis with strong chemicals and sol-vents is performed at extreme temperatures andpH and generally yield products with reducednutritional 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 consumedin the U.S. are prepared by acid hydrolysis, mostlyfrom inexpensive vegetable protein sources thatotherwise would have poor nutritive and littlefunctional value in foods. Although the process isharsh and hard to control, it is still the preferredmethod for hydrolyzed vegetable proteins. Hy-drolyzed vegetable protein, which are widely usedfor flavor and taste enhancement properties, re-quire extensive acid hydrolysis.38 Applications ofhydrolyzed vegetable proteins are primarily asflavoring agents in processed meat, crackers, andsoup mixes. Acid hydrolysis of fish protein hasusually involved reacting fish proteins with hy-drochloric acid, or in some cases sulfuric acid,and the proteins are completely hydrolyzed athigh temperature, and often high pressure. Thehydrolysate is then neutralized to pH 6.0 to 7.0and concentrated to either a paste or further dried.41

Because the product is hydrolyzed extensively,its primary functional property is high solubility.Total hydrolysis of fish protein substrate can beachieved in 18 h at 118°C in 6N hydrochloricacid.42 In addition, following the neutralization of

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the digest, the hydrolysate contains large amountof salt (NaCl), which can make the product unpal-atable and interferes with functionality in foodsystems. Another drawback of acid hydrolysis isthe destruction of tryptophan, which is an essen-tial amino acid. Orlova et al.43 proposed an acidhydrolysis process of whole fish, where steamdistillation is used to remove aromatic substancesfollowed by filtration then concentration. Theconcentrate was used in dehydrated soup cubesand as a microbial media.43 The acid hydrolysis isalso widely utilized to convert underutilized andsecondary raw material from fish into fertilizerdue to the low production cost and resulting ex-tensive hydrolysis.

b. Alkali Hydrolysis

The use of alkali reactants, primarily sodiumhydroxide, to hydrolyze protein often results inpoor functionality and more importantly can ad-versely affect the nutritive value of the hydroly-sate. Despite this, limited alkali treatment is usedin the food industry to recover and solubilize abroad range of proteins. For example, mechani-cally deboned turkey residue (MDTR) includes asignificant proportion of alkali-soluble proteinsthat can be recovered by alkali treatment and usedin food applications. Fonkwe and Singh44 dis-cussed the use of alkali extraction to recoverMDTR with an alkaline sodium chloride solutionbut found it to be unsuitable due to low recovery.Alkaline hydrolysis of fish proteins has primarilyused FPC as the starting substrate. During alka-line hydrolysis of fish protein, rapid cleavage tolarge 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.24 Tannenbaum et al.45,46

have studied the alkaline process for hydrolyzinginsoluble FPC and its applications. They devel-oped a small-scale batch process that utilizes highpH (12.5) and 95°C for 20 min. The productconsisted of large peptides, some relatively in-soluble at the isoelectric point, but with an overallimprovement in functionality with respect to theoriginal FPC. Use of the solubilized FPC as amilk substitute gave a product far superior to that

obtained with FPC starting material, which hadpoor 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 ofL-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 andformations of lysinoalanine, ornithinoalanine,lanthionine, and β-amino alanine can also oc-cur.31 Some of these elimination and addition re-actions may lead to the formation of toxic sub-stances (e.g., lysinoalanine) that are highlyundesirable in foods.47,48 Alkaline hydrolysis re-action products have an inhibiting effect on pro-teolytic enzymes, reducing the rate of hydroly-sis.49 Some of the possible reaction products thatmay form during alkali hydrolysis are shown inFigure 3.24 High collagen solubility is also ob-served with alkali treatment.50

B. Biochemical Methods for Fish ProteinHydrolysis

Biochemical hydrolysis to produce fish orother food protein hydrolysates is performed byutilizing enzymes to hydrolyze peptide bonds.This can be done via proteolytic enzymes alreadypresent in the fish viscera and muscle (endog-enous proteases), or by adding enzymes from othersources. To understand the process of enzymatichydrolysis, it is very important to understand thenature and activity of proteolytic enzymes.

1. Proteolytic Enzymes

Enzymes are biochemical catalysts vital forliving beings, because they accelerate chemicalreactions between organic constituents within thecells that otherwise would take an extremely longtime to complete. In food science and technologyenzymes are exploited to perform desired func-tions in processing and analysis and to facilitatethe conversions of raw materials into higher-qual-ity, more desirable foodstuffs.51 Enzymes makethis possible because the active site of an enzyme

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

is highly specific for certain substrates. Enzymescatalyze only one specific reaction and functionby forming a complex with the substrate whosetransformation they catalyze.

Enzymes used by the food industry and infood research are predominantly hydrolases, mostof which are carbohydrases, followed by pro-teases and lipases. Proteases are among the bestcharacterized enzymes. Proteolytic enzyme prepa-rations are economically the most important groupof enzymes, and their use is well established inthe food industry.52 Proteases are categorized ac-cording to the specificity of the peptide bondsthey attack (hydrolyze) and the mechanism bywhich they act.48 Four major classes of proteasesare known. They are designated by the principalfunctional group in their active site: serine, thiol,carboxyl and metallo.53 Proteases are character-ized further by their hydrolyzing mechanism intoendoproteinases or exopeptidases. The endo-proteinases cleave/hydrolyze the peptide bondswithin protein molecules, usually at specific resi-dues to produce relatively large peptides. Theexopeptidases systematically remove amino acidsfrom 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 arealways used, but occasionally endoproteinases arecombined with exopeptidases to achieve a morecomplete degradation.20

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 proteinhydrolysis. 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 primarilyas three consecutive reactions: (1) the formationof the Michaelis complex between the originalpeptide chain and the enzyme, (2) cleavage of thepeptide bond to liberate one of the two peptides,and (3) a nucleophilic attack on the remains of thecomplex to split off the other peptide and to re-constitute the free enzyme.20,54 The hydrolysis ofpeptide bonds leads to an increase in the numbersof ionizable groups (NH3

+ and COO-), with a con-comitant increase in hydrophobicity and netcharge, 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 Eand the peptides in the reaction P, the overallmechanism can be presented as:

E S ES EP H P

E OH P H P

k

k

k

k

H O

+ ← → → + − ′

→ + − + − ′

1

1

2

3

2

–

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This enzyme-substrate complex may dissoci-ate back to reactant substrate and free enzyme, orto free enzyme and product molecules. In otherwords, classic Michaelis-Menten kinetics apply.20

The generally accepted mechanism for proteasesindicates that the second step is the rate-determin-ing step, thus k2 primarily determines the overallreaction speed, and Km is more or less equal to thetrue dissociation constant. This simple mecha-nism does not, however, deal with the detailedquestion of how the enzyme and substrate arebound or what molecular configurations lead toproduct 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 complexprocess because of several peptide bonds and theirspecific accessibility to enzymatic reactions.47 Thespecificity of enzymes is not the only factor thataffects the peptide profile of the final product.Environmental factors such as temperature and pHplay an important role. Both temperature and pHcan greatly affect the enzyme reaction kinetics, andthe effect of these factors is different for eachenzyme. Generally, there is an optimum combina-tion of both pH and T, where an enzyme is the mostactive. Temperature and pH extremes deactivatethe enzymes by denaturing them.

2. Autolytic Hydrolysis

Biochemical production of fish protein hy-drolysates may be carried out by employing anautolytic process. An autolytic process dependson the action of the digestive enzymes of the fishitself. There are no enzyme costs involved, and itis a simple operation.59 The end product of au-tolytic hydrolysis is generally a fairly viscousliquid rich in free amino acids and small peptides.The digestive enzymes in question are primarilythe serine proteases trypsin and chymotrypsin,and the thiol protease pepsin, all major enzymesof fish viscera and digestive tract. Lysosomalproteases, or catheptic enzymes, present in fishmuscle 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 resultin end products of different molecular profiles.Another complication is that the presence of cer-tain digestive enzymes and their concentrationmay be highly seasonal, gender and age specific,and can vary tremendously within a species aswell as between species. These variations make itvery hard to control the hydrolytic process, anddirect the production of hydrolysates with spe-cific molecular properties. Autolytic methods suchas chemical methods often result in a final prod-uct with bad functionality. Despite these prob-lems, endogenous proteolytic enzymes are usedto produce hydrolyzed products, specifically fishsauces and fish silage.

The production of fish sauce preceded fishsilage production and is the major fermented fishproduct presently consumed in the world. Its pro-duction has thousands of years of tradition inAsia, and it is also known to have been producedin Mediterranean countries in ancient times. Pres-ently, fish sauce is used mainly as a condiment onrice dishes like the popular Nuoc-Nam producedin Vietnam, and the annual production in South-east Asia is about 250,000 metric tons.60 Theproduction of fish sauce does not require elabo-rate processing equipment. The substrate is usu-ally fish from one or more pelagic species, suchas anchovies or sardines, or minced whole fish oflow commercial value. The substrate is immersedin a solution containing high concentrations ofsalt (20 to 40%) and at relatively high tempera-tures, preferably ambient tropical temperatures.In the case of whole fish, the visceral proteolyticenzymes start by hydrolyzing the stomach con-tents, then work their way through the stomachwall, and finally reach the muscle tissue, wherethey join with catheptic enzymes to hydrolyzefish muscle proteins. The pH of the fish sauceprocess is usually neutral, because no base or acidis added to the reaction mixture. The acid-depen-dent proteolytic enzymes, such as pepsin and thecatheptic enzyme, contribute little to fish sauceproduction. The endogenous serine proteases veryslowly breakdown the fish muscle for 6 to 12months (Nuoc-Mam) under anaerobic conditions.This slow but extensive breakdown results in aliquefied fish sauce composed predominantly offree amino acids, with up to 50% nitrogen recov-

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ery. The high concentration of salt is responsiblefor the slow hydrolysis because this slows theactivity 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 thetissue.61 Lower concentrations of salt, however,result in sauces with higher yield, lower levels ofvolatile acids, and better balanced composition ofamino acids.62 During autolysis to produce fishsauce, a lipid phase, an aqueous soluble phasethat contains much protein but little lipid, andinsoluble sediment of protein and lipid is formed.63

After the hydrolysis is completed, the liquid pro-tein hydrolysate is tapped, filtered, and bottled,with the final product containing up to 10% freeamino acids and low-molecular-weight peptides,and 25% salt.26,60 Although the production of fishsauce does not improve the nutritive value of theprotein, the keeping quality is greatly increasedand organoleptic characteristics are generallyimproved.64

The process of manufacturing fish silage isdifferent, 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 employedas fish sauce production. The application of fishsilage is primarily for animal feed productioninstead of food applications. The process is rapidand enzymes involved are very effective in pro-ducing oil and proteins fractions that are readilyseparated. The substrate is usually secondary rawmaterial from fish processing or underutilized fishspecies. The substrate is mixed with strong min-eral acids or organic acids such as formic acid toacidify the mixture below pH 4. At this pH theserine proteases are generally inactive, but pepsinand the catheptic enzymes are highly active. Thepepsin content can be very high in the visceralportion of fish, and the enzyme is primarily re-sponsible for fish silage production. Under acidicconditions the active endogenous enzymes par-tially hydrolyze the fish over several weeks toproduce a slurry containing up to 12% aminoacids and peptides. Usually about 80% of theprotein in acid fish silage become solubilized af-ter 1 week at temperatures around 23 to 30°C.65

The rate is primarily dependent on conditions

such as ambient temperature and relative amountsof the visceral organs present. The processingtime is much shorter than for fish sauce becauseno salt is added.26,60 The production of fish silageusing lactic acid bacteria as the hydrolyzing agenthas been reported.66,67 The bacterial fermentationis initiated by mixing minced or chopped fishwith a fermentable sugar that favors the growth oflactic acid bacteria, which is advantageous be-cause the bacteria produces acid and antimicro-bial factors that inhibit competing bacteria.65

The feed applications of fish silage are prima-rily limited to young animals due to the extensivehydrolysis of the proteins. For fish silage to beincorporated successfully in animal feeds, it hasto contain the majority of the nitrogen fraction asintact proteins or peptides rather than as free aminoacids, which are less well absorbed. A shorterprocessing time and added commercial proteasesmay be useful in such instances. Also, problemsconnected with the development of bitterness inthe hydrolyzed silage can make the product highlyunpalatable not only for humans but also animalsfed feeds rich in fish silage. The utilization of fishsilage as a primary protein source in fish feeds hasbeen neither fully investigated nor commerciallysuccessful; however, it is incorporated into dietsfor pigs, poultry, and mink.67

Research on fish hydrolysates made with en-dogenous enzymes for human food applicationshas been very limited. Fish sauce is almost theonly 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 knowledgeof the enzymes involved is crucial. Endogenousenzymes in fish are a complex and highly variablemixture, and thus the properties of functional pro-tein hydrolysates so prepared may vary greatlyunder the same reaction conditions. Also, in theU.S. the sale of processed fish foods containingvisceral material of any kind is prohibited by theFDA. Despite this, some work on developing fishsilage for human consumption has been conducted.In 1972, Malcolm B. Hale and a group of scien-tists at NMFS and the University of Marylandconducted a comprehensive study on making func-tional FPH by enzymatic hydrolysis for humanconsumption.33 This study involved autolysis of

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red hake (Urophycis chuss), a relatively lean fish,and alewife (Alosa pseudoharengus), a very fattyfish. The autolysis of raw hake was conducted atoptimal conditions for the native enzymes, 50°Cand pH 7.0, for 24 h where fish was 50% of thetotal slurry. The resulting hydrolysate was eitherspray dried or tested as a concentrate. The aver-age yield of dry solids for hake was lower than forany other proteolytic enzyme employed, 10.0%(dry solubles/wet fish), compared with the high of14.3% from using Alcalase. Similarly, the chemi-cal score attained for soluble hydrolysate werequite low due to low tryptophan content that iscorrelated with a low recovery. The protein effi-ciency ratios for both red hake and alewife hy-drolysates were essentially equivalent to that ofcasein. Also, the inclusion of insoluble solids inthe final product resulted in very high fat content.The results for lipid pressed alewife were similar.However, by lowering the reaction period to 4 hand raising the temperature to 55°C, a satisfactoryproduct with good nutritional value was prepared.Both the red hake and alewife were only 50 to70% soluble, requiring substantial additions ofcommercial enzymes, at uneconomic levels, tobecome fully soluble. The alewife hydrolysatealso suffered from very fishy taste; however, thered hake hydrolysate had a less fishy taste andodor. Food applications of the products obtainedby Hale and co-workers were limited and couldbe used primarily as a protein supplement in cul-tures where its taste would be acceptable and thecaloric value of the lipid desirable.33

Shahidi et al.59 hydrolyzed ground capelin(Mallotus villosus) by endogenous enzymes andfound that it enhanced the overall extraction ofthe fish protein at both acid and alkaline pH, asboth acid and alkaline proteases are present infish muscle and viscera. The protein recovery ofhydrolysates produced autolytically was, however,considerably lower compared with commercialenzymes, 22.9% compared with 70.6% withAlcalase. A recent study by Cui68 with chumsalmon (Oncorhynchus keta) mince and visceralcontent showed a surprisingly extensive and rapidhydrolysis 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 themajority of peptides of lower molecular weightthan the pepsin hydrolysate under the same ex-perimental conditions. This indicates that proteinhydrolysates can be obtained through autolysisvery efficiently at relatively mild temperatures.The functional properties of the product were,however, not investigated.

The main limitation of work performed onautolytic hydrolysis of food proteins is the lack ofresearch on functional properties. Studies haveshown that protein recovery can be adequate andthat nutritional requirements are good, but infor-mation on functional properties of the resultinghydrolysate is very important to successfully evalu-ate its use in formulated foods.

3. Enzymatic Hydrolysis of Fish MuscleProteins with Added Enzymes

Using added enzymes to hydrolyze food pro-teins is a process of considerable importance usedto improve or modify the physicochemical, func-tional, and sensory properties of the native pro-tein without jeopardizing its nutritive value, andoften protein absorption is improved. These en-zyme-based processes occur under mild condi-tions over a series of stages and do not producehydrolytic degradation products via racemizationreactions observed with both acid and alkalinehydrolysis.69 The process of using added enzymesinstead of chemicals or endogenous enzymes of-fers many advantages because it allows good con-trol of the hydrolysis and thereby the properties ofthe resulting products.59 Processes can be designedto take advantage of substrate specificity and therelative reaction rates of different enzymes underthe reaction conditions employed. The physico-chemical and functional properties of hydrolyzedfish proteins are discussed separately in a latersection.

Enzymatic hydrolysis has been employed ona variety of different proteins derived from live-stock and poultry meat,39,44,47,70,71 milk,57,72–76 andplants.77,78 Hydrolysis of fish and other aquaticfoods is also being seen more frequently in theliterature. Several different aquatic protein sourceshave been investigated for the production of func-

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tional fish protein hydrolysates. These includeRastelliger canagurta and Barbus carnaticusboth Indian tropical fishes,79,80 hake (Urophycischuss),10,25,33 shark (Isurus oxyrinchus),81,82

sardine (Sardina pilchardus),35,36,83,84 herring(Clupus harengus),5 crayfish,85 lobster (Panulirusspp.),86 pollack (Theragra chalcogramma),87 cape-lin (Mallotus villosus),59 dogfish (Squalusacanthias),88 chum salmon (Oncorhynchus keta),68

Pacific whiting (Merluccius productus),89 andAtlantic salmon (Salmo salar).90 It can readily beseen from the list above that the majority of thesources represent underutilized species or areconnected to utilization of processing wastes.

Enzymatic hydrolysis of fish protein has beenemployed primarily as an alternative approach forconverting underutilized fish biomass, which iscommonly used in making feed or even fertilizerinto edible protein products.15,88 More recently,fish processing waste, or, more appropriately,secondary raw material, has been connected toFPH studies. In many cases this is due to strictgovernment waste regulations. Many processorsare no longer allowed to discard their offal di-rectly to sea, resulting in a very high cost ofrefining the material before discarding. Second-ary raw material is the material remaining afterfillets are removed, and if viscera is included, thiscan represent something on the order of 64% ofthe weight of whitefish, the protein content of thiswaste being about 10%.3 Hydrolysis of fish pro-tein with selected proteolytic enzymes providesthe possibility of controlling cleavage degree ofprotein in the substrate. Using suitable enzyme/substrate ratios and reaction times, this permitsthe production of hydrolysates with differentmolecular structures and different functional prop-erties that could find applications in various foodformulations.82

The hydrolytic process and reaction condi-tions differ between different substrates and en-zymes used and also depend on the propertiesdesired for the hydrolysate. Most of the describedprocesses are conducted under research condi-tions and may have limited applications in theindustry. Commercial production of fish proteinhydrolysates is still limited on a worldwide basis,but has reached a significant level in a few coun-tries, including France, Japan, and SoutheastAsia.60 Commercial batch protein hydrolysis has

several disadvantages such as (1) high cost ofusing large quantities of enzymes, (2) difficulty incontrolling the extent of reaction that can result innonhomogenous products consisting of fractionsof varying molecular weight, (3) low yields, and(4) the need to inactivate enzymes by pH or heattreatment at the end of the reaction, which adds tothe processing costs.91 Also, the enzymes em-ployed in the process cannot be reused.20 Figure 4outlines a fairly typical process for producing fishprotein hydrolysates. Each step is given a detaileddiscussion 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 bepreferred. The small pelagics comprise 23% ofthe world’s catch,92 of which only 42% is used ashuman food. These are mostly fatty species suchas herring, sardines, anchovies, and mackerel, andFPH prepared from them would contain highamounts of lipid, which would require additionaltreatments such as centrifugation to remove ex-cess fat.93 The fewer steps that are involved in theproduction, the more economically viable theoperation becomes. If a whole fish is used, it iseviscerated and washed, then ground in a meatgrinder, usually mixed with an equal amount ofwater and homogenized in a blender until a vis-cous homologous mixture is achieved. In someinstances a buffer solution is added to the mincedfish, for example, phosphate buffer1,88 and boricacid-NaOH buffer.85 The presence of buffer saltsmay affect the final properties of the hydroly-sates. In a study of whey protein hydrolysis,Kuehler and Stine72 decided not to buffer thesolutions because of the influence buffer saltsmight have on foaming or emulsifying properties.

Processes for fatty and lean species are differ-ent. If the FPH contains more than 1% fish fat, thefat must either be removed by solvent extractionor stabilized by antioxidants3,60 such as butylatedhydroxytoluene, butylated hydroxyanisol,5 orpropylgallate.10 A fish protein hydrolysate withhigh lipid content may darken. The formation of

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brown pigments may result from aldol condensa-tion of carbonyls produced from lipid oxidationafter reaction with basic groups in proteins.5 Re-searchers have developed many means of mini-mizing the lipid content in FPH. To obtain aproduct of a lipid content not exceeding 0.5% byweight, as established by the Protein AdvisoryGroups of FAO for a fish protein hydrolysatesuitable for human consumption,94 Quaglia andOrban36,84 defatted ground sardines by extractionwith isopropanol three times (solvent: substrateratio 1:1) at 46°C for 30 min, and then homog-enized the mixture with water. Hoyle and Merritt5

used an ethanol (90%) extraction directly onminced herring at the fish/ethanol ratio of 1:2 at70°C for 30 min, then mixed with equal volumeof water, hydrolyzed the mixture, then spray driedit. Through this procedure the lipid content was

reduced to 0.9 from 4.0% of raw herring. Alsobefore placing the treated substrate in the reactionvessel, chemical agents such as NaCl, sorbic acid,or ethanol are occasionally added to the mincedfish to minimize bacterial degradation,24 espe-cially if reaction conditions are at neutral or alka-line pH. However, adding NaCl can reduce therate of hydrolysis, increasing reaction time. Etha-nol can also adversely affect the reaction processin too high concentration by inhibiting proteaseactivity, although sorbic acid has not been foundto affect hydrolysis in concentrations up to 0.5%.24

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

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ten a flask, ranging from 0.5 to 3 L with a close-fitting multisocket lid, has been used. The socketsin the lid usually carry: a stirrer, driven by aoverhead variable speed motor to ensure adequatemixing of the system, a thermometer to monitortemperature, a pH electrode to monitor pH, and a“pH-stat” device, where acid or base is added tomaintain a constant pH (Figure 5). The tempera-ture of the reaction vessel is controlled. After therequired temperature is achieved, the pH of theslurry 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 foruniform distribution of the added acid or base.Processing temperature and pH is normally se-lected to optimize the kinetics of the selectedenzyme or enzyme mixture.48 A commercial pro-tease is added in varying concentrations depend-ing on the rate of hydrolysis needed. Given aparticular enzyme and a particular substrate, anyhydrolysis process involves at least five indepen-dent variables. These are S (protein substrate con-centration: %N × 6.25), E/S (enzyme-substrateratio in % or in activity units per kg N × 6.25), pH,T (temperature), and t (time).95

A wide variety of commercial enzymes existthat have been used successfully to hydrolyze fishand other food proteins. Proteolytic enzymes fromplants and microorganisms are most suitable toprepare fish protein hydrolysates.26 Enzymes usedto hydrolyze fish protein have at least one com-mon characteristic: they have to be food grade,and, if they are of microbial origin, the producingorganism has to be non-pathogenic.96 The choice

of enzyme(s) is usually determined by a combina-tion of efficacy and economics.48

The screening for a suitable enzyme in a pro-cess or experiment is very important if the prod-uct is to have predetermined properties. Thescreening process can be conducted in a variety ofways, and there is no standard methodology forthis selection, leaving it primarily up to the indi-vidual researcher what method is most appropri-ate. Good examples of selection procedures arefound in studies by Hale,97 Cheftel et al.,25 Arzuet al.,98 Rebeca et al.,1 Baek and Cadwallader,85

and Kristinsson and Rasco.99 In the comprehen-sive study by Hale,97 the relative activities ofmore than 20 commercially available proteolyticenzymes were measured for the hydrolysis of awashed and freeze-dried fish protein substratefrom haddock. Preliminary tests at 1 h, 40°C, andpH 7 resulted in the plant enzyme ficin to be mostactive, but with papain, also a plant enzyme, hav-ing a much higher relative ranking. When theenzymes were tested at 24 h the picture changed,60% digestion (the set limit) was achieved fastestby Pronase, which exhibited the greatest activityper unit weight. The enzymes pepsin, papain, andpancreatin were most suitable if the lowest costper unit of proteolytic activity was to be followed.These cost estimates are less valid today due tothe commercial availability of bacterial enzymes.

Although many would prefer to use acid pro-teases so microbial growth could be more easilylimited, they usually yield a product with lowprotein yield and too excessive hydrolysis forfood use.39,84,86 Therefore, milder enzymes at neu-

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

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tral and slightly alkaline conditions have beenused more frequently in recent years. In somecases, a high level of solubilization is desired.The acid enzyme pepsin has been most successfulin solubilizing fish protein. Liu and Pigott100 pro-duced a high-quality, fluffy, water-soluble fishprotein hydrolysate by pepsin hydrolysis of rock-fish fillets. Tarky et al.101 used pepsin at 37°C andpH 2.0 to hydrolyze the entire fish waste resultingfrom filleted English sole. The final product, afterultrafiltration and spray drying, was a creamywhite, nonhygroscopic, water-soluble hydrolysatewith low lipid content but very poor nutritionalvalue.

Alcalase, an alkaline enzyme produced fromBacillus licheniformis and developed by NovoNordisk (Bagsvaerd, Denmark) for the detergentindustry, has been proven repeatedly by manyresearchers to be one of the best enzyme used toprepare functional FPH and other protein hydroly-sates.20,36,59,84,88,89 Shahidi et al.59 successfully usedAlcalase to optimize processing conditions toproduce capelin protein hydrolysates. Alcalase-treated hydrolysates exhibited superior proteinrecovery (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 andOrban36 studied the same three enzymes at opti-mal conditions on enzymatic solubilization ofsardine proteins. Hydrolysates produced usingAlcalase and papain were almost identical in ni-trogen recovery, which increased with increasingenzyme concentration (70% recovery at a en-zyme/substrate ratio of 4%). Neutrase-treatedhydrolysates at the same ratio had only over 20%nitrogen recovery. Hydrolysates from Alcalaseand papain also exhibited better functional prop-erties and high nutritional value than those fromNeutrase. Improved nitrogen recovery of fish pro-tein hydrolysates with increase of protease con-centration has been reported elsewhere.1,79 Alcalaseand Neutrase were studied further recently byBenjakul and Morrissey89 on Pacific whiting solidwaste 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 were20 Anson Units (AU)/kg, 1 h reaction time, and

waste:buffer ratio of 1:1 (w/v) at 60°C and pH9.5. The resulting hydrolysate had a high proteincontent with excellent nitrogen yield (up to 70%)and an amino acid composition comparable tofish muscle. Further, Alcalase was found to bethe most cost-effective enzyme out of five en-zyme preparations tested to hydrolyzed salmonmuscle proteins.99 Other new enzyme prepara-tions have shown excellent potential for hydro-lyzing fish proteins to make highly functionalFPH, including Flavourzyme 1000L (NovoNordisk, Bagsvaerd, Denmark), Corolase 7089(Rohm Enzymes; Somerset, NJ) and CorolasePN-L (Rohm Enzymes, Somerset, NJ).99

In an extensive paper,33 Hale reported theeffects of various processing conditions and com-mercially available proteolytic enzymes on yieldand composition of water-soluble fish proteinhydrolysates. He concluded that the hydrolysis ofraw hake (Urophycis chuss) with Alcalase at pH8.5 or above gave the best balance of essentialamino acids and a high yield of soluble product,followed by pancreatin (a mixture of serine pro-teases). One of the first studies on added enzymehydrolysis of fish protein was with papain, due toits favorable properties of pH and temperatureoptima for activity.79,80 Two fish species wereused as substrate, one freshwater, Barbuscarnaticus, and the other marine, Rastrelligercanagurta, and studied at 40 and 55°C, and pH 5and 7. Total solids and nitrogen recovery for bothspecies was high, with pH 7 having the highesttotal solids and nitrogen recovery (69.7% at 55°Cfor freshwater species) compared with pH 5, pos-sibly attributed to better hydration at pH 7.

To properly compare enzyme activity on thesame substrate, it is necessary to determine thegeneral proteolytic activity units at specific reac-tion conditions. Unfortunately, very few research-ers have done this, and most compare enzymeactivity on a weight basis of enzymes used in thereaction mixture. Adding enzymes on the basis ofweight is meaningless if relative enzyme activityis to be compared, because enzyme activity perweight is different for each enzyme under experi-mental conditions used. However, there exist fewstudies that use and compare enzymes on thebasis of their proteolytic activity. Gonzalez-Telloet al.69,102 studied three proteases on whey protein

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substrate by adding them to the system based onAnson Units (AU). Unfortunately, they did notuse the three enzymes at the same AU. In addi-tion, the activity units were obtained from themanufacturer and not assayed by the researchers.Also, reaction conditions used in the hydrolysisexperiments were different from the conditionsused in the enzyme assays, thus making the activ-ity units very unreliable.

Benjakul and Morrissey89 studied the hydroly-sis of Neutrase and Alcalase on Pacific whitingsolid wastes by adding the enzymes to the systembased 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 thoseused to assay the enzyme for proteolytic activityand 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 fromthese same limitations.59,82 A study by Beddowsand Ardeshir103 is the most carefully conductedstudy in the literature with respect to using stan-dardized relative enzyme activity. They assayedthree proteases, bromelain, ficin, and papain, byusing BApNA (Benzoyl-Arg-para-Nitroanilide) toobtain some indication of the relative proteolyticactivities of these enzymes. They then added theenzymes 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 conditionsthan the hydrolysis experiment, which limited itsreliability. Assaying enzymes with BApNA alsoonly estimates the trypsin activity of the enzymepreparation, not their general proteolytic activity.Using BapNA, therefore, gives a less accurateand and probably underestimated value of en-zyme activity. In a research conducted byKristinsson and Rasco,99 where salmon muscleproteins were hydrolyzed, the enzymes were as-sayed under the same reaction conditions as theywere used in the hydrolysis experiment. A syn-thetic protein substrate for proteolytic activity,Azocoll, was used to obtain a uniform level ofproteolytic activity for all enzymes used. Azocollis an insoluble cowhide preparation consistinglargely of collagen. The method is based on dyerelease from the insoluble substrate Azocoll when

a proteolytic enzyme cleaves the peptide linkagesin it. The rate at which the dye is released can beused to quantitatively measure the amount of pro-teolytic enzyme(s) activity working in a solutionby measuring the absorbance at 520 nm. Theenzymes were then used to hydrolyze the sub-strate, all at the same activity unit according to theAzocoll assay, thus comparing them at the sameactivity level on the same substrate. No reports inthe literature have taken this approach; however,the use of Azocoll in fish protein hydrolysis ex-periments has been reported by Ferreira andHultin.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 arehydrolyzed, after this rate of enzymatic hydroly-sis decreases and reaches a stationary phase whereno apparent hydrolysis takes place59 (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 Km value for the solublepeptides that act as effective substrate competi-tors to the unhydrolyzed fish protein,1 and possi-bly autodigestion of the enzyme.105 Shahidi etal.59 found that a high concentration of solublefish peptides in the reaction mixture, releasedduring the initial phase of hydrolysis, reducedboth the rate of hydrolysis and the recovery ofsoluble proteins. Thus, removal of hydrolysatefrom the reaction mixture should enhance thehydrolysis rate and the protein recovery.

By increasing the protease concentration, andthereby increasing the extent of hydrolysis, re-covery of soluble nitrogen increases,1,71,103 al-though increasing enzyme concentrations may notbe cost effective. Substrate concentration has alsonegative effects on protein recovery. Linder etal.47 found that more than 8% protein concentra-tion in the system, regardless of enzyme concen-tration, seemed to have an inhibiting effect onprotein recovery. Baek and Cadwallader85 reportedusing Optimase to hydrolyze crayfish processingbyproducts the %DH increased as substrate con-

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centration decreased to 45% (w/v), suggestingthat high %DH did not coincide with a high amountof hydrolysate. Similarly, Surowka and Fik,70 whomeasured the production of protein hydrolysatewith Neutrase from chicken heads, reported thathydrolysis increased as substrate concentrationdecreased. Ferreira and Hultin,104 using NewlaseA to hydrolyze cod (Gadus morhua) frames, foundthat enzyme autolysis can be reduced at highersubstrate 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 thissystem.60 A kinetic study of the process is alsoquite complicated due to the various types ofpeptide bonds involved and their differing vulner-ability to attack by enzymes during the hydrolyticprocess.69 Very few studies on kinetics of fish andfood protein enzymatic hydrolysis are reported.Sakai et al.87 conducted a kinetic study on thehydrolysis of pollack surimi protein using a acidprotease derived from Aspergillus niger, deter-mining the effects of temperature, pH, initial sub-strate concentration, and enzyme concentration

on the kinetics of protein solubilization. Theirexperimental data followed Michaelis-Mententype kinetics. Michaelis-Menten kinetics has alsobeen observed with whey protein hydrolysis.69

Kristinsson and Rasco99 studied the kinetics offive different enzyme preparations during thehydrolysis of salmon muscle proteins and foundthat the initial rate of the reaction for all enzymesshowed a linear relationship to enzyme activity.These experiments indicated that the initial rateconstants for each enzyme tested were in the sameorder. This confirmed previous studies by Heviaet al.106 and Cheftel et al.25 on menhaden fishprotein as a substrate.

A kinetic model of whey protein hydrolysiswith Alcalase has been proposed, where the hy-drolytic reaction is zero-order for substrate. Theenzyme denatures simultaneously via a second-order reaction due to free enzymes attacking theenzyme bound to the substrate.69

Moreno and Cuadrado107 hydrolyzed vegetableproteins with Alcalase and found reaction mecha-nism consistent with substrate inhibition and asecond-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).

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pendent on the substrate concentration. Cheftel etal.25 used Pronase to hydrolyze fish protein con-centrate and found that the rate constant decreasedwith time, and that proteolysis did not followfirst-order kinetics with respect to concentrationof the peptide bonds. Cheftel et al.25 suspectedthis to be due to the multitude of possible sub-strates in FPC, the number of different proteolyticenzymes present, the inhibitory effects of sub-strate or self-digestion, as well as the differentspecificities that are known to be present in Pro-nase. Archer et al.108 also studied the kinetics ofthe enzymatic hydrolysis of FPC. Their researchfound that the enzyme is initially adsorbed to thesurface of the protein, with the initial rate ofreaction being proportional to the surface area ofsubstrate exposed to the aqueous phase. The over-all kinetics were described by a sequence of twofirst-order processes, an initial, fast reaction inwhich loosely bound polypeptide chains arecleaved from an insoluble protein particle, and asecond, slower reaction in which a more com-pacted core protein is digested.108 Langmyhr109

studied the kinetics of the hydrolytic breakdownof cod muscle with acetyltrypsin. The results in-dicated that enzyme molecules are rapidly andfirmly bound to fish proteins and that the maxi-mum binding occurred under the optimum pHand T conditions for the enzyme. Results alsosuggested that muscle proteins are hydrolyzed inthe same way and to the same extent whether theyoccur separately or are integrated into intactmuscle.

d. Measuring the Extent of EnzymaticHydrolysis

To follow the reaction kinetics and get ameasure 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.96 The degree of thehydrolysis is most commonly used to describehydrolysis of food proteins. The advantage ofusing %DH as a process parameter is that %DHappears to determine unambiguously the proper-

ties of a protein hydrolysate for a given protein-enzyme system.96 The degree of hydrolysis dem-onstrates both theoretically and empirically thatfour processing variables, S, E/S, T, and t, can beleft uncontrolled, provided that %DH is con-trolled.95 From this, it is obvious that %DH is avery simple and rapid method of measuring theextent of protein breakdown.

Two methods for measuring %DH have beenstudied thoroughly and shown to be satisfactory,the pH-stat technique and osmometer technique.The pH-stat method is more commonly used andmore useful for industrial applications. The prin-ciple behind the pH-stat method is relatively simpleand is based on maintaining a constant pH duringthe reaction. By pH-stat, the %DH is calculatedfrom the volume and molarity of base or acid usedto maintain a constant pH. The degree of hydroly-sis is defined as the percent ratio of the numbersof peptide bonds broken (h) to the total numbersof bonds per unit weight (htot; meq/kg protein,calculated from the amino acid composition ofthe substrate): %DH = (h/htot) × 100.%DH can also be expanded to:

%DHB N

h MPB

tot

=⋅

⋅ ⋅⋅

α100

where B = base consumption in ml (or acid incase of acid proteases), NB = normality of the base(or acid), α = average degree of dissociation ofthe –NH groups or COOH groups, MP = mass ofprotein in grams (%N × 6.25). The degree ofdissociation is found by the following equation:

α =+10

1 10

pH pK

pH pK

–

–

This equation for %DH is valid when hydrolysisis conducted at a pH above the pK of the α-NHgroup (for hydrolysis at neutral or alkaline pH).Under these conditions, the reaction will result ina net release of protons (H+) when peptide bondsare cleaved and the base consumption is propor-tional to the number of peptide bonds split.110

Above pH 6.5, the dissociation of the protonatedα-NH group becomes significant. Therefore, it is

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important to know the pK values of the α-NHgroups, both to determine the reaction pH and tocalculate the dissociation constant correctly. ThepK value varies significantly with temperature.The pK values at different temperatures can becalculated according to Steinhardt and Beychok:111

pKT

T= +

⋅⋅7 8

298298

2400.–

where T = temperature in Kelvin.If samples are drawn during the experiment,

it is crucial to correct for the actual value of baseconsumed. Actual base consumption (B′) will besmaller than the theoretical base consumption (B)if this is the procedure. To correct for this, B canbe calculated from the following formula:20

∆ ∆T K Cf= ⋅ ⋅ω

where Kf is 1.86 K/mol for water, ω the osmoticcoefficient, and C the osmolality of solution. Fromthe increase in osmolality, ∆C, %DH can be cal-culated according to the following formula:

%%

DHC

S f w hosm tot

=⋅

⋅ ⋅

⋅∆ 1 1100

where ∆C = depression of freezing point mea-sured in milli-osmol, S% × fosm = g protein/1000ml H20, 1/w = calibration factor for the osmom-eter: 1.04, htot = total number of peptide bonds inthe substrate.

The fosm factor is calculated by knowing thepercent substrate dry matter, D%, present in thereaction mixture:

fDosm = 1000

100 – %

Although the osmometer technique is a verygood method to determine the degree of hydroly-sis, it is being less employed frequently. The de-gree of hydrolysis is now being more commonlydetermined by using either the trichloroacetic acid(TCA) method or the trinitrobenzenesulfonic acid(TNBS) method. Both of these methods are veryuseful when working within the pH 3 to 7 range,where the pH-stat is unusable. Several variationsof these methods exist. The most commonly usedTCA method is based on determining the ap-proximate degree of hydrolysis (%DH) of proteinhydrolysates by the ratio of percent 10% TCAsoluble nitrogen in the hydrolysate compared tototal 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. Thesemixtures are then centrifuged and the supernatantanalyzed for nitrogen. The degree of hydrolysis isthus calculated from the following equation:

%DH = (10% TCA-soluble N in sample/Total Nin sample) × 100

where B′1, B ′2, etc. are the actual base consumptionat the drawing of samples number 1, 2, etc. Thevalue n is the sample number and m is the samplesize (ml). The pH-stat method, however, has somelimitations. Use of pH-stat as a means of controlis only practical outside the approximate pH in-terval 3 to 7.96 This has to be taken into consider-ation when deciding on pH values for the hy-drolysis reaction. Within this pH range, processcontrol may depend on other methods. Therefore,the pH-stat method for %DH determination isuseful mainly under alkaline conditions.

The other commonly used method to measurehydrolysis, the osmometer technique, is moreuniversally applicable. This technique measuresthe freezing point of samples drawn at regularintervals during hydrolysis to construct a hydroly-sis curve. For this method, the freezing pointdepression (∆T) is measured and converted tomilli-osmol (∆C) as follows:95

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This method has been used successfully in fish5

and other food protein hydrolysis.44,70 The TNBSmethod is based on the concentration of primaryamino groups in the hydrolysate. It is a spectro-photometric assay of the chromophore formed bythe reaction of TNBS with liberated amino groups(420 nm), at slightly alkaline conditions.112 Thedegree of hydrolysis can then be calculated aspresented by Baek and Cadwallader,85 andBenjakul and Morrissey,89 for crayfish hydrolysisand whiting hydrolysis, respectively:

%–

–max

DHL L

L Lt= ⋅0

0

100

where Lt = the amount of a specific liberatedamino acid at time t, L0 = the amount of thespecific amino acid in original substrate (blank),Lmax = the maximum amount of the specific aminoacid in the substrate obtained after hydrolysis.

New methods and modifications of previousmethods for determining the degree of hydrolysisare being developed. In the special case of fish, asimple method for monitoring the enzymatic hy-drolysis of fish protein was developed by Ukedaet al.113 The degree of hydrolysis was monitoredby an amino group determination method withgluteraldehyde (GA). The method is based on theconsumption of dissolved oxygen during the re-action between gluteraldehyde and the liberatedamino groups of the protein substrate. The resultsby the GA method were in agreement with theTNBS 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 isvery important as otherwise the enzymes wouldremain active in the substrate and further hydro-lyze the protein and peptides. Deactivation ofenzymes is achieved either by chemical or ther-mal means. Usually the slurry of hydrolysate andenzymes are transferred to a heat bath, where theenzymes are deactivated by exposing them totemperatures ranging from 75 to 100°C for 5 to30 min, depending on the type of enzyme. Forexample, papain is very heat tolerant, and has

been reported to need at least 90°C for 30 min tobe fully inactivated.5 Terminating the reaction bythermal means is undesirable114 because of theeffects of heat denaturation on the protein thatleads to exposure of hydrophobic residues andsubsequently protein aggregation. Diniz and Mar-tin88 suggest that this form of heating can have theadvantage of being very effective in the separa-tion of oil from the fish protein substrate, al-though Webster et al.39 found that some protein-fat interaction occurred at elevated temperaturesthat prevented their separation, when using bo-vine lungs as the protein substrate.

The temperatures at which various proteinsdenature and unfold vary enormously. Becausefish muscle proteins are adapted to function atlower temperatures, they may not be particularlyheat stable. Denaturation is usually undesirablebecause it results in altered physicochemical prop-erties, particularly a loss in protein solubility andfunctionality.31 Spinelli et al.34 are among fewresearchers 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 theprotein fractions, they reacted enzymatically hy-drolyzed muscle proteins from rockfish with 5%sodium hexametaphosphate (HP), following slightacidification to form an insoluble protein-phos-phate complex. By isoelectric precipitation, theyrecovered up to 90% of the protein. In addition,the complex could be washed free of occludednonprotein nitrogen components with no loss ofprotein nitrogen.

Chemical inactivation would be to either loweror raise the pH of the slurry to a point where theenzyme deactivates. Some enzymes are more sen-sitive to pH changes than they are to temperaturechanges. Alcalase is a relatively thermostableenzyme, but it is very sensitive to acid pH. Com-plete inactivation of Alcalase therefore is obtainedby lowering the pH to 4.0.20,59 Neutralizing theslurry to pH 7.0 would inactivate pepsin and mostother 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 than10. Unfolding at such extremes of pH usuallyoccurs because the folded protein (or oligopeptide)has groups buried in nonionized form that can

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ionize only after unfolding.52 Stabilizing salt-bridges between ionizing groups can also be dis-rupted by extreme pH values. The termination ofthe enzymatic reaction thus is a formidable ob-stacle in any FPH operation. The choice of inac-tivation method should be carefully made andbased up on what the enzyme under study issensitive to, whether it is heat or pH. In somecases a combination of elevating temperaturesand lowering pH have been used.82

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 themiddle, lipid-protein fraction between aqueouslayer and sludge, aqueous and oil layers and oillayer on the top.68 A single centrifugation step caneliminate the vast majority of the lipids present.The oil layer overlying the aqueous layer is thenremoved and the soluble fraction collected. Be-cause lipid in the final hydrolysate is a majorconcern for FPH, it is important to remove it.Lipid residues in FPH must be lower than 0.5% toprevent alteration of the lipid fraction during stor-age.34 More than one centrifugation step is oftenrequired to separate the soluble proteins from thelipids 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 sludge82 and filtering theslurry by passing it through a 2-mm mesh screen.86

The removal of colored and odiferous matter hasalso been reported by treating the first solublefraction obtained by centrifugation with 1% w/vcharcoal at 55°C and 30 min before the nextseparation step.59

In a commercial operation, the final solublefraction is generally spray dried to convert thehydrolysate to a powdered form, which can beincorporated into food formulations. The insolublefraction or the sludge precipitated during cen-trifugation may be used as animal feed. Spraydrying of the soluble fraction is one of the mostenergy consuming and expensive steps in the pro-duction of protein hydrolysates. In industrial pro-duction a compromise must be made between thehydrolysate yield and the amount of water thatmust be removed to obtain a dry product.60 Asuspension of equal amounts of water and fishsubstrate appears to be convenient from an eco-nomic point of view. Rebeca et al.1 performed astudy 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 higherdry matter content, with high protein recovery, ofthe soluble fraction and the cost of drying wasreduced. 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

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sates are neutralized and freeze dried. By neutral-ization the final product can have a fairly high saltcontent, which is undesirable (HCl + NaOH →H2O + NaCl). This can be mostly avoided if thesoluble fraction is passed through an ion exchangecolumn before freeze-drying. Dialysis to desaltthe soluble fraction can also be a useful process.

Increasingly, ultrafiltration membranes havebeen introduced into the production process forhydrolysates. The hydrolysate after enzyme inac-tivation is filtered directly through a membranewith a specific molecular weight cut-off value.The ultrafiltration process can contain more thanone filter, yielding several molecular weight frac-tions depending on the product desired. A majoradvantage for such a process is the control ofmolecular size of selected peptides so that a uni-form product is possible,76 as the molecular sizeof the hydrolyzed proteins is a key factor in deter-mining functional properties of hydrolysates.115

The method has, however, not found its way intofish protein hydrolysate production. The reasonprobably being that in order to successfully usethe ultrafiltration, the sample has to be very pureand free of lipids. It is, however, possible that themethod could be applied to highly purified anddefatted FPH powders. The method has found tobe very useful in soy,115 whey,75,116 and caseinprotein hydrolysis.76 In the case of whey protein,large-molecular-weight fractions are primarilyresponsible for allergic reactions. However, smallpeptides and free amino acids are less well ab-sorbed by humans. Therefore, protein hydrolysisshould not be more extensive than the minimumrequired to eliminate allergic responses. By ultra-filtration therefore it is possible to exclude boththe too large and too small peptides and collectthe molecular weight in between by passing thehydrolysate solution through two filters, the firstwith a higher cut-off value than the second.116

Applying this technique to FPH would be an inter-esting task to undertake.

IV. PHYSICOCHEMICAL ANDFUNCTIONAL PROPERTIES OF FISHPROTEIN HYDROLYSATES

As previously mentioned, one of the majoradvantages and goals of enzymatically hydrolyz-ing fish proteins is to modify and improve their

functional properties. The functional propertiesof fish protein hydrolysates are important, par-ticularly if they are used as ingredients in foodproducts.60 Enzymatic hydrolysis of fish proteinsgenerates a mixture of free amino acids, di-, tri-,and oligopeptides, increases the number of polargroups and the solubility of the hydrolysate, andtherefore modifies functional characteristics ofthe proteins, improving their functional qualityand bioavailability. The choice of substrate andproteases employed and the degree to which theprotein is hydrolyzed affect the physicochemicalproperties of the resulting hydrolysates.105 En-zyme specificity is important to peptide function-ality because it strongly influences the molecularsize and hydrophobicity of the hydrolysate.117

Thus, the peptides obtained have different mo-lecular profiles, and the surface energy of thehydrolysate is different, depending on the en-zyme used; these variations have a bearing on thefunctionality of the mixture.114,118 The more nar-row the specificity, the are the larger peptidesproduced; the broader the specificity, the smallerare the peptides generated. As the range of enzy-matic activities within commercial preparationsis increased, the hydrolysate becomes more com-plex.4 The chain length of peptides or breaking oflinkage is also dependent on the extent of hy-drolysis; conditions of hydrolysis; concentrationof enzyme and the type of protein to be hydro-lyzed.41 Due to the complex peptide profile, it isoften useful to calculate the average peptide chainlength (PCL) introduced by Adler-Nissen andOlsen119 to express the composition of the proteinhydrolysate. PCL is calculated from the degree ofhydrolysis in the following way:

PCLDH

= 100%

This approximation is acceptable in nearly allpractical cases.20 The relationship between PCLand %DH is shown in Figure 8.

Manipulating the reaction conditions duringenzymatic hydrolysis of food proteins produceshydrolysates with different solubility and emulsi-fying characteristics, foaming properties, or tastecharacteristics.4 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

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peptides, completely lacking the functional prop-erties of the native proteins. This may promotethe formation of undesirable bitter peptides. Bycontrolled hydrolysis it is possible to eliminatethese drawbacks and to obtain hydrolysates withdifferent physicochemical and functional proper-ties, in some cases even better than the originals.36

The physical and chemical properties that governprotein functionality include size, shape, aminoacid composition and sequence, net charge anddistribution of charges, hydrophobicity/hydrophi-licity ratio, peptide structures, molecular flexibil-ity/rigidity, and the ability to interact/react withother components.120 Functionality of food pro-teins has been defined as: “those functional andchemical properties which affect the behavior ofproteins in food systems during processing, stor-age, preparation and consumption”.31 The follow-ing sections discuss the main functional proper-ties that fish protein hydrolysates exhibit, that is,solubility, water holding, emulsifying, foamingand sensory properties, and research aimed to-ward evaluating these properties for various FPHpreparations.

A. Solubility

Solubility is probably the most important ofprotein and protein hydrolysate functional prop-erties. Many of the other functional properties,

such as emulsification and foaming, are affectedby solubility,121 and therefore it is an excellentindicator of the protein hydrolysate functionality,and its potential (and limitations of) applica-tions.31,58 Hydrophobic and ionic interactions arethe major factors that influence the solubility char-acteristics of proteins. Hydrophobic interactionspromote protein-protein interactions and result indecreased solubility, whereas ionic interactionspromote protein-water interactions and result inincreased solubility. Ionic residues on the surfaceof peptides and proteins introduce electrostaticrepulsion between protein molecules and repul-sion between hydration shells around ionic groups,both major contributors to increased solubility ofproteins. Solubility of protein and protein hy-drolysates is generally measured by employingthe nitrogen solubility index (NSI), a standard-ized method developed by AOCS and later modi-fied by Morr et al.122 The NSI is determined bysuspending a protein hydrolysate sample in waterthen stirring and centrifuging the mixture. Thesupernatant is then analyzed for nitrogen contentby the Kjeldahl procedure, and the NSI calculatedas a percentage of the soluble nitrogen to thepercentage of total nitrogen in the sample.20

Intact fish myofibrillar proteins have the prob-lem of the lack of solubility in water over a widerange of pH,13,123 and enzymatic hydrolysis is veryimportant in increasing the solubility of these

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

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proteins. The effects of enzymatic hydrolysis onfish protein are straightforward. Enzymatic break-down of the protein involves a major structuralchange in that the protein is gradually cleavedinto smaller peptide units, and as the degree ofenzymatic hydrolysis increases the solubility offish proteins increases. The enhanced solubilityof the hydrolysates is due to their smaller molecu-lar size compared with the intact protein, and thenewly exposed ionizable amino and carboxylgroups of the amino acids, that increase the hy-drolysate hydrophilicity.58,117 To effectively bindto water molecules, the peptides have to have theability to form hydrogen bonds between its hy-drophilic polar amino acid side groups and thewater molecules. Hydrolysis exposes some of thehydrophobic groups to the surface, but at thesame time converts even more hydrophobic groupsto hydrophilic groups by generating two end car-bonyl and amino groups. Thus, the smaller pep-tides from myofibrillar protein hydrolysis haveproportionally more polar residues, with the in-creased ability to form hydrogen bonds with wa-ter. This increases protein solubility to that of theintact protein. In addition, ion-dipole interactionsbetween water and simple ions such as Na+ andCl- are also important in the interactions betweenthe polar or charged groups on biomolecules andwater.18 Biomolecules tend to be very soluble atfavorable NaCl conditions. The increased solubil-ity is partly through the formation of sodium saltsof carboxyl groups of proteins, COONa.13 This istrue for fish myofibrillar proteins, but the hydro-phobicity of globular proteins, such as milk pro-teins, may increase with exposure of apolar aminoacid residues after hydrolysis.117 Although in-creased solubility has a positive relationship tothe extent of hydrolysis, care has to be taken thatthe substrate is not too extensively hydrolyzed. Avery high degree of hydrolysis may lead to highsolubility, but this can have very negative effectson the rest of the functional properties. To main-tain or improve functionality, generally low de-grees of hydrolysis are necessary.4

Sugiyama et al.84 studied enzymatic hydroly-sis with several alkaline, neutral, and acid pro-teases on defatted sardine meal. The result showedthat all the alkaline proteases had higher ability toproduce highly soluble protein hydrolysates com-

pared with the neutral and acid ones. Gel chroma-tography showed that hydrolysates prepared withthe alkaline proteases had a lower average mo-lecular weight. The increased hydrophilicity ofthese preparations could be due to their largercharge to size ratios compared with the longerproteins such as those found in nonhydrolyzedproteins from a similar source.44 Interestingly,pepsin had lower solubilized protein ratio than allof the alkaline proteases, but pepsin is generallyconsidered one of the best enzymes to solubilizefish protein.35,36,68,97,100 Quaglia and Orban35,36 alsostudied the properties of hydrolysates producedfrom sardine and concluded that Alcalase andpapain, at optimum pH and temperature, bothgave hydrolysates characterized by high solubil-ity. Hydrolysates made with Alcalase at higherdegrees of hydrolysis showed a decrease in high-molecular-weight fractions, and increased solu-bility.35 This connection between %DH and solu-bility is also reported for other food protein.124 Asignificant increase in solubility was observedwith an increase in %DH from 2.5 to 15% for anenzymatically hydrolyzed single cell microbialprotein (“Pruteen”), from less than 10% solubilityto over 90%.124 Similar results were observedwith soy protein hydrolysates from 1 to 8.3%DH.96 Yu and Fazidah125 hydrolyzed Aristichthysnoblis, a Chinese freshwater fish, with protease P“Amano” 3, and reported excellent solubility at15% DH after a 3-h digestion.

Hoyle and Merritt5 found that enzymaticallyhydrolyzed ethanol-extracted herring FPH hadhighest solubility compared with nonextractedenzymatically hydrolyzed FPH. This finding sug-gests that the lower lipid content of ethanol-ex-tracted herring FPH may have resulted in lesscompetitive water binding of proteins comparedwith hydrolysates with higher lipid contents. Vieiraet al.86 studied the functional properties of hy-drolysates from lobster processing wastes andfound these to be highly soluble, pepsin yieldinga fraction with higher solubility than papain or afungal protease. The highest nitrogen solubilityindex (NSI) for these hydrolysates was found atextremes 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 itprecipitates. Shahidi et al.59 also found that cape-

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lin protein hydrolysates (CPH) prepared by dif-ferent enzymes had different solubility profiles atdifferent pH conditions. However, in contrast tothe findings of Vieira et al.,86 Shahidi et al.59

found lower solubility at pH 7 to 8, and with closeto 100% solubility for Alcalase at pH 5. Thelowest solubility value at pH 7 to 8 was, however,84%, thus CPH had excellent solubility over therange of pH 2 to 11. Hydrolyzed salmon muscleproteins exhibited excellent solubility at 5 to 15%DH, between 92 to 100%, with over 96% solubil-ity in the pH range of 2 to 11.90

The solubility of enzymatically hydrolyzedfish protein is generally much higher than forchemically produced fish protein concentrate(FPC). A FPC type-B air-dried fish powder hadextremely poor solubility at a wide range of NaClmolarity and pH,126 and alkaline hydrolysis of redhake exhibited lower than 30% solubility at pH 1to 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 isa very useful property for many food applica-tions, including beverage applications. The goodsolubility and good nutritive value of enzymati-cally hydrolyzed fish protein hydrolysates alsomakes them well suited to produce milk replacersfor 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 enzymaticdigestion of sardines that is used in feeds as amilk replacer for calves and piglets.60,126 Menha-den hydrolysate produced by pancreatine wasfound to be an excellent milk replacer with highPER value with the process cost half as much asdried skimmed milk.127 Soluble FPH is an excel-lent amino acid source for supplementing cerealproteins10 and to be used in bakery products, soups,and infant formulas.1 Pasta enriched up to 3.5% offish protein hydrolysate increased the proteincontent by 5%, with a dramatic increase in thedietary essential amino acids lysine (37.5%), va-line (31%), and threonine (18%).128 Soluble FPHfrom extensive hydrolysis is an excellent sourceof nitrogen for microbial growth. Extensive hy-drolysis results in a product of free amino acidsand 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 weanlingfeed for piglets, and increasingly as a pellet coat-ing for pet food.60

B. Water-Holding Capacity

Water-holding capacity refers to the abilityof the protein to imbibe water and retain it againstgravitational force within a protein matrix, suchas protein gels or beef and fish muscle, and it ispositively correlated with water-binding capac-ity.120 Water-holding capacity of proteins addedto muscle tissue is of great importance to the foodindustry because retaining water in a food systemoften improves texture. The functional propertiesof proteins in a food system depend in part on thewater-protein interaction, and the final outcomegreatly depends on how well the protein bindsand holds water in a food system. However, asuccessful specific application of a functionalprotein ingredient or additive for enhancing asingle specific functional property may not betransferable to other food systems. Fish proteinhydrolysates are highly hygroscopic, and this hasto be considered when producing them. Properpackaging and low relative humidity of air duringprocessing is an important consideration. Thepresence of polar groups such as COOH and NH2

that increase during enzymatic hydrolysis have asubstantial 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 than15% RH.130

Fish protein hydrolysates have excellent wa-ter-holding capacity, and thus useful propertiesfor certain food formulations. Kristinsson90 madesalmon FPH with several enzymes to 5, 10, and15% 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 with3% for the control. In this study there was noconnection observed between %DH and waterloss, 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-

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trate. Onodenalore and Shahidi82 increased cook-ing yield of comminuted pork by 2.4 to 9.3% byadding 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 myofibrillarshark proteins were more effective than hydroly-sates made from untreated shark muscles. Shahidiet al.59 found similar results with capelin proteinhydrolysates (CPH) when added at a 3% level tocommunited pork, CPH increased cooking yieldof approximately 4%. At even lower addition lev-els there was a large reduction in the amount ofdrip 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 17.7to 60.4%, suggesting that CPH may have antioxi-dant properties, perhaps due to chelation effects.Hatate et al.131 also found that sardine myofibrilprotein hydrolysates exhibited antioxidant activ-ity. More importantly, hydrolysates appeared toact synergistically with several commercial anti-oxidants. These antioxidant effects are highlydependent on the amino acid composition andmolecular size of the hydrolysate peptides.

Fish protein concentrate (FPC) produced fromsardine with an ethanol process has also beenstudied in a meat model system with respect towater holding. FPC was added to a hamburger-type product made from beef at 20, 40, and 60%addition, and increased percentage of FPC in theformulations significantly improved the cookingyields of the products.37

C. Emulsifying Properties

The emulsifying properties of FPH are di-rectly connected to their surface properties, orhow effectively the hydrolysate lowers the inter-facial tension between the hydrophobic and hy-drophilic components in food. Proteins adsorb tothe surface of freshly formed oil droplets duringhomogenization and form a protective membranethat prevents droplets from coalescing.6 Hydroly-sates are surface active and promote oil-in-wateremulsions because they have hydrophilic and hy-drophobic functional groups and are watersoluble.121 Hydrolysates orient their hydrophobic

loops in the apolar oil phase, while the polarsegments extend into the aqueous phase. Desir-able surface active proteins and protein hydroly-sates have three major attributes: (1) ability torapidly absorb to an interface, (2) ability to rap-idly unfold and reorient at an interface, and (3) anability, once at the interface, to interact with theneighboring molecules and form a strong cohe-sive, viscoelastic film that can withstand thermaland mechanical motions.120,132

Emulsifying capacity and emulsifying stabil-ity are two methods generally used to measure theability of protein hydrolysates to form and stabi-lize emulsions. Emulsifying capacity is usuallydefined as the volume of oil (ml) that can beemulsified by the protein hydrolysate (g), beforephase inversion or collapse of emulsion occurs.31

Because FPH produces emulsions of low viscos-ity, the most accurate way to measure this is touse an oil titration method,133 and measure theelectrical resistance during oil titration. A suddenincrease in resistance is observed when the maxi-mum emulsifying capacity is reached. Emulsionstability refers to the ability of an emulsion toresist changes in its properties over time134 andcan be determined simply using gravitationalmethods. Measurement involves blending thehydrolysate with oil and water, centrifuging, andmeasuring total volume of emulsion, its stabilityexpressed as the difference between total volumeof an emulsion and the aqueous volume to totalvolume.135,136

The emulsifying properties of hydrolyzedprotein are improved by carefully controlling theextent of hydrolysis. Extensive hydrolysis resultsin a drastic loss of emulsifying properties.58 Al-though small peptides are highly stable and dif-fuse rapidly and adsorb at the interface, they areless efficient in reducing the interfacial tensionbecause they cannot unfold and reorient at theinterface, like proteins with higher molecularweight.137 Solubility seems to play an importantrole in emulsification because rapid migration toand adsorption at the interface are critical.138

However, complete solubility is not an absoluterequirement. 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

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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.57 Smaller and smaller peptidesare formed as the enzymatic hydrolysis progressesand this impacts emulsifying properties.138 Pro-tease specificity also plays a key role in the emul-sifying properties of protein hydrolysates becausethis strongly influences the molecular size andhydrophobicity of the resulting peptides. Kuhlerand Stine72 found that whey protein hydrolyzedwith Prolase yield large-molecular-weight pep-tides with excellent emulsifying stability and ac-tivity. In contrast, Pronase, which has a broadspecificity, produced much smaller peptides andyielded 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 andemulsifying 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 sardineprotein hydrolysates83 and salmon protein hydroly-sates.90 Different molecular weight distributionsof the hydrolysates show that a higher content ofhigh-molecular-weight protein fractions plays animportant role in stabilizing emulsions. Hydroly-sates with lower degrees of hydrolysis have highersurface hydrophobicity and sardine hydrolysatesat low %DH have better emulsifying capacitythan commercial sodium caseinate.83 Cui68 ob-tained similar results for enzymatically hydro-lyzed chum salmon muscle.

Hydrophobicity plays an important positiverole in determining emulsifying properties. Katoand Nakai139 reported that effective hydrophobic-ity determined fluorometrically showed signifi-cant correlation with interfacial tension and emul-sifying activity of a wide variety of proteinsstudied. Li-Chan et al.140 also found that surfacehydrophobicity predicted the emulsifying proper-ties of meat proteins. A positive correlation be-tween surface activity and peptide length has beenfound,73 and it has since been generally acceptedthat a peptide should have a minimum length of>20 residues to possess good emulsifying andinterfacial properties.141

Various emulsification properties have beenfound for hydrolyzed seafood protein products.Hydrolyzed processing wastes from lobster usingpapain and an undefined fungal protease had arather poor capacity for oil emulsification.86 Also,relatively poor emulsifying capacity and stabilitywere reported for enzymatically produced capelinprotein hydrolysate59 and shark protein hydroly-sates;82 however, neither of these studied definedthe %DH of the products. Enzymatically hydro-lyzed myofibrillar proteins from rockfish filletsshowed an increase in emulsifying capacity andstability with hydrolysis compared to the intactprotein.34,123 Emulsifying capacity reached a pointat which there was no change with %DH.34 Hy-drolysates from rockfish muscle made with bro-melain had relatively poor emulsifying capacityand yielded unstable emulsions.136 Removing lip-ids from rockfish protein hydrolysates resulted ina dramatic loss in emulsifying capacity; the lossincreased with increasing extraction temperature.34

Fish protein derivatives with good emulsify-ing properties can also be prepared by acetyla-tion.60 Acylating agents such as acetic or succinicanhydrides can react with the amino groups ofproteins in an alkaline environment, forming amidegroups.24,142 The acetyl residues participate in theformation of additional hydrogen bonds or elsecan contribute to hydrophobic adherences, thusincreasing emulsifying properties. Groninger andMiller136,142 are among researchers that reportedan increase in emulsifying properties of fish pro-tein by acetylation. Miller and Groninger136 foundthat emulsifying properties of FPH prepared bybromelain increased until 43 to 59% of the freeamino groups were acetylated, with further acety-lation having no effect.

Emulsifying properties of fish protein con-centrates have also been reported.27 Fish proteinconcentrate produced by isopropanol extractionhad a decreased emulsifying capacity with in-creased solvent extraction. This loss of emulsifi-cation capacity was believed to be tied to a loss insolubility.

The emulsifying ability of other food pro-teins, primarily milk protein, has been studiedextensively. Enzymatically hydrolyzed whey pro-tein had excellent emulsifying capacity and sta-bility when made with the protease Prolase (~3.0 g

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oil/mg protein).72 Pepsin and Pronase hydrolyzedwhey protein yielded emulsions of less capacityand stability, both decreasing with time of hy-drolysis and %DH.72 The degree of hydrolysiswas, however, not specified. Each of these en-zymes have different substrate specificities andaction patterns. Pepsin and Pronase hydrolysateshad 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 smalldecrease at 67% DH.57 A drop in emulsifyingcapacity was highly correlated with a drop inhydrophobicity, and reduced molecular size, andthus in agreement with our findings.90 Trypsinhydrolysis was found to greatly improve the emul-sifying properties of casein and whey protein andwas highly influenced by pH.138

Chicken head hydrolysates70,71 and turkeywaste hydrolysates44 have also been tested foremulsifying capacity, and both had poor emulsi-fication ability. A significant drop in emulsifyingproperties as hydrolysis progressed was observedwhen alpha-chymotrypsin was used to hydrolyzepeanut protein fractions, reaching a stationaryphase after a certain time.78

There are many different factors that mayaccount for the difference observed between hy-drolysates in both the ability to form an emulsionand emulsion stability. Peptides molecular char-acteristics and peptide chain length are the majorreason for the different emulsification ability ofhydrolysates. Environmental conditions such aspH, ionic strength, temperature, etc. also have aneffect on the emulsification properties. Also, somesynergistic effects of peptides on emulsifyingproperties have been noted.117 In short, the fore-going indicates that peptide behavior is complexand not easy to explain.

D. Foaming Properties

The chemistry underlying foaming propertiesof protein and protein hydrolysates have manythings in common with emulsifying properties.Both rely on the surface properties of protein.Food foams consist of air droplets dispersed inand enveloped by a liquid containing a soluble

surfactant lowering the surface and interfacialtension of the liquid.31 The amphiphilic nature ofproteins makes this possible; the hydrophobicportion of the protein extends into the air and thehydrophilic portion into the aqueous phase.Townsend and Nakai143 showed that total hydro-phobicity, or the hydrophobicity of exposed orunfolded protein, have a significant correlation tofoaming formation. The surface hydrophobicity,which is important for emulsification, does notcorrelate with foam formation.

Many different methods have been developedto measure foaming properties of proteins andprotein hydrolysates. Most, if not all, of thesemethods have the drawback of poor experimentalreproducibility, thus making it hard to comparefindings between laboratories. Foaming proper-ties are usually expressed as foam formation andfoam stability. Phillips et al.144 developed a methodwhere the ability of protein to form foams isdescribed as overrun. Overrun is the percentageof excess volume produced by whipping a proteincontaining liquid compared with the initial vol-ume of the liquid. Foam stability is measured bywhipping the protein solution and measuring thetime required to decrease half of the volume.Foam stability is a highly empirical method, butis simple to perform and has found useful appli-cations with FPH.

Very few studies have been performed onfoaming properties of fish protein hydrolysates.Shahidi et al.59 and Onodenalore and Shahidi82

studied the foam formation, or whippability, andfoam stability of shark and capelin protein hy-drolysates, respectively. In both processes Alcalasewas the protease used for hydrolysis. The capelinprotein hydrolysate had good whippability, 90%,at a 12% DH. Shark protein hydrolysates fromshark meat had higher whippability, 106%, andhydrolysates from washed shark myofibrillar pro-teins had whippability of 130%. Capelin hydroly-sates had higher foam stability after 30 s thatdropped significantly compared with hydrolyzedshark 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 higherthan for shark and capelin protein hydrolysates.

There is a connection between the degree ofhydrolysis and foaming properties. Kuehler and

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Stine72 concluded that whey proteins hydrolyzedto a limited degree had a increased foaming abil-ity but reduced foam stability. This was attributedto more air being incorporated into solution ofsmaller peptides, but the smaller polypeptides donot have the strength required to give a stablefoam. This was seen in the initial 30 min ofhydrolysis with further hydrolysis resulting inpeptides that lack the ability to stabilize the aircells in the foam. Weak foams are commonlyobserved when food proteins are hydrolyzed.However, the advantage of using hydrolyzed pro-teins as foaming agents is their insensitivity tochange in pH. The pH of the dispersing mediummarkedly affects foaming, particularly foam sta-bility, with foaming properties being highest closeto 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 foamformation of FPC is provided by the soluble pro-teins, only 1% of total FPC, while the remainingdenatured proteins act as foam stabilizers.30

Foaming properties of fish protein hydroly-sates can be improved by acylation. Groningerand Miller147 succinylated hydrolysates producedby hydrolyzing rockfish fillets with bromelain.These foams were significantly more stable com-pared with foams from modified soy protein andegg white; however, they had smaller foam vol-ume. The succinylated protein foams were morestable over a wide range of pH (3 to 9) comparedwith the other protein. The succinylated FPH wasincorporated into a dessert topping, a soufflé, andboth a chilled and a frozen dessert, and the prod-ucts were as acceptable to a taste panel as similarfoods containing no fish protein. Ostrander etal.148 incorporated succinylated FPH, as preparedby Groninger and Miller,147 into whipped gelatindesserts. The succinylated FPH formed a stablefoam with smooth texture, highly acceptable withrespect to flavor and mouthfeel.

E. Fat Absoption

Various methods for the measurement of fatabsorption have been described. Commonly, hy-

drolysates are mixed with a specified amount ofexcess 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 by1 g protein hydrolysate59,90 for FPH. The mecha-nism of fat absorption, as assessed by this method,is attributed mostly to physical entrapment of theoil, and thus the higher bulk density of the proteinthe more fat absorption.31 Fat-binding capacity ofproteins 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.90 TheFPH at all %DH exhibited excellent fat absorp-tion, greater than both egg albumin and soy pro-tein concentrate. The hydrolysates at 5% DH hadsignificantly higher fat absorption (5.98 to 7.07 mloil/g FPH) than 10% DH (3.22 to5.12 ml oil/gFPH) and 15% DH (2.86 to 3.86 ml oil/g FPH)due to the larger peptide sizes. Only two otherpublications dealing with fat absorption for FPHcould be found, for capelin protein hydrolysates59

and shark protein hydrolysates.82 Both studies failto define the units used to express fat absorption,and thus it is impossible to compare them to otherstudies.90

Other protein hydrolysates have been studiedwith respect to fat absorption. Casein hydroly-sates with excellent oil-holding capacity, madewith papain, were dissociated by SDS, indicatingthat hydrophobic interactions were primarily re-sponsible. The substrate specificity of enzymesalso seemed to play a major role in this.114 Thiswas further confirmed by Kristinsson,90 whoshowed that different enzymes used to hydrolyzesalmon muscle protein had different fat absorp-tion ability. The capacity of a hydrolysate to ab-sorb fat/oil is an important attribute that not onlyinfluences the taste of the product, but it is also animportant functional characteristic that is requiredespecially for the meat and confectionery indus-try. Fish protein hydrolysates therefore could verywell be used in such applications.

F. Sensory Properties

Although enzymatic hydrolysis of proteinsdevelops desirable functional properties, it has

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the disadvantage of generating bitterness. This isa common problem with fish protein hydroly-sates, and the major reason for its slow accep-tance as a food ingredient. The mechanism ofbitterness is not very clear, but it is widely ac-cepted that hydrophobic amino acids of peptidesare a major factor. In fact, the hydrophobicity ofpeptides can be measured, and it has been foundthat most bitter peptides have values over 1400kcal/mol, whereas nonbitter peptides have valuesbelow 1300 kcal/mol. This principle is, however,only valid for molecular weights below 6000 D.149

Peptides with a molecular weight from 1000 to6000 D and with hydrophobic characteristics haveshown most likely to be bitter.102 Hydrolysis ofprotein results in exposing buried hydrophobicpeptides, which will be readily able to interactwith the taste buds resulting in detection of bittertaste. An extensive hydrolysis to free amino ac-ids, however, decreases the bitterness of thesebitter peptides because hydrophobic peptides arefar more bitter compared with a mixture of freeamino acids. Free amino acids, are however, un-desirable from a functional standpoint. Strict con-trol of any hydrolysis experiment and terminationat low %DH values therefore is desirable to pre-vent the development of a bitter taste and theretention of functional properties.

Common features found in the structure ofbitter peptides are, for example, N, or C, terminalbasic amino acid residues, C, or N, terminal hy-drophobic peptide fragments and peptide chainfolding due to proline residues within the peptidestructure.150 Bitterness is due to particular arrange-ments of certain chemical groups in the peptides.Two functional groups are necessary to producebitterness, such as a pair of hydrophobic groupsor a hydrophobic or a basic group.151 The sensa-tion of bitterness may also be exerted by chemicalcompounds having a hydrophobic region and onehydrophilic group spaced 0.3 nm apart.95 Becauseenzymes have different preferences for aminoacids, choosing the most appropriate enzymepreparation for hydrolysis can control the bitter-ness. Enzymes with a high preference for hydro-phobic amino acids such as Alcalase are oftenpreferred and frequently yield products of lowbitterness.20,96 The use of exopeptidases, as op-posed to endoproteinases, can also be helpful inovercoming the bitterness in fish protein hydroly-

sates, particularly exopeptidases that split offhydrophobic amino acids from bitter peptides.However, for an enzyme preparation to be effec-tive for hydrolyzing protein, both exopeptidasesand endoproteinases are required. Many studieshave shown that proteolytic preparations contain-ing exopeptidases and endoproteinases produceless bitter peptides than single proteases.152-155

Trials have shown that by the additional action ofexopeptidases in proteolytic processes, it is pos-sible to move the bitterness point up to higherdegree of hydrolysis.156

Despite the problem with bitterness of fishprotein hydrolysates, little research has been con-ducted. While FPH is highly nutritious and safeand functional properties are good, the sensoryproperties are extremely important for the suc-cessful adaptation and acceptance by the foodindustry and the consumer. Yu and Fazidah125

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 thebitterness, and after a 5-h hydrolysis sampleswere described as “very bitter”. Interestingly, nobitterness was observed until after 4 h of hy-drolysis. Papain-hydrolyzed herring had higherbitterness scores than Alcalase-hydrolyzed her-ring.5 Alcalase hydrolyzed samples at higherdegree of hydrolysis were less bitter than pa-pain-hydrolyzed samples at the same %DH. Thegreater hydrophobic specificity of Alcalase com-pared with papain could result in a reduced bitter-ness. Enzymatically hydrolyzed ethanol-extractedherring, with either Alcalase or papain, had verylow bitterness, suggesting that reduced bitter-ness could be due in part to lower lipid content.Lalasidis et al.157 hydrolyzed cod filleting offalwith Alcalase, which produced a product with abitter taste. This bitter taste was eliminated whenthe hydrolysate was treated further withpancreatine, which is rich in exopeptidase activ-ity.

Fish protein hydrolysates have been in-corporated into food systems to evaluate theiracceptability, and sometimes with satisfactoryresults.147,148 Proteins from Oreochromismossambicus, a freshwater fish, were hydrolyzedwith Alcalase to produce a soluble hydrolysatethat was incorporated into crackers.158 Sensory

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evaluation showed the fried crackers with up to10% addition of FPH were highly acceptable.

Vieira et al.86 hydrolyzed lobster processingwaste for 5 h with several commercial enzymesand found no difference in taste between the hy-drolysates and a control sample, and detected nobitterness. This is perhaps due to the extensivehydrolysis. The hydrolysate possessed a distinct,pleasant smell, and it was proposed for use invarious formulated food products such as flavorenhancers. Is has long been known that proteasescan be used for recovery of flavor.159 Baek andCadwallader85 attempted to fully utilize crayfishprocessing 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 productionof value-added seafood flavor extracts. Fujimakiet al.160 reported that a fish protein concentratetreated with Pronase had a flavor potentiatingactivity like that of monosodium glutamate (MSG)but was accompanied by an unfavorable bitterflavor. A low molecular acidic peptide fractioncontributed significantly to this MSG-like flavor.The acidic peptides isolated later from the samefish protein hydrolysate showed that at least fourdipeptides (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 ofMSG, but with weaker intensities.161 Hevia andOlcott162 analyzed basic tripeptides that were re-sponsible for bitter taste in fish protein hydroly-sates. The tripeptides contained asparagine andlysine as the second and C-terminal residues, re-spectively, with the N-terminal residue leucine orglycine.

Many techniques have been suggested to re-duce or mask bitterness in hydrolysates, but fewof them applied to FPH. These include treatinghydrolysates with activated carbon that partlyremoves 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 plasteinreaction.164 Chakrabarti165 reported success indebittering fish protein hydrolysate using ethylalcohol. Higher concentrations of alcohol low-ered the bitter fraction in the hydrolysate.

The plastein reaction is among few that havebeen studied with respect to fish protein hydroly-sates. Figure 9166 outlines the steps involved in theplastein reaction. Heck164 investigated the plas-tein reaction in FPH from peptic hydrolysis andfound that it reduced the bitterness but with anaccompanied loss of FPH solubility. Some suc-cess using the plastein reaction has been reportedwith fish waste167 and sardines.168

Plastein can be defined as a protein substratethat may be prepared by reversing hydrolysis witha protease such as pepsin or papain in a concen-trated protein hydrolysate.60,169 The plasteins are amixture of peptides of varied composition andmolecular weight between 1000 and 500.000D.24,166 Three factors are necessary for completionof the plastein reaction: (1) a low-molecular-weightprotein 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 highconcentration of hydrolysate (30 to 50%) is incu-bated with an enzyme resulting in a condensationof the peptides. As new polypeptides are formed,they aggregate via hydrophobic associations. Thefinal product has a low lipid content and specialhydrophobic functional properties.31,41,60 Tanimotoet al.170 have studied the mechanism of plasteinsynthesis with α-chymotrypsin and found that itproceeds 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 undergoesaminolysis as a result of a nucleophilic attack ofa second peptide. The plastein reaction has alsobeen found useful for protein recovery from ex-tensively autolytically produced fish silage.171 Itis strongly recommended that this process be stud-ied further for fish protein hydrolysis.

V. FUTURE DEVELOPMENTS ANDPOTENTIAL APPLICATIONS

The use of FPH as a functional food ingredi-ent still has a long way to go until it becomeseconomically feasible and accepted by industryand consumers. However, there is a wonderfulopportunity for this to happen, due to regulations

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

of processing waste and the abundance of smallpelagic underutilized species that will be the fron-tiers of fisheries in this century. These factorsshould stimulate the conversion of low value fishmaterials into more valuable and palatable prod-ucts. Other applications outside the food industrymay also be a feasible option for FPH. Soluble

FPH from extensive hydrolysis is an excellentsource of nitrogen for maintaining the growth ofdifferent microorganisms. Previously, it was notedthat extensive hydrolysis of fish protein results ina product containing free amino acids and low-molecular-weight peptides, which is very promis-ing for microbial peptone production.129

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The beneficial chemical composition of FPHand FPC has also led to using these materials asfertilizer with good results. The FPH might be tooexpensive for this purpose, as added enzymes areused, and the product is usually dried. However,Ferreira and Hultin104 developed an enzymaticprocess using the acid protease Newlase A toextensively hydrolyze cod frames. This process iscurrently being used. The use of FPC as fertilizeris more common, either from chemical hydrolysisor autolysis. A successful process using second-ary raw material from processing plants (framesand viscera) and using the acid endogenous en-zymes to extensively hydrolyze the material isbeing operated in Washington State. The finalproduct is sold in bulk to local farmers, mainlycranberry growers, and with superior results com-pared with other commercially available fertiliz-ers.172 The mechanism behind the effect of theFPH and FPC to stimulate growth and develop-ment better than synthetic fertilizers need to bestudied further. Novel applications taking advan-tage of plant growth-stimulating effect of FPHand FPC could possibly be developed. An exten-sive research program supervised by Dr. KalidasShetty at the University of Massachusetts atAmherst examines the effect of FPH on plants. Inone study the effects of using FPH to stimulatesomatic embryogenesis in Anise (Pimpinellaanisum) when compared with proline, a knownstimulator, was examined.173 The conclusion wasthat FPH could well become a proline and aminoacid substitute in plant tissue culture applications.The positive effects of FPH due to proline and itsprecursor glutamate on plant growth was con-firmed in a recent study by the same group, wheremelon (Cucumis melo L.) shoot organogenesiswas stimulated.174 Proline and glutamate can beobtained from FPH and potentially can be usedfor value-added applications in the plant propaga-tion industry,174 a new arena of application forFPH and possibly a very valuable one.

Using FPH and FPC for animal feed applica-tions due to its good amino acid balance and highprotein content could be quite feasible. As men-tioned previously, fish silage is used primarily forthis purpose and is essentially limited to younganimals, due to the extensive hydrolysis of theproteins.67

Another little-studied property of FPH is itsantioxidant potential. Studies have found power-ful antioxidant activity in intact fish protein131 andin hydrolysate.59 These effects are probably highlydependent on the amino acid composition andmolecular size of the FPH peptides and deservefurther study.

VI. CONCLUSION

There have been several attempts to makefunctional fish protein hydrolysates with enzymes,some successful and some not. Most of thesestudies are rather crude, perhaps due to the ap-plied nature of the field, and have many short-comings, specifically failing to compare enzymesat the same activity level, controlling the %DHproperly and characterizing the chemical and func-tional properties of the final products. There ispotential for these products to be produced andsold as functional food ingredients, but at presentother applications such as plant nutrients, fertiliz-ers, and animal feeds might be more feasible.Standardized procedures to examine the functionalproperties are needed, as well as more studies onusing endogenous enzymes to make functionalFPH. With more basic research on the molecularlevel the future of FPH could be bright, especiallyin light of the environmental problems facingfisheries. It is time for FPH research to take anew, fresher, and more scientific direction, be-cause the field has been more or less stagnant forthe last 20 years or so.

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Biochemical, and Functional Properties Fish Protein ......Enzymatic modification of proteins using selected proteolytic enzyme preparations to cleave specific peptide bonds is widely