CAPÍTULO 3: REQUERIMIENTOS DEL SISTEMA
3.9. Conclusiones
Fabienne Guérard1(u) · Daniel Sellos2· Yves Le Gal2
1ANTiOX-UBO, Pôle universitaire P.J. Helias, Creac’h Gwen, 29000 Quimper, France [email protected]
2Marine Biology Station, Muséum National d’Histoire Naturelle, BP 225, 29182 Concarneau cedex, France
1 Introduction. . . . 129 2 Enzymes From Fish and Other Marine Creatures . . . . 130
3 Fish and Shellfish Protein Hydrolysates. . . . 131 3.1 From Fish Silage to Fish Protein Hydrolysates . . . . 131 3.2 Advantages of Commercial Exogenous Enzyme Addition . . . . 133 3.3 Quantification of the Proteolysis Extent . . . . 137 3.4 Mechanism of Hydrolysis and General Properties of Hydrolysates . . . . . 138
4 Recent Developments in Fish Protein Hydrolysates . . . . 140 4.1 Biologically Active Substances in By-Product Hydrolysates . . . . 140 4.1.1 Neuroactive Peptides . . . . 140 4.1.2 Enzyme Regulators and Inhibitors . . . . 141 4.1.3 Immunoactive Peptides . . . . 142 4.1.4 Hormonal and Hormonal-Regulating Peptides . . . . 142 4.1.5 Antioxidant Activities . . . . 143 4.2 Marine Waste as a Nutrient Source in Fermentation Processes . . . . 145 4.3 Other Applications of FPHs . . . . 146
5 Genetic Traceability of Fish and Shellfish Species and By-Products . . . . 146 5.1 Choice of Marker Sequences . . . . 147 5.2 Protocols for Fish and Fish Coproduct Identification . . . . 150 6 Conclusion . . . . 157 References . . . . 158
Abstract Recognition of the limited biological resources and the increasing environmen-tal pollution has emphasised the need for better utilisation of by-products from the fisheries. Currently, the seafood industry is dependent on the processing of the few selected fish and shellfish species that are highly popular with consumers but, from eco-nomic and nutritional points of view, it is essential to utilise the entire catch. In this review, we will focus on recent developments and innovations in the field of underutilised marine species and marine by-product upgrading and, more precisely, on two aspects of the bioconversion of wastes from marine organisms, i.e. extraction of enzymes and preparation of protein hydrolysates. We will deal with the question of accurate determin-ation of fish species at the various steps of processing. Methods of genetic identificdetermin-ation applicable to fresh fish samples and to derived products will be described.
128 F. Guérard et al.
Keywords Marine by-products· Hydrolysates · Bioactivity · Genetic traceability
Abbreviations
MWDP Molecular weight distribution of peptides N Nitrogen content
UF Ultrafiltration UV Ultraviolet
1
Introduction
In the preparation of sea food products for today’s consumer market, up to 50% of the whole animal is commonly discarded. The remainder is consi-dered as a waste or by-product, even though often high in protein. The wastes from most fishes comprise the skeletal structure, intestinal organs and also a large amount of edible fish muscle that cannot be easily removed from the bone structure of the fish by conventional fish filleting processes. The prin-ciple use for the waste has been for fish meal production, with some specific types of waste being utilised for human consumption, e.g. cod livers for cod liver oil [1].
Recognition of the limited biological resources and the increasing envi-ronmental pollution has emphasised the need for better utilisation of by-products from the fisheries. Currently, the seafood industry is dependent on the processing of the few selected fish and shellfish species that are highly popular with consumers, but, from economic and nutritional points of view, it is essential to utilise the entire catch [2]. The increasing demand for protein on a global scale also turns the focus on underutilised protein sources.
According to the 2002 FAO annual report [3], the world inland and marine aquatic resources were estimated to be 128.8 million tonnes in 2001. Global production from capture fisheries, aquaculture and the food fish supply is currently the highest on record and remains very significant for global food security, providing more than 15% of total animal protein supplies. Among these resources, marine captures accounted for 86 million tonnes while ma-rine aquaculture accounted for 14.2 million tonnes. This results in about 50 million tonnes of wastes available as a source of raw material including fish guts, which could be further processed for recovering useful enzymes as will be discussed below. Solid wastes generated by seafood plants range from about 30 to 85% of the weight of the landed fish, that is, the portion left after the fillets have been removed, depending upon the type of fishery. Processing of fin fish, crab and shrimp can result in 30–60%, 75–85% and 40–80% waste, respectively.
There are many ways in which the fish and shellfish waste could be better utilised, including the following [1]:
• Extraction of chitin, enzymes, oils, vitamins, pigments, flavour material
• Production of gelatine, chitosan, fish leather
Production of novel food ingredients from such underutilised aquatic species or by-products is desirable. According to Shahidi [4], fish proteins possess
ex-130 F. Guérard et al.
cellent amino acid scores and digestibility characteristics and as such, may be used to enhance the nutritive value of cereal-based foods.
In this chapter, we will focus on recent developments and innovations in the field of underutilised marine species and marine by-product upgrad-ing and, more precisely, on two aspects of the bioconversion of wastes from marine organisms, i.e. extraction of enzymes and preparation of protein hy-drolysates.
We will deal with the question of accurate determination of fish species at the various steps of processing. Methods of genetic identification applicable to fresh fish samples as well as to derived products will be described.
The potential uses of marine hydrolysates in the near future may be in the production of bioactive substances as this process is currently under control, in the case of casein and soya hydrolysates. We will not deliberately discuss fish meal for animal nutrition, fish protein concentrates as a cheap nutritious protein source for developing countries, and fish silage product development using mince from low-cost fishery resources (e.g. sausages, etc.) (see the re-views of Sikorski & Naczk [5], Raa & Gildberg [6], Venugopal and Shahidi [7], respectively).
2
Enzymes From Fish and Other Marine Creatures
In recent years, a number of enzymes from fish processing wastes have be-come commercially available for food and other applications, as reviewed by Haard [8]. Aquatic organisms include a wide and extensive taxonomic di-versity and many organisms occupy unusual environmental habitats, thus conferring to enzymes some unique characteristics such as psychrophilic properties. The most extensively studied enzymes from the marine envi-ronment include pepsin, trypsin, chymotrypsin, elastase, collagenase and alkaline phosphatase isolated from Atlantic cod (Gadus morhua) viscera [9–
11], polar cod (Boreogadus saida) [12], dogfish [13–15], salmon [16, 17] and tropical tuna [18]. Several of these enzymes from poikilothermal organisms are cold-active and have a catalytic activity equal or higher than mammalian enzymes.
For example, the temperature optimums of trypsin and alkaline phophatase from cold-water fish are about 30◦C lower than the homologues from warm-water fish or mammals [19, 20]. This property is advantageous in applications where it is desirable to inactivate the enzyme with a mild heat treatment [8].
Nevertheless, a trypsin purified from the pyloric caeca of white craker (Micro-pogonias furnieri) exhibited a temperature optimum of 60◦C. This could be related to the warm water in which the fish lived [21]. Enzymes like LDH from deep-water fish that live at high pressure have a tighter polypeptide struc-ture than homologues at normal atmospheric temperastruc-ture, thus making them
more resistant to proteolytic degradation and more suitable for applications where proteases might interfere with their activity [22]. The gastric proteases of fish that take in saltwater during feeding are salt-activated, in contrast to homologues from mammals that are inhibited by NaCl. This property may be advantageous in applications, such as fermentations, silage and fish sauce, where significant amounts of salt are present [23]. Lysozyme from Artic scal-lop [24] and other fish have a unique ability to attack both Gram-positive and negative bacteria.
According to Vilhelmsson [25], research in this area dates back at least to the early 1970s and the literature was sparse on the subject. However, there has recently been a surge of interest in enzymes from the marine environment and their potential use in food processing, mainly in North European coun-tries. These include a commercially available collagenase preparation from crab hepatopancreas that may have several applications, such as the deskin-ning of squid (Loligo sp.,), production of caviar, and ripedeskin-ning of salt fish.
Cold-active fish pepsins from species such as Atlantic cod (G. morhua) and orange roughly (Hoplostetus atlanticus) have been used for caviar produc-tion from the roe of various species. These proteolytic enzymes are used to ease the riddling process, increasing the yield from 70% to 90% for salmon (S. salar) for example. A protease preparation from Novo Nordisk was found to double the yield of roe from rainbow trout O. mykiss [26].
In conclusion, these examples illustrate the fact that enzymes from aquatic organisms will never fit more than a niche in food processing because of the cut-throat competition with enzymes of microbial origin. However, with the advent of recombinant DNA technology, there is also growing interest in cloning genes for unique biochemicals from exotic aquatic organisms for mass production by microbial or other expression systems [8].
3
Fish and Shellfish Protein Hydrolysates 3.1
From Fish Silage to Fish Protein Hydrolysates
Fish silage is a liquid product, made from whole fish or parts of fish, to which no material has been added other than a mineral acid to lower the pH to values be-low 4.5. Liquefaction is carried out by endogenous enzymes naturally present in the fish. Acid aids in accelerating the process by creating the right conditions for the enzymes to work and by helping to break down bone. This procedure efficiently prevents growth of spoilage bacteria [6]. After liquefaction, it is con-venient to remove the oil coming from the raw material. The protein in the aqueous layer may thereafter be dried or semi-dried. The main advantages of fish silage are the recovery of fish offal and waste fish, low cost, good nutritional
132 F. Guérard et al.
value of the resulting product and long storage life. The main inconvenience is the impossibility of regulating the degree of hydrolysis achieved.
In recent years the enzyme-catalysed process of hydrolysis, as applied to protein-containing raw materials used by the food industry, has been the ob-ject of numerous studies. It has been found that hydrolysis of the proteins themselves may increase yields in recovery processes, improve functional properties or improve process methodology, e.g. with regards to possible means of control. In more general terms, in the mild conditions that charac-terise enzymatic processes, a protein tends to retain its nutritive value better than in traditional acidic or alkaline hydrolysis [27].
Fish protein hydrolysates (FPHs) are products with high protein content and a wide variety of uses, as reviewed by Mackie [28] and Kristinsson and Rasco [29]. There are several methods of FPHs production, which include utilisation of acids, bases [30], endogenous enzymes or exogenous proteases.
Research during the past 20 years has greatly increased the understanding of how to process fish or shellfish by-product hydrolysates [25, 31–34]. Gene-rally, underutilised fish, fish frames or crustacean wastes are suspended in water and enzyme is added to the slurry. In some cases, the meat is first heated in order to denature the endogenous proteases [35]. The reaction is al-lowed to proceed from under 1 h to 1 week, depending on the activity of the enzyme employed, process temperature and other factors. After separation of solids, pH is adjusted and the aqueous layer is clarified, and then dehydrated.
Figure 1 outlines the main steps of the production of protein hydrolysates from the raw material.
Cassia et al. [36] described the obtention of FPHs using an autolytic pro-cess. The authors concluded that enzymatic autolysis might be a simple and efficient process for upgrading fish filleting wastes. However, although the FPHs had high protein and low lipid content, together with an amino acid composition similar to FAO/WHO standards, the process yield was rather low (< 7%). Shahidi et al. [4] showed that endogenous enzyme alone pro-duced hydrolysates with a protein recovery of approximately 23%, whereas a yield of 51.6–70.6% was obtained for commercial enzymes. Shahidi & Syno-wieck [30] described an alkali-assisted extraction of proteins from meat and bone residues of harp seal with a high recovery of proteins, ranging from 57%
to 64%, together with a high level of taurine, excellent emulsifying capacity and emulsion stability.
In addition, processing by-products from shellfish is made up of pro-tein residues from body sections such as heads, carapace and exoskeleton.
Enzyme-assisted proteolysis of shellfish processing discards may be used to recover the chitin and nutritionally valuable protein hydrolysate containing up to 64% of protein and 81% of total nitrogen in the product [37], or to ex-tract proteins with flavour-enhancing effects, including carotenoids and/or carotenoproteins [38]. A new process for advanced utilisation of shrimp wastes that includes enzymatic hydrolysis was recently described. The authors
Fig. 1 Flowsheet for the enzymatic hydrolysis of fish or shellfish proteins to make fish or shellfish protein hydrolysates
demonstrated the recovery of amino acids, nitrogen and astaxanthin by Al-calase pre-treatment of shrimp waste before further processing in chitosan.
The nitrogen recovery was about 70% as compared to only 15% by conven-tional methods. The yield and quality of chitosan was not affected by the enzymatic treatment. In addition, a concentrate of astaxanthin was recovered and could constitute a valuable supplement in salmon feed, improving both the growth and the disease resistance of the fish [39].
3.2
Advantages of Commercial Exogenous Enzyme Addition
The solubilisation of fish tissue in traditional silage production is a time-consuming process. After 3–10 days, depending on the storage temperature, the degree of hydrolysis (DH) is around 20–70%. Addition of commercial exogenous enzymes to the fish tissue reduces the time needed to obtain a similar DH and allows a control of the DH, and subsequently of the peptide size obtained. The choice of hydrolysis process will depend on the targeted applications. For dietary use, or in order to obtain a hydrolysate with a high nutritional and therapeutic value, it has been shown that protein hydrolysates should be rich in low molecular weight peptides, with as few free amino acids as possible [40]. On the other hand, large molecular weight peptides (more than 20 amino acid residues) are presumed to be associated with the improve-ment functionality of hydrolysates.
134 F. Guérard et al.
Table 1 Comparison between chemical and enzymatic hydrolysis [41, 42, 48]
Specificity Advantages Disavantages
Acid/ Random Fast reaction High temperatures
alkaline process Complete hydrolysis Molecular weight
hydrolysis out of control
Low cost Large amount of salt
High solubility Undesirable side reactions (destruction of tryptophan, racemisation, etc.)
Enzymatic Unique Control of the Higher cost
hydrolysis specificity molecular weight
Digestion under mild Subsequent deactivation
conditions of the enzyme
Attractive functional Time consuming product characteristics
Few side reactions No destruction of amino acids
Higher nutritional value
Enzymatic hydrolysis using commercial exogenous proteases presents a lot of advantages compared to chemical hydrolysis (Table 1).
Most commercial proteases can be used to solubilise marine wastes. They are obtained from animal viscera, plants and GRAS microorganisms. Selected examples of proteolytic enzymes used to hydrolyse marine by-products, are presented in Table 2. With regards to the effect of the concentration of enzyme in the reaction solution, it has been found that the percent hydrolysis in-creases with higher concentrations of enzyme, but only up to a certain point.
When using an endopeptidase, such as Alcalase 2,4 L for hydrolysis, so as to moderate DH values (i.e. below DH 20%), the relative content of free amino acids and dipeptides is likely to be low [43].
By selecting both the enzyme and the conditions of digestion, various de-grees of hydrolysis or breakdown of the proteins can be achieved in order to obtain products with a range of functional properties. Much of the work is still at the laboratory or pilot-scale level, but there is good reason to be con-fident that these biological processes will make some of this “waste” protein available as hydrolysates containing bioactive substances (see Sect. 4).
In some cases, experimental designs were employed to optimise hydro-lysis conditions. Simpson et al. [56] used a 3× 3 factorial central composite
Table 2 Selected examples of proteolytic enzymes used to hydrolyse marine wastes Enzymes Suppliers Substrates Appli- Evaluation
Refe-cations of hydrolysis rences
Papain Solvay Herring 1 TCA soluble N, [44]
Enzymes (Clupea harengus) total N, color, sensory, MWDP
Sigma Lobster cephalothorax 1 TL, NP, NSI [31, 45]
(Palinurus sp.)
Sigma Capelin (Mallotus 1, 2 pH-st, FP, NP [4, 46]
villosus)
Merck Sardine 1 pH-st, NR [33]
(Sardina pilchardus)
Pepsin Sigma Lobster cephalothorax 1 TL, NP, NSI [31, 45]
(Palinurus sp.)
Sigma Atlantic cod 1 pH-stat, MWDP [46]
(Gadus morhua)
Fungal Sigma Lobster cephalothorax 1 TL, NP, NSI [31, 45]
protease (Palinurus sp.) type II
from A. oryzae
Alcalase Novo Capelin 1, 2 pH-st, FP, NP [4, 32, 46]
2, 4L Nordisk (Mallotus villosus)
Shrimp wastes 1 pH-stat, NP [37]
(Crangon crangon)
Salmon muscle 1 pH-stat, NSI, FP [48]
(Samon salar)
Tuna stomach 1, 2 pH-stat, MWDP [35, 49]
(Tunus albacora)
Herring 1 TCA soluble N, [44]
(Clupea harengus) total N, colour, sensory, MWDP Pacific whiting 1 DH-α-amino acid, [50]
(Merluccius productus) NR, colour
Dogfish fillet np pH-stat, RSM,NR [34, 51]
(Squalus acanthias)
Atlantic cod 1 pH-stat, MWDP [47]
(Gadus morhua)
and salmon (Salmo salar)
Shrimp waste 1 NR [39]
(Pandalus borealis)
Sardine 1, 2 pH-stat, MWDP [52]
(Sardina pilchardus)
Harp seal 1,2 pH-stat, FP [4, 53]
(Phoca groenlandica)
136 F. Guérard et al.
Table 2 (continued)
Enzymes Suppliers Substrates Appli- Evaluation Refe-cations of hydrolysis rences
Alcalase Novo Sardine 1 pH-st, NR [33]
0, 6L Nordisk (Sardina pilchardus)
Neutrase Novo Capelin 1, 2 pH-st, FP, N, [4, 32, 46]
0.5L Nordisk (Mallotus villosus)
Sardine 1 pH-st, NR [33]
(Sardina pilchardus)
Pacific whiting 1 DH-α-amino [50]
(Merluccius productus) acid, NR, colour
Harp seal 1 pH-stat, FP [4, 53]
(Phoca groenlandica)
PTN 3.0 Novo Capelin 1 pH-st [32]
type special Nordisk (Mallotus villosus)
Corolase 7089, Novo Salmon muscle 1 pH-stat, [48]
Corolase PN-L, Nordisk (Salmo salar) NSI, FP Flavourzyme
1000L
Umamizyme Novo Tuna stomach 1 pH-stat, [54]
Nordisk (Tunus albacora) MWDP
Protamex Frames of Atlantic np NR [55]
Salmon (Salmo salar L)
1 dietary protein source, 2 biological activities, np not precised, TL tyrosine level, pH-st pH-pH-stat method, SC soluble content, N nitrogen content, TCA TCA soluble protein, FP functional properties, NP nutritional properties, NSI nitrogen solubility index, NR nitro-gen released, RSM response surface methodology, MWDP molecular weight distribution of peptides
design to optimise hydrolysis of shrimp for recovery of amino acids. The second order polynomial models they proposed could be used to predict the content of specific amino acids to a reasonable degree of accuracy. The response surface regression procedure of the statistical analysis system was used by Shahidi et al. [46, 53] in order to fit a quadratic polynomial equation to the experimental data. The three-dimensional response surface indicated that both the Alcalase concentration and the treatment temperature affected the DH and thus the protein recovery. Response surface methodology was used by several authors in order to study the effects of pH, temperature, enzyme/substrate ratio and substrate concentration on the degree of hydro-lysis of crayfish by-products [57] and dogfish muscle [51, 58]. The resulting equations were adequate for predicting the DH under any combination of values of the variables.
3.3
Quantification of the Proteolysis Extent
When using exogenous proteases, the hydrolysis reaction must be carefully controlled in order to maintain uniform quality of the end products. The hy-drolysis degree (DH), which is defined as the percentage of cleaved peptide bonds, serves as the controlling parameter for the hydrolysis reaction. The pH-stat technique consists in adding acid or base in order to titrate the re-leased α-amino and α-carboxyl groups, thus maintaining the pH constant.
Equation 1 relates the DH to alkali consumption [59]:
DH = B× Nb1
whereα–1is the calibration factor for the pH-stat, and is the reciprocal of the degree of dissociation:
α = 10pH-pK
α = 10pH-pK